IA"RC HO~CXPAPHS

ON TH
EVALATION OF TH CA~CINCXEniC RISK
OF CHENICAS 'I MA:

Sorn Hiscellaneous Pharnceutical Substances

Volur 13

This publication reDresents the views of an

IA Working Group on the
Evaluation of the Carcinogenic Risk of Chemcals to .l'l
which rnt in Lyon,
18-25 October 1976
IARC i~RKNC CROU ON TH EVATION OF TH CAI~CINOSENIC PJSK OF CHElITCA
'I MA: sa MISCELIANEOUS PHARr1ACEICA SUBTANS
Lyon, 18-25 October 1976

Mers1
Dr L. Baa, Head, Departrnt of Chemcal Carcinogenesis, Cancer Research
Institute, Slovak Academ of Sciences, ul. Cs. ardy 21,
880 32 Bratislava, Czechoslovakia

Professor L. Fiore-Donati, Director, Istituto di Antomia e Istologia

Patologica, Policlinico Borgo Ram, 37100 Verona, Italy

Dr P. Grasso, Deputy Director and Chef Pathologist, The British Industrial

Biological Research Association (BIBRA), Wosteme Raad, Cashalton,
Surrey SMS 4DS, OK

Dr T. Hirayam, Chief, Epidemolog Division, National Cacer Center Research

Institute, Tsukji 5-chor, Chuo-ku, Tokyo, Japa

Professor D.B. Ludlum, Aly Meical College, Depant of Phacolog

and Exrimntal Therapeutics, Aly, New York 12208, USA

Dr L. Massé, Ecole Nationale de la Saté Pulique, 35043 Rees Cmex,

France (Vice-Chairman)

Dr J. McCan, Departrt of Biochemstr, University of Californa, Bekeley,

Califomia 94720, USA

Dr V. B. Okulov, N. Petrov Research Insti tute of Oncolog, Leingradskaya

Street 68, Pesochny-2, Leingrad 188646, USSR

Professor R. Preussm, Deutsches Krebsforschungszentr, Institut für

Toxiologie und Cheitherapie, lm Neueneimr Feld 280, Postfach 101949,
6900 Heidelbrg l, FRG (Chairman)

Professor A. Somoi, Bundesgesundheitsam, Thelallee 88/92, Postfach -

1000 Berlin 33, FRG

Dr G.M. William, Chief, Division of Exrimtal Patholog, Naylor Dana

Institute for Disease Prevention, Arrican Health Foundation, Valhalla,
New York 10595, USA

lUnale to attend: Professor D.B. Clayson, Deputy Director, The Eppley

Insti tute for Research in Cacer, Uni versi ty of Neraska ~ical Ceter,
42nd and Deey Avenue, Cm, Neraska 68105, USA; Professor S. ('"aattini,
Director, Istituto di Ricerche Farcologiche "Maio Neri:l, Via Eri trea 62,
20157 Milan, Italy

3
Invited Guest

Dr K.E. HcCaleb, Director, Chemcal-Environrtal Program, Chemcal Industries

Center, Stanford Research Institute, !1elo Park, California 94025, USA
(Rapporteur sections 2.1 and 2.2)

Representative
~
froID the National Cancer Insti tute
Dr S. Siegel, Coordinator, Technical Inforntion Acti vi ties, Bioassay and
Carcinogenesis Program, Division of Cacer Cause and Prevention,
National Cancer Institute, Bethesda, .lßrland 20014, USA

Secretariat
Dr C. Agthe, Chief, Foo Addi ti ves Unit, ílmO, Geeva
Dr H. Batsch, Unit of Chemcal Cacinogenesis (Rapporteur section 3.2)
Dr J.F. Bertaux, l1eical Officer, Pharnceuticals, WHO, (':ineva
Dr A. Davis, Chief, Schistosorasis and Oter Helmthic Infections,
WHO, Geneva
Dr L. Griciute, Chief, Unit of Environrntal Carcinogens
Mrs D. l1ietton, unit of Chemcal Carcinogenesis (Library Assistant)
Dr R. Montesano, Unit of Chemcal Cacinogenesis (Rapporteur section 3.1)
Mrs C. Partensky, Unit of Chemcal Cacinogenesis (Technical editor)
Mrs I. Peterschm tt, Unit of Chemcal Cacinogenesis, WHO, Geneva
(Bibliographie researcher)
Dr V. Ponomakov, Uni t of Chemcal Cacinogenesis
Dr R. Saracci, Unit of Epidemolog and Biostatistics (Rapporteur
section 3.3)
Dr L. Tomatis, Chief, Unit of Chemcal Cacinogenesis (Head of the
Programe and Secretary)
Iv.. E.A. Walker, Unit of Environrtal Cacinogens (Rapporteur
sections 1 and 2.3)
Mrs E. Ward, Montignac, France (Editor)
Mr J .D. Wilbum, Unit of Chemcal Carcinogenesis (Co-secretary)

4
 Note to the reader

Every effort is made to present the IInographs as accuately as

possible wi thout undul y delaying their publication. Neverteless,

mistaes have occured and are still likel y to occu. ln the interest of
all users of these IInogaphs, readers are reqested to communicate any
errors observed to the Unit of Chemcal Carcinogenesis of the International
Agency for Research on Cacer, Lyon, France, in order that these ca be
included in corrigenda which will appe in subseqent volurs.

Since the IInographs are not intended to be a review of the li terature

and contain onl y data considered relevant by the Working Group, i t is not

possible for the reader to determne whether a certain study was considered
or not. However, reseach workers who are awae of imrtant published
data that may chage the evaluation are reqested to mae ther available
to the abve-rntioned address, in order tht they ca be considered for
a possible re-evaluation by a future l'iTorking Group.

5
 CONTENTS

BACKGRO AN PUROSE OF TH IA PR(X ON TH EVATION

OF TH CAINX.:IC RISK OF CHECA 'I MA ............. 9

SCOPE OF TH MONCHS 9

MEHAISM FOR PRODUCING TH MONcxRAES 10

GEN PRINCIPLES FOR TH EVATION 11

EXIA'IRY NOIS ON TH M)cxRAHS 14

GE RE ON TH SUTANES CONSIDER 23

TH MONcxRAHS

Acriflavinium chloride ...................................... . 31

Aurothoglucose . .... ..... ...... .... ...... ... ... ... ... ........ 39
ChlorCXine .................................................. 47
Diazeoa and oxazeoa ........................................ 57
Di thanol .... ...... ....... ..... .......... .................. 75
Ethionamde ....................... 83
Hycanthone and hycanthone ræsylate 91
8-Hydroxyquinoline ............ .......... ........... .... ...... lOi
l'1etronidazole ..... ...... ............. ........... ..... .... .... 113
Niridazole .... ............ ......... .... ........ ... .... ..... 123
Oxtholone ....... ...... ... ... .... ..... .... ....... .......... 131
Phencetin . ....... ........ ... ...... ... ....................... 141
Phenobarbi tal and phenobarbital sodum ....................... 157
Phenylbutazone and oxyhenutazone ..... ..... ........... ...... 183
Phenytoin and phenytoin sodum ............................... 201
Pronetalol hydrochloride .. ..... ...... ... ............. ........ 227
Pyimthamne .. ........................ ....... ..... .... ...... 233

SUPPLEAR CORRGENA 'I VOLUS 1-12 243

CUTIV INEX 'I MONcxRAHS .................................. .. 245

7
 BACKCROUND lì...l\ PUP-POSE OF TH IlmC PECY:?P.. ON TH
EVATION OF TH CACINCXENIC RISK OF [Link] 'I l-WJ

The International AgenoJ for Research on Cacer (IAC) initiated in

1971 a program to evaluate the carcinogenic risk of chercals to ma.
This :orogam was sUDported by a Resolution of the ('-overning Council at
i ts Ninth Session concerning the role of IAC in providing governnt
authorities with exprt, indeoendent scientific opinion on environrntal
carcinogenesis .
In view of the irrtace of the project and in order to expite
production of rrnographs, the National Cacer Insti tute of the United
States has provided IAC wi th addi tional funds for this purse.

The obj ecti ve of the program 1S to elabrate and publish in the

forr of rrnographs critical reV1ews of carcinogenicity and related data
in the light of the present state of knowledge, with the final air of
evaluating the data in terr of -pssible hum risk, and at the sar tim
to indicate where additional research efforts are neeed.

SCOPE OF THE MONChRAHS

The rrnogaphs su:ize the ev.:_dence for the carcinogenici ty of

indi vidual chercals and other relevant informtion on the basis of data
compiled, reviewed and evaluated by a ílvorking Group of exorts. No reccr-
rnndations are gi ven concerning preventive measures or legislation, S1nce
these matters denend on risk-benefit evaluations, which see best made by
individual governnts and/or international agencies such as WHO and ILO.

Since 1971, when the program was started, twelve volurs have be
published 1-1 L. As new data on chemcals for which IInographs have already
been prepared and new principles for evaluation become available, re-
evaluations will be made at future meetings, and revised IInogaphs will
be published as necessary.

The rnnographs are distributed to international and governntal

agencies, are available to industries and scientists dealing wi th these
chemcals and are offered to any interested reader though their world-wide

9
distribution as a vllO publication. Trey also forr the basis of advice
fror IA on carcinogenesis fror these substances.

MEISM FOR PRODUCING TH MJOGHS

As a first step, a list of chemcals for possible consideration by
the Working Group is estalished. IAC then collects Pertinent references
regarding physico-chercal characteristics*, production and use*, occurrence*
and anlysis* and biological data** on these comunds. The material on
biological aspects is suirized by an exrt consultant or an IA staff
rnr, who prepaes the first draft, which in sor cases is sent to another
exprt for corrts. Th drafts are circuated to aii rrrs of the
Working Group abut one rnnth before the meting. During the meting,
further additions to and deletions fror the data are agree upn, and a
final version of commts and evaluation on each corund is adopted.

Priori ty for the preparation of monographs

Priori ty is gi ven mainl y to chemcals belonging to paticular chemcal

groups and for which there is at least sorr suggestion of carcinogenici ty
froID observations in animls and/or ma and evidence of hum expsure.
Hawever, the inclusion of a particular compound in a volume does not
necessari ly me an that i t is considered to be carcinogenic. Equally ~ the
fact that a substance has not yet been considered does not imply that it
is wi thout carcinogenic hazard.

Data on which the evaluation is based

wi th regard to the biological data, onl y published aricles and papers

already accepted for publication are reviewed. The IInographs are not
intended to be a full review of the literature, and they contain only data
considered relevant by the Working Group. Research workers who are áware

*Data provided by Chemcal Industries Ceter, Staford Research

Insti tute, Menlo Park, California, USA

**In the collection of original data reference was made to the series
of publications 'Surey of Compunds which have ben Tested for Carcinogenic
Activity' 13-18. l'lost informtion on rntagenicity was provided by The
Environrntal Mutagen Inforntion Center, Oakridge, Ten., USA.
10
of imrtt data (published or accepted for publication) that rry influence
the evaluation are invi ted to mae ther available to the Unit of Chercal
Cacinogenesis of the International li~gency for Research on Cacer, Lyon,
France.
The Working Group

The tasks of the Working Group are five-fold: (1) to ascertain that
all data have be collected; (2) ta select the data relevant for the
evaluation; (3) to determne whether the data, as sumized, will enale
the reader to follow the reasoning of the car ttee; ( 4 ) to j udge the
signficance of results of exrirtal and epiderological studies; and
(5) to mae an evaluation of carcinogenici ty .
The rrrs of the Working Group who paicipated in the consideration
of particular substances are listed at the beinning of each publication.
The rrrs serve in their indi vidual capaci ties as scientists and not as
representati ves of their governts or of any organization wi th which
they are affiliated.

GEN PRINIPLES FOR TH EVTION

The general principles for evaluation of carcinogenici ty were
elabrated by previous Working Groups and also applied to the substances
covered in ths volur.

Terrnolog
The terr 'chemcal carcinogenesis' in i ts widel y accepted sense is
used to indicate the induction or enhancernt of neolasia by chercals.
It is recogized that, in the strict etyilogical sense, this terr Iæans
the induction of cancer; howver, camn usage has led to i ts emloyrt
to denote the induction of various tys of neoplasm. The terr ' tururigen' ,
'oncogen' and 'blastoimen' have all been used syonyiusl y wi th 'carcinogen',
al though occasionall y 'tUlurigen' has ben used specif icall y to denote the
induction of beign tururs.

11
Response to carcinogens

In general, no distinction iS made between the induction of tUIurs

and the enhanceInt of tUIur incidence, although it is noted that there
rny be fundartal differences in mechanisr that will eventuall y be
elucidated. The response of exprirntal anirls to a carcinogen may take
several forr: a significant increase in the incidence of one or IIre of
the sar typs of neoplasrn as found in control anirls; the occurrence
of typs of neoplasrn not observed in control animls; and/or a decreased
latent oeriod for the production of neoplasr as compared with that in
control animls.
Puity of the corunds tested
In any evaluation of biological data with respect to a possible
carcinogenic risk, paticular attention mut be paid to the purity of the
chercals tested and to their stability under conditions of storage or
admnistration. Informtion on purity and stability is given, when
available, in the IInographs.

Quali tati ve aspects

In may instances, bath benign and malignant tururs are induced by
chercal carcinogens. There are so far few recorded instances in which onl y
benign tururs are induced by chercals that have ben studied extensively.
Their occurrencein exprimntal system has been taken to indicate the
possibili ty of an increased risk of malignant tUIurs also.
In exprirntal carcinogenesis, the typ of cancer seen may be the
sam as that recorded in hum studies, e. g ., bladder cancer in ma, monkeys,
dogs and hamters after admistration of 2-naphthy lamne. In other instances,
however, a chercal may induce other typs of neoplasr at different si tes
in various species, e. g ., benzidine induces hepatic carcinoma in rats but
bladder carcinom in ma.
Quantitative aspects

Dose-resPOnse studies are imrtant in the evaluation of hum and

animl carcinogenesis: the confidence wi th which a carcinogenic ef fect
can be established is strengthened by the observation of an increasing

12
incidence of neoplasr wi th increasing eXPsure. ln addition, such studies
forr the only basis on which a miniml effective dose can be established,
allowing sor corrison wi th data for hum expsure.
Corarison of corunds wi th regard to potency can onl y be made [Link]
the substaces have been tested sirul taneousl y.
Animl data in relation to the evaluation of risk to ma
At the present tir no attemt can be made to interpret the animl
data directly in terr of hum risk, since no objective criteria are
available to do so. The cri tical assesSTnts of the validi ty of the animl
data given in these IInographs are intended to assist national and/or
international authorities in rning decisions concerning preventive measures
or legislation. In ths connection attention is drawn to í'\1HO recomrndations
in relation to foo additives 19, drgs20 and occupational carcinogensL 1.

Evidence of hum carcinogenici ty

Evaluation of the carcinogenic risk to ma of suspected environrntal
agents rests on purel y observational studies. Such studies must cover a
sufficient variation in levels of hum expsure to allow a meaningful
relationship between cancer incidence and expsure to a given chercal to
be established. Difficulties arise in isolating the effects of individual
agents, however, since people are usuall y expsed to multiple carcinogens.
The initial suggestion of a relationship between an agent and disease
often corns froID case reports of -ptients wi th simlar expsures. Varia-
tions and tir trends in regional or national cancer incidences, or their
correlation wi th regional or national 'expsure' levels, may also provide
valuable insights. Such observations by therel ves carot 1 however 1 in rrst
circurtances be regarded as conclusive evidence of carcinogenici ty .
The rrst satisfactory epidemological method is to compare the cancer
risk (adjusted for age, sex and other confounding variables) amng groups
or cohorts, or amng indi viduals expsed to various levels of the agent in
question, and aIng control groups not so expsed. Ideally, this ls accom-
plished directly, by following such groups forwd in tim (prospectively)
to determne tim relationships, dose-response relationships and other
aspects of cancer induction. Lage cohorts and long observation periods
13
 are reqired to pravide sufficicnt cases for a statistically valid
comparison.
An alterntive to prospective investigation is to assemle cohorts
froID past records and to evaluate their subseqent IIrbidity or IIrtality
by means of rnical histories and death certificates. Such occupational
carcinogens as nickel, ß-naphthylarne, asbestos and benzidine have ben
confirr by this method. Another rrthod is to compe the past expsures
of a defined group of cacer cases with those of control samles froID the
hospital or general population. Ths does not provide an absolute measure
of carcinogenic risk but ca indicate the relative risks associated with
different levels of expsure. Indirect rrans (e.g., interviews or tissue
residues) of rrasuring exsures which may have comrnced rny years before
can constitute a major source of error. Neverteless, such 'case-control'
studies can often isolate one factor froID several suspected agents and can
thus indicate which substance should be followed up by cohort studies.

EXlA'IRY NOS ON TH MON(XHS

In sections l, 2 and 3 of each ffnograph, except for mior remks,
the data are recorded as gi ven by the author, whereas the comrnts by the
Working Group are gi ven in section 4, headed 'Cornts on Data RelXrted
and Evaluation 1 .
Chemcal and Physical Data (section 1)
The Chemcal Abstracts Reistry Serial Numr and the latest Chemcal
Abstracts Nar are recorded in this section. Oter synonyr and trade
nars are given, but this list ls not intended to be comprehensive. It
should also be noted that sorn of the trade nars are those of mixtures in
which the compund being evaluated is only one of the active ingredients.

Chemcal and physical properties include, in paticular, data that

might be relevant to carcinogenicity (for exale, lipid solubility) and
those that concern identification. All chemcal data in this section refer
ta the pure substance, unless otherwise speified.

14
Production, Use, Occurence and Anl- vsis (section 2)
The purse of this section is to indicate the extent of possible
hum expsure. wi th regard to data on production, use and occurrence,
rAC has collabrated wi th the Staford Research Insti tute, USA, wi th the
support of the National Cancer Insti tute of the USA. Since cancer ls a
delayed toxic effect, past use and production data are also provided.

The United States, Euope and Japan are reasonabl y representati ve

industrialized areas of the world, and if data on production or use are
available froID these countries they are reported. It should not, hCMever,
be inferred tht these nations are the sole or even the major sources of
any indi vidual chercal.
Production data are obtained froID bath governntal and trade publi-
cations in the thee georaphic areas. In sar cases, separate production
data on chemcals maufactured in the US v-re not available, for proprieta
reasons. However, the fact that a maufacturer acknowledges production of a
chercal to the US International Trade Comssion imlies that anual produc-
tion of that chemcal is greater than 450 kg or that its anual sales excee
$1000. Inforntion on use and occurence ls obtained by a review
of published data, complemnted by direct contact wi th maufacturers of the
chemcal in question; however, inforntion on onl y sar of the uses ls
available, and this section canot be considered to be comprehensive. In an
ef fort to provide estimtes of production in sorn Euope countries, the
Stanford Research Insti tute in Zurich sent general questionnaires to sorn of
those Euopean compies thought to produce the cornunds being evaluated.
Inforntion froID the replies to these questionnaires has been compiled by
country and included in the indi vidual IInographs.
Staterts concerning reglations in sorn countries are ITntioned as
exales onl y . They may not reflect the most recent situation, since such
legislation is in a constant state of change; nor should it be taen to
imply that other countries do not have simlar regulations. ln the case
of drgs, metion of the therapeutic uses of such chemcals does not
necessaril y represent present 1 y accepted therapeutic indications, nor does
it imly judgernt as to their clinical efficacy.

15
 The purse of the section on anal ysis is ta gi ve the reader a
general indication, rather than a colete review, of rrthods cited
in the literature. No attert is made ta evaluate the rrthods quoted.

!3iological Data Relevant ta the Evaluation of Cacinogenic

Risk to l-:1 (section 3)

The rrnographs are not intended to consider all reported studies.

Sorn studies were pursely omitted (a) because they were inadeqte, as
judged from previously describ criteria22-;¿ 5 (e.g., too short a duration,
tao few animls, por suri val or tao srll a dose) ; (b) because they onl y
confinr findings which have already ben fu11y describ¡ or (c) because
they were judged irrelevant for the purse of the evaluation. However, in
certain cases, reference is made to studies whch did not meet estalished
criteria of adeqacy, particularly when ths inforntion was considered a
useful supplernt to other reports or when it was the only data available.
Their inclusion does not, however, irly acceptance of the adeqcy of
their exprirtal design.

In general, the data recorded in this section are sumized as gi ven

by the author; however, certain shortcorgs of reporting or of exri-
mental design that were comrnted upon by the Working Group are gi ven in
square brackets.
Carcinogenicity and related studies in anls: M2tion is rrde of
all routes of admistration by which the camund has been adeqatel y
tested and of all species in which relevant tests have ben carried out.
In rrst cases, animl strains are given¡ general chaacteristics of mouse
strains have been reviewed26. Qutitative data are given to indicate
the order of magnitude of the effective doses. In general, the doses are
indicated as they appear in the original pa:oer; sometirs conversions
have ben made for better comparison. When the carcinogenici ty of know
rnetablites has been tested this also is reported.

Oter relevant biological data: ID data are given when available,

5 a

and other data on toxici ty are included when considered relevant. The
rntablic data included is restricted to studies shawing the metablic
fate of the chercal in animls and ma, and coarisons of animl and
hum data are made when possible. Oter rntablic inforntion (e.g.,
16
absorption, storage and cxcretion) is given when the hbrking Group considered
that it would be useful for the reader ta have a better understanding of the
fate of the comund in the boy.
Teratogenicity data fror studies ln exrimntal animls and ln hums
are included for san of the substances considered, hawever, they are not
meant to represent a thorough review of the li te rature .

Mutagenicity data are also included; the reasorts for including them
and the principles adopted by the Working Croup for their selection are
outlined below.

May, but not all, mutagens are carcinogens and vice versa; the exact
level of correlation is still under investigation. Neverteless, practical
use may be made of the available mutagenicity test procedures that combine
rncrobial, malian or other animl cell system as genetic targets wi th an
in vitro or in vivo rntablic activation system. The results of relatively
rapid and inexpnsi ve mutagenici ty tests on non-hum organisrn may help to
pre-screen chemcals and may also aid in the selection of the most relevant
animl species in which to carry out long-terr carcinogenici ty tests on
these chercals.
The role of genetic alterations in chemcal carcinogenesis ls not yet
fully understoo, and therefore consideration must be given to a variety
of changes. Although nuclear DNA has been defined as the main cellular
target for the induction of genetic changes, other relevant targets have
been recogized, e.g., mitochondrial DNA, enzyrs involved in DNA synthesis,
repair and recombination, and the spindle apparatus. Tests to detect the
genetic acti vi ty of chemcals, including gene mutation, strctural and
nurrical chrolIsoml changes and mitotic recomination, are available for
non-hum mcels; but not all such tests can be applied at present to hum
cells .
Ideall y, an appropriate mutagenici ty test system would include the full
metablic comtency of the intact hum. Since the developrænt or appli-
cation of such a system appears to be llssible, a battery of test system
is necessar in order to estalish the mutagenic potential of chemcals.
There are may genetic indicators and rætablic activation system available

17
 for detecting rnutagenic acti vi ty; they all, howver, have indi vidual advan-
tages and limtations.

Since may chemcals reqire rntalism to an active forr, test

system which do not tae this into account may fail to reveal the full
range of genetic damge. Futherrre, since sarr reactive rrtalites
with a limted lifespan may fail to reach or to react with the genetic
indicator, either because they are further rrtablized to inactive corrunds
or because they react wi th other cellular consti tuents, rntagenici ty tests
in intact animls may gi ve false negati ve resul ts.
It is difficult in the present state of knowledge to select specific
rnutagenicity tests as being the most appropriate for the pre-screening of
substances for possible carcinogenic acti vi ty . Hc:ever, greater reliance
may be placed on data obtained from those test system which (a) permt
identification of the nature of induced genetic changes, and (b) demnstrate
that the changes are transmtted to subseqent generations. Mutagenicity
tests using organisrn that are well -understoo geneticall y, e. g ., Escheri-
chia coli~ Salmonella typhimurium~ Saccharomyces and Drosophila, rret these
reqirernts.
Al though a correlation has often ben observed between the abili ty of
a chemcal to cause chrorrsorr breakage and i ts abili ty to induce gene
mutation, data on chrolIsoml breakage alone do not provide adequte
evidence for mutagenici ty, and therefore less weight should be gi ven to
pre-screening that is based on the use of peripheral leucoce cultures.
Because of the complexi ty of factors that can contribute to reproductive
failure, as well as the insensitivity of the rrthod, the dominant lethal
test in the m:l does not provide reliable data on mutagenici ty .

A large-scale systemtic screening of corrunds to assess a correlation

between mutagenici ty aDd carcinogenici ty has so far ben carried out onl y
wi th the bacterial/m:lian li ver microsom system. Notwi thstanding the
deinstra tion of the rntagenici ty of may know cacinogens to Sa lmone lla
typhimurium in the presence of liver rncrosonl system, the possibility
of false-negative and false-positive results must not be overlooked.
False negati ves might arise as a conseqence of rntagen spcifici ty or

18
frorn failure to achieve optiml conditions for activation in vitro.
Alternative test system must be used if there appear to be substantial
reasons for suspecting that a chemcal which is apparently non-mutagenic
in a bacterial test system may nevertheless be potentiall y carcinogenic.
Conversel y, sar chemcals found to be mutagenic in this test may not in
fact have rnutagenic activity in other system.

For more detailed informtion, see references 27-34.

Observations in ma: Case reports of cancer and epiderological

studies are sumrized in this section.
Commnts on Data Reported and Evaluation (section 4)
This section gi ves the cri tical view of the Working Group on the data
reported .

Animl data: The animl species rrntioned are those in which the
carcinogenicity of the substances was clearly deinstrated. TIe route of
admnistration used in exprimntal animls that is simlar to the possible
hum expsure (ingestion, inalation and skin expsure) is given particular
rnntion. Tuur sites are also indicated.
Exrimnts involving a possible action of the vehicle or a physical
effect of the agent, such as in studies by subutaneous injection or
bladder imlantation, are included; however, the results of such tests
reqire careful consideration, particularly if they are the only ones
raising a suspicion of carcinogenici ty . If the substance has produced
tumurs after pre-natal expsure or in single-dose exrimnts, this also
is indicated. This subsection should be read in the light of c0Tnts
made in the section, 'Anl Data in Helation to the Evaluation of Risk
ta Ma' of this introduction.

Hum data: In sorr cases, a brief staternt is made on possible

hum expsure. The significance of epidemological studies and case
reports is discussed, and the data are interpreted in terr of possible
hum risk.

19
 P,eferences

1. IAC (1972) IA Monographs on the Evaluation of Cacinogenic Risk

of Chercals to Ma, l, Lyon

2. IAC (1973) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to VEn, 2, Sa Inorganic and Organorrtallic
Compunds, Lyon

3. IAC (1973) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 3, Certain Pol ycyclic Aromatic Hydrocarbons
and Heterocclic Comunds, Lyon

4. IAC (1974) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 4, Sa Aromatic Amnes, Hvdrazine and
Related Substances, N=Nitroso Compunds and Miscellaneous
Alkylating Agents, Lyon

5. IAC (1974) lAC Monographs on the Evaluation of Cacinogenic Risk

of Chemcals to Ma, 5, Sorr Organochlorine Pesticides, Lyon

6. IAC (1974) IA Monographs on the Evluation of Carcinogenic Risk

of Chemcals to Ma, 6, $e HOrInes, Lyon

7. IAC (1974) IA Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 7, So Anti-thyroid and Related Substaces,
Nitrofurans and Industrial Chemcals, Lyon

8. IAC (1975) IA Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 8, Sorn Aromatic Azo Compunds, Lyon

9. IAC (1975) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 9, Sorn Aziridines, N- ~ 5- and Q-Mustards
and Selenium, Lyon

10. IAC (1976) lAC Monographs on the Evaluation of Cacinogenic Risk

of Chemcals to Ma, 10 ,So Naturall y Occurring Substaces,
Lyon

11. IAC (1976) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 11, Cadmum, Nickel, Sorr Eoxides, Miscella-
neous Industrial Chemals and Geeral Considerations on Volatile
Anaesthetics, Lyon

12. IAC (1976) IAC Monographs on the Evaluation of Carcinogenic Risk

of Chemcals to Ma, 12, Sa Carbamtes, Thiocarbamtes and
Carbazides, Lyon

13. Hartwell, J.L. (1951) Surey of Compunds which have ben Tested
for Carcinogenic Acti vi ty, Washington DC, US Governnt Printing
Office (Public Health Service Pulication No. 149)

20
14. Shubik, P. & Hartwell, J.L. (1957) Surey of Compunds which have
ben Tested for Cacinogenic Acti vi ty, i"¡ashington DC, US
Governnt Printing Office (Pulic Heal th Service Pulication
No. 149: Supplemt 1)
15. Shubik, P. & Harwell, J.L. (1969) Surey of COIunds which have
been Tested for Cacinogenic Acti vi ty, Washington DC, us
Governt Printing Office (Pulic Health Service Pulication
No. 149: Supplemnt 2)

16. Cacinogenesis Program National Cacer Intitute (1971) Surey of

Compunds which have been Tested for Cacinogenic Acti vi ty ,
Washington DC, us Governnt Printing Office (Pulic Health
Service Pulication No. 149: 1968-1969)

17. Carcinogenesis Program National Cacer Intitute (1973) Surey of

Compunds which have been Tested for Carcinogenic Acti vi ty ,
Washington DC, us Governt Printing Office (Public Heal th
Service Pulication No. 149: 1961-1967)

18. Carcinogenesis Program National Cacer Institute (1974) Suey of

Compunds which have been Tested for Cacinogenic Acti vi ty ,
Washington DC, us Governt Printing Office (Pulic Health
Service Pulication No. 149: 1970-1971)

19. WHO (1961) Fifth Report of the Joint FAO/WO Exrt Commttee on
Foo Additives. Evaluation of carcinogenic hazard of foo
addi ti ves . wid Hl th Org. techn. Rep. Ser., No. 220, Pp. 5, 18, 19
20. WHO (1969) Report of a WHO Scientific Group. Principles for the
testing and evaluation of drgs for cacinogenicity. wid Hlth
Orge techn. Rep. Ser., No. 426, pp. 19, 21, 22

21. WHO (1964) Report of a WHO Exprt Commttee. Prevention of cancer.

Wld Hlth Org. techn. Rep. Ser., No. 276, pp. 29, 30

22. WHO (1958) Second Rert of the Joint FAO/WO Exrt Commttee on
Foo Addi ti ves. Procedures for the testing of intentional
foo addi ti ves to establish their safety for use. wid Hl th Org.
techn. Rep. Ser., No. 144

23. WHO (1961) Fifth Report of the Joint FAO/WO Exrt Commttee on
Foo Additives. Evaluation of carcinogenic hazard of foo
additives. wid Hlth Org. techn. Rep. Ser., No. 220

24. WHO (1967) Scientific Group. Procedures for investigating intentional

and unintentional foo addi ti ves . Wld Hl th Org. techn. Rep. Ser.,
No. 348

25. Berenlum, 1., ed. (1969) Cacinogenicity testing. UICC techn. Rep.
Ser.,2

21
26. Corttee on Standadized C':ietic NOlTenclature for Hice (1972)
Stadadized no:rnclature for inred strains of rnce. Fifth
listing. Cacer Res., 32, 1609-1646

27. Bartsch, H. & Grover, P.L. (1976) Chemcal carcinogenesis and rnta-
genesis. In: Syrngton, T. & Carer, R.L., eds, Scientific
Foundations of Oncolog, Vol. IX, Chemcal Carcinogenesis,
lDndon, Heinem Meical Boks Ltd, pp. 334-342
28. Holländer, A., ed. (1971) Chemcal Mutagens: Principles and Method
for Their Detection, Vols 1-3, Ne York, Plenum Press

29. Montesano, R. & Tartis, L., eds (1974) Chemcal Cacinogenesis

Essays, Lyon (IA Scientific Pulications No. 10)

30. Ral, C., ed.(1973) Evluation of genetic risks of environrntal

chemcals: report of a symsium held at Skokloster, Sweden,
1972. Amio Special Report, No. 3
31. Stoltz, D.R., Poirier, L.A., Irving, C.C., Stich, H.F., Weisburger,
J.H. & Grice, H.C. (1974) Evluation of short-terr tests for
carcinogenicity. ToxicoL. appL. Phacol., 29, 157-180

32. WHO (1974) Report of WHO Scientific Group. Assess:rt of the

carcinogenici ty and rntagenici ty of chemcals. wid Hl th Org.
techn. Rep. Ser., No. 546

33. Montesano, R., Batsch, H. & Tartis, L., eds (1976) Screening Tests
in Chemcal Carcinogenesis, Lyon (lAC Scientific Pulications
No. 12)

34. Commttee 17 (1975) Environrntal rntagenic hazards. Science, 187,

503-514

22
 GE RENARK ON SUBTAS CONSIDER

This volum includes IInCXraphs on a numr of miscellaneous pharn-

ceutical substaces for which epidemological reports and/or animl studies
suggest a possible carcinogenic activity. Although the tenu 'drug' is often
used to describ compunds which are used illici tl y, for the purses of
this volur of rrnographs i t has been used synonyIusl y wi th the tenu
'pharceutical substance'. Since onl y a lim ted numr of compunds can
be evaluated at any one meting, the list of drgs reviewed here is not
rnt to be exhaustive. Certain other drgs have already been considered
in previous volurs of rrnographs in ths series (Vols 2, 4, 6, 7, 9 & 10).
A numr of drgs ~re considered but eventuall y not included
beause the available data were inadeqate. These drgs are:
acety lsalicy lic acid, ambabi tal, chlorpromzine, dipheny 1 th iohydantoin ,
lysergide (LSD), nitroxoline, petobarbital sodium, prednisone, pyrazinamde,
rifamicin and spironolactone. On-going carcinogenici ty studies in expri-
rnntal animls are in progess for rny of these substaces (IA, 1976).
A paticular case is that of acetylsalicylic acid (aspirin), for which
the available hum and exrimntal data were insufficient to mae
an evaluation: th results of only tw inadeqte cacinogenicity tests on
this very conm::m drg were available. The W:rking Group was aware, however,
of several on-going carcinCXenici ty studies on this compund. Al though
evidence of teratCXenicity is not necessarily related to an evaluation of
carcinogenici ty, i t is also worth noting that the exprirntal evidence of
a teratogenic action of aspirin has not been follaw up by adequate studies
to confinu or exclude a simlar effect in hums.

Azathioprine was also considered by ths Working Group. Al though

exprirtal evidence for its carcinogenicity existed, it was felt by soræ
mers of the Working Group that it would be IIre appropriate to consider
this compund in the context of other imosuppressi ve drgs.
Soræ of the compunds considered are amnes which, in principle, could
be converted to carcinogenic N-ni troso counds by reaction wi th ni tri te
under mildly acid conditions. Tt is well know that nitrate and, to a
lesser extent, nitrite occur in rny fc:s and that nitrate can be reduced

23
 to ni tri te in the gastrointestinal tract. Thus, N-ni trosodialky lames
could be forr in the stomach frorn a numr of drugs wi th tertiar arno
groups. Drugs which contain secondar arno groups could also forr the
corresponding N-ni troso drgs.

Regulatory reqirernts that include assay, identification and lirt

tests for imurities in pharceutical grade drgs or pharmceutical
products are gi ven in various national and international pharnco¡:ias,
such as the British Pharrcopoia, The US Phacopeia, WHO International
Pharncopoia and Euope Pharncopoia. Such inforntion is gi ven for
a numr of drgs considered in this volur. No attert was made to gi ve
such reqiremts in detail in the section on anal ysis; however, these
ITnographs describ simle and effective rrthods for testing the purity
of bath pure and forrulated drgs.

When evaluating drg-cancer relationships, the following points, in

addition to the possibility that the drug itself may cause the cancer,
should also be considered:

(1) The pathological condition may predispose to cacer (see Table I).

(2) The cancer may predispose to the pathological condition (see

Table l - Epilepsy).

(3) The pathological condition or the treatmnt may increase the

chance of diagnosis of cancer but not cause an increase in i ts
incidence.
(4) Sor third factor (e.g., a genetic factor) may cause bath the
pathological condition and cacer, which are not othe:rise related.

(5) The pathological condition and the cancer are, in reali ty ,

phases of the sam process.
(6) Patients may be expsed to rrre than one drug either segentially
or in combina tion.

(7) Patients admnistered the drg may, as a conseqence, surive longe

24
 TAB l
Exeomples of associations between sor pathologica1 conditions
(in which drugs included in the present rrnograph are used) and cancer

Original Drug used Independent associations of the original patho1ogica1 condition

patho1ogica1 wi th cancer which may confound the assessment of the re1ationship
condi tion between drg Use and cancer

Epilepsy Phenytoin Brain tumurs cou1d cause epi1eptic seizures and cou1d therefore
coexist wi th the disease at a higher freqency than the expcted
Phenobarbi ta1 rate (C1emsen et al., 1974).
Fanconi 1 S Oxtho1one One case of hepatoma has ben reported in a patient with Fanconi 1 s
anaema anaema who was never treated with androgenic steroids (Cattan
et al., 1974). Patients with Fanconi 1 s anaema have a high risk
of deve10ping acute 1eukaema (Ibsik et al., 1970).
Malaria Ch10rcqine There is an apparent connection between malaria infection and
Burkittls 1ymhoma (Burkitt, 1969; Da11dorf et al., 1964; lAC,
Pyri1thamne 1975; 0' Connor, 1970).
Schistosomiasis Niridazo1e There is an association between infection by Schistosoma haemato-
bium and urinar b1adder cancer (Edington, 1956; Hashem, 1961;
Hycanthone Mustacchi & Shimkin, 1958). One report has discussed the association
between infection by s. mansoni and 1ymphoma in ma (Andrade & Abreu, 1971).

Trichormniasis Metronidazo1e The incidence of trichormna1 infection in 3682 non-gyaeco1ogica1

in-patients with negative (C1ass l and II) smears was 10%.
Trichormna1 infection was present in 35% of patients with
dysp1asia of the cervix, carcinoma in si tu or adenocarcinoma of the
uteru (Wachte1 et al., 1972). Berggren (1969) and Bertini &
Hornstein (1970) found an increased incidence oftrichormna1 infection
in patients with ma1ignant and prem1ignant diseases of the cervix.
An association between herps sim1ex virus typ 2 and cervical carcinoma
N in situ has a1so been estab1ished. (Nahas et al., 1974).
lJ
 References

Andrade, Z.A. & Abreu, W.N. (1971) Follicular lymhoma of the spleen in
patients wi th hepatosplenic schistosamasis masoni. Amr. J. trop.
Me. Hyg., 20, 237-243

Berggren, O. (1969) Association of carcinoma of the uterine cervix and

Trichomonas vaginalis infestations. Amr. J. Obstet. Gyec., 105,
166- 168

Bertini, B. & Hornstein, M. (1970) The epiderolog of trichomoniasis

and the role of this infection in the developmt of carcinoma of
the cervix. Acta cytol., 14, 325-332

Burkitt, D.P. (1969) Etiolog of Burkitt's lymhoma - an alternative

hypthesis to a vectored virus. J. nat. Cacer Inst., 42, 19-28

Cattan, D., Kalifat, F., Wautier, J.-L., ~~ignan, S., Vesin, P. & Piet, R.
(1974) Maladie de Fanconi et cancer du foie. Arch. franç. MaL.
App. Dig., 63, 41 -48

Clersen, J., Fuglsang-Frederiksen, V. & Plum, C.M. (1974) Are anticon-

vulsants oncogenic? Lacet, i, 705-707

Dalldorf, G., Linsell, C.A., Baart, F .E. & Maryn, R. (1964) An epi-
derologic approach to the 1 ymhorns of African children and Burkitt' s
sarcor of the jaws. Perspect. BioL. M2., -l, 435-449

Dosik, H., Hsu, L. Y., Todaro, G.J., Le, S. L., Hirschhorn, K., Selirio, E. S.
& Alter, A.A. (1970) Leukera in Fanconi's anema: cyogenetic and
tumr virus susceptibility studies. Bloo, 36, 341-352

Edington, G.M. (1956) Malignant disease in the Gold Coast. Brit. J.

Cancer, 10, 595-608

Hashem, M. (1961) The aetiolog and pathogenesis of the bilharzial bladder

cancer. J. Egt. rrd. Ass., 44, 857-966

lAC (1975) Anual Report, 1975, Lyon, International Agency for [Link]
on Cancer, pp. 15-16

IAC (1976) IAC Informtion Bulletin on the Surey of Chemcals Being

Tested for Carcinogenicity, No. 6, Lyon

Mustacchi, P. & [Link], M.B. (1958) Cancer of the bladder and infestation
with Schistosoma hematobium. J. nat. Cacer Inst., 20, 825-842

Nahas, A.J., Naib, Z.M. & Josey, W.E. (1974) Epidemological studies
relating genital herptic infection to cervical carcinoma. Cacer
Res., 34, 1111-1117

26
O'Connor, G.T. (1970) Persistent imologic stimlation as a factor in
oncogenesis, wi th spcial reference ta Burkitt' s turr. Arr. J. Me .
48, 279-285

Wachtel, E., Wycherley, J. & Le, C.N. (1972) Screeing for cancer of the
femle genital tract in general meical and surgical wards.
Practitioner, 208, 505-508

27
TH MONcxRAHS
 ACRIFIA VINIù'M CHIDRIDE

1. Checal and Physical Data

1. 1 Synonyr and trade nams

Cher. Abstr. Reg. Serial ~b.: 8048-52-0

Cher. Abstr. Nam: 3, 6-Diamo- 10-rrthy lacridiniur chloride mixture

wi th 3, 6-acridinediamne
Acriflavine mixture with proflavine; acriflavinii chloridum;
3,6-diamnoacridine mixture with 3, 6-diamno-10-rrthylacridinium
chloride; 2, 8-diamno- 1 O-rrthylacridiniur chloride mixture with
2,8-diamoacridine; flavacridinur hydrochloricum; neutral
acriflavine; neutroflavine; traflavine; traflavine neutral;
tryflavinur; xanthacridinum

Acriflavon; Angiflan; Assiflavine; Avlon; Bialflavina;

Bioacridin; Bovoflavin; Buol; Buroflavin; Choliflavin;
ChrolIflavine; Diacrid; Euflavin; Eulavine; Flaviforr;
Flavine; Flavinetten; Flavipin; Flavisept; Glyco-Flavine;
Gonacin; Gonacrine; Isravin; Meiflavin; Neutroflavin;
Panflavin; Pantonsiletten; Tolivalin; Trachosept; Tripla-Etilo;
Tryaflavin; Vetaflavin; Zoriflavin
1. 2 Chercal formlae and molecular weight

3, 6-Diamo- 10-rrthy lacridiniur chloride

NH2
J CL-

3,6-Acridinediamne
NH2

C14H14N3 .Cl + C13Hii N3 MoL. wt: 469.0

31
1. 3 Chemca1 and physical properties of the pure substance
From Gupta et al. (1967), unless othei:ise specified

(a) Description: Red microcrystals

(b) Melting-point: 295-2980C

(c) Spectroscopy data:

in ethanol
~ 1 1
À 261 nm (El - 1337) and 465 nm (El = 1531)

(d) Solubility: Soluble in water, 33 g/lOOrn; slightly soluble in

ethanol; near1y insoluble in chloroforr, ether and fixed oils
(Stecher, 1968)

1.4 Technical products and imurities

Acriflavinium chloride is a mixture of acriflavine and proflavine

and can be obtained in the us in a mixture stated to contain as rnuch as
30% proflavine (Gupta et al., 1967).

2. Production, Use, Occurrence and Anal ysis

For imrtant background informtion on this section, see preamle,

p. 15.

2. 1 Production and use

(a) Production

Acriflavinium chloride was synthesized in 1910 by Ehlich and Benda.

It can be prepaed by heating meta-phenylenediame with oxalic acid,
glycerol and a condensing agent, follawed by iæthylation with dimthyl
sulphate or iæthy 1 para-toluenesulphonate (The Soiety of Dyers and
Colourists, 1971).

Acriflavinium chloride has been produced camrciall y in the US for

over 50 years (US Tariff Conssion, 1927); only one us company repJrted
production (see preamle, p. 15) in 1972 (US Tariff COssion, 1974a).
US imrts of acriflavinium chloride through the principal cutorn districts
were repJrted to be 380 kg in 1972 (US Tariff Comssion, 1973), 255 kg in
1973 (US Tariff Cossion, 1974b) an 240 kg in 1974 (US International
Trade Commssion, 1976).

32
 No evidence ms been found tht acriflavinium chloride is produced
comrcially in Japa; 250 kg were Ì1rted froID the Federal Republic of
Germy in 1975. It is produced by a-t least one coy in Euope;
however, anual production estirtes were not available.

(b) Use

Acriflavinium chloride has been used as a topical antiseptic, due to

its bacteriostatic action (Klarm, 1963), and as a urinar antiseptic
(Stecher, 1968); for topical use a 0.01-0.1% solution is usually applied
(Stecher, 1968).

It has been used in Japan in the treatrt of gonorrhoea, and in the

USSR in the treatrnt of rningitis, endocarditis and St Anthony' s Fire
(herps zoster) (Mashkovski, 1972).

Acriflavinium chloride was fonærly used in veterinary rrdicine as an

udder infusion in bovine masti tis, ta treat trichomnal infections in bulls
and in piroplaslIsis. l t has reportedl y ben used locall y in wounds
(Stecher, 1968).

It has also been used as a basic dye but is no longer in commrcial

use for that purse (The Soiety of Dyers and Colourists, 1971).

2. 2 Occurrence
Acriflavinium chloride is not know to occur in nature.

2.3 Analysis

Determations of acriflavinium chloride invol ve chromtogaphic

separation techniques, including colu: and paper chromtogaphy and paper
electrophoresis, prior to spectrophotometric analysis (Gupta et al., 1967).
Thin-layer chromatographic ræthods have been develope (Theler, 1973)
and include a method using partition and ion-exchange thn- layer
chromtography on cellulose (Gill, 1967).

33
 3. Biological Data Relevant to the Evluation
of Cacinogenic Risk to Ma
3.1 Carcinogenicity and related studies in animls

Subutaneous admnistration

Rat: A group of 24 young, adult, random-bred white rats of bath sexes

received s.c. injections of 1 mg acriflavinium chloride/animl in 0.4 ml
olive oil at l5-day intervals (total dose, 40 mg/animl). At 12 IInths,
14 animls were still ali ve, and the [Link] was termated 20 IInths
after the first injection, at which tim 3 rats remined alive. One
animl develope a sarcom at the site of injection 14 Irnths after the
begining of the treatrnt. lb tururs occurred in 20 controls receiving
olive oil alone (Ezeyza, 1952).

3.2 oter relevant biological data

(a) Exrimntal system

The s. c. lethl dose of acriflavinium chloride in mice is 250 mg/kg bN
(Stecher, 1968). After admistration of i.p. doses of 10 or 30 mg/kg bN
or of 50 mg/kg li oral 1 y in male rnce, the compund could be detected in
gerr cells (Balderm et al., 1967).

Acriflavinium chloride interacts with DNA in HeL cells and in

SV40 viru-transfor1d lIuse cells (Smth et al., 1971). Studies with
isolated DNA on the mechaism of acriflavinium chloride-DNA interaction
have included flow dichroisr studies with calf thymus DNA (Nagata et al.,
1966) and fluorescence-quenching studies wi th synthetic polynucleotides
(Schreibr & Daune, 1974).

Acriflavinium chloride induces point mutations in Escherichia coli

(Derec et aL., 1947). It is a weak inducer of frar-shift mutations in
Salmonella typhimurium his-C3076, without metablic activation (Tosk, 1974),
and a stronger frar-shift mutagen in TA98 and TA537 in the presence of
rat liver hOIIenates (Brow & Brow, 1976). It induces respiratory-
deficient petite mutats in Saccharomyces cerevisiae (Bie~, 1972; Bi~ &
Konrad, 1972). At high oral doses (50 mg/kg bN) ìn male rnce, it was weakly

positive in the donnant lethal test (Balderm et al., 1967); at 10Wer

34
doses given by Lp. injection it was negative (Baldern et aL., 1967;
Epstein et al., 1972).

(b) Ma
Acriflavinium chloride induces altered sedimtation velocity of DNA
frorn cultured hum fibroblasts (Kleijer et aL., 1973) and binds to
DNA in cultured buccal cells (Roth, 1971; Roth & Majon, 1969).

ChOISOIl abnornli ties in HeL cells and cul tured peripheral hum
leucocyes have be reported at a concentration of 10-6 Wl in the
absence of light; at 10-9-10-7 M/l, ChrOIISor abnorrlities were observed
ln roth HeL cells and in peripheral leucocyes on exsure for 1 hour
to light (Buchinger, 1969).

3. 3 Case reports and epiderological studies

No data were available to the Working Group.

4. Cornts on Data Reported and Evaluation

4. 1 Animl data
No evaluation concerning the carcinogenicity of acriflaviniur
chloride can be made fror the onl y lim ted study in rats gi ven the
compund bl' subutaneous injection. Futher testing of this cornund
would appear to be desirable, also in view of the resul ts obtained in
rnutagenici ty studies.

4.2 Hum data

No case reports or epidemological studies were available to the
Working Group.

35
 5. References

Balderm, K. H., RÖhrborn, G. & Schroeer, T .M. (1967) Mutagenitäts-

untersuchungen mit Trflavin und Heaithylentetramn am S~uger
in vivo und in vi tro. Hugenetik,!, 112-126
Bie~, M. (1972) Sensitivity of Saccharomyces cerevisiae in various phases
of the life cycle to induction by acriflavine. II. Induction of
mutats by acriflavine in diploid cultures. Acta rncrobiol. pol.,
Ser. A, 4, 91-98

Bie~, M. & Konrad, B. (1972) Sensitivity of Saccharomyces cerev&s&ae in

various phases of the life cycle to induction by acriflavine. III.
Induction of mutants during sporuation. Acta microbiol. pol., Ser. A,
4, 99-106

Brown, J.P. & Brow, R.J. (1976) Mutagenesis by 9,10-anthaqunone

derivatives and related compunds in Salmonella typhimurium.
Mutation Res., 40, 203-224

Buchinger, G. (1969) Die Wirkug von Tryflavin allein und in Komination

mit sichtben Licht auf die ChOIISOff von HeLa-Zellen und rnsch-
liche Leukocen. HlUgenetik, 7-- 323-336

Derec, M., Witkin, KM., Necom, B.B. & Beale, G.H. (1947) The gene.
Carnegie Institution Wash. Yearbok, 46, 127-135

Epstein, S.S., Arold, E., Andrea, J., Bass, W. & Bishop, Y. (1972)
Detection of chemcal mutagens by the dominant lethal assay in the
lIuse. Toxicol. appL. Pharncol., 23, 288-325

Ezeyza, S. (1952) Neosalvarsán, sulfato de atropina y tripaflavina en

ratas inyectadas subtáneainte: caencia de poer cacerÍgeno
de los dos primros y producción de un sarcoma en el lugar de la
inyección de tripaflavina. Sea rn., 100, 778-780

Gill, J .E. (1967) Parition and ion-exchange thn-layer chromtogaphy

of water-soluble fluorescent comunds. J. Chomat., 26, 315-319
Gupta, V.S., Kraft, S.C. & Samelson, J.S. (1967) Puification and
properties of acriflavine, proflavine and related compunds.
J. Chromat., 26, 158-163

Klarm, E.G. (1963) Antiseptics and disinfectants. In: Kirk, R.E. &
Otr, D.F., eds, Encyclopeia of Chemcal Technolog, 2nd ed.,
VoL. 2, New York, John Wiley and Sons, p. 639

Kleijer, W.J., Hoesem, J .L., Sluytr, M.L. & Botsm, D. (1973) Effects
of initors on repair of DNA in nomil hmn and xerodern pigrto-
sum cells after exsure to X-rays and ultraviolet radiation.
Mutation Res., 17, 385- 394

36
Mashkovski, H.D. (1972) Drug Caunds, VoL. 2, M:scCM, M2izina, p. 465
Nagata, C., Kodar, M., Tagashira, Y. & Ima, A. (1966) Interaction of
polynuclea aromtic hydrocarbons, 4-nitroqin01ine l-oxides, and
various dyes with DNA. Biopolyirs,!, 409-427

Roth, D. (1971) An acridine labl for thyme photodiirs in intact cells.

EM Newslett., 4, 38- 39

Roth, D. & Majon, M.L. (1969) Studies of a speific association betw

acriflavine and DNA in intact cells. Biopolymrs, 2, 695-705

Schreibr, J.P. & Daune, M.P. (1974) Fluorescence of complexes of acridine

dye wi th synthetic pol ydeoxyribnucleotides : a physical imel of frai-
shift imtation. J. 1I1. BioL., 83, 487-501

Smth, C.A., Jordan, J.M. & Vinograd, J. (1971) In vivo effects of inter-
calating drgs on the superhelix density of mitochondrial DNA isolated
fran hur and lIuse cells in culture. J. rrl. BioL., 59, 255-272

The Soiety of Dyers and Colourists (1971) Colour Index, 3rd ed., VoL. 4,
Bradford, Yorks, p. 4431

Stecher, P.G., ed. (1968) The ~ck Index, 8th ed., Raway, ID, Merck &
Co., p. 16

Thieler, H. (1973) Dfuschichtchramtogaphische Trennung und

Identifizierug von Acriflavin (3, 6-Diamo- 10-methylacridiniur
hydroxyd) und Athcridin (2-Athoxy-6, 9-diamoacridin- lactat) .
Sci. Pha., 41, 338-339

'Isk, J. (1974) Chlorpromazine protection against acridine-induced

reversion of a histidie-reqiring imtant of Sa lmone lla typhimurium.
Mutation Res., 24, 1-3

US International Trade Cossion (1976) Irrts of Bezenoid Chercals

and Products, 1974, USITI Pulication 762, Washington OC, us Covernt
Printing Office, p. 79

us Tariff Carssion (1927) Census of Dyes and Oter Synthetic Organic

Chemcals, 1926, Tariff Inorntion Series No. 35, Washington OC,
us Governt Printing Office, p. 70
us Tariff Corssion (1973) Irrts of Bezenoid Chemcals and Products,
1972, TI Publication 601, Washington OC, us Covernnt Printing Office,
p. 82

us Tariff Cossion (1974a) Sythetic Oranic Chemcals, us Production

and Sales, 1972, TI Pulication 681, Washington OC, us Coverrt
Printing Office, p. 110

us Tariff Commssion (1974b) Irrts of Beenoid Chemcals and Products,

1973, TI Pulication 688, Washington OC, US Covernnt Printing Office,
p. 78
37
 ATJROlICGWCOSE

1. Checal and Physical Data

1. 1 Synonyr and trade nas

Cher. Abstr. Reg. Serial No.: 12192-57-3

Cher. Abstr. Nar: (1 -Tho-D-glucopyranosato) gold

1 -Aurotho-D-g lucopyranose; (D-g lucopyranosy 1 thio ) gold; (1 -D-

glucosylthio) gold; gold thioglucose; (1 -thio-D-glucopyrano-
sato)gold; l-thio-D-glucopyranose, gold colex; l-tho-gluco-
pyranose, IInogold (1+) salt; 1 -tho-D-glucopyranose, rrogold (1+) salt

Aureotan; Auromyose; Aure i Authon; Brenol; Gl ysanol B;

Oronol; Roisol; Solganl; Solganal B; Solganol B

1. 2 Chemcal formla and 1I1ecular weight

vP~\\
HO~AU
H OH

C6Hii Au05S MoL. wt: 392.2

1. 3 Chemcal and physical properties of the pure substance

From Stecher (1968)

(a) Descri~tion: YellCM crystals

(b) Solubility: Soluble in water with decorsition; slightly

soluble in propylene glycol; insoluble in ethanol, in IIst
other organic sol vents and in vegetable oils
1.4 Technical ~Droducts and irurities

Aurothoglucose is available in the US as a USP grade containing

95-105.0% active ingredient on a dried basis; it is stailized with
small arunts of no IIre than 5% soium acetate (Harey, 1975). Amules

39
contaL~ing 50 or 100 mg auroG~oglucose/ITl as a sterile susoeion in a
suitable vegetable oil are also available and contain 90-110% of the stated
aiunt of aurothioglucose. Suitable thickening agents may alš6 be present
in the suspesion (US Pharrcopeial Convention, Inc., 1975).

2. Production, Use, Cccuence and Anal ysis

For lirtat background inforntion on this section, see preamle,
p . 15 .

2.1 Production and use

(a) Production

Aurothioglucose ca be prepared by refluxing gold tribrorde with

an aqueous solution of thioglucose in the presence of sulphur dioxide
(Harey, 1975).

Commrcial production of aurothioglucose in the US was first reoorted

in 1940 (US Tariff Conssion, 1941); onlv one US company reported
production (see preamle, p. ) in 1974 (US International Trade Comssion,
1976). No evidence was found that it has ever ben produced c0Ircially
or imrted in Japa. No data were available concerning its production
in Euope.

(b) Use

The chief theraoeutic application of aurothoglucose is in rhelitoid

arthitis (Harey, 1970). HCAever, the vast majority of such patients
are treated with acetylsalicylic acid and newer anti-inflantory drgs;
aurothoglucose is reserved for the treatrt of ITre severe cases
resistat to other rres of treatrnt. For active rhelitoid artitis, a
suggested dose schedule involves i.m. admnistration of 10, then 25, then
50 mg weekl y until a total dose of 800-1,000 Ir has ben gi ven, fOllCAed by
50 mg every second or third week for 4 doses, then ITnthl y thereafter
(Harvey, 1975).

Aurothoglucose 1S used ta a lesser exent in the treatrt of non-

dissemnated lupus eryemtosus (Harey, 1970).

40
 It has be used exrirntally in veterinar.i practice to produce
obesity (Stecher, 1968).

2. 2 Occurence
Aurothioglucose is not knaw to occu in nature.

2 . 3 Anal ysis
Aurothioglucose in bulk and injection forr can be determed gravi-.
rntrically (US Pharcopeial Convention, Inc., 1975). It can be analysed
by anodic stripping voltatry (Schriid & Bolger, 1973), and a colorimtric
deterrnation has ben develope (Janik & Rzeszutko, 1969).

3. Biological Data Relevant to the Evaluation

of Cacinogenic Risk to .l1a
3.1 Carcinogenicity and related studies in anirls

Intraperi toneal admnistration

!'use: A group of 88 8- l2-wee old virgin femle C3H rnce recei ving
an unrestricted diet we give single i.p. injections of 10 mg/aniIl
aurothioglucose; 78/88 mice becar obse following this treatrt. At
295 days, 50% of these rnce develope rr tururs, carared with 354
days in 50% of untreated controls; after 295 days only 19% of 66 controls
had ma tururs. The 10 treated rnce that did not becoræ obese
developed ma tururs within a tim simlar to that in controls
(Waxler et al., 1953).

Ma tururs develope ln 38/38 virgin femle RIIIxC mice that

becar obese after receiving a single i.p. injection of 400 m:jkg lM at
70-80 days of age, comped with 29/34 untreated virgin femles (controls)
and 10/10 untreated breeers. Fifty percent of the anirls in the
three groups develope ma tururs at 240, 350 and 250 days, respecti vel y.
The average m:iibrs of tururs per animl were 3.2 in aurothioglucose-treated
virgin femles, 1.9 ln controls an 2.2 in untreated breeders (Liebelt, 1959).

In groups of male and femle AK rnce, thyictomized at the age of 4

wees and given single Lp. injections of 750 m:jkg lM aurothioglucose
at 10 weeks of age, 1/9 (11%) males and 5/12 (41%) fera les develope benian

41
osteomas of t.l-e sku1l, corcd wit.1i 0/15 male and 1/23 (4%) femle AKR
thyrtomized controls. Of 31 thyrtomized males given a s.c. irlant
(arunt unstated) of a cholesterol p2llet containing 10% oestradiol at 14
weeks of age, 14 (45%) developed osteomas. Of 11 sirlarly treated males
receiving in addition single i.p. injections of 750 mg/kg hw aurothioglucose,
8 (72%) develope osteomas. No osteomas occured in simlarly treated
C57BL rnce (Rudali, 1968).

Groups of intact (34 male and 23 femle) or castrated (25 male and
22 femle) CBA rnce were given 400 rr/kg bw aurot.1iioglucose in saline by
i.p. injection at the age of 13-14 wees and observed until they were 56
wees of age; simlar groups of intact (36 male and 40 femle) and castrated
(16 male and 15 femle) controls were used. In treated animls a significant
increase in the incidence of hepatomas was observed in intact males (21/34
versus 7/36) but not in intact ferles. Treated castrated males had no
significant increase in the incidence of hepatomas. The 2 treated intact
femles that develope hepatomas also had androgen-secreting adrenal
cortical tururs. The 3 untreated castrated and the 8 treated castrated
males that develope hepatomas also had adrenal cortical adenOIs, which
were associated with androgenic activity in 8 cases (Gray et aL., 1960).

3.2 Oter relevant biological data

(a) Exrimntal system
The i.p. LD of aurothioglucose in stock rnce is 2000-2500 mg/kg hw
50
(Brecher & Waxler, 1949). Doses of 350 rr/kg hw to CB mice cause
[Link] hypthalamc lesions associated with obesity; in C57BL rnce
doses of 1200 mg/kg bw are reqed ta cause simlar changes in weight
gain (Lieblt & Perry, 1957). In Swss rnce, doses of 650 rn/kg hw cause
hypthlarc les ions , but onl y 30 % of the animls becar obese (Brecher
et al., 1965).

Wista rats of bath sexes and of various ages ShCMed no SignS of

obesity withn 60-70 days after single i.p. or i.m. injections of
0.5-1.6 g/kg bw (Talbrt & Hamlton, 1954). In Lona-Evans ~ats given
400 mg/kg bw i.p., no obesity occurred, but severe hypthalamc les ions
were seen (Wagner & de Groot, 1963).

42
 In rats given i.m. injections of aurothioglucose daily for 14 days
(total dose, 14 mg gold/animl), retention of aborbed gold wa greatest
in the kidney an then in the liver and spleen; 15% of the dose was
retained in the boy after 85 days (Block et al., 1944).

(b) Ma
Water-soluble gold sal ts are absorbe rapidl y after their i. m. inj ection
ln hurs. Approximtely 85% of the injected gold is retained over 7 days.
It is excreted mainly by the kidney, but a smll aiunt appes in the
faeces (Harey, 1970).

3.3 Cae refDrts and epideIological studies

No data were available to the Working GrouP.

4. Contnts on Data Rerted and Evaluation 1

4 . 1 Animl data
Aurothoglucose is carcinogenic in mice after its admnistration
by single intrapeitoneal injection: it produced an increased incidence
of hepatoms in male mice.

4. 2 Hum data
No case reports or epidemological studies were available to the
Working Group.

1 See also the section 'Anl Data in Relation to the Evaluation of

Risk to Ma' in the introduction to this volur, p. 13.

43
 5. P-eferences

Block, W.D., Buchanan, G.H. & Freyberg, R.H. (1944) M:talisr, toxicity,
and nier of action of gold COunds used in the treatrnt of
arthi tis. V. A coarative study of the rate of absorption, the
retention, and the rate of excretion of gold admistered in different
cOITunds. J. Phacol. ex. Thr., 82, 391-398

Brecher, G. & Wæ.::er, S.H. (1949) Obsity in albino rnce due to single
injections of goldthioglucose. Proc. So. exp. Biol. (N.Y.), 70,
498-501
Brecher, G., Laqueur, G.L., Cronkite, E.P., Edelm, P.M. & Schwartz, LL.
(1965) The brain lesion of goldthioglucose obesity. J. exp. M:d.,
121, 395-401

Gray, G.F., Lieblt, R.A. & Lieblt, A.G. (1960) The developrænt of li ver
tuirs in goldthioglucose-treated CB rnce. Cacer Res., 20, 1101-1104

Harey, S.C. (1970) Heavy rætals. In: Go, L.S. & Gilm, A., eds,
The Pharcological Basis of Therapeutics, 4th ed., Ne York,
Macmllan, pp. 969-974

Harvey, S.C. (1975) Honrnes. In: Osol, A. et al., eds, Rengton's

Pharceutical Sciences, l5th ed., Eaton, Pa, Mack, p. 899

Janik, B. & Rzeszutko, W. (1969) Colorimtric determation of gold in

Solganal B. Acta p:Ü. pha., 26, 339-342

Lieblt, R.A. (1959) Effects of goldthoglucose-induced hypthalarc

lesions on ma tuirigenesis in RIIIxC mice. Proc. Arr. Ass.
Cacer Res., 3, 37-38

Lieblt, R.A. & Perr, J. H. (1957) Hythalarc lesions associated with

goldthog lucose- induced obesi ty . Proc. So. ex. BioL. (N . Y .), 95,
774-777
Rudali, G. (1968) Aparition d 'ostéors intracraniens chez des souris
injectées avec du thoglucose d'or. Rev. franç. Etud. clin. biol.,
13, 40-48

Schmd, G.M. & Bolger, G.W. (1973) Determation of gold in drgs and
seru by use of anodic stripping voltartr. Clin. Chem., 19,
1002- 1005

Stecher, P.G., ed. (1968) Th Meck Index, 8th ed., Ray, ID, M2rck &
Co., p. 112

Talbrt, G.B. & Hamlton, J.B. (1954) Failure to produce obesity in the
rat following gold thoglucose injection. Proc. So. exp. BioL. (N. Y.) ,
86, 376-378

44
us Interntional Trade Cossion (1976) Synt.i-ctic Organic Chcrcals, vs
Production and Sales, 1974, USITC Pulication 776, Washington DC,
US Governnt Printing Office, p. 105
US Pharcopeial Convention, Inc. (1975) The US Phacopeia, 19th rev.,
Rockville, Md, pp. 41-42

US Tariff Commssion (1941) Synthetic Organic Chemcals, US Production and

Sales, 1940, Report No. 148, Seond Series, i'Jshington DC, US Covernnt
Printing Office, p. 40

Wagner, J.W. & de Groot, J. (1963) Effect of goldthoglucose injections on

suri val, organ damge and obesi ty in the rat. Proc. So. exp. Biol.
(N.Y.), 112, 33-37

Waxler, S.H., Tab, P. & Melcher, L.R. (1953) Obesity and the tim of
appearance of spontaneous ni carcinom in C3H mice. Cacer Res.,
13, 276-278

45
 CHRCXUINE

1. Chemcal and Physical Data

1. 1 Synonyr and trade nas

Chem. Abstr. Reg. Serial No.: 54-05-7

Cher. Abstr. Nar: N4 - (7-Chloro-4-qinolinvl) _Nl ,Nl -diethyl - 1,4-

pentanediarne
Chloraquine; 7 -chloro-4- (4-diethy larno- 1 -methy lbuty larno ) quinoline;
7 -chloro- 4- -i ( 4- (diethy lamo ) - 1 -methy lbuty 1 J arno lquinoline ; chloro-
quinium
Aikin; Aralen; Arechin; Arochin; Arrichin; Avlochlor;
Avloclor; Beco; Bephate; Besulph; Beaquin; Bipiqun;
Cheihin; Chingarn; Chorochin; Chlorquin; Cidanchin; Clorochina;
Chloroqina; Cocartrit; Delagil; Dichinalex; Elestol; ('.,ntochin;
Heliopar; Irgon; Iroqine; Klorokin; Lapaquin; Malaquin; Malaren;
Malarex; Mesylith; Neohin; Nivachine; Nivaquine B; OUnachlor;
Quinercyl; Quingarn; Quingame; Qunilon; Qunoscan; Resochen;
Resochin; Resoqina; Resoqine; Reumchlor; Reumquin; Roqine;
RP 3377; Sanoqin; SilbsaIl; Siragan; SN 6718 ¡ SN 7618; Solprina;
Sopaquin; Tanakan; Tresochin¡ Trochin; W 7618; Win 244

1. 2 Chemcal formula and molecular weight

C~I N
/" L "
:: ~
/CH2 -CH3
HN - CH -CH2 -CH2 -CH2 -N "-
1 CH2 -CH3
CH3

C18H26C1N3 t'bl. wt: 319.9

47
 1.3 Chemcal and physical properties of the pure substance
From US Phacopeial Convention, Inc. (1975), unless otherwise
specified
(a) Description: Wh te crystal line paer
o
(b) Mel ting-point: 87 C (Stecher, 1968)

(c) Spectroscopy data: ma

À 220 nm (Viala et al., 1973), 255, 329
and 343 nm (WHO, 1967) in rnneral acids

(d) Solubility: Very slightly soluble in water; soluble in dilute

acids, chloroforr and ether

1.4 Technical products and imurities

Various national and international phaco~ias gi ve specifications

for the purity of chloroqine and its salts ln pharceutical products. For
exanle, chloroqine is available ln the US as a USP grade containing
98- 102% active ingredient on a dried basis, as chloroqine hydrochloride
injection, as chlorCXne phosphate on a dried basis and in talets
(US Pharcopeial Convention, Inc., 1975). Chloroqine phosphate and
sulphate are available in the OK, containing no less than 98% active
ingredient on a dried basis, for injection (95-100% of the stated arunt)
and in tablets (92.5-107.5% of the stated arunt) (British Pharcopoia
Commssion, 1973). Combinations of chlorCXne with other anti-malarial
agents, e. g., pyrimtharne, are also available in sorn countries.

2. Production, Use, Occurrence and Anl ysis

For imrtant background inforntion on this section, see preamle,

p. 15.

2. 1 Production and use

(a) Production

Chloroqine was synthesized ln Germy as early as 1934 (Rollo, 1975).

It can be prepaed by condensing 4,7-dichloroqnoline with l-diethylamo-
4-amopentae (Harey, 1975).

48
 In the US, canrcial production of chloroqine was first reported in
1949 (US Tariff Commssion, 1950); howver, it is usually maufactured
as the phosphate or chloride salt. In 1974, only one us compy reported
production (see prearle, p. 15) of the phosphate salt (US Interntionl
Trade Commssion, 1976a). US imrts of chloroqine phosphate were
reported to have been abut 100 kg in 1972 (US Tariff Comssion, 1973),
500 kg in 1973 (US Tariff Comssion, 1974) and 1300 kg in 1974 (US
Interntional Trade Commssion, 1976b).

Production, imrtation and use of chloroqine in Japan are believed

to have ben miiml due to the risk of adverse effects involving optical
retinopathy.
India was reported to have two producers of chloroqine, wi th a
combined production of 23,820 kg in 1972. Indian imrts were reported
ta be 99,330 kg for the period 1972-1973 (Anon., 1974).

(b) Use

Chloroqine was first develope and is currently used as an anti-

mlarial agent in hum rnicine. Partially ime adults infected with
chloroqine-sensitive parasites may need only a single dose of 600 mg
chloroqine ta termnate a mild attack. Non-ime adults are given
1. 5- 2. 4 g chloroqine or amiaque in di vided doses over 3- 5 days.
May dosage variations exist, but ITst are resed on the admnistration of
900 mg chloroqine in di vided doses on the first day, followed by smaller
doses on subseqent days (WHO, 1973).

Caninations of chloroqine wi th other drgs, such as qunine, dapsone,

pyrimthamne, sulphafurazole and sulphadiazine have been used against
chloroqine-resistant Plasmodium falciparum malaria (WHO, 1973).

Chloroqine is also used as an efficient suppressive in aii forr of

malaria. The adult dose is either 300 rr chloroqine weekly or 100 mg
dail y on 6 days of each week depeding on the transmssion level in the
area. Protective doses for children range from 37 mg chloroqine base
for ages under one year to 225 rr chloroqine for ages 11-16 years, given
weekly (Covell et al., 1955).

49
 As a lupus eryemtosus suppressant, chloroqine is admistered
orally in tv 150 mg doses daily for 1 to 2 weeks and then 150 rr daily
the rea f ter (Harey, 1975). It can also be emloyed in the treatrnt of
extra-intestinal amiasis (Kastrp, 1971).

2. 2 Occurence
Chloroqine ls not known to occur in nature.

2. 3 Anal ysis
Methods for the estimtion of chloroqine have ben reviewed (Vykydal
et al., 1967). Those tht iæt reguatory reqerts for phaceu-
tical products include aqueous titration, non-aqueous titration and 1J1
spectrophotorrtry. Determnation of chloroqe in biological sanles is
rnst freqently carried out by spctrofluorimtric rrthods, although gas
chromatography and colorimtric rrthods are also emloyed.

In spectrofluorimtric determnations, pH conditions for Inirum

sensitivity have been investigated (Schulm & Young, 1974). A comarison
of thee photochemcal fluoriItric rrasuring techniques shcwed tht
digital integration of the fluorescence of chloroqine was the most sensi-
ti ve, gi ving a lim t of detection of 0.1 Wg/l (Lukasiewicz & Fitzgerald,
1974). Both spectrofluorirtric and UV spectrophotometric methods have
been emloyed for determnation of chloroqne after separation by thin-
layer chromatogaphy. Th lim ts of detection by UV spetrophotometry
were 0.17 Wg/rn for bloo, 0.05 Wg/rn for urine and 1.07 Wg/g for tissue;
these values were abut 9 tims higher than the corresponding lim ts using
spectrofluorimtry (Viala et al., 1973).

Lits of detection for gas chromtography using flam ionization

detection were 0.15 Wg/rn for urine, O. 25 Wg/rn for bloo and 1. 50 Wg/ g
for tissue (Viala et aL., 1975).

A system of coloriItric tests for the presence of chloroqne in

autopsy material, which is claiId to be speific, can detect 10 Wg of
the drg (Farushnyi, 1967).

50
 3. Biological Data Relevant to the Evaluation
of Cacinogenic Risk to Ma
3.1 Carcinogenicity and related studies in animls

Oral admnistration
Rat: Groups of 10 male and 10 femle 2l-day old Osborne-Mendel rats
were given 0 (control), 100, 200, 400, 800 or 1000 mg chloroquine per kg of
diet for up to 2 years. Inhibi tion of grCM was severe at the 800 and
1000 mg/kg levels but temrar at 400 mg/kg. The toxici ty of chloroqine
becar progessi vel y IIre severe wi th increasing dosage, and 100% IIrtli ty
was observed at the two highest dose levels at 35 and 25 weeks, respectively.
No tUlurs were reported in 86 treated rats or in 15 control rats examned
rncroscopically (Fitzhugh et aL., 1948; Nelson & Fitzhugh, 1948) (The
Working Group noted the lcw numr of suri vors. )

3. 2 Oter relevant biological data

(a) Exrimntal system
The oral LD of chlorogine phosphate is 620 mg/kg in mice, 970 mg/kg
50
in rats and 136 mg/kg in rabbits (Sunshine, 1969). In a 2-year chronic
toxicity study in rats fed diets containing from 100 to 1000 mg chloro-

quine/kg of diet, progressive retardation of grCM and an increase in

rnrtality were observed with levels of 800 and 1000 mg/kg of diet.

Myocardial and voluntary muscle darge, centrilobular necrosis of the

liver and testicular atrophy were also observed (Fitzhugh et al., 1948).

Orally admistered chloroqine is well and rapidly absorbe by rny

species (McChesney & McAuliff, 1961). It is deposited at up to 1000 tims
the plasm concentration in spleen, liver, kidney and lung and to a lesser
degree in other tissues (HcCesney et al., 1967a). Extensive accumulation
occurs in rælanin-containing tissues (Lindquist, 1973; l'cChesney et aL.,
1967a) . ln rats, up to 88% of extractable tissue-bound material was
unchanged chloroqine (McChesney et aL., 1965); in IInkeys, the main
metablite is 4- (7-chloroqinolyl)-y-amo-n-valeric acid (Rollo, 1970).
Chloroqine rapidly crosses the placenta in rnce (Lindquist, 1973).

51
 The drg interacts non-covalently with double-stranded DNA ~n vitro
(Cohen & Yielding, 1965a; 0' Brien et al., 1966; Waring, 1970) and inibits
the activity of DNA polyrrase (Cohen & Yielding, 1965b), DNA synthesis

- ..
in malian cells (Cleaver & Painter, 1975) and DNA repair bath in
bacteria (Yielding et al., 1970) and in malian cells (l1chael & William,
1974) .

(b) Man

High-dose, long-terr therapy (IIre than one year) may result in

retinopathy with doses exceeding 100 g (Voipio, 1966) and in ototoxicity
(Hart & Naunton, 1964).

Chloroqine is well absorbe froID the gastrointestinal tract and

avidl y retained in tissues. The main metabli te is de-ethy lchloroqine
(Rollo, 1975), but 70% is excreted as unchanged chloroqine (McChesney
et al., 1967b). When treatmnt with chloroqine alone or in combination
with hydroxychloroqine is discontinued, chloroqine and its metablites
can be detected in the urine for up to 5 years (Rubin et al., 1963).

3. 3 Case reports and epiderological studies

No data were available to the Working Croup.

4. Comrnts on Data Reported and Evaluation

4.1 Animl data

The negative results obtained in the only available study in rats

given chloroqine by oral admnistration did not forr an adeqate basis
on which to mae an evaluation of the carcinogenici ty of this COrrund 1 .

4.2 Hum data

No case reports or epidemological studies were available to the
Working Croup.

1Th-' Working Group was aware of an on-going carcinogenicity study in

rnce (IAC, 1976).

52
 5. :Rferences

Anon. (1974) Production and irrts of selected drgs and pharceuticals

ln India. Chemcal Industry News (India), July

British Pharrcopoia Comssion (1973) British Pharmcopoia, London,

HMO, pp. 100-102
Cleaver, J.E. & Painter, R.B. (1975) Absence of spcificity in inhibition
of DNA repair replication by DNA-binding agents, cocarcinogens and
steroids in hum cells. Cacer Res., 35, 1773-1778

Cohen, S.N. & Yielding, K.L. (1965a) Spectrophotomtric studies of the

interaction of chloroqine wi th deoxyribnucleic acid. J. biol. Cher.,
240, 3123- 3131

Cohen, S. N. & Yielding, K. L. (1965b) Inhibition of DNA and RN polymrase

reactions by chloroqine. Proc. nat. Acad. Sci. (Wash.), 54, 521-527

Covell, G., Coatney, G.R., Field, J.\'i. & Singh, J. (1955) Cheitherapy
of malaria. Wld Hlth Org. M::mogr. Ser., No. 27, p. 87

Fartushnyi, A.F. (1967) Detecting chloroqne in autopsy matter. Sudebno-

Med. Ekspertiza, Mi. Zdravaokh. SSSR., 10, 45-48

Fitzhugh, O.G., Nelson, A.A. & Holland, O.L. (1948) The chronic oral
toxicity of chloroqine. J. Pharrcol. exp. Ther., 93, 147-152

Hart, C. W. & Naunton, R. F. (1964) The ototoxici ty of chloroqne phosphate.

Arch. OtolarygoL., 80, 407-412

Harvey, S.C. (1975) Antircrobial drgs. In: Osol, A. et al., eds,

Remngton' s Pharnceutical Sciences, l5th ed., Easton, Pa,
Mack, pp. 1155- 1156

rAC (1976) IA Inforntion Bulletin on the Surey of Chemcals Being

Tested for Carcinogenici ty, No. 6, Lyon, p. 233

Kastrup, E.K., ed. (1971) Facts and Comparisons, St IDuis, Missouri,

Facts and Comparisons, Inc., p. 378a

Lindquist, N.G. (1973) Accumulation of drugs on melanin. Acta radioL.,

Suppl. 325, 1-92

Lukasiewicz, R.J. & Fitzgerald, J.M. (1974) Comparison of thee photo-

chemcal -fluorimtric methods for deterrnation of chloroqine.
Appl. Spetr., 28, 151-155

Mchesney, E.W. & McAuliff, J.P. (1961) Laratory studies of the 4-

amoqinoline antimlarials. I. Sorne biochemcal characteristics
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11, 800-810
53
 McChesney, E.W., Bas, W.F., Jr & Sullivan, D.J. (1965) Metablism of
chloroqine and hydroxychloroqe in albino and pigInted rats.
'IxicoL. appL. PharncoL., "l, 627-636

Mchesney, E.W., Bas, W.F., Jr & Fabian, R.J. (1967a) Tissue distribution
of chloroqine, hydroxychloroqe and desethy lchloroqine in the rat.
Toxicol. appl. Pharmcol., 10, 501-513

McCesney, E. W., Fasco, M. J. & Bas, W. F ., Jr (1967b) The rrtablism of

chloroqine in ma during and after repeated oral dosage. J. Pharcol.
exp. Thr., 158, 323-331

Michael, R.O. & William, G.M. (1974) Chlorcqne inibition of repair of

DNA damge induced in malian cells by rrthyl ræthanesulfonate.
Mutation Res., 25, 391-396

Nelson, A.A. & Fitzhugh, O.G. (1948) Chlorcqne (SN-76l8). Pathologic

chages observed in rats which for tw years had been fed various
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O'Brien, R.L., Allison, J.L. & Hah, F.E. (1966) Evidence for intercalation
of chloroqine into DNA. Biochir. biophys. acta, 129, 622-624

Rollo, LM. (1970) Drugs used in the cheitherapy of malaria. In: G:,
L.S. & Gilm, A., eds, The Pharncological Basis of Therapeutics,
4th ed., New York, Macmllan, pp. 1101-1105

Rollo, LM. (1975) Drugs used in the cheitherapy of malaria. In: COod,
L.S. & Gilm, A., eds, The Pharncological Basis of Therapeutics,
5th 00., New York, Macmllan, pp. 1049-1053

Ruin, M., Bernstein, H.N. & Zvaifler, N.J. (1963) Studies on the pharn-
colog of chloroqine: recomrndations for the treatrnt of chloroqine
retinopathy. Arch. OphthaiL., 70, 474-481

Schulm, S.G. & Young, J.F. (1974) A imified fluorirtric deterrnation

of chloroqne in biological samles. Analyt. chir. acta, 70, 229-232

Stecher, P.G., 00. (1968) The Merck Index, 8th ed., Raway, NJ, .Mrck &
Co., p. 247

Sunshine, L, ed. (1969) CRC Handbk of Analytical 'Ixicolog, Cleveland,

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US Production and Sales, 1974, USITe Publication 776, Washington De,
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54
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RockvilM, pp. 81-83

US Tariff Commssion (1950) Synthetic Organic Chemcals, us Prouction and

Sales, 1949, Report No. 169, Second Series, v-Jashington OC, US Goverrnt
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999- 1001

55
 DIAEPAM AN OXAEPAM

1. Chercal and Physical Data

Diazepa
1. 1 Synonyr and trade nars
Chem. Abstr. Reg. Serial No.: 439-14-5

Chem. Abstr. Nar: 7 -Chloro- l, 3-dihydro- 1 -methy 1 - 5-pheny 1 - 2H-

l, 4-benzodiazepin- 2-one
7-Chloro- 1 -methy 1- 5- 3H - l, 4-benzodiazepin- 2 (lH) -one; 7-chloro- 1-
methyl -2-oxo-5-phenyl -3H- l, 4-benzodiazepine; 7-chloro- 1 -methyl-5-
phenyl -2H- i, 4-benzodiazepin-2-one; 7-chloro- l-methyl-5-phenyl-3H-
l, 4-benzodiazepin- 2 (lH) -one; 7-chloro- 1 -rnthy 1 -5-pheny 1 - l, 3-dihydro-
2H-l,4-bezodiazepin-2-one; diacepan; diazepan; methyldiazepinone;
l-methy 1 -5-pheny 1- 7-chloro- l, 3-dihydro- 2H- l, 4-benzodiazepin- 2-one
Albral; Aliseur; Arprol; Ansiolin; Aniolisina; Apaurin;
Apozepa; Assi val; Atensine; Atilen; Bialzepam; Ca1ii tene;
Calmse; Cercine; Ceregart; Condi tion; Diapam; Diazetard;
Dienpax ; Dipam; Dipezona; IXliur; Dusen; Duen; E-Pam;
Eridan; Faustan ; Freudal; Gihita; Horizon; Kiatrium; IA III;
Lerol; Levium; Liretas; Morosan; Noan; Pacitran; Paranten;
Paxte; Paxel; Plidan; Quetinil; Quatril; Quevita; Relanium;
Relax; Renbrin; Ra 5-2807; S.A. R.L.; Sarornt; Sedipam; Seduxen;
Serenack; Serenam ; Serenzin; Setonil; Sonacon; Stesolid;
Stesolin; Tensopam; Tranirul; Tranqdyn; Tranquirit; Unriur;
Unisedil; User AP; Valeo ; Valitran; Valium; Vatran; Velium;
vi val; Vivol; Wy 3467 ; Zipan
1. 2 Chercal formla and molecular weight

C16H13C1N20

:Ml. wt: 7.84.8

57
1. 3 Chemcal and physical properties of the pure substance
From Stecher (1968), unless otherwise speified

(a) Description: Plates

(b) Melting-point: l25-l260C

(c) Spectroscopy data: Àrr 242, 285 and 368 nr in acidified

ethanol; infra-red, nuclear magnetic resonance and rnss
spectra have been reported (MacDonald et aL., 1972).

(d) Solubility: Slightly soluble in water; soluble in ethol;

freely soluble in chloroform (US Pharncopeial Convention, Inc.,
1975)

1.4 Technical products and imuri ties

Various national and international pharcopoias gi ve specifications

for the puri ty of diazepa in pharceutical products. For examle,
diazepa is available in the US as a National Formulary or USP grade con-
taining 98.5-101.0% active ingredient on a dried basis with a maimum of
0.002% heavy metals. Injections of a sterile solution of diazepa in water
or other suitable meia (such as solutions of propylene glycol, ethanol,
sodium benzoate, benzoic acid or bezyl alcohol (Kastrup, 1976)) are
available in a dose of 5 rr diazepam per ml containing 90-110% of the
stated arunt. Diazepam talets are also available in 2, 5 and 10 mg doses
containing 90.0-110.0% of the stated arunt of diazepar (National Formlary
Bod, 1970; US Pharncopeial Convention, Inc., 1975). In Japan,
diazepa is also available in poders and capsules. ln the OK, diazepa is
available on a dried basis, containing 99-101% active ingredient, and in
capsules and tablets containing 92.5-107.5% of the stated arunt (British
Pharmcopoia Comssion, 1973).
Oxzepa
1. 1 Synonym and trade nars
Chem. Abstr. Re. Serial Ib.: 604-75-1

Chem. Abstr. Nar: 7-Cloro- l, 3-dihydro- 3-hydroxy-5-pheny 1 - 2H- 1,4-

benzodiazepin- 2-one

58
 7-Chloro- l, 3-dihydro-3-hydroxy-5-phenyl -2ll- l, 4-benzodiazepin-2-one;
7-chloro-3-hydroxy-5-phenyl -2H- l, 4-benzodiazepin-2-one; 7-chloro- 3-
hydroxy-5-phenyl - l, 3-dihydro-2H- l, 4-bezodiazepin-2-one
Adumran; Ansioxacepa; Aniolit; Aplakil; Astress; Bonare;
Enidrel; Isodin; Liial; Nesontil; Praxiten; Propax; Psicopax;
Quen; Quilibrex; Ro 5-6789; Rondar; Serax; Serenal; Serenid;
Serenid-D; Serepa; Seresta; Serp; Tazepa; Vabn; \rJ-3498;
Z 10 TR

1.2 Chercal fonna and 1I1ecular weight

C15Hii C1N202
1 L\-OH
-N
MoL. wt: 286.7

1. 3 Chercal and physical properties of the pure substance

From Stecher (1968), unless otherwise specified

(a) Description: Crystals from ethanol

(b) Melting-point: 205-2060C

(c) Spetroscopy data: Àma

230 and 318 nr in ethanol; 236, 284
and 362 nr in 0.1 N hydrochloric acid; infra-red, nuclear
magnetic resonance and mass spectra have ben reported
(Shearer & Pilla, 1974).

(d) Solubility: Soluble in ethanol (0.45 g/lOO ml), chloroforr

(0.4 g/lOO ml) and dioxane; practicall y insoluble in water
(0.003 g/lOO ml) (Shearer & Pilla, 1974)

(e) Staility: Stable as a solid or in neutral solutions but

hydrolysed by acids (Shearer & Pilla, 1974)

59
 1. 4 Technical products and inuri ties
OXazepa is available in the US as a National Forrlar grade containing
98-102% active ingrerient on a dried basis. Capsules are available in
10, 15 and 30 rr doses containing 90-110% of the stated arunt qi
oxazepa (National Formulary Bod, 1970). Tablets are also available,
in 15 rr doses (Kastrp, 1974). Oxzepam is available in Japan in poder
forme

2. Production, Use, O:currence and Analysis

For imrtant background informtion on this section, see preamle,

p. 15.

2. 1 Production and use

(a) Production

A method of prepaing diazepam was first reported in 1961; benzoyl

chloride is reacted wi th para-chloroaniline in the presence of a zinc
chloride catalyst. The resulting 2-arno-5-chlorobenzophenone is converted
to the oxim with hydroxylarne and then cyclized to the chlororæthyl-
quinazoline- 3-oxide by treatrnt wi th chloroacety 1 chloride. When this
is trea ted wi th alkali, ring enlargemnt occus, gi ving 7 -chloro- l, 3-
dihydro-5-pheny 1 - 2H- l, 4-benzodiazepin- 2-one-4-oxide. Raey nickel reduction
of the oxide followed by methylation with dimthyl sulphate gives diazepa
(Sternach & Reeder, 1961; Sternch et aL., 1961). Diazepam has also
been prepared by the reaction of 2-methy larno- 5-chlorobenzophenone wi th
ethyl glycinate (Reeer & Sternch, 1963), or with brolIacetyl brornde
followd by reaction with arnia (Reeder & Sternach, 1964).

Preparation of oxazepa was first reported in 1962; in this method

6-chloro-2-chloromethyl-4-phenylquinazoline 3-oxide is treated with sodium
hydroxide to gi ve 7 -chloro- 5-pheny 1 - l, 3-dihydro- 2H- l, 4-benzodiazepin- 2-
one-4-oxide. This rearranges to the 3-acetyloxy derivative when treated
with acetic anydride. Reaction of this with sodit:rn hydroxide forr a
precipitate, which, after dissolution in water and acidification with
acetic acid, gives oxazepa (Bell & Childress, 1962).

60
 Corrcial production of diazepam was first reported in the US in 1963
(US Tariff Conmssion, 1964) i only one US company reported production (see
preamle, p. 15) in 1974 (US Intertionl Trade Comssion, 1976a). US
Ì1rts of diazepam though the principal custorn districts were 33 kg
in 1974 (US International Trade Commssion, 1976b).

Coirrcial production of oxazepa wa first reported in the US in

1965 (US Tariff Commssion, 1967); only one US company reported production
(see preamle, p. 15) in 1974 (US International Trade Commssion, 1976a).
US Ì1rts of oxazepa through the principal custorn districts were
560 kg in 1973 (US Tariff Comssion, 1974).
No evidence was found that diazepam or oxazepa ls produced conircially
in Japa. In 1975, Japan Í1rted 135 kg diazepa, principally from
Argentin, with smller arunts from Dek and Caada, and over 200 kg
oxazepa, principal 1 y fror Tukey.
Anual production of diazepam ls less than 1,000 kg in each of the
following countries: Austria, Beelux, France, Scandinavia and the UK;
1-100 thousand kg are produced in the Federal Republic of Germy, in Italy,
in Spa and in Swi tzer land. Anual production of oxaze~am is less than
1,000 kg in Austria, in Benelux, in the Federal Republic of ('::nny, in
France, in Scandinavia, in Spain, in Switzerland and in the OK; 1-100
thousand kg are produced in Italy. Anual production of bath diazepam and
oxazepa in eastern Euope is estimted to be in the range of 1-100
thousand kg.

(b) Use

Diazepar is widel y used for the treatrnt of aniety in a usual dose

range of 5-20 m~-¡day. It has also been used as a skeletal muscle relaxant
in various spastic disorders (2-20 rr intravenously at intervals of 2-8
hours), as a pre-anaesthetic meication in childbirt and cardioversion
(i. v. doses of 5-30 mg) an as a hypnotic antiepileptic agent (Eyck, 1975).
The usual route of admnistration for treatmnt of chronic diseases is oral,
but an injectable solution of 5 mg/ml is available for acute disorders
(Wour & Fingl, 1975).

61
 Oxzepam, a rrtablite of diazepam, is primrily used in hur rrdicine
for the treatrnt of aniety and tension (Grol1r & Grol1r, 1970);
it has also ben used as an antiepileptic agent (Woour & Fingl, 1975).
The usual dose range is 30-90 rr/day given in capsules of 10 to 30 rr
(Eyck, 1975).

Because of potential abuse of bath diazepam and oxazepa, leading to

1imited physical or psychological depedence on these drgs, on 1 April 1976,
the US Drug EnorceInt Agency placed diazepa and oxazepam in Schedule IV
of the Controlled Substances Act, which reqires all maufacturers and
distributors of these compunds to register and report to the agency any
changes in the quanti ty maufactured and distributed and reqires physicians
to review their patients' status -priodicall y (US Drug Ehforcert
Adistration, 1975, 1976).

It has been reported that abut thee billion tablets of diazepa were
sold in the US in 1974 (Anon., 1975). Total US sales of oxazepam for us in
hur meicine are estimted to be less than 3,000 kg anuall y .
2. 2 CXcurrence

Neither diazepam nor oxazepam is knCM to occur in nature.

2.3 Analysis

Compilations of anal ytical rrthods for the deterrnation of diazepa

(MacDonald et aL., 1972) and of oxazepa (Shearer & Pilla, 1974) have be
published. Methods of assaying diazepam that rrt regulatory reqirernts
for pharceutical products include non-aqueous titration and UV spectro-
photorrtry .
A rrthod using thn- layer chromtography prior to spectrophotorrtric
analysis ca separa te diazepa in pharceutical formulations from its
degradation product (Sbarbati Nudelm & De Waisbaum, 1975). A t.-
dimnsional thin- layer chromatographic rrthod can separa
te and detect
five sirlar tranquilizer drgs (including diazepam) (Schuetz et al., 1972).

A gravimtric and complexorrtric rrthod for the determnation of

diazepa is based on the forntion of the hexathiocanatochromate (III)
(Grecu & Macu, 1973).

62
 A colorimtric method has ben develope to determne diazepam as the
pure drg and in fonnations by hydrolysis in chloroforr to 2-methylamo-5-
chlorobezophenone, a deep-yellow cound (Baggi et aL., 1975). Another
colorimtric rrthod, develope for the detection of prim amnes on
thin-layer plates, was found to be applicable to diazepam, apparently
because diazepa decompses on the plate (Haefelfinger, 1970).
A polarographic method allCMs sirtaneous determnation of thee
1,4-bezodiazepine drgs (including diazepa) (Oelschlaeger & Volke, 1966).
A rapid-pulse polarographic rrthod to determe diazepa in seru has
a detection limt of 0.01 llg/rn (Jacobsen et al., 1973).

One gas- liquid chromtographic method uses electron capture detection

capable of detecting the metabli tes of nrzepam (such as diazepa ) in
bloo and urine at the 0.04-0.05 llg/rn level; the rrthod invol ves hydrol ysis
to the benzophenone derivatives (de Silva & Puglisi, 1970). A simlar
rrthod for i ts detection in seru using flar ionization detection has been
describd (Steyn & Hunt, 1975). Agas chantogaphic method ca
determe diazepam and its major rrtalites in biological materials
qutitatively, with a limt of detection of 0.003 llg/rn (Heidbrin et aL.,
1975). Gas chromatographic-mass sptrortric methods are used to
determne diazepa and its rrtalites in biological materials (Horning
et al., 1974) and to analyse the staility of bulk diazepam at gas
chromatogaph teratures (Sadée & van der Kleijn, 1971). A high-pressure
liquid chr0rtographic rrthod separates and determes diazepam at the
rncrogram level (Scott & Bommr, 1970).

OXzepa can be assayed by gravimtric anal ysis using silicotungstic

acid (Bal tazar & Ferreira Braga, 1967); non-aqueous ti trimtric rrthods
can be used to determe bulk oxazepa (Baltazar & Ferreira Braga, 1967;
Beyer & Sadée, 1967; National Forrular Bord, 1970; Salim et aL., 1968),
and W spectrophotortry ca be used ta determne oxazepa in capsules
(National Forrlar Board, 1970; Salim et aL., 1968).
OXzepa and i ts metabli tes can be assayed colorirtricall y in
biological materials by hydrolsis to 2-aro-5-chlorobenzophenone and
diazotizing and coupling with a-naphthol (Pelzer & Maass, 1969), N-a-

63
naphthyl-N'-diethyl propylenediarne (Lafargue et al., 1970) or N-(l-
naphthyl)ethylenediarne hydrochloride (Kam & Kelm, 1969).

A fluorirtric rrthod ca de termne oxazepam in biological fluids

(Walkenstein et al., 1964). Polarogaphic rrthods are used to analyse
bulk oxazepam in a rrthanol -rnthy lene chloride sol vent system (Fazzari &
Rigglem, 1969) and to analyse oxazepam talets in Britton-Robinson buffer
solutions containing 20% dimthylformde (Oelschlaeger et al., 1969).
Gas chromatogaphic-mass spectrorrtric rrthods to analyse oxazepa,
in which oxazepa is quanti tati vel y rearranged to the quinazoline carbox-
aldehyde (Sadée & va der Kleijn, 1971) 1 and to detenne the rrtalite of
oxazepa, oxazepa glucuronide, in urine (Macucci et al., 1975) have ben
developed.
A high-pressure liquid chromatogaphic procedure to separate mixtures
of pure benzodiazepines (including oxazepam) has been describd (Scott &
Bol1r, 1970).

3. Biological Data Relevant to the Evaluation

of Carcinogenic Risk to Ma
3.1 Cacinogenicity and related studies in animls

Oral admistration
fuuse: Thee groups of 14 male and 14 femle Swiss-Webster mice
received oxazepam in the diet from 3 to 12 IInths of age at concentrations
of 0.05 or 0.15%, or ~re given a control diet. At 12 IInths of age all
mice recei ved the control diet for a further 2 IInths; then, all suri ving
animls were killed. Li ver tUlurS describ as li ver-cell adenomas were
found in 3/12 (25%) males receiving the lower dose and in 8/13 (61%) males
and 5/8 (62%) femles receiving 0.15% oxazepam. No liver tUIurs were seen
in 13 male and 10 ferle survi ving controls (Fox & Lacen, 1974).

3.2 Oter relevant biological data

(a) Exrimtal system
The oral ID of diazepam in nuce is 278 ID:./kg l: and that of oxazepam,
~ 0
1540 mg/kg hw (Marcucci et al., 1968).

64
 1 3
N -Dethylation and C -hydroxylation of diazepa lead to forntion
of N-derthyldiazepam, 3-hydroxydiazepam and oxazepa, which are excreted
as glucuronides in rnce (Macucci et aL., 1968), rats (Schwartz et al.,
1967), guinea-pigs (.l1acucci et al., 1971), rabbits (Jomm et al., 1964) and
dogs (Ruelius et al., 1965; Schwartz et al., 1965). In addition, para-
hydroxylation of the 5-phenyl ring occurs in rats (Schwartz et al., 1967)
and rabbits (Jomm et al., 1964).

Oxazepam has also been identified as a rntablite of chlordiazepoxide

(libriur) in dogs (Kiml & Walkenstein, 1967). Orally admistered
oxazepa is metablized to 6-chloro-4-phenyl-2- (lH) -qunazolinone, 5-
para-hydroxyoxazepa and the thee ope-ring rntalites (free or conju-
gate), 2-amo-5-chlorobezophenone, 2' -bezoyl-4' -chloro-2,2-dihydroxy-
acetailide and 2' -bezoyl -4' -chloro-2-hydroxy-2-ureidoacetailide, in
miiature swine (Sisenwine et al., 1972).
In vitro studies showed that liver of dogs (Schwartz & Postr, 1968),
rats and mice (Kvetina et al., 1968) rætablizes diazepam to fonn the thee
metablic products, N-derthyldiazepam, 3-hydroxydiazepam and oxazepa.

By whole boy autoradiography, rapid and intense accumulation of

14C-lablled diazepam was observed ir brain, kidney, liver, rnocardiur and

boy fat of mice (van der Kleijn, 1969) and of newbm rhesus monkeys (van
der Kleijn & Wijffels, 1971). Follawing i.v. admistration of diazepa it
was found that N-derthy ldiazepam accumulated in the bloo and brain of
mice (Marcucci et al., 1968), in bloo, brain and adipose tissue of

guinea-pigs (Marcucci et al., 1971) and in very smll aiunts for a short
tim in the bloo, brain and adipose tissue of rats (Macucci et al., 1970).

Dcs given diazepam intravenously excrete 61% of the compund and its
metablites in the urine and 34% in the faeces (Schwrtz et aL., 1965).
Transplacental transfer of diazepam occurred in mice, hamters and IInkeys
(Id~pä~-Heikkilä et al., 1971a,b).

When pregnant A/J rnce were gi ven a single i .m. dose of 100 rn/kg li
diazepa on the l4th day of pregnancy, no changes in the incidence of
cleft palate were observed in the offspring (Walker & PattersOl, 1974).

65
 OXzepa did not cause non-disjunction and crossing-over in Aspergillus
nidu lans (Bignam et al., 1974); malian rntalic acti vating system
were not used in ths test.

(b) Ma
The major rntalites of diazepa in hurs are forr by N-derthyla-
tion and C -hydroxylation (Schwarz et al., 1965). The main bloo rntalite,
3
N-demthyldiazepa (Kaplan et al., 1973), appears in the plasm withn
1-1.5 hours after i.m. admnistration (Baird & Hailey, 1973). Diazepar
rntablites are alist completely conjugated before excretion; oxazepa
glucuronide is a major urinary rntalite. Schwarz et al. (1965) found 10%
N-derthyldiazepam, 10% 3-hydroxydiazepam, and sorrat IIre than 30% oxazepa
in the urine; approximtely 71% of orally admnistered diazepa and its
rntalites are excreted in the urine and 10% in the faeces.

OXzepam is rntablized ta 6-chloro-4-phenyl-2 (lH)-qunazolinone,

5-para-hydroxyoxazepa and the 3 ope-ring rntali tes, 2-amno-5-chlorobenzo-
phenone, 2' -benzoy 1 -4' -chloro- 2, 2-dihydroxyacetanilide and 2' -bezoy 1 -4' -
chloro-2-hydroxy-2-ureidoacetanilide in hum (Sisenwine et al., 1972).
Diazepam and its rntablite N-derthyldiazepam cross the placenta
readil y when diazepa is gi ven to IIthers during labur (Erkkola et al.,
1973) or at the end of the first trimster of pregnancy (Erkkola et al.,
1974; Idänpään-Heikkilä et aL., 1971c). The tissue concentration of
diazepa in the foetus was higher than that in the IIther; the concentration
of N-derthy ldiazepa in foetal li ver was exceptionall y high (Errkola et
aL., 1974).

A significant association was found between maternal intake of diazepa

during the first trimster of pregnancy and the freqency of oral clefts
in the children. Of 709 WJrnn interviewed, 21 reported first-trirster
expsure to diazepa. A computer screen identified a statistically signifi-
cant four-fold relative risk for first-trimster exsure to diazepam amng
the IIthers of infants wi th cleft lip wi th or wi thout cleft palate as compred
with the IIthers of infants with all other defects. Simlar results were
obtained using Ilngolism as the control, but the significance levels VIre
lowr due to smller samle size. In no other defect category did first-

66
trirster use of diazepa differ significantly frar that of the rest of
the group (Safra & Oaley, 1975).

In the years 1967-71 informtion was collected on the mothers of 599

children with oral clefts; for 590 matched pair controls the data were
considered adeqate. The material was di vided into three groups - cleft
palate (CP), cleft lip with or without cleft palate (CL:!CP) and clefts
wi th addi tional defects. When the anti -aniety drgs were di vided into
tw groups - benzodiazepines (diazepam, oxazepa, nitrazepa, chlordiaz-
epoxide) and 'other' (iæprobate, chlorrzanone) - a significant association
was found (P-cO. 05) l:twen intae of benzodiazepines during the first tri-
rnster of pregnancy and oral clefts: the association was significant in
the CP group; an increased intake of these drugs was also seen in the
CL:!CP group when compared to controls, but the difference was not signi-
ficant (Saxén & Saxén, 1975).

3. 3 Case reports and epiderological studies

No data were available to the oorking Group.

4. Corts on Data Reported and Evaluation 1

4. 1 Animl data
In the only report available, oxazepar, a major metalite of diazepam,
was carcinogenic in rnce after its oral admistration: it produced liver-
cell tUIurS.

4. 2 Him data
No case reports or epiderological studies were available to the
Working Group.

ISee also the section 'Animl Data in Relation to the Evaluation of

Risk to Ma' in the introduction to this volur, p. 13.

67
 5. References

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Baggi, T.R., Maj an 1 S.N. & Rao, G.R. (1975) Colorimtric determnation
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Baird, E.S. & Hailey, D.M. (1973) Plasm levels of diazepa and its major
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Baltazar, J. & Ferreira Braga, M.M. (1967) Sa physico-chemcal charac-

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Bell, S. C. & Childress, S. J . (1962) A rearrangement of 5-ar 1 - l, 3-diydro-

2H-l,4-benzodiazepine-2-one 4-oxides. J. org. Chem., 27, 1691-1695

Beyer, K.H. & Sadée, W. (1967) Chestr and anlysis of bezodazepine

derivatives. III. Titration of bezodiazepine derivatives in any-
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British Phacopoia comssion (1973) British Pharmcopoia, London,

HMO, pp. 154-155
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Erkkola, R., Kangas, L. & Pekarinen, A. (1973) The transfer of diazepam

across the placenta during labur. Act obstet. gyec. scad., 52,
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Erkkola, R., Kanto, J. & Sellr, R. (1974) Diazepa ln ealy hur pregancy
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Fazzari, F .R. & Rigglem, O.H. (1969) Polarographic determnation of

oxazepam. J.. pha. Sci., 58, 1530-1531

Fox, K.A. & Lacen, R.B. (1974) Liver-cell adenomas and peliosis hepatis
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8, 481-488

68
Grecu, 1. & Macu, P. (1973) Anionic complexes of Cr (III) in the analysis
and the control of drgs. Aplications, to the gravimtric and
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Grol1m, A. & Grol1m, E.F. (1970) Pharncolog and Therapeutics, 7th ed. ,
Philadelphia, Lea & Febiger, p. 236

Haefelfinger, P. (1970) Emfindlicher Nachweis von Substanzen mit primen

Arnogruppen auf DÜschichtplatten. J. Chomat., 48, 184-190

Heidbrink, V., Mallach, H.J. & .fsmayer, A. (1975) Zur gaschromatographischen

Anal ytik der Benzcxiazepine. II . Diazepam und seine Metalite.
Arzneimttel-Forsch., 25, 516-517

Horning, M.G., Stillwell, W.G., Ncwlin, J., Lertratanangkoon, K., Carroll, D.,
Dzidic, I., Stillwell, R.N. & Horning, E.C. (1974) The use of stable
isotopes in gas chromatography-mass spectrorrtric studies of drug
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Idänpään-Heikkilä, J.E., Taska, R.J., Allen, H.A. & Schoolar, J.C. (197la)
Placental transfer of diazepam-14C in mice, hamters and IInkeys.
J. Pharcol. exp. Ther., 176, 752-757

Idänpään-Heikkilä, J.E., Taska, R.J., Allen, H.A. & Schoolar, J.C. (197lb)
Autoradiographic study of the fate of diazepam-C l 4 in the IInkey brain.
Arch. int. Pharmcodyn., 194, 68-77

Idänpään-Heikkilä, J.E., Jouppila, P.I., Puolaka, J.O. & Vorne, M.S. (197lc)
Placental transfer and fetal metablism of diazepa in early hum
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Jacobsen, E., Jacobsen, T.V. & Rojah, T. (1973) Determnation of diazepam

in seru by differential pulse polarography. Anal yt . chir. acta, 64,
473-476

Jomm, G., Maitto, P. & Silanos, M.A. (1964) Metablism of diazepam in

rabbits. Arch. Biochem. Biophys., 108,334-340

Ka, G. & Kelm, R. (1969) Qutitativer Nachweis von 7-Chlor-l,3-dihydro-

3-hydroxy- 5-pheny 1 - 2H- l, 4-benzodiazepin- 2-on ir Plasma bei einmliger
und längerer Verabreichung. Arzneimttel-Forsch., 19, 1659-1662

Kaplan, S.A., Jack, M. L., Alexander, K. & Weinfeld, R.E. (1973) Phar-
cokinetic profile of diazepam in ma follawing single intravenous
and oral and chronic oral admnistrations. J. phar. Sci., 62,
1789- 1 796

Kastrup, E.K., ed. (1974) Facts and Compisons, St lDuis, Missouri,

Facts and Ccrarisons, Inc., p. 26lc

Kastrup, E.K., ed. (1976) Facts and Comparisons, St lDuis, lv1issouri,

Facts and Comparisons, Inc., p. 288f
69
Kil, H.B. & Walkenstein, S.S. (1967) OXzepa excretion by chlordiaze-
poxide-14C-dosed dogs. J. pha. Sei., 56, 538-539

van der Kleijn, E. (1969) Kietics of distribution and :rtalisr of

diazepar and chlordiazepoxide in mice. Arch. int. Phacoyn.,
178, 193-215 .
van der Kleijn, E. & Wijffels, C.C.G. (1971) Whle-l:y and regional brain
distribution of diazepam in newbrn rhesus IInkeys. Arch. int. Pha-
codyn., 192, 255-264

Kvetina, J., Macucci, F. & Fanelli, R. (1968) MetaJ:xÜisr of diazepa in

isolated perfused liver of rat and lIuse. J. Pham. Pharncol., 20,
807-808

Lafargue, P., Pont, P. & Meunier, J. (1970) Etude des dérivés de la

benzo-(f)-diazépine-l,4 utilisés en thérapeutique. 1. Etude
spéctroscopique et pa chrantogaphie en couche mince de ces dérivés,
de leurs principaux métalites et des casés forms pa leur
hydrolyse acide. An. pham. franç., 28, 343-354

MacDonald, A., Michaelis, A.F. & Senowki, B. Z. (1972) Diazepa. In:

Florey, K., ed., Analytical Profiles of Drg Sustances, VoL. l,
New York, Academc Press, pp. 80-99

Macucci, F., .Guaitai, A., Kvetina, J., !'ussini, E. & Garattini, S. (1968)
Speies difference in diazepa rntablisr and anticonvulsat effect.
Euop. J. Phacol., 4, 467-470

~..cucci, F., Fanelli, R., Mussini, E. & Gaattini, S. (1970) Fuer

studies on speies difference in diazepa rntablisr. Euop. J.
Pharcol., 9, 253-256

Macucci, F., Guaitai, A., Fanelli, R., Hussini, E. & Gaattini, S. (1971)
Metalisr and anticonvulsant acti vi ty of diazepa in guinea-pigs.
Biochem. Pharcol., 20, 1711 - 1 713

Macucci, F., Bianchi, R., Airoldi, L., Sana, H., Fanelli, R., Chabrando,
C., Frigerio, A., Mussini, E. & Gaattini, S. (1975) Gas chræito-
graphic-rss spetrcrtric determation of intact C3-hydroxylated
benzodiazepine glucuronides in urine. J. Chomt., 107, 285-293

National Forrlar Board (1970) National Forrlar XIII, Washington Le,

American Phaceutical Association, pp. 220-223, SOl -502

CÆlschlaeger, H. & Volke, J. (1966) Polarography of therapeuticall y imr-

tat 1,4-bezodiazepine derivatives. In: Hanc, O., ed., Proceeings
of the 25th International Pharceutical Congress, 1966, Vol. 2,
Iondon, Butte~rt, pp. 101-104

70
Oelschlaeger, H., Volke, J., Lim, G. T. & Spag, R. (1969) Pharceutical
analysis by polarography and oscillopolarogaphy. X. Reduction of
oxazepa at the dropping rrrcu elecode. Arch. Phar. (Weinheim),
302, 946-951

Pelzer, H. & Maass, D. (1969) Phaokinetik und Metalisrus von 7-clor-

l, 3-dihydro- 3-hydroxy-5-pheny 1 - 2H- l, 4-bezodiazepin- 2-on un des
sen
Hemsuccinat beim lvsche. Arzneimttel-Forsch., 19, 1652-1656

Reeer, E. & Sternch, L.H. (1963) 5-Phenyl-l,2-dihydro-3H-l,4-bezo-

diazepines. US Patent 3,109,843, Novemr 5, to Hoffr-La Roche Inc.
Reer, E. & Sternch, L.H. (1964) 5-Arl-3H-l,4-benzodiazepin-2 (lH)-
ones. US Patent 3,136,815, June 9, to Hoffr- La Roche Inc.

Ruelius, H. W., Le, J .M. & Alum, H. E. (1965) Metalism of diazepa

in dogs: transforntion to oxazepam. Arch. Biocher. Biophys., 111,
376-380
Sadée, W. & van der Kleijn, E. (1971) Therilysis of 1,4-bezodiazepines
during gas chrantography and mass spetroscopy. J. phar. Sei., 60,
135- 137

Safra, M.J. & Oakley, G.P., Jr (1975) Association between cleft lip with
or without cleft palate and prenatal expsure to diazepam. Lacet,
ii, 478-480

Salim, E.F., Deuble, J.L. & Papariello, G. (1968) Qualitative and quanti-
tative tests for oxazepam. J. pham. Sei., 57, 311-313

Saxén, I. & Saxén, L. (1975) Association betwen maternal intae of diazepa

and oral clefts. Lacet, ii, 498

Sbabati Nudelm, N. & De Waisbaum, R.G. (1975) Method to deterre

diazepa and nitrazepa stability. Farco, Ed. prat., 30, 488-495

Sehuetz, C., Post, D., Sehewe, G., Sehuetz, H. & Muskat, E. (1972) Micro-
chemcal detection and sepaation of five 5-phenyl-l,4-bezodiazepine
tranquilizers by thin- layer chromatogaphical SRS-technique. Fresenius '
Z. analyt. Cher., 262, 282-286

Schwatz, M.A. & Postm, E. (1968) Metablism of diazepa -in vltro. Biochem.
Pharmcol., 17, 2443-2449

Schwartz, M.A., Koechlin, B.A., Postm, E., Palmr, S. & Krol, G. (1965)
Metablisr of diazepam in rat, dog, and ma. J. PhacoL. ex. Ther.,
149, 423-435

Schwatz, M. A., Bor, P. & Vane, F. M. (1967) Diazepa rrtali tes in the
rat: characterization by high-resolution mass spectrortry and nuclear
magnetic resonance. Arch. Biocher. Biophys., 121, 508-516

71
Scott, C.G. & Bor, P. (1970) The liqud chromtogaphy of sor benzo-
diazepines. J. chromt. Sci., 8, 446-448

Shearer, C.M. & Pilla, C.R. (1974) OXzepa. In: Florey, K., ed.,
Analytical Profiles of Drug Substaces, VoL. 3, New York, Academc
Press, pp. 442-464

de Silva, J .A.F. & Puglisi, c.v. (1970) Determation of rnzepam

(nobrium), diazepam (valium) and their major biotransformtion
products in bloo and urine by electron capture gas- liquid chromto-
graphy. Analyt. Chem., 42, 1725-1736

Sisenwine, S.F., Tio, C.O., Shrader, S.R. & Rulius, H.W. (1972) The bio-
transformtion of oxazepa (7-chloro-l,3-dihydro-3-hydroxy-5-phenyl-
2H-l,4-benzodiazepin-2-one) in ma, miniature swine and rat.
Arzneirttel-Forsch., 22, 682-687

Stecher, P.G., ed. (1968) The Herck Index, 8th ed., Raay, ID, M2rck &
Co., pp. 341, 772-773

Sternch, L.H. & Reeder, E. (1961) Qunazolines and 1,4-bezodiazepines.

IV. Transformtions of 7-chloro-2-methylamno-5-phenyl-3H-l,4-benzo-
diazepine 4-oxide. J. org. Chem., 26, 4936-4941

Sternch, L.H., Reeder, E., Keller, O. & Metlesics, W. (1961) Quinazolines

and 1,4-benzodiazepines. III. Substituted 2-amno-5-phenyl-3H-l,4-
benzodiazepine 4 oxides. J. org. Chem., 26, 4488-4497

Steyn, J.M. & Hundt, H.K.L. (1975) Qutitative gas chromatographic deter-
mination of diazepam an its major rntalite in hum ser.
J. Chromt., 107, 196-200

US Drug Enforcemnt Admistration (1975) Schedules of controlled substaces.

Federal Reister, 40, 4016-4017

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Production and Sales, 1974, USITC Pulication 776, Washington OC,
US Governnt Printing Office, p. 106
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and Products, 1974, USITC Pulication 762, í'iashington OC, US Goernnt
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Rockville, M:, pp. 135-136

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Sales, 1963, TC Pulication 143, Washington OC, US Goernnt Printing
Office, p. 129

72
US Tariff Commssion (1967) Synthetic Organic Chercals, US Production and
Sales, 1965, TC Publication 206, Washington DC, US Governt Printing
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1973, TC Publication 688, Washington DC, us Governnt Printing Office,
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Walkenstein, S.S., Wiser, R., Gudmdsen, C.H., Kiml, H.B. & Corradino, R.A.
(1964) Absorption, metablism, and excretion of oxazepar and its suc-
cinate half-ester. J. pharr. Sci., 53, 1181-1186

Walker, B.E. & Patterson, A. (1974) Induction of c1eft pa1ate in mice by

tranquilizers and barbi turates . Teratolog, 10, 159-164

Woour, D.M. & Fingl, E. (1975) Drugs effective in the therapy of the
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73
 DITHL

1. Chcal and Physical Data

1. 1 Synonyr and trade nars

Cher. Abstr. Reg. Serial l'.: 480-22-8

Cher. Abstr. Nar: 1,8, 9-Anthracenetriol

Anthalin; 1,8, 9-anthatriol; l, 8-dihydroxyanthanol; l, 8-dihydroxy-

9-anthanol ¡ dioxyanthranol; 1,8, 9-trihydroxyanthracene

Antha-Derr; Batidrol; Chsoderil¡ Cignolin; Cigthanol;

Cygnolin¡ Dernline; Derobin; Lasan; Psoriacid-Stift
1.2 Chemcal formla and 1I1ecular weight

HO HO HO

C14H1003 M::Ü. wt: 226.2

1. 3 Chemical and physical properties of the pure substace

From Stecher (1968), unless otherwise specified

(a) Description: Yellaw leaflets or needles

(b) Melting-point: 176-181 °c

(c) Spectroscopy data: Àma 256 and 288 nr (E: = 504 and 411) in
rnthol ¡ infra-red and nuclear magnetic resonance spectra are
also given (Grasselli, 1973)

(d) Solubility: Practically insoluble in water¡ freely soluble in

chloroforr¡ soluble in acetone, benzene, pyridine, oils and
dilute sodium hydroxide¡ slightly soluble in ethanol, ether
and glacial acetic acid

(e) Reactivity: When oxidized it produces dithranol dir and

1,8-dihydroxyanthraquinone in acetone or irthanol (Segal et aL.,
1971)

75
1.4 Technical products and imurities

Various national and international phacopoias gi ve specifications

for the puri ty of di thanol in pharnceutical products. For exarle,
dithanol is available in the US as a USP grade containing 95.0-100.5%
active ingredient on a dried basis. It is available in ointrt fonn
containing 0.1, 0.2, 0.25, 0.4, 0.5 or 1.0% dithanol in a petrolatum or
other suitable base Which contains 90-115% of the stated aiunt of dithanol
(US Phacopeial Convention, Inc., 1975).
In the OK, dithanol is available as an ointmt containing 0.1% dithanol
in paraffin and containing 90-110% of the stated aiunt of active
ingredient (British Pharmcopoia Commssion, 1973).

2. Production, Use, Ocurrence and Anal ysis

For imrtant background inforntion on this section, see prearle,

p. 15.

2. 1 Production and use

(a) Production

A rrthod for the synthesis of di thanol by the reduction of l, 8-di-

hydroxyanthaquinone wi th hydrogen and nickel catal ysts at high pressure
was reported in 1938 (Zah & Koch, 1938). Dithanl can also be prepaed
by sulphonating anthaquone and heating the resulting 1,8-disulphonic
acid with a calcium hydroxide/calcium chloride mixture to give 1,8-dihydroxy-
anthaquinone, which is reduced with tin and hydrochloric acid (Haey, 1975a).

Camrcial production of dithanol was first reported in the US in

1936 (US Tariff Comssion, 1938). Hoever, since 1957, when it was reported
for th lasttiI, dithranol has not be prouced in [Link] quantities
in the US (US Tariff Comssion, 1958). US imrts though the principal
custom districts were 30 kg in 1972 and in 1973 (US Tariff Comssion,
1973, 1974) and 120 kg in 1974 (US International Trade Commssion, 1976).

(b) Use

Dithanol is a polyphenol with mild skin irritat and antibcterial

activity. It is used in hum rrdicine for the treatrt of psoriasis
76
and chronic derntoses (Harey, 1975b). Dithanol is applied to the skin
as an ointrt containing frar 0.1-1.0% di thanol. The usual dosage
regirn begins with the lCMer available concentration and is increased
gradually as neeed to obtain the desired therapeutic effect (Arrican
Soiety of Hospital Pharmcists, 1959).

Total US use of di thanol in hum rricine is estimted to be less

thn 25 kg anuall y .
Di thanol has also ben used in veterinary rricine to treat ringwonn
(Stecher, 1968).

2. 2 Occurrence
Di thanol is not known to occur in nature. A chemcal closel y related
to dithanol, 1,8-dihydroxy-3-ræthyl-9-anthone, constitutes abut 30% of
a natural product, chrsarobin (purified Goa poder, purified araroba) ,
which rny produce conjunctivitis if used in the treatrt of psoriasis
(Stecher, 1968).

2. 2 Anl ysis
Method of assay of dithanol that ræt regulatory reqirernts for
pharceutical products include W spctrophotorætr and colorimtry.
One assay procedure is based on the bromination of dithanol with ¡:tassium
brornde and ¡:tassium brornte (Ionescu-SololIn et al., 1966).

3. Biological Data Relevant to the Evaluation

of Cacinogenic Risk to Ma
3.1 Carcinogenicity and related studies in animls

Skin admistration
MJuse: Groups of 10-16 ICR Swiss or C57/St adult mice, 55 days of age,
received a single application of 125 ~g 7,12-dimthylbnz(a)anthacene
(DMB) in acetone as an initiator¡ 25 days later they received repeated skin
paintings of 0.25 ml of a 0.01% or 0.033% solution of dithanol in acetone
5 tims per [Link] for 32 or 48 [Link]. In Swiss rnce painted 5 tims weekl y
for 48 [Link], the incidences of skin papillors were 14% (2/14) in anirls
receiving 0.01%, and 87% (13/15) in animls receiving 0.033% dithanol.

77
C57/St rnce panted with a 0.033% solution develope tururs, with an
incidence of 46% (6/13) (P,[Link]. Th first tUlur appeared 14 wees after
the first application of di thanol. No tururs were observed in CS 7/St
rnce painted with a 0.01% solution or in 29 control rnce of bath strains
combined painted with DMB follCMed by acetone without the initiator.

Applications of di thanol in acetone gave rise to a single skin papilloma

arng 30 Swiss rnce, and no skin tUlurS occured arng 39 CS 7/St mice
treated with dithanol in acetone. In Swss rnce given one application
of dithanol (0.033 or 0.1%) follCMing DM initiation, the incidences
of skin tUlurS were 2/15 and 5/15, respetively. Of 36 tuiur-bering
rnce treated with av and dithanol, 8 develope squarus-cell carcinomas
(Bok & Burs, 1963).
A group of 20 7-week old femle ICR/Ha mice were painted with a
single application of 20 llg DM in 0.1 ml acetone, follCMed 2 wees later
by repeated paintings 3 tirs wely for 490 days with 0.1 ml of a 0.08%
solution of dithanol in acetone. The first papilloma appeared 59 days
after the first application of dithanol. In all, 18 rnce developed a
total of 94 skin tururs, including 9 sqarus-cell carcinomas. In control
animls painted with DM follCMed by acetone or by tranforntion products
of di thanol , i. e., di thanol dimr or l, 8-dihydroxyanthaquinone , no tUIurs
were found. Applications of dithanol in acetone without DMB induced a
skin papillam in 1/20 rnce 287 days after the first application of
dithanol (Segal et al., 1971).

Groups of 40 and 20 ICR and 20 CF mice (6 or 8 wes of age) received

i. p. injections of urethane (ethyl cabate) as initiator in a single dose
of 50 mg or as thee daily doses of 40 mg, foilCMed 3 days later by repeated
skin paintings, 5 tirs w=ekly for 27-28 wes, with 0.25 ml of a 0.033% or
0.1% solution of dithanol in acetone. The culative percetages of skin
papilloma-bearing rnce of bath strains pretreated with 3 injections of
urethane follCMed by 0.1% dithanol in acetone were 40% in ICR and 75% in
CF mice. ünly one skin tUlur was observed in a group of 20 CF mice
treated only with 0.1% dithanol in acetone, and no tUlurS were observed
in a group treated with urethane follCM by acetone. Lymhomas were also
found in ICR mice: 21/40 mice (52.5%) treated with 50 mg urethane, followed

78
by topical application of 0.033% dithanol in acetone, had developed
such tururs when th animls ~re killed 27 wes after the stat of the
[Link]; 6/40 (15%) and 4/20 mice (20%) develope lymhomas following
topical applications of 0.033% and 0.1% dithanol in acetone without
urethe pretreatrnt; 3/40 (7%) and 4/20 rnce (20%) develope lymhomás
after treatrnt with urethane alone at a single dose of 50 ID or thee
doses of 40 ID (Yasuhira, 1968a,b).

3.2 oter relevant biological data

(a) Exrimntal system

Dithanol forr nolecuar complexes with calf thymus DNA in vitro
(Swanck & Zetterberg, 1971). It induces petite mutants in Saccharomyces
~erevisiae (Gillbrg et al., 1967; Siebrt et al., 1970) but no gene
conversion in Saccharomyces cerevisiae (D4 strain) (Siebrt et al., 1970).
At the rn non-toxic dose, no ¡:int mutations were found in Sa lmone lla
typhimurium TA98, ThlOO, Th1535 or Th1537 in the presence or absence of
rat liver harenate (McC et aL., 1975). In a large mutagenicity study
on anthaquinone deri vati ves, di thanol at near toxic concentrations was
negative (or very weak) in inducing ¡:int mutations ln Salmonella
typhimurium Th1537, TA98 and Th1538 in the presence of rat li ver hoimenate
(Brow & Brow, 1976).
(b) Ma
No chages in li ver or kidney function or in the bloo picture were
observed in 40 patients treated with 0.1-0.4% dithanol paste or ointrnt
topically during the treatrt of psoriasis over a 3-lInth period (Gay
et aL., 1972). Inhibition of DNA repair after UV irradiation ha ben
reported in cutured peripheral hum leucoes (Gaudin et al., 1972),
but this appears to be the result of general cellular toxicity (Cleaver &
Painter, 1975; Poir~er et al., 1975).

3. 3 Case reports and epidemological studies

No data were available to the WJrking Group.

79
 4. Col1nts on Data Reported and Evaluation l

4.1 Animl data

Di thanol is a tuiur-prarting agent in lIuse skin carcinogenesis

exrirts following initiation wi th ei ther 7, l2-dimthy lbenz (a) anthacene
or urethane. An increased incidence of 1 ymhomas was also observed in
nuce painted with dithanol after urethane initiation.

4.2 Hum data

No case reports or epiderological studies were available to the
Working Group.

ISee also the section 'Animl Data in Relation to the Evaluation of

Risk to Man' in the introduction to this volur, p. 13.

80
 5. References

Amrican Soiety of Hospital Pharmcists (1959) Amrican Hospital Formlar

Service, Section 84: 28, Washington CC

Bok, F.G. & Burs, R. (1963) Tur-prorrting properties of anmralin

(1,8,9-anthatriol). J. nat. Cacer Int., 30, 393-397

British Pharmcopoia Commssion (1973) British Pharcopoia, London,

HMO, p. 176
Brow, J.P. & Brown, R.J. (1976) Mutagenesis by 9,10-anthaquinone deri-
vatives and related compunds in Salmonella typhimurium.
Mutation Res., 40, 203-224

Cleaver, J.E. & Painter, R.B. (1975) Absence of spcificity in inibition

of DNA repair replication by DNA-binding agents, coarcinogens and
steroids in hum cells. Cacer Res., 35, 1773- 1778

Gaudin, D., Gregg, R. S. & Yielding, K.L. (1972) Inhibition of DNA repair
by cocarcinogens. Biochem. biophys. Res. Commun., 48, 945-949

Gay, M. W., More, W. J., lvrgan, J. M. & Montes, L. F. (1972) Anthalin toxi-
city. Arch. Derntol., 105, 213-215

Gillberg, B.O., Zetterberg, G. & Swck, G. (1967) Petite mutats induced

in yeast by di thanol (1,8, 9-trihydroxyanthacene), an imrtat thera-
peutic agent against psoriasis. Nature (Lond.), 214, 415

Grasselli, J.G., ed. (1973) CRC Atlas of S¡:~ctral Data and Physical Constants
for Organic COI1unds, Cleveland, Ohio, Chemcal Rubber Co., p. B-166

Harvey, S.C. (1975a) Topical drugs. In: Osol, A., et al., eds,
Remngton 1 s Pharceutical Sciences, l5th ed., Easton, Pa, Mack,
p. 720

Harey, S.C. (1975b) Antiseptics and disinfectants; fungicides; ectoparasi-

ticides. ln: Godm, L. S. & Gilm, A., eds, The Pharmcological
Basis of Therapeutics, 5th ed., New York, Macmllan, p. 1006

Ionescu-Solol1n, L, Constantinescu, T., Enache, S. & Garti, V. (1966)

Determnation of the product Cignolin - a volumtric rrthod for the
determation of l, 8-dihydroxy-9-anthanol. Rev. Chir. (Buc.), 17,
490-493

McC, J., Choi, E., Yamsaki, E. & Ams, B.N. (1975) Detection of
carcinogens as mutagens in the SalmoneZ la/rncrosorr test: assay of
300 chemcals. Proc. Nat. Acad. Sci. (Wash.), 72, 5135-5139

81
Poirier, M.C., De Cicco, B.T. & Lieb, H.W. (1975) Ibnspeific inition
of DNA repair synthesis by turr pronters in hmi diploid fibroblasts
daged with N-acetoxy-2-acetylamofluorene. Cancer Res., 35, 1392-1397

Seal, A., Katz, C. & Van Duuren, B.L. (1971) Structure and tuir-pronting
activity of anthalin (1,8-dihydroxy-9-anthone) and related compunds.
J. me. Cher., 14, 1152-1154

Siebert, D., Zimrm, F.K. & Lerle, E. (1970) Geetic effects of

fungicides. Mutation Res., 10, 533-543

Stecher, P. G., ed. (1968) The Merck Index, 8th ed., Raay, NJ, Merck &
Co., pp. 260, 394

Swanck, G. & Zetterberg, G. (1971) Studies on dithranol and dithanol-

like cc:unds. I. Binding to nucleic acids. Acta derme venereol.
(Stokh.), 51, 41-44

US International Trade Commssion (1976) Imrts of Benzenoid Chercals

and Products, 1974, USI'I Pulication 762, Washington OC, US Governt
Printing Office, p. 79

US Phacopeial Convention, Inc. (1975) The US Pharmcopeia, 19th rev.,

Rockville, Md, p. 33

US Tariff Cæission (1938) Oyes and Oter Synthetic Organic Chemcals in

the US, 1936, Report No. 125, Seond Series, Washington De, US
Governt Printing Office, p. 39
US Tariff Cassion (1958) Synthe tic Organic Chercals, us Production and
Sales, 1957, Report Ib. 203, Seond Series, Washington De, US
Governt Printing Office, p. 103
US Tariff Cæission (1973) Imrts of Bezenoid Chercals and Products,
1972, 'I Pulication 601, Washington OC, US Governt Printing
Office, p. 82

US Tariff Cæission (1974) Imrts of Bezenoid Chercals and Products,

1973, 'I Pulication 688, Washington OC, US Governt Printing
Office, p. 78

--
Yasuhra, K. (1968a) Skin papillan production by anthalin painting after
ureth initiation in rnce. Ga, 59, 187-193

Yasuhra, K. (1968b) Induction of :rlignt lyrhoma and other turrs during

exprimnts with anthalin painting of mice. Ga, 59, 195-200

Zah, K. & Koch, H. (1938) Zur Kentns der katalytischen Reuktion und
Hydrierug einiger Ox-anthachinone. 13r. dtsch. chem. (':is.,
71, 172-186

82
 EIIONAHIDE

1. Chemcal and Physical Data

1. 1 Synonyr and trade nas

Cher. Abstr. Reg. Serial No.: 536-33-4

Cher. Abstr. Nar: 2- Ety 1 -4-pyridinecarbothioamde

Ardazine; ethna; ethioniamde; a-ethylisonicotinic acid thioade;

2-ethylisonicotinic acid thioamde; 2-ethylisonicotinic thoamde;
a-ethy lisonicotinoy 1 thioamde; ethylisothiamde; a-ethy lisothioni-
cotinarde; 2-ethy lisothionicotinamde; 2-ethy 1 -4-thioamdy lpyridine;
2-ethy 1 -4-thiocarbay lpyridine; a-ethyl thioisonicotinarde; 2-ethy 1-
thioisonicotinamde; ethyonomde; thianid; thianide; thioade

Aetina; Aetiva; Ardazin; Bayer 5312; EI; Ethinamde; Etimde;

Etird; Etiocidan; Etionid; Etionizina; Etionizine; ET; Fatoli-
amd; F.I. 58-30; Iridocin; Iridozin; Isothin; Isotiamda;
Itiocide; Nicotion; Nisotin; Nizotin; Pigenicid; Sertinon;
Tebrus; 1314 TH; TH 1314; Th 1314; Thanid; Thoarde;
Thiord; Thioniden; Tio-Mid; Trecator; Trescatyl; Trescazide;
Turmn; Turoid; Turoson
1. 2 Chercal formla and molecular weight

56'
N CHi-CH3

C8H10N2S MoL. wt: 166 .2

1. 3 Chemcal and physical properties of the pure substance
From Stecher (1968), unless otherwise soecified

(a) Description: Yellaw crystals fror ethanol

(b) Melting-paint: DecOIsition at l64-l660C

83
 (c) Spectroscopy data: À
ni290 nr in rrthanol (US Pharncopeial
Convention, Inc., 1975)

(d) Solubility: Insoluble in water and ether; sparingly soluble in

rnthanol, ethanol and propylene glycol; soluble in hot acetone and
ethylene dichloride; freely soluble in pyridine

1. 4 Technical products and imuri ties

Various national and international nharmconoias give specifications
for the purity of ethionade in oharceutical nroducts. Por [Link],
ethionarde is available in the US as a USP grade containing 98.0-102.0%
active ingredient on an anydrous basis, a maim of 0.003% selenium and
2% water. Tablets are available in a 250 ID dose which contains 95.0-110.0%
of the stated aiunt of ethionamde (US Pharncopeial Convention, Inc., 1975).
In Japan, ethionarde is also available in supPOsitory forme In the UK,
it is available in forr containing not less than 98.5% active ingredient
on a dried basis and in 125 ID talets containing 95.0-105.0% of the stated
aiunt of active ingredient (British Pharmcopoia Commssion, 1973).

2. Production, Use, Occurrence and Anal ysis

For imrtant background inforntion on this section, see preamle,

p. 15 .

2 . 1 Production and use

(a) Production

A rnthod of synthesizing ethionarde ootented in the OK in 1958

involves dehydration of 2-ethylisonicotinamde to 2-ethylisonicotinonitrile
which is reacted wi th hydrogen sulphide in the presence of triethanolarne
to give ethionarde (Chime et Atomistique, 1958).

Commrcial production of ethionamde was first reported in the US in

1968 (US Tariff Commssion, 1970); only one US company reported production
(see preamle, p. 15) in 1972 (US Tariff Corssion, 1974a). US irrt of
ethionamde though the principal custorn districts were abut 40 kg in
1972 (US Tariff Commssion, 1973), 125 kg in 1973 (US Tariff Commssion,
1974b) and 276 kg in 1974 (US International Trade Comssion, 1976).

84
 (b) Use

Ethionarde 1S an analoge of thioisonicotinarde and shows strong

antibacterial activity. It is used in hum rncine for the treatInt of
tubrculosis. l t comletel y suppresses the multiplication of hum strains
of Mycobacterium tuberculosis in vitro and is usually effective against
bacilli resistant to other tubrculostatic agents. However, since i t 1S
less patent and more toxic than prim antitubrculosis drgs, it 1S used
only as a secondary antitubrculosis drug, usually in comination therapy
with prim agents, such as isonicotinic acid hydrazide or streptomycin,
or in cases of prim drg resistance (Weinstein, 1975).

Ethionarde is adrnistered orally at an initial adult dose of

250 rn twice a day. The dosage is increased by 125 rn per day for 5 davs
until one gram is adrnistered daily (Weinstein, 1975). The highest tolerated
dose, usually between 750 mg and 1 g per day, is recommnded to prevent the
develoomnt of resistance (Amrican Soiety of Hospital Pharcists, 1963).

2 . 2 Occurence
Ethionarde 1S not know to occur ln nature.
2 . 3 Anal ysis
Methods of assay of ethionarde that rnt regulatory reqirernts for
pharceutical products include titration and W spctrophotometry.

A simle colorimtric method for its detection and analysis involves

deterrnation of its reaction product with 2, 3-dichloro- l, 4-naphthoqinone

- ... -
(Devani et al., 1974). Other colorimtric methods for the determnation
of ethonarde in plasm have also been reported (Harnanansingh & Eidus,
1970; PUtter, 1972).
A coulometric assay has been describ (Sernt et aL., 1972).

85
 3. Biological Data Relevant to the Evluation
of Cacinogenic Risk to Han

3.1 Cacinogenicity and related studies in animIs 1

Oral admistration
Mouse: A group of 36 8-wee old femle BA/c/Cb/Se mice were given
daily intragastric instillations of 0.1 ml of a 2% solution of ethionarde
in propylene glycol (2 rr/animl) on 6 days per we for 50 weeks; 7/33 rnce
suriving 69 weeks develope thyroid tUIurS (5 papillar and 2 epideriid
carcinors) between 28 and 69 wees. After 69 weeks none of 18 suriving
controls receiving propylene glycol alone and none of 47 untreated controls
had develope thyroid tururs. Fi ve cases of diffuse hyrtropy and 3
of nodular hyprtrophy were observed in the thyroids of treated mice.
Pulinary tUIurs occurred in 7/33 treated rnce that died between 60-69
wees, in 10/38 untreated controls that died betwee 80-109 weeks and in
2 that died betwen 40-69 weeks (Biancifiori et aL., 1964).
3.2 Oter relevant biological data
(a) Exrimtal system
The i. p. ID of ethionarde in rnce is 1350 rrjkg tw (Dlufniewski &
~ 0
Gastol-lewiriska, 1971). Intragastric and s.c. admnistrations of 54 or
270 mg/kg bw ethionarde ta pregant rats on days 6-14 of pregnancy
prcxuced skeletal malforntions in their offspring. No malforntions were
seen in the offspring of pregnant rabbits treated by gavage with 13.5 or
27.0 rrjkg tw on days 7-14 of pregnancy (DruZniewski & Gastor-Leir~ska,
1971) .

(b) Ma
Toxic hepatitis has been associated with use of ethionarde and is
accompied by elevated levels of li ver enzyms ln the plasma (Phillips &
Tashm, 1963; Simn et al., 1969).

IThe Working Group was also aware of on-going studies in mice and
rats (IA, 1974).

86
 Oral admistration of 1 g ethionarde results in peak plasma levels of
abut 20 llg/rn after 3 bours (Weinstein, 1975). Thee rætablic pathways
are knO\: a sulphoxide is forr; the thoketone group is hydrol ysed
to give 2-ethylpyridine-4-carboxylic acid amde and the free carboxylic
acid; or the pyridiniur N-atom is ræthylated, followed by hydroxylation
at the 6-carbon atom ta give N-rnthyl-2-ethylpyrid-6-one-4-thiocarboxylic
acid amde (Bieder & Mazeau, 1962, 1964; Bieder et al., 1966; Iwainsky ,
1964; Iwainsky et al., 1965; Kae, 1962; Ritter, 1973). The rætablites
were excreted in the urine (Bieder & Bruel, 1971).

3. 3 Case reports and epidemological studies

J: data were available to the WJrking Group.

4. Cornnts on Data RefXrted and Evluation 1

4 . 1 Animl data
Ethonade 1S carcinogenic in rnce after its oral admnistration,
the only speies and route of admistration tested: it produced thyroid
carcinonas.
4 . 2 Hur data
J: case refXrts or epidemological studies were available to the
WOrking Group.

1 Se also the section 'Anl Data in Relation to the Evaluation of

Risk to Ma' in the introduction ta this volur, p. 13.

87
 5. References

Arrica Soiety of Hospital Phacists (1963) Arrican Hospital Forrlar

Service, section 8: 16, Washington OC

Biancifiori, C., Milia, U. & Di Le, F.P. (1964) Turi della tiroide indotti
meiante etionade (Er) in topi femna vergini BA/ c/Cb/Se substrain.
Lav. Ist. Anat. Univ. Perugia, 24, 145-166

Bieder, A. & Bruel, P. (1971) Application de la polarographie rapide à

courant alternatif surÌIsé à l'étude de l'élimnation urinaire des
métali tes de l' éthonarde et du protionarde chez l' hoIl . An.
phar. franç., 29, 461-476

Bieder, A. & Mazeau, L. (1962) Etude du métablisi de l' éthionarde chez

1 'hoIl. 1. Séparation des métablites par chromatographie. An.
phar. franç., 20, 211 - 216

Bieder, A. & Mazeau, L. (1964) Reherches sur le rrtablisrn de l' éthionamde

chez 1 'hom. Sépaation et identification de certains rrtablites par
chromatographie sur couche mince. Thérapie, 19, 897-907

Bieder, A., Bruel, P. & Mazeau, L. (1966) Identification de trois nouveaux

métalites de l' éthionarde: chromatographie, spctrophotométrie,
polarogaphie. An. phar. franç., 24, 493-500
British Pharmcopoia Cossion (1973) British Pharmcopoia, London,
HM, pp. 193-194
Chime et Atamstique (1958) a-Substituted isonicotinic thioamdes.
British Patent 800,250, August 20

Devani, M.B., Shishoo, C.J., Moy, H.J. & Raja, P .K. (1974) Detection of
thioamdes : determnation of ethionarde wi th 2, 3-dichloro- l, 4-naphtho-
quinone. J. phar. Sei., 63, 1471-1473

Dluž'niewski, A. & Gastol-Leinska, L. (1971) The search for teratogenic

acti vi ty of sorn tubrculostatic drgs. Diss. pha. (Krakow), 23,
383-392
Harnanansingh, A.M. T. & Eidus, L. (1970) Determnation of ethionamde in
seru. Int. Z. klin. Pharmol. Ther. Toxikol., 3, 128-131

IAC (1974) lAC Informtion Bulletin on the Surey of Chercals Being

Tested for Carcinogenicity, No. 3, Lyon, P. 23

Iwainsky, H. (1964) Zum Stoffwechsel des a-Athylisonikotin8äurethoades

(Athioniard) . Z. Turkul., 122, 127-130

Iwainsky, H., Sehrt, 1. & Gruert, M. (1965) Zum Stoffwechsel des Athioniards
nach intravenöser Verabreichung. Arzneimttel-forsch., 15, 193-197

88
Kane, P.O. (1962) Identification of a fftalite of the antitubrculous
drg ethionamde. Nature (Lond.), 195, 495-496
Phillips, S. & Tashr, H. (1963) Ethionamde jaundice. AIL Rev. resp.
Dis., 87, 896-898

PUtter, J. (1972) Bestimg von Prothionamd und Athionamd scwie den

entsprechenden Sulfoxiden ir Blutplasm. Arzneirtte1 -Forsch., 22,
1027- 1031

Ritter, W. (1973) Neue Ergebnisse tfr Biotransformtion und Pharmo-

kinetik von Antitubrkulotika. Prax. Pneurl., 27, 139-145

Sert, E., Rousselet, F., Girard, M.L. & Chera, ~1. (1972) les méthodes
électrochiques dans 1 ' analyse pharceutique. IV. Applications
de la coulométrie à intensité constate au dosage automatique de
compsés organiques soufrés. An. pham. franç., 30, 691-700

Sirn, E., Veres, E. & Bái, G. (1969) Chages in SGO activity during
treatrnt wi th ethionamde. Scand. J. resp. Dis., 50, 314-322

Stecher, P .G., ed. (1968) The Merck Index, 8th ed., Raway, NI, Berck &
Co., pp. 427-428

us International Trade Comssion (1976) Imrts of Bezenoid Chercals

and Products, 1974, USI'l Pulication 762, Washington OC, us
Governnt Printing Office, p. 82
us Pharcopeial Convention, Inc. (1975) The us Pharcopeia, 19th rev.,
Rockville, Md, pp. 187-188

us Tariff Comssion (1970) Synthetic Organic Chercals, us Production and

Sales, 1968, 'l Pulication 327, Washington OC, us ('.,vernt Printing
Office, p. 117

us Tariff Commssion (1973) Imrts of Bezenoid Chercals and Products,

1972, 'l Pulication 601, Washington OC, us C:ivernnt Printing Office,
p. 84

us Tariff Commssion (1974a) Synthetic Organic Chercals, us Production and

Sales, 1972, 'l Publication 681, Washington OC, us Governnt Printing
Office, p. 110

us Tariff Commssion (197 4b) Imprts of Bezenoid Chercals and Products,

1973, 'l Publication 688, Nashington OC, us Goverrnt Printing Office,
p. 80

Weinstein, L. (1975) Drugs used in the cheitherapy of tubrculosis and

leprosy. In: Go, L. S. & Gilm, A., eds, The Pharmcological
Basis of Therapeutics, 5th ed., Ne York, Macmllan, pp. 1212-1213

89
 HYCAONE and HYCAONE NESYITE

1. Chercal and Physical Data

Hycanthone
1. 1 Synonyr and trade nas
Chem. Abstr. Reg. Serial No.: 3105-97-3

Chem. Abstr. Nar: 1-1:2- (Diethylamo)ethyl)am01-4- (hydroxy-

methy 1) -9H-thioxanthen-9-one
1- t ( 2- (Diethy lamno) ethy 1) amno 1-4- (hydroxythy 1) thioxathen-9-one;
lucathone rntabli te

Win 24933

1. 2 Chercal formla and molecular weight

/CH2-CH)
NH-CH2 -CH2 -N,
CHr CH)

CH20H

C20H24N202S MoL. wt: 356.5

1. 3 Chemcal an physical properties of the pure substace

From Rosi et al. (1967) an Blaco (1972)

(a) Description: Orange p:er

(b) Melting-point: 100.6-l02.80C

(c) Spetroscopy data: Àma 233, 258, 329 and 428 nm (E~ - 544.2,
1037.9, 272.1 and 185.1) in ethanol

(d) Solubility: Slightly soluble in water

1.4 Technical products and imurities

No inforrtion was available to the Working Group.

91
Hycanthone rnsylate

1. 1 Synonyr and trade nars

Chem. Abstr. Reg. Serial No.: 23255-93-8

Chem. Abstr. Nar: 1 - r (2- (Diethy larno) ethy 1 L amo J-4- (hydroxythy 1) -
9H-thoxanthen-9-one, monorrthanesulphonate (salt)
l-r (2- (Diethylarno)ethyllarnoJ-4- (hydroxythyl) thioxathen-9-one,
IInorrthanesulphonate (salt); hycanthone rrthanesulphonate; hycanthone
monorrthanesulphonate
Etrenol
1. 2 Chemcal formla and molecular weight

+ CH2 -CH3
NH -CH2 -CH2 -NH(
CH2-CH3
~ CH3 -5°3-
CH20H

C2l H28N205S2 M::Ü. wt: 452.6

1. 3 Chercal and physical properties of the pure substance

(a) Description: Yellow-orange :ider

(b) Melting-point: Abut l430C

(c) Spectroscopy data: À 232-234, 257, 327-331 and 428-435 nm

ma
in rrthanol

(d) Solubility: Very soluble in water; freely soluble in 95%

ethanol; slightl y soluble in chloroform; very slightl y soluble
in acetone¡ practically insoluble or insoluble in benzene and
ether
(e) Staility: The cCJund is qute stable; it degrades very
rapidl y in aqueous-acid solution.

92
1.4 Technical products and imurities

Hycanthone rnsylate ls not available cOlrcially in the us but ca

be obtained in injection forr from the Parasitic Disease Drug Service,
National Conmunicable Disease Center, Atlanta, Ga (Swinyard, 1975).

2. Production, Use, Occurrence and Anal ysis

For imrtant background inforntion on this section, see preamle,

p. 15 .

2. 1 Production and use

(a) Production

Hycanthone is produced by Aspergi llus sc lerotiorum oxidation of the

synthetic schistosorncidal drg, lucanthone, and i ts isolation was first
reported in 1965 (Rosi et al., 1965). Hycanthone irsylate was also first
produced in 1965 (Rosi .et aL., 1965); it can be prepared by reacting
hycanthone with an eqiilar quantity of rnthanesulphonic acid (Swinyard,
1975) .

No evidence was found that hycanthone or hycanthone rrsylate have ever

ben produced comrrcially in the us or Japan. Both are produced in the OK.

(b) Use

Hycanthone ls a schistosomicide effective against hum Schis tosoma

haemotobium and S. mansoni infections. Given as an intramuscular injection
of the irsylate salt, the recmrnded dose is a single iniection of
3.0i:0.5 mg hycanthone per kg hw (Friedheim, 1973) to a maimum of 200 mg
per injection (Blaco, 1972). Worldwide, over 300,000 patients had been
treated with hycanthone rnsylate up ta June, 1972 (Friedheim, 1973). It
is believed that the present numr of patients treated is 1.2 million.
2 . 2 Occurrence

Oxidation of the synthetic drg lucanthone by Aspergillus sclerotio~um

produces hycanthone, but is not otherwise known to occur in nature (Rosi
et aL., 1965).

93
2 . 3 Anl ysis
Hycanthone has been determned in urine by thn- layer chromatogaphy
(Rosi et al., 1967).

3. Biological Data Relevant to the Evaluation

of Cacinogenic Risk to Ma
3. 1 Cacinogenici ty and related studies in animls 1
Intramuscular admnistration
r-use: A group of 66 5-6-wee old femle CF mice, infected percuta-
neously by tail irrsion with 20 S. mansoni cercariae, surived for at least
53 weeks¡ at 54-102 wee, 19 (29%) had deve10pe diffuse hepatic hyp1asia,
10 (15%) nodular benign hyprplasia and 1 (1. 5%) a hepatocellular carcinoma.
When a group of 47 rnce were simlarly infected and treated 56 days later
with a single Lm. injection of 3 or 60 mg/kg l: hycanthone rrsylate, 33
(70%) develope diffuse hepatic hyprplasia, 12 (26%) nodular benign
hyprplasia and 4 (8.5%) hepatocellular cacinomas (P?0.051. No hepatic
hyprplasia or hepatocellular cacinomas develope in any of 45 uninfected
mice treated with the san dose of hycanthone or in 47 uninfected, untreated
controls (Haese et al., 1973).

Groups of 80-100 4-week old femle Swiss wester rnce were infected
with either 0, 40 or 80 S. mansoni cercariae by i.p. injection; 46 days
post-infection the mice w=re given single Lm. injections of 0, 12.5 or
50 rn/kg l: hycanthone rrsy late. Whle the resul ts proved that hycanthone
rnsylate was effective in suppressing \\rr, no evidence was found that
the . treatrnt alone or combined wi th schistosaTl infection had any effect
on turur incidence. Only 0-45% of the infected animls surived the 18
IInths of the exprimnt (Yarinsky et al., 1974). (The low surival rates
in this exprimnt were noted by the Working Group 1 .

IThe Working Group was aware of ongoing studies in whch hycanthone

rnsylate is being tested in mice and haters by i.p. and i.m. injection
(lAC, 1976).

94
 Thee groups of 144, 59 and 103 femle [Link] COl mice ~re
infected by tail [Link] with 16 S. mansoni cercaiae; 8, 12 and 16
weeks after infection they wee given thee Lm. injections of 0, 3 or
60 rn/kg l: hycanthone. Of 101, 38 and 74 rnce in the thee groups that
surived 60 or rrre ~eks, 0, 1 and 9 (12.2%) develope hepatocellular
carcinoma. The increased incidence at the higher dose level wa reJ?rt
to be statistically signficant (P=[Link]) (Haese & Buedg, 1976).

3.2 Oter relevant biological data

(a) Exrimntal system
The Lv. LD of hycanthone in dCXs was 40 rn/kg l:; rrnkeys shCMed
50
a greater sensitivity th dogs (Hen, 1974). Various adverse biolCXical
effects related to hycanthone have ben reviewed (de Serres, 1975).
It induced hepatoxicity and imired function of hepatic drg-rntalizing
enzyrs in rats (Lucier et aL., 1973).

In male Sprague-Daley rats and in rhesus IInkeys of bath sexes

receiving single i.m. injections of randony tritiated hycanthone rrsylate
at doses in the range of those therapeutically recoIlded for ma (3 rn/kg
l:), pe bloo and tissue levels ~re found abut 30-60 miutes after
admistration. Highest concentrations ~re observed in the li ver, splee,
kidneys and adrenals but decreased to less than 20% of the admnistered
dose in 48-72 hours. Unchaged drg was found in bloo and tissues, except
in the li ver where rapid conversion to hycathone sulphoxide occued in
rats and to the N-de-ethylated rrtalite in IInkeyS (Hemandez et ai., 1971).

ln mice, single Lm. doses of 35 or 50 rn/kg l: hycathone rrsylate

admistered on day 7 of gestation caused malforntions in rrre th 73%
of the litters (More, 1972). ln rabits, hycanthone rrsylate shCMed
erryolethl and teratogenic effects at an i .m. dose of 50 ng/kg l: on
days 7, 8 and 9 of gestation (Siebr et al., 1974). The rrst freqent
malforntions were exencephal y, hydrocephal y, rncrophthalma and abnorrli-
ties of the axial skeleton.

The mutagenic properties of hycathone rrsy late have be reviewed

(Ha & Hu1rt, 1975). It induces J?int mutations in Salmonella
typhimurium TA538 in the absence of liver horente (McC et aL., 1975),

95
sex-lined recessive lethals in Drosophila melanogaster (Knaap & Kramrs,
1973), mitotic recamination and other mutations in Saccharomyces cerevisiae
(Meadows et al., 1973; Shan & de Serres, 1973) and specific locus muta-
tions in the ad-3 region in Neurospora crassa (Ong & de Serres, 1975). In
ræls, hycanthone has ben reported to induce forwd mutations at the
thymdine kinase locs in lIuse lyrhoma cells treated with 5-50 llg/rn
in vitro (Clive et aL., 1972). Negative results have ben reported in the
speific locus test in mice treated with 150 irjkg bv by Lp. injection
(Rusell, 1975; Russell & Kelly, 1973) and in the dominant lethal test
(cited in Geeroso & Cosgrove, 1973; Rusell, 1975). Donat lethals
have ben reported in rats treated with 40-80 irjkg bv daily for 5 days
by Lp. injection (Green & Springer, 1975; Green et aL., 1973a). Cyto-
genetic effects have also been reported in vivo in rat bone-marrow cells
(Green et al., 1973b). Hycanthone causes malignant transforntion of
Rauscher leukema virus-infected rat erryo cells but not of non-infected
ceiis (Hetrick & Kos, 1975).

(b) Ma
Two of 8 patients treated with a single Lm. injection of 3 ir/kg bv
hycanthone for Schistosoma haematobium infestation develope severe hepato-
cellular injur, with the histological pattern of acute toxic hepatitis
(Farid et al., 1972).

Cyogenetic effects have been reported in cultures of hum .rripheral

leucoes treated in vitro with hycanthone rrsylate (Obe, 1973; Siebr
et al., 1973). No chrOlIsor abnornlities were observed in lymhoctes
from patients treated with a single injection of 2.5 irjkg hw hycanthone
(Frota-Pessoa et al., 1975).

3. 3 Case reports and epidemological studies

No data were available to the Working Group.

96
 4. Cots on Data Rerted and Evaluation 1

4.1 Animl data

Hycanthone iæsy late 1S carcinogenic in rnce previousl y infecte wi th

Schistosoma mansoni: a significant increase in the incidence of hepato-
cellular carcinors was observed in these mice follawing repeated intra-
rncular injections of this colTund. The limted data available are
not sufficient to evaluate whether hycanthone rnsylate is carcinogenic in
non-infected animls.
4.2 Hum data
No case reports or epidemological studies were available to the
Working Group.

lSe also the setion 'Anl Data in Relation ta the Evaluation of

Risk to Ma' in the introduction to ths volur, p. 13.

97
 5. References

Blacow, N.W., ed. (1972) Maindale, The Exra Phacopoia, 26th ed.,
London, Pharmceutical Press, p. 1617

Clive, D., Flam, W.G. & Mëchesko, M.R. (1972) Mutagenicity of hycanthone
in rnlian cells. Mutation Res., 14, 262-264

Farid, Z., Smth, J.H., Bassily, S. & Spaks, H.A. (1972) Hepatotoxici ty
after treatrnt of schistosomiasis with hycanthone. Brit. iæd. J.,
ii, 88-89

Friedeir, E.A.H. (1973) Chtherapyof schistosorasis. In: Cavier, R.,

ed., Interntional Encyclopeia of Phacolog and Therapeutics,
VoL. l, Setion 64, Cheitherapy of Helmthiasis, Ne York, Pergain;
pp. 110-114

Frota-Pessoa, O., Ferreira, N.R., Pedoso, M.B., rbro, A.M., otto, P.A.,
Chaine, D.A.F. & Da Silva, L.C. (1975) A study of chrolIsoiæs of
lymhoces froID patients treated with hycanthone. J. Toxicol.
environr. Hlth, l, 305-307

Generoso, W.M. & Cosgrove, G.E. (1973) Total reproductive capacity in ferle
rnce: chemcal effects and their analysis. In: Hollaender, A., ed.,
Chemcal Mutagens: Principles and Methods for Their Detection, Vol. 3,
Ne York, Plenum, pp. 241-258

Green, S. & Springer, J.A. (1975) Additional statistical evaluation and

phacological considerations of hycanthone rnthesulfonate-induced
dominat lethality. J. Tbxicol. environr. Hlth, l, 293-299

Green, S., Carr, J.V., Sauro, F.M. & Leator, M.S. (1973a) Effects of hycan-
thone on sperntogonial cells, deoxyribnucleic acid synthesis in bone
maraw and domiant lethality in rats. J. Pharncol. ex. Ther., 187,
437-443

Green, S., Sauro, F.M. & Leator, M.S. (1973b) Cyogenetic effects of
hycanthone in the rat. Mutation Res., 17, 239-244

Haese, W.H. & Bueding, E. (1976) Long-tenu hepatocellular effects of hycan-

thone and of tw other antischistosoml drgs in mice infected wi th
Schistosoma mansoni. J. Pharmcol. ex. Ther., 197, 703-713

Haese, W.H., Smth, D.L. & Bueding, E. (1973) Hycanthone-induced hepatic

changes in rnce infected with Schistosoma mansoni. J. Pharncol. exp.
Ther., 186, 430-440

Hatr, P.E. & Hulrt, P.B. (1975) Geetic activity spectra of sorn anti-
schistosal comunds, with paticular emhasis on thioxathenones and
bezothopyranoindazoles. J. Tbxicol. environr. Hlth, l, 243-270

98
Hen, H.C. (1974) Preclinical toxicolog of hycanthone in dogs and
rrnkeys. NSC 142982, Report 11'I 'IX 32 PH 43671141, Washigton De,
US Governnt Printing Office
Hemadez, P., Deis, E.W. & Farah, A. (1971) M2talism of the schisto-
sorcidal agent hycanthone by rats and rhesus rrnkeys. Bull. wid Hl th
Org., 45, 27-34

Hetrick, F.M. & Kos, W.L. (1975) Transforntion of cell cultures as a para-
rrter for detecting the potential carcinogenici ty of antischistosomal
drgs. J. 'IxicoL. environr. Hlth, l, 323-327

IA (1976) rAe Inforntion. Bulletin on the Surey of Chemcals Being

Tested for Carcinogenicity, No. 6, Lyon, p. 240

Knaap, A.G.A.C. & Krarrs, P.G.N. (1973) Mutagenic effects of hycanthone in

Drosophila melanogaster. Mutation Res., 21, 38-39

Lucier, G.W., McDaniel, O.S., Bed, J.R. & Faeder, E. (1973) Effects of
hycanthone and t\ of its chlorinated analogs on hepatic microsorrs.
J. Phacol. ex. Thr., 186, 416-424

Mc, J., Choi, E., Yamsaki, E. & Ars, B.N. (1975) Detection of cacino-
gens as mutagens in the Sa lmone lla/microsorr test: assay of 300
chemcals. Proc. nat. Acad. Sei. (Wash.), 72, 5135-5139

M2adows, M.G., Qu, S-K. & von Porstel, R.C. (1973) Mutagenic action of
hycanthone and IA-4 on yeast. J. Phacol. exp. Ther., 187, 444-450

M:re, J.A. (1972) Teratogenicity of hycanthone in mice. Nature (Iond.),

239, 107-109

Obe, G. (1973) Action of hycanthone on hum chrolISors in leukoce

cultures. Mutation Res., 21, 287-288

Ong, T-M. & de Serres, F. J. (1975) Mutagenic evaluation of antischistosoma1

drgs and their deri vati ves in Neurospora cras sa. J. 'Ixicol. environr.
Hlth, l, 271 -279

Rosi, D., Peruzzotti, G., Deis, E.W., Berberian, D.A., Freele, H. &
P..cher, S. (1965) A~, active rrtalite of lmiracil Dl.
Nature (Iond.), 208, 1005-1006

Rosi, D., Peruzzotti, G., Deis, E.W., Berberian, D.A., Freele, H.,
Tui.!.ar, B. F. & Archer, S. (1967) Hycanthone, a new active rntablite
of lucanthone. J. ræ. Cher., 10, 867-876

Russell, W.L. (1975) Results of tests for J:ssible transrtted genetic

effects of hycanthone in m:ls. J. ToxicoL. environr. Hlth, l,
301-304

99
Russell, W.L. & Kelly, E.M. (1973) Ineffectiveness of hycathone in
inducing specific- loc mutations in rnce. Mutation Res., 21, 14
de Serres, F. J ., ed. (1975) long-terr toxici ty of antischistospmal drgs.
J. ToxicoL. environr. Hlth, 1

Shahn, M.M. & de Serres, F.J. (1973) Th effect of pH on hycanthone

iæthanesulfonate-induced inactivation and mitotic recorrination in D5,
a new diploid strain of Saccharomyces cerevisiae. Mutation Res., 21,
234

Siebr, S.M., Whg-Peng, J., Johns, D.G. & Adaon, R.H. (1973) Effects
of hycanthone on rapidly proliferating cells. Biocher. PharcoL.,
22, 1253-1262

Siebr, S.M., Whg-Peng, J. & Adon, R.H. (1974) Teratogenic and cyo-
genetic effects of hycanthone in rnce and rabi ts. Teratolog, 10,
227-236

SWinyard, E.A. (1975) Parasiticides. In: Osol, A. et al., eds,

Rengton' s Phaceutical Sciences, l5th ed., Easton, Pa, Mack r
pp. 1174-1175

Yarinsky, A., Drobeck, H.P., Freele, H., Wiland, J. & Guer, K.r. (1974)
An l8-rrnth study of the paasitologic and ttlrigenic effects of
hycanthone in Schistosoma mansoni-infected and noninfected rnce.
ToxicoL. appL. PharncoL., 27, 169-182

100
 8-HYROXYQUINOLINE

1. Chcal and Physical Data

1. 1 Synonyr and trade ris

Cher. Abstr. Re. Serial No.: 148-24-3

Cher. Abstr. Nai: 8-Quinolinol

Hydroxybezopyridine; 8-hydroxy quinoline; oxin; oxine; oxybezo-

pyridine; oxychinolin; 8-oxyquinoline; phenopyridine; 8-quinol
8-(X; Bicxin; Qunophenol; Tu
1. 2 Chercal formla and rrlecular weight

::L "=
ÓO°H ~
N

C9Hl'¡) MoL. wt: 145.2

1. 3 Chercal and physical properties of the pure substace

From Stecher (1968), unless otherwise speified

(a) Description: Whte crystals or crystal line pcer

(b) Boiling-point: Abut 2670C

(c) ~lting-point: 760C

(d) Spetroscopy data: Àrr 318 and 243 nr in cyclohexe;

infra-red and nuclear magnetic resonance spetra are also
given (Grasselli, 1973).

(e) Solubility: Alst insoluble in water and ether; freely soluble

in acetone, aqueous mineral acids, bezene, chloroforr and
ethanol
(f) Rectivity: Readily forr stale netal chelates

lOi
1.4 Techncal products and imurities

8-Hydroxyquinoline is available in the US in techncal and reagent

grades. l t is also available as a O. 5 % solution (or aerosol) sui tale for

topical use (Harey, 1975).

2. Production, Use, O:currence and Analysis

For imrtt background informtion on ths section, see preamle,

p. 15.

2.1 Production and use

(a) Production

8-Hydroxyquinoline was first prepared by the decarboxylation of

8-hydroxyquinoline-4-caboxylic acid (Prager & Jacobson, 1935). It has
also been prepaed by heating 2-arnophenol, glycerine and 2-nitrophenol
in concentrated sulphuric acid (Prager & Jacoson, 1935) and by the fusion
of quoline-8-sulphonic acid wi th caustic sod and water (Bedall &
Fischer, 1881). 8-Hydroxyquo1ine ca a1so be prepaed by the sulpho-
nation of qu01ine with oleum and fusion of the sodum salt with sodium
hydroxide at 2250C (Kulka, 1968).

In the US, comrcial production of 8-hydroxyquinoline was first

reported in 1933 (US Tariff Comssion, 1934); only two US cOIDanies
reporte production (se preale, p. 15) in 1974 (US Interational Trade
Commssion, 1976a). Production of the sulphate salt was reported to be
4,000 kg in 1974 (US International Trade Corrssion, 1976a). US imrts
of 8-hydroxyquinoline though the principal cutorn districts were 25,000 kg
in 1972 (US Tariff Comssion, 1973), 8000 kg in 1973 (US Tariff Commssion,
1974) and 47,000 kg in 1974 (US International Trade Commssion, 1976b).

One compy produces 8-hydroxyquinoline in Japan, and its anual

production is estimted to be in the range of 100-500 thousand kg. Anual
production of 8-hydroxyquinoline in western Euope is estirted to be in
the range of 0.5-1 million kg, of which 1-100 thousand kg are produced in
the Federal Republic of Gerry and 100-500 thousand kg in France and in
the OK.

102
 (b) Use

8-Hydroxyquinoline 1S an antimcrobial drg used in hum rrdicine

for the treatrnt of rnnor burs and haeirrhoids (Harey, 1975) and has
reportedl y ben used as a bacteriostatic addi ti ve in hairdressing pre-
parations to control dadrff (Markland, 1966). Total US sales of the
compund for use ln hum rrdicine as an antiseptic spray are estimted
to be less than 400 kg anually.

As 8-hydroxyquoline forr a chelate wi th may rrtal ions, i t lS used

as an anl ytical colorimtric reagent and for the precipitation and
separation of rætals (Stecher, 1968).

8-Hydroxyquinoline is also used in the prepaation of a numr of

deri vati ves used in rrdicine and in a variety of indus
trial applications.
The benzoate salt is used in the treatrt of minor burs and haerrrhoids
and was reportedl y a compnent of a conrcial sprrcidal preparation
(Hoch-Ligeti, 1957). The sulphate salt is used in the treatint of
dermtophytosis, vaginitis and as an eyewash and gargle (Harey, 1975).

8-Hydroxyqinoline is a chemcal interriate for pre:oation of the

aicide, diiodohydroxyquin, which may be used in the treatrnt of
intestinal amiasis, Trichomonas vaginalis vaginitis, infections caused
by Trichomonas hominis, and in the topical treatmnt of certain fungal
cutaneous infections. Iodochlorhydroxyquin, another deri vati ve of
8-hydroxyquinoline, is used as an antibcterial and antifungal agent in
derntologic preparations and for the treatrt of intestinal aiiasis
(Harvey, 1975).

8-Hydroxyquinoline 1S also used ta rne its copper chelate, cooper

8-hydroxyquinolate, which lS registered in the US by the Environrntal
Protection Agency for use as a fungicide in agricultural and indus
trial
applications.
8-Hydroxyquinoline can also be used in the maufacture of the follaving
dyes: CI Acid Orange 61, CI Mordant Orange 26, CI Direct Black 104, and
CI Direct Red 174 (The Society of Dyers and Colourists, 1971) i havever, no
evidence was found that these dyes are produced corrrciall y in the US.

103
2. 2 Occurrencc
8-Hydroxyqunoline 1S not know to occu in nature.

2. 3 Anal ysis
Of 6 colorimtric rrthods for the determation of 8-hydroxyquinoline
in pharceutical products and tissues, one using Gibs reagent (2, 6-dibroII
benzoqinone chlorirne) was reported to be the IIst sensitive, with a
detection lirt of 0.5 lJg/l (Galea & Popa, 1971). A colorintric rnthod
for its determation in galenical preparations containing other phenols
is based on its corlex with a vanadium ion (Peteri, 1969).

Non-aqueous ti tration rnthods have been describ to determe 8-

hydroxyquinoline and its oxidation products (Kondratov et al., 1967) and
to determe 8-hydroxyquinoline and saæ of its rntal chelates (Grey &
Cave, 1969). An aqueoUS titration rrth for the determation of 8-
hydroxyquinoline sulphate has been outlined (Horwitz, 1970), and it can
be determned fluorimtrically as its chelate comund with tin (2+)
(Khabov et aL., 1971). A fluorimtric determation of 8-hydroxyqunoline
is based on its lumescence in sulphuric acid at 770K and has a
sensitivity of O. 12 ~ (Golovina et al., 1974).

A spetrophotorntric rrthod for the determnation of 8-hydroxyquinoline

as the copper chelate can be used in the presence of sorr other rrtals
(Winde, 1970).
Paper, thn- layer and gas- liquid chromatography ca also be used to
determe 8-hydroxyquinoline (Clarke, 1969).

3. Biological Data Relevant to the Evaluation

of Cacinogenic Risk to Ma
3.1 Carcinogenicity and related studies in animls

(a) Oral admnistration

[Link]: A group of 49 CC57W mice were given 1.5 ff/animl 8-hydroxy-

quinoline in sunflCMer oil 6 tis IEr W2ek for 660 days (total dose,
852 rr/animl). Of 21 rnce suriving at the tim of appearance of the
first tUlur (460 days), 4 develope 2 lynhoms, 2 lung adenomas and 1

104
haemgioi of the li ver. Th tu:ur incidence in untreated CCS 7W mice
in that labratory was reported ta be 17% (Pliss & Volfson, 1970) (The
absence of concurrent controls was noted).

Rat: A group of 15 6-we old male Fischer F344 rats received 0.8%
8-hydroxyquinoline in the diet 'for 78 weks, at which tim 13 surived.
The onl y tuiurs observed were 4 Leydig-cell tuiurs of the testis. Hypr-
plasia of the interstitial cells of the testis was observed in 2/8 suriving
controls (Yamto et aL., 1971) (The smll numr of anÍ1ls and the short
duration of the exprirt were noted).

Groups of male and femle Fischer rats were treated with 8-hydroxy-
quinoline at the following dose levels: 12 males at 0.1 rr/animl/day,
12 femles at 3 rr/anirl/day, 15 males and 14 femles at 10 rr/animl/day
and 2 males and 3 femles at 30 rr/animl/day. Maimum surival ranged
from 384-563 days. One hepatoma with lung irtastases occurred after 456
days in one male rat given the highest dose level. No liver tuiurs
occured in a large numr of vehicle or untreated controls. Thee cystic
adenomas of the lung, 15 testicular tuiurs and 4 niry tuiurs occurred
alng rats in the other groups; these tuiurs also occurred in controls
suriving up to 600 days. The incidence in treated animlswas slightly
but not significantly increased (Hadidian et al., 1968).

A group of 39 random-bred rats were given 15 rr/animl 8-hydroxyquinoline

in sunflower oil 6 tirs per week for 729 days (total dose, 9.36 g/animl).
Of 21 rats suriving at the tim of appearance of the first tuiur (589 days) ,
11 developed 2 lymphorns, 1 lung tuiur (plaslIcytoma), 2 parasitic sarcomas
of the li ver, 1 reticulosarcom of the intestine, 1 adenocarcinorn of the
uterus and 5 ma fibroadenomas. In untreated rats of that labratorv,
no tuiurs of the uterus, lung or intestine or 1 ymhorns were observed
(Pliss & Volfson, 1970) (The absence of concurrent controls was noted).

(b) Subutaneous admnistration

MJuse: A group of 32 CC57í\T rnce received. s.c. injections of 1. 5 rr/

animl 8-hydroxyqunoline in sunflower oil 3 lis per IInth for 660 days
(total dose, 97.5 mg/animl). Of 23 rnce surviving at the tim of
appeance of the first tuiur (148 days), 10 develooed a total of 2 lyrhomas,

105
5 lung adenoras, 1 subutaneous haergioma, 1 follicular adenom of the
ovar and 2 haergioms of the li ver. In untreated ü:57W rnce of that
labratory the turur incidence was reported ta be less than 17% (Pliss &
Volfson, 1970) (The absence of concurrent controls was noted).

Rat: A group of 70 randombred rats received væekly s.c. injections of

100 mg/animl 8-hydroxyquinoline in sunflowr oil for 730 days (total dose,
10.5 g/anirl). Of 24 rats suri ving at the appearance of the first tUlur
(624 days), 4 develope 3 lymhomas and 1 fibrosarcoma at the injection site
(Pliss & Volfson, 1970) (The absence of concurent controls was noted).

(c) Skin application

Mouse: A group of 46 ü:57W rnce received 3 applications per wee of

1.2 mg 8-hydroxyquinoline as a O. 75 % solution in benzene for 656 days (total
dose, 335 mg/animl). A control group of 22 rnce received benzene alone for
656 days. Of 27 treated rnce suri ving at the appearance of the first tUlur
(350 days), 8 develo!? 2 lymhomas, 2 subutaneous haergiomas, 1 subcu-
taneous lymhangiom, 1 follicular adenorn of the ovar and 2 haergioms
of the liver; 2/22 control rnce receiving benzene alone develo~d lung
adenoms after 427 and 619 days (Pliss & Volfson, 1970) (P/O. 05) .
(d) Oter exprirtal system
Bladder imlantation: A group of 20 stock rnce (sex unpecified)
received a bladder imlant of a 9-11 mg cholesterol pellet containing 20%
8-hydroxyquinoline. Of 13 mice suri ving 40 or IIre væks, 5 develope
bladder tururs (3 cacinoms and 2 oapillomas), coared wi th 1 carcinoma
in 21 controls alive at 40 væeks (P.(O. 01) (Allen et al., 1957; Boland &
Watson, 1956). Sirlar results were obtained by Clayson et aL. (1958) who
observed 6 carcinoms in 25 treated mice compared wi th 5 in 55 controls.
In a further study in which paaffin-wa pellets containing 20% 8-hydroxy-
qunoline were imlanted into the bladders of 8- l3-we old fe~le Swiss
rnce, 1 papilloma and 1 carcinom of the bladder were found aIng 35 treated
rnce (average surival, 410 days), comed with 1 papilloma and 1 carcinom
arng 47 controls given imlants of paaffin wa alone (average surval,
366 days) (Bryan et al., 1964).

106
 Vaginal instillation: A group of 20 femle stock mice recei ved vaginal
instillations of 0.1 ml of a 1% solution of 8-hydroxyquinoline in polyethy-
lene glycol twice weekly for 18 IInths. Ten rnce surived 12 IInths and 2
for 18 IInths; 7 develope cacinorns of the vagina and cervix. In
controls given polyethylene glycol alone, 11 rnce surived for 12 IInths
and 7 for 18 IInths; 5 develope carcinors of the vagina and cervix
(Boyland et al., 1961). In a later test, 20 BA/c rnce received sirlar
twice weekly instillations of 0.1 ml of a 1% solution of 8-hydroxyqunoline
in gu tragacanth for 50 weeks. One lIuse develope a squarus papilloma
of the uterine cervix after 20 TInths. No increase in the incidence of
tururs at other sites was observed (Baland et aL., 1966).

A group of 46 CC57W rnce received twice wely intravaginal applications

of 2 mg/animl 8-hydroxyqunoline as a 20% suspesion in distilled water
applied to a smll piece of cotton wool; treatrnt continued to 812 days
(total dose, 428 rr/animl); 27 controls received an insertion of cotton
wool and distilled water. Of 42 treated rnce still alive at the tii of
the appeaance of the first turur (242 days), 11 develope 4 lymhoms,
4 lung adenomas, 5 follicular adenomas and thecors of the ovar and 1
haemgioma of the li ver; 2 1 ymhomas, 1 lung adenoma and 1 haemgioma
of the liver occured in 27 controls (Pliss & Volfson, 1970) (P?0.05).

A group of 30 femle Bethesda black rats recei ved vaginal instillations

of 0.2 ml of a 20% suspension of 8-hydroxyquinoline in 20% aqueous gelatin
twice weely for 2 years; 16 rats surived 22-24 IInths. Four sqarus-cell
carcinomas or adenocarcinomas of the endaitriur were observed in 22 rats
tht died after 19 TInths; 1 uterine cacinoma was seen in a group of 21
controls that died between 19 and 24 TInth (Hueper, 1965) (p?O. 05) .

A group of 62 randornbred rats received twice weekly intravaginal

instillations of 20 mg/animl 8-hydroxyquinoline as a 20% suspesion in
distilled water; treatrnt continued for 729 days (total dose, 3.86 g/
animl). Of 24 rats still alive at the appearance of the first turur
(647 days), 4 developed 1 lynhoma, 1 follicular adenom of the ovar, 1
carcinoma of the uterus, 2 rrry fibroadenors and 1 thyroid cacinom.
In untreated rats of that labratory, no tururs of the uteru or lymhom
were reported to occur (Pliss & Volfson, 1970) (The absence of concurrent
controls was notedJ.
107
3.2 Oter relevat biological data

(a) Exrimtal system

ln rnce, s . c., oral and painting ID 's of 8-hydroxyquinoline are
50
7.5 m:, 7.5 ff and 6 mçi/ani.l, respetively. ln rats, s.c. and oral ID 's
50
are 500 and 75 mg/ani.l, respetively (Pliss & Volfson, 1970). The oral
ID20in guea-pigs is 1.2 gjkg l: (Stecher, 1968).
In rats, orall y admistered 8-hydroxyquinoline produced a deposi tion
of iron in rny tissues (Yamto et al., 1971); this effect was increased
by increasing the anunt of available iron in the diet (William &
Yamto, 1972).
8-Hydroxyqunoline, at a dose of 20-40 lJg/plate, caused point mutations
in Salmonella typhimurium TA100 in the presence of rat liver homoenate
(Talcott et al., 1976). In a brief note, in which control values were not
reported, a variety of chrOrnsOI abnornlities were reoorted in rruse bone-
marON cells after treatmt with 40 ffjkg bt by i.p. injection (Das &
Maa, 1970).
(b) Ma
No data were available to the Working Group.

3. 3 Case reports and epiderological studies

No data were available to the Working Group.

4. Carts on Data [Link]

- and Evaluation l

4. 1 Animl data
8-Hydroxyquinoline has æen tested in mice and rats by oral, subcu-
taeous and intravaginal admnistration and in mice by skin application
and bladder imlantation. l'st of these exrirts were of limted value,
for the reasons m2ntioned in the texte Within these limtations, the

l Data on the coppr chelate of 8-hydroxyounoline were discussed, but

beause its chemcal propies and uses are different from those of
8-hydroxyquinoline it will be considered by a further Í'70rkinq (;roup.

108
studies in rnce and rats by oral admnistration and subutaneous injection
or in rnce by skin application gave positive or negative results of
border line signif icance.

Positive results ~re obtained in bladder imlantation exprimnts

when 8-hydroxyquinoline was incorprated in cholesterol pellets but were
negative when paaffin wa pellets ~re emloyed (see also introduction,
p. 19). Its application intravaginally in different vehcles to mice and
rats did not significantly increase the incidence of tUIurS when compared
wi th that in appropriate controls.
No evaluation of the cacinogenicity of 8-hydroxyqunoline can be
made on the basis of the available data (however, se also the results
obtained in mutagenici ty studies) .
4 . 2 Hum data
No case reports or epidemological studies were available to the
Working Group.

109
 5. References

Allen, M.J., Boyland, E., Dues, C.E., Horning, E.S. & Watson, J.G. (1957)
Cacer of the urinar bladder induced in mice wi th rntablites of
aromatic ames and trtophan. Brit. J. Cacer, 11, 212-228

Bedall, K. & Fischer, O. (1881) Obr Oxychinolin aus Chinolinsulfosäure.

Ber. dtsch. chem. C:is., 14, 442-443, 1366-1369

Boyland, E. & Watson, G. (1956) 3-Hydroxyanthranilic acid, a carcinogen

produced by endogenous rntablisrn. Nature (Lond.), 177, 837-838

Boyland, E., Charles, R.T. & Gcing, N.F.C. (1961) The induction of tllurS
in mice by intravaginal application of chemcal cornunds. Bri t. J.
Cacer, 15, 252-256

Boyland, E., Roe, F.J.C. & Mitchley, B.C.V. (1966) Test of certain consti-
tuents of spermcides for carcinogenici ty in geni tal tract of femle
rnce. Brit. J. Cancer, 20, 184-189

Bryan, G.T., Brow, R.R. & Price, J.M. (1964) Incidence of lIuse bladder
tllrs following imlantation of paraffin pellets containing certain
trytophan :rtablites. Cacer Re., 24, 582-585

Clarke, E.G.C., ed. (1969) Isolation and Identification of Drugs in Phar-

ceuticals, Boy Fluids and Post-rnrter Material, London, Pharceutical
Press, pp. 375-376

Clayson, D.B., Jull, J.ítV. & Bonser, G.M. (1958) The testing of ortho hydroxy-
amnes and related comunds by bladder imlantation and a discussion of
their structural reqrernts for cacinogenic acti vi ty . Bri t . J.
Cancer, 12, 222-230

Das, R.K. & Maa, G.K. (1970) Chornso:r abrrations in the bone rnrraw
cells of rnce induced by 8-hydroxyqunoline. In: Proceedings of the
57th Indian Science Congress, part III, Setion VII, Zoolog and
Entorlogy, p. 354

Galea, V. & Popa, L. (1971) Anlysis of oxine and its derivatives.

Farmcia (Buc.), 19, 17-23
Golovina, A.P., Runov, V.K. & Alimrin, I.P. (1974) Lumnescence of 8-
hydroxyquinoline. Izv. Akd. Nauk SSSR, Sere Kh., 6, 1425-1427

Grasselli, J.G., ed. (1973) CRe Atlas of Spectral Data and Physical
Constants for Organic Compunds, Cleveland, Ohio, Chemcal Rubber Co.,
p. B-876

Grey, P. & Cave, G.C.B. (1969) Chelate-exchange titrimtr in non-aqueous

solvents. II. Determation of 8-quolinol and :rtal 8-quolinates.
Canad. J. Cher., 47, 4555-4562

110
Hadidian, Z., Fred ickson, T. N., Weisburger, E. K., Weisburger, J. H. ,
Glass, R.M. & Matel, N. (1968) Tests for chemcal carcinogens.
Report on the acti vi ty of deri vati ves of aromatic amnes, ni trosames ,
quinolines, ni troalkanes , amdes, epoxides, aziridines and pure anti-
rntablites. J. nat. Cancer Inst., 41, 985-1036

Harey, S.C. (1975) Antimcrobial drgs. In: Osol, A. et aL., eds,

Remngtn' s Phaceutical Sciences, l5th ed., Eaton, Pa, Mack,
pp. 1093, 1103, 1159

Hoch-Ligeti, C. (1957) Effect of prolonged admnistration of spermcidal

contraceptives on rats kept on low-protein or on full diet. J. nat.
Cancer Inst., 18, 661 -685

Horwitz, W., ed. (1970) Official Methcxs of Analysis of the Association

of Official Anlytical Chemsts, llth ed., Washington DC, Association
of Official Analytical Chersts, p. 697

Hueper, W.C. (1965) Exrimtal studies on 8-hydroxyqunoline in rats and

mice. Arch. Pathol., 79, 245-250

Khabov, A.A., Shemakin, F.M. & Fakeeva, O.A. (1971) Fluororntric deter-
rnnation of 8-hydroxyquinoline, 4- and 8-amoqoline, and cincho-
ninic acid derivatives. Novye tftcxy Kh. AnaL. Mater., ~, 95-97

Kondratov, V.K., Rus'yanova, N.D., Malysheva, N.V. & Yurkina, L.P. (1967)
Determation of pyridine and quinoline basis in mixtures wi th their
oxidation prcxucts. Zh. analyt. Kh., 22, 1585-1589

Kulka, M. (1968) Quinoline and isoqinoline. In: Kirk, R.E. & Otr, D.F.,
eds, Encyclopeia of Chemcal Tehnolog, 2nd ed., Vol. 16, Ne York,
John Wiley and Sons, pp. 865-886

Markland, W.R. (1966) Hair preparations. In: Kirk, R.E. & Otr, D.F.,
eds, Encyclopeia of Chemcal Technolog, 2nd ed., Vol. 10, New York,
John Wiley and Sons, p. 780

Peteri, D. (1969) Photorntric deterrnation of 8-hydroxyquinoline in the

presence of other phenols in galenical preparations. Fresenius' Z.
analyt. Cher., 248, 38-39

Pliss, G.B. & Volfson, N. l. (1970) On cacinogenic action of 8-hydroxy-

quinoline.
.
Vop. Onol., 16, 67-71

Prager, B. & J acobson, P., eds (1935 ) Beilsteins Handbuch der organischen
Cheme, 4th ed., VoL. 21, Syst. No. 3114, Berlin, Springer-Verlag,
p. 91

Th Society of Dyers and Calourists (1971) CaloUl Index, 3rd ed., Vol. 4,
Bradford, Yorks, pp. 4134, 4264

111
Stecher, P.G., ed. (1968) Th l~rck Index, 8th ed., P-aay, NT, .l~rck &
Co., p. 555

Talcott, R., Hollstein, M. & Wei, E. (1976) Mutagenicity of 8-hydroxyquno-

line and related counds in the Salmonella typhimurium bioasay.
Biocher. Phacol., 25, 1323-1328

US International Trade Cossion (1976a) Sythetic Organic Chercals, US

Production and Sales, 1974, USI'I Pulication 776, Washington OC,
US Governnt Printing Office, pp. 95, 101, 103
US International Trade Cossion (1976b) Irrts of Bezenoid Chercals
and Products, 1974, USI'I Pulication 762, Washington OC, US Governt
Printing Office, p. 26

US Tariff Comssion (1934) Production and Sales of Oyes and oter Synthetic
Organic Chercals, 1933, Report tb. 89, Send Series, Washington OC,
US Governt Printing Office, p. 32
US Tariff Commssion (1973) Imrts of Bezenoid Chercals and Products,
1972, 'I Publication 601, Washington OC, US Governt Printing Office,
p. 22

US Tariff Commssion (1974) . Imrts of Bezenoid Chercals and Products,

1973, 'I Publication 688, Washington OC, us Governt Printing Office,
p. 22

William, G.M. & Yamto, R.S. (1972) Abence of stainale iron from pre-
neoplastic and neoplastic lesions in rat liver with 8-hydroxyqunoline-
induced siderosis. LJ. nat. Cacer Inst., 49, 685-692

Winde, E. (1970) Qutitative determnation of 8-hydroxyqunoline. Dtsch.

Apoth. -Ztg., 110, 123-124

Yamto, R.S., William, G.M., Franel, H.H. & Weisburger, J.H. (1971)
8-Hydroxyqunoline: chonic toxici ty and inibi tory effect on the
carcinogenicity of N-2-fluorenylacetarde. 'IxicoL. appl. PharcoL.,
19, 687-698

112
 MEONIDAZOLE

1. Checal and Physical Data

1. 1 Synonyr and trade nas

Cher. Abstr. Reg. Serial No.: 443-48-1

Cher. Abstr. Nam: 2-Methv 1 -5-ni tro- lH-imdazole- 1 -ethanol

1- (ß-Ethylol) -2-rnthyl-5-nitro-3-azapyrrole; l-hydroxyethyl-2-
methyl-5-nitroimdazole; 1- (2-hydroxyethyl) -2-methyl -5-nitroimdazole;
1- (ß-hydroxyethyl) -2-methyl-5-nitroimdazole; 2-methyl-5-nitro-l-
imdazoleethanol; 2-methyl -5-nitroimdazole- 1 -ethanol
AcrolIna; Angiardil; Atrivyl; Bayer 5360; Beon; Clont; Cont;
Danizol; Deflam:m-Wirkstoff; Efloran; Elyzol; Entizol¡ Ein;
Flageina; Flagesol; Flagil; Flagyl; Flegyl¡ Giatricol; Gineflavir;
Klion; Maibol' silanes'; Meronidal; Metronidaz; .l1etronidazol;
Monagyl; Nalox; NeoTric; Nida; Novonidazol; Oragil; RP 8823;
Sanatrichom; SC 10295; Takimtol; Trichazol; Trichex; Trichocide;
TricholI1; TricholInacid' Pharnchir' ; Trichopal; Trichopol;
Tricocet; Tricom; Tricavas B; Trikacide; Trikain; Trikojol;
Trikozol; Trimks; Trivazol; Vagilen; Vagimd; vertisal
1. 2 Chemcal fonna and 1I1ecular vvight

CH2 -CH20H
o,~(tH,
1

C6H9N303 MJ1. wt: 171.2

1. 3 Chercal and physical prooerties of the pure substance

From Stecher (1968), unless other specified

(a) Description: Cream crystals

(b) Melting-point: l600C

(c) Spctroscopy data: Infra-red soetru is given by Staugh

et al. (1968)

113
 (d) Solubility: Soluble at 200C in water (1 g/lOO ml), in ethanol
(0.5 g/lOO ml), in ether (~0.05 g/lOO ml) and in chlorofoIT
(~0.05 g/lOO ml) i sparingly soluble in dimthylformde;
soluble in dilute acids

1.4 Technical products and imuri ties

Various national and international pharco~ias gi ve speifications

for the purity of rntronidazole in pharnceutical products. For exarle,
rrtronidazole is available in the US as a USP grade containing 99-101%
active ingredient on a dried basis and rDt rrre than O. 005% heavy rntals.
Suppositories in 500 mg doses and talets in 250 mg doses contain 95-105%
of the state aIunt of rntronidazole (US Pharncopeial Convention, Inc.,
1975) .

It is available in the OK on a dried basis containing 99- 101% active

iigredient and as 200 mg talets containing 95-105% of the stated aIunt
(British Phacopoia Commssion, 1973).

2. Production, Use, O:urrence and Anal ysis

For imrtt background inforntion on this section, see prearle,

p. 15.

2.1 Production and use

(a) Production

A i:thod for the synthesis of i:tronidazole was patented in the US in

1960 in whch 2-rnthyl-4 (or 5) -nitroimdazole is heated with ethylene
chlorohydrin, and the isolated crde rrtronidazole is recrysta1lized fran
ethyl acetate (Jacob et al., 1960).

CoITrcial production of rntronidazole was first reported in the US in

1963 (US Tariff Cossion, 1964); only one US company reported production
(see preamle, p. 15) in 1974 (US Interational Trade Cossion, 1976).
Japaese production of rrtronidazole was first reported in 1961. In
1975, one Japaese coy reported production of 2100 kg, and [Link]
fran France and Caada w=re rei:rted to be 385 kg. Anual production lS
estirtedto be 1-100 thousand kg in bath Italy and the OK.

114
 India was reported to have one producer. of rrtronidazole in 1972, with
a production volur of 6,922 kg. Indian Ìlrts for the period 1972-1973
were reported to be 16,.250 kg (Ann., 1974).
(b) Use

Metronidazole is effective on oral adistration in infections due to

Entamoeba histolytica~ Trichomonas vaginalis and Giardia lamblia and has
been used in Vincent' s infection (Blaco, 1972). It is prescrib for
invasive intestinal aiiasis or aiic hepatic abscess. One recomrnded
oral dose regim is 750 rr thee tirs per day for 5 to 10 days (Rollo, 1975a).

Metronidazole was shaw to be an effective systerc trichomonacidal

agent in 1960. It imarts tricholInacidal activity to sern and urine, and
a high cure rate can be obtained in bath male and ferle patients infected
with Trichomonas vaginalis. A curently accepted dose regim is 250 rr
irtronidazole orall y three tims per day for 7 days. Stubbarn infections
can be treated by a repeated course, but an interval of 4 to 6 weeks is
recoIInded between treatrts (Rollo, 1975b; us Foo & Drug Admistration,
1976). It can also be applied in pessaries, in a dose of 500 rr daily
for 10-20 days (Blacow, 1972).

In giardiasis, a dail y dose of 500 mg for 5 days, repeated if necessar,

is usually effective (Blacow, 1972).

Metronidazole has ben evaluated for use in the treatrnt of alcoholism

(Blacaw, 1972), and in the USSR, indications for such use are given
(Mashkovski, 1972).
The use of irtronidazole in the treatrnt of acne rosacea has been
suggested recently (Pye & Burton, 1976), and a goo clinical response was
also reoorted to occur in a small numr of patients wi th acne vulgaris
(Carne, 1976).

In early 1976, the US Foo and Drug Admnistration anounced that, in

future, all packages of irtronidazole offered for sale in the us would
be reqired to carry a warning labl stating that unecessar use of the
drug should be avoided in view of the resul ts of animl carcinogenici ty
studies (see section 3.1) (US Foo & Drug Admnistration, 1976).

115
 Total US sales of rætronidazole for use in hum ræicine are
estimted to be less than 13, 000 kg anuall y.

Tt ca be used as a trichornacide in veterinar ræicine (Stecher,

1968) .

2. 2 Occurence
Although rætronidazole 1S closely related to the natural antitricharnal
agent, azancin, it is not knCM ta occu in nature (Rollo, 1975b).

2. 3 Anal ysis
Methods of assay of rætronidazole to iæt reglatory reqrernts for
phaceutical products corrm::ml y emloy non-aqueous ti tration. Ths has
be comped to potentiortric ræthods for determation of rætronidazole
in pharceutical products (Tuckerm & Bican-Fister, 1969).
Bioassay procedures using agar diffusion techniques have ben reported
(Levison, 1974; Ralph & Kiby, 1975). Ga chromtogaphy of the silyl
derivative has ben used for its determnation in plasm (Midha et aL.,
1973; WO, 1975).
A UV spetrophotorætric ræthod for the determnation of rætronidazole
in pharceutical products has been describ (Korantseva et aL., 1973),
and colorimtric (Populaire et al., 1968; Sanghavi & Chandrarhan, 1974)
and corlexorætric determations (Gajewska, 1972) have been reported.

3. Biological Data Relevant to the Evluation

of Cacinogenic Risk to Ma
3.1 Carcinogenicity and relate studies in animIs

Oral admistration 1

l'use: Metronidazole was adrnistered for lifetim in the diet of

groups of 6-8-wee old Swiss rnce at levels of 0.06% (effective numrs:
9 males an 10 femles), 0.15% (19 males and 20 femles), 0.3% (18 males

1 The WOrking Group was also aware of ongoing tests in rats (Illl ,
1976) .

116
and 20 femles) and 0.5% (35 males and 36 femles) ¡ 70 male and 70
femle mice were used as untreated controls. Suri val was sirlar in all
groups. The incidence of lung tururs rose fror 19% in untreated males to
33, 58, 67 and 77% in treated males and from 20% in untreated femles to
40, 50, 70 and 44% in treated femles. Femle rnce also exhibited a signi-
ficantly increased incidence of lymhomas at the tw highest dose levels,
whereas no significant increase was observed in the two other groups of
treated femles and in none of the groups of treated rnles (Rustia &
Shubik, 1972).

Rat: Metronidazole was admnistered at a concentration of 0.135% in

the diet to wealing femle Sprague-Dawley rats for 66 weeks, follawed by
a 10-week observation period; 36 rats surived for IIre than 10 weeks.
'lel ve rats develope benign rr fibroadenomas and 3 rr adeno-
carcinomas. In an untreated control group, 12 rats develope fibroadenom
and 6, adenoccinans arg 71 rats suriving for nore th 10 wees.
Whereas untreated rats develope no IIre than one rr turur, those
developing rr tumurs after rætronidazole treatrt had an average
of 2.8 tururs per tumur-bearing rat (Cohen et aL., 1973).

3.2 Otr relevant biological data

(a) Exrimtal system

After its oral admnistration, rætronidazole is readily absorbe from
the stomach and duodenum in rats (Populaire et aL., 1971). Follawing i. v.
admnistration of (1,2-1~C)-metronidazole ta rnce, activity was found in
liver and kidney and in heart, brain, salivar gland, gastrointestinal
tract, splee and skeletal mucle and was shaw to cross the placenta to
the foetus (Placidi et al., 1970). ln rats, rætronidazole was conjugated
in the liver and excreted in the bile (Populaire et aL., 1971) and in
the urine (Mészaros & Szporny, 1968).

Metronidazole is reduced in rats by caecal flora in the absence of

oxygen (Searle & Willson, 1976) and by guea-pig liver preparations
&n vitro (Mitchard, 1971).

. ..
Metronidazole caused point mutations in Salmonella typhimurium TAlOO
without addition of liver hom:enate (McCan et al., 1975). lt is mutaaenic

117
only under anaerobic conditions in a mutat of TA100 tht is deficient in
aerobic nitroreductase activity (Rosenranz & Spek, 1975). NJ mutagenic
activity was found in the urine of mice treated for 4 days with daily doses
of up to 400 IT/kg li rntronidazole, and rrrginal acti vi ty was reported in
the host-ræiated as say when mice were treated with 400 IT/kg tM for 5 days.
S. typhimuriwn was used as the genetic indicator in bath tests (Leator
et aL., 1975). In a fluctuation test, rætronidazole induced streptoIIcin-
resistat mutats ln Klebsiella pnewnoniae (Vood et aL., 1974).

(b) Ma
Transient neutropeia was observed in 10 (3%) of 386 netronidazole-
treated patients (Lefebvre & Hesseltine, 1965).

In 4 healthy male subjects given 750 IT 14C-lablled rætronidazole,

14% of the activity was excreted in the faeces and 77% in the urine withn
5 days (Schwarz & Jeunet, 1976). In ma, the rrjor urinar netablite
is 1- (2-hydroxyethyl)-2-hydroxythyl-5-nitroimdazole (Stamaugh et al.,
1967). In addition, four other nitro-group-containing netalites have
been identified, each derived from the side-chain oxidation of the ethyl
and/or rnthyl groupe They included l-acetic acid-2-rnthyl-5-nitroimdazole
and 1- (2-hydroxyethyl) -2-carbaxylic acid-5-nitroimdazole salt (Staaugh
et al., 1968).

In wom gi ven 250 mg netronidazole orall y during pregancy or

lactation, the drg was found in 10W concentrations (0.25 mg/kg tM) in
erryonic tissue and in milk (Ain et aL., 1972).

In hur patients receiving 750 mg/day, mutagenic activity was found

in the urine, using Salmonella typhimurium as a genetic indicator ([Link]
et al., 1975). A 2-4-fold increase in the occurence of chrolIsorn
abnormli ties was observed in cul tured ¡:ripheral leucocytes froID patients
with Crohh's disease being treated with 200-1200 mg/day rætronidazole for
1-24 ITnths (Mitelm et al., 1976).

3. 3 Case reports and epiderological studies

NJ data ~re available to the Working Group (see also 'General Remks
on Substaces Considered', pp. 24-25).

118
 4. Conts on Data RerxKted and Evaluationl

4 . 1 Animl data
Metronidazole 1S carcinogenic ln mice after its oral admnistration:
it significantly increased the incidence of lung tUlurS in bath sexes
and the incidence of lymhoms in ferles. Its oral admnistration to
rats increased the incidence and rnltiplicity of m: fibroadenomas.
4. 2 Hum data
No case reports or epiderological studies were available to the
Working Group.

lSe also the section 'Animl Data in Relation to the Evaluation of

Risk to Ma' in th introduction to this volur, p. 13.
119
 5. References

Arn::m, K., AIn, 1. & Hueller, H. (1972) Maternal-fetal passage of iætroni-

dazole. In: Hej zlar, M., ed., Advances in Antimcrobiolog and Anti-
neoplastic Chertherapy, Proeeings of International Congress of
Cheitherapy, 7th, 1971, Batiire, M:, University Park Press,
pp. 113-115

Anon. (1974) Production and imrts of selected drgs and pharceuticals

in India. Chemcal Industr"Ns (India), July
Blacow, N.W., ed. (1972) Matindale, The Exa Phacopoia, 26th ed.,
London, Pharceutical Press, pp. 1095-1098

British Phacopoia Commssion (1973) British Phai:copoia, London,

HM, p. 309
Cae, S. (1976) [Link] and acne. Lacet, ii, 367
Cohen, S.M., Ertürk, E., Van Esch, A.M., Crovetti, A.J. & Bryan, G. T.
(1973) Carcinogenicity of 5-nitrofurans, 5-nitroimdazoles, 4-nitro-
bezenes and related counds. J. nat. Cacer Int., 51, 403-417

Gajewska, M. (1972) Comlexoiætric determnation of various diazole

derivatives with cupric picrate. Acta, pol. phar., 29, 399-404

IA (1976) rAC Inforntion Bulleti on the Surey of Chemcals Being

Tested for Cacinogenicity, No. 6, Lyon, p. 244

Jacob, R.M., Régnier, G.L. & Crisan, C. (1960) Nitroimdazolealkanols and

acyl derivatives. us Patent 2,944,061, July 5, to Société des
usines chiques Rhne-Poulenc

Koantseva, E.V., Vergeichi, E.N. & Belikov, V.G. (1973) Differential

spetrophotoiætric deterrnation of rrtronidazole. Fartsi ya
(Mosco), 22, 45-48

Lefebvre, Y. & Hesseltine, H.C. (1965) The peripheral white bloc cells
and iætronidazole. J. Amr. me. Ass., 194, 127-130

Leator, M.S., Connor, T.H. & Stoekel, M. (1975) Detection of mutagenic

activity of iætronidazole and niridazole in boy fluids of hums and
rnce. Science, 188, 1118-1119

Levison, M.E. (1974) Microbiological agar diffusion assay for iætronidazole

concentrations in seri. Antimcrob. Agents Cheither., ~, 466-468

Mashkovski, M.D. (1972) Drug Compunds, VoL. II, MJsco, Meizina,

pp. 390-392

120
Mc, J., Choi, E., Yarsaki, E. & Ars, B.N. (1975) Detection of
carcinogens as ITtagens in the Sa lmone lla/rncrosoi test: assay of
300 chemcals. Proc. Nat. Acad. Sei. (Wash.), 72, 5135-5139

Mêszaros, C. & Szporny, L. (1968) Urinar excretion of metronidazole and

isamtronidazole in the rat. Acta physiol. acad. sci. hung., 34,
103- 106

Midha, K.K., McGilveray, LJ. & CcX)per, J.K. (1973) Determation of

therapeutic levels of metronidazole in plasm by gas- liqud chroma-
togaphy. J. Chomat., 87, 491 -497

Mitchard, M. (1971) Bioreduction of organic nitrogen. Xenobiotica, l,

469-481
Mitelm, F., Hartley-Asp, B. & Ursing, B. (1976) ChOlIsome abrrations
and metronidazole. Lacet, ii, 802

Placidi, G.F., Masuoka, D., Alcaraz, A., Taylor, J.A.T. & Earle, R. (1970)
Distribution and metablisr of 14C-rntronidazole in rnce. Arch. int.
Pharcodyn., 188, 168-179

Populaire, P., Deouvelaere, B., Lereton, G. & Pascal, S. (1968) Ibsage

colorimtrique de l' (hvdroxy-2-pthvl) -1 nÉthyl-2 ni-tro-5 iridazole
(rntronidazole). An. pha. franç., 26, 549-556

Populaire, P., Beazet, F., Pascal, S., Lereton, G., Deouvelaere, B. &
Guillaum, L. (1971) Circulation et sort du rntronidazole dans le
tractus digestif chez le rat après admnistration pa voie orale:
absorption du rntronidazole par les ITqueuses digestives. Thérapie,
26, 581-594

Pye, R.J. & Burton, J.L. (1976) Treatrnt of rosacea by metronidazole.

Lacet, i, 1211-1212

Ralph, E.D. & Kirby, W.M.M. (1975) Bioassay of metronidazole with either
anaerobic or aerobic incubtion. J. infect. Dis., 132, 587-591

Rollo, LM. (1975a) Drugs used in the cheitherapy of aiiasis. In:

Go, L.S. & Gilm, A., eds, The Phacological Basis of
Therapeutics, 5th ed., New York, Macmllan, pp. 1072-1073

Rollo, LM. (1975b) Miscellaneous drgs used in the treatrt of proto-

zoal infections. In: Go, L.S. & Gilm, A., eds, The
Pharncological Basis of Therapeutics, 5th ed., New York, Macmllan,
pp. 1086-1088

Rosenanz, H. S. & Spek, W. T. (1975) Mutagenici ty of metronidazole:

activation by rrlian li ver rncrosors. Biocher. biophys. Res.
Comm., 66, 520-525

121
Rustia, M. & Shubik, P. (1972) Induction of lung tuirs and malignant
lynhomas in mice by rrtronidazole. J. nat. Cacer Inst., 48,
721-729
Sanghavi, N.M. & Chdramha, H.S. (1974) Methods of estimtion of
rrtronidazole. Ind. J. Pharr., 36, 151 - 152

Schwartz, D.E. & Jeunet, F. (1976) Compative pharcokinetic studies of

ornidazole and rntronidazole in ma. Cheitherapy, 22, 19-29
Searle, A.J.F. & Willson, R.L. (1976) Metronidazole (Flagyl): degradation
by the intestinal flora. Xenobiotica, 6, 457-464

Staugh, J.E., Feo, L.G. & Mathei, R.W. (1967) Isolation and identifi-
cation of the major urinary rrtablite of rrtronidazole. Life Sci.,
6, 1811-1819

Starugh, J.E., Feo, L.G. & Mathei, R.W. (1968) The isolation and identi-
fication of the urinar oxidative rrtablites of rrtronidazole in ma.
J. Phacol. exp. Ther., 161, 373-381

Stecher, P. G., ed. (1968 ) The M2rck Index, 8th ed., Raway, ID, Merck &
Co., p. 695

Tuckerm, M.M. & Bican-Fister, T. (1969) Anlysis of rrtronidazole.

J. phar. Sei., 58, 1401-1403

US Foo & Drug Adistration (1976) M=tronidazole (Flagyl) bo waing.

£DA Drug Bull., ~, 22-23

US International Trade Cornssion (1976) Synthetic Organic Cherncals, US

Production and Sales, 1974, USITe Pulication 776, Washington De,
US Governt Printing Office, p. 101
US Pharcopeial Convention, Inc. (1975) Th US Phacopeia, 19th rev.,
Rokville, Md, pp. 327-328

US Tariff Commssion (1964) Synthetic Organic Chercals, US Production and

Sales, 1963, Te Pulication 143, Washington De, US Governt Printing
Office, p. 134

Vood, C.E., van der Stel, J.J. & Jacobs, J.J.J.A.A. (1974) The mutagenic
action of nitroirdazoles. 1. Metronidazole, nirazole, dirtri-
dazole and ronidazole. Mutation Res., 26, 483-490

W:, N.F. (1975.) GL anlysis of rrtronidazole in hum plasm.

J. phar. Sci., 64, 1048-1049

122
 NIRIAZOLE

1. Chercal and Physical Data

1. 1 Synonym and trade nars

Chem. Abstr. Reg. Serial No.: 61 -57-4

Chem. Abstr. Nar: 1- (5-Nitro-2-thiazolyl)-2-irdazolidinone

Ni trothiardazol; ni trothiardazole; ni trothiazole; 1 - (5-ni tro- 2-
thiazol y 1) - 2-irdazolinone; 1- (5-ni tro- 2-thiazol y 1) - 2-oxotetrahydro-
irdazole
Amilhar; 32644-Ba; Ba 32644; Ba 32644 CIBA: Ciba 32644;
Ciba 32644-Ba; N'I
1. 2 Chercal formula and molecular weight

o N~
O,NL(r~
N

C6H6N403S MoL. wt: 214.2

1. 3 Chercal and physical properties of the pure substance

From Stecher (1968), unless otherwise specified

(a) Description: Yellow crystals from dimthylformde

(b) Melting-ix:ánt: 260-2620C

(c) Spectroscopy data:

1968 )
max 1
À 370 nm (El - 536) (Antaki & Tewfik,

1.4 Technical products and imurities

Niridazole is not commrcially available in the US, but 500 mg tablets

can be obtained from the Parasi tic Disease Drug Service, Center for Disease
Control, Atlanta, Ga (Rollo, 1975). In certain Euope countries, it is
available in 100 mg tablets (Blaco, 1972).

123
 2. Production, Use, Ocurence and Anlysis

For imrtt background inforntion on this section, see prearle,

p. 15.

2.1 Production and us

(a) Production

A iæthod for the prepaation of niridazole was first patented in

Belgimn in 1963 (Cib Ltd, 1963). Niridazole can be prepared by the
condensation of 2-chloroethyl isocanate with 2-amo-5-nitrothazole
follow by elirnation of hydrogen chloride (Archer & Yarinky, 1972).

No evidence has been found that niridazole was ever produced COr-
cially in the US or Japa, although Japan has imrted smll aiunts for
labratory use. It is produced by one coy in Switzerland.

(b) Use

Niridazole 1S effective against the thee connn schistosolIs tht

may infect ma, agaist Entamoeba histolytica, and against Dracunculus
medinensis and is used clinicall y in all these infections. l t is particu-
larly useful in the treatrnt of Schistosoma haematobium infections but
less so in infections with S. mansoni or S. japonicum. The recoITnded
total doses for adul ts are 175 rr niridazolejkg l:, gi ven orall y over 5
days, for S. haematobium and S. mansoni and 100 rrjkg l: over 5 days for
S. japonicum (WHO, 1973).

Although niridazole is an effective aricide, its use in the

treatIt of intestinal or extra-intestinal ariasis has largely been
suprceded by tht of alterntive drgs.

In dracontiasis, total doses of 250 rrjkg l: given over 10 days have

freqently ben erloyed.

2. 2 O:curence
Niridazole 1S not kncw to occur in nature.

124
2. 3 Anl ysis
A spetrophotortric irthod has been used for the deterrtion of
niridazole in urine (Antai & Tewik, 1968). A radiochemcal rrthod
emloying IlfC_ lablled niridazole was used to determne the comund and
i ts metabli tes in biological sales after their separation on a
Sephadex G- 25 colum (Faigle & Kebrle, 1969).

3. Biological Data Relevant to the Evluation

of Cacinogenic Risk to Ma
3.1 Cacinogenicity and related studies in animls

Oral admistration
M'use: Adistration of initial concentrations of 0.1, 0.05, 0.025
or 0% niridazole in the diet to groups of 30 nale and 30 femle 10-we old
Swiss mice led, at the highest dose level, to significat graw retadation
by 30 wees. All groups of rnce ~re therefore fed a basal diet for 6
week and were subseqentl y retured ta diets containing niridazole at half
the original concentrations. In treated rnce, signficatl y elevated
incidences of lung adenoms (37-87% versus 4-28% in oontrols) and of the
mmibr of adenOl pe lung tUIur-bearing IIuse (3-5 versus 1), of
carcinomas (all grolis: 8 versus 0) and papillams (85 versus 1) of the
forestomach (combined: 26-63% versus 4%), and, in femle mice, of m:
carcinomas (10-20% versus 4%) and ovarian granulosa-cell tUIurS (7-20%
versus 0%) were observed. Thre ~re also 4 transitional-cell cacinom
of the bladder in femles recei ving the highest dose level and 1 in a
femle recei ving the next highest dose level. Lymhoms were observed in
treated but not in untreated male mice and in treated and untreated femle
mice, the latency period being reduced in treated femles. The ræ
ninr of all tUIurs per animl was proportional to the dose gi ven,
except in the group receiving the highest dose level where surival was
considerably reduced (Urm et al., 1975). In a later report of the sai
exprirt, tw subcosal atypical cellular nasses were found in th wal
of the urinar bladder of males of the tw lower dose groups and were
identified as srth-rncle tUIurs; these ocured after 52 and 72 weeks
(Jacobs et al., 1976).

125
 Groups of at least 29 rrle and 29 femle Swss rrce infected with 40
Schis tosoma mansoni cercariae by Yarinsky' s method (Yarinky, 1975) and
non-infected mice ~re fed 0.05, 0.025, 0.0125 or 0% niridazole in the
diet 46 days after infection. After 25 wees the dose levels were doubled.
The incidence, distribution and latency of tururs in bath infected and
non-infected rnce were sirlar to those describ in the abve exrimnt
and invol ved lung adenomas, papillomas and cacinomas of the forestamch
and bladder and carcinomas of the ma gland and ovary (Bulay et aL.,
1976) .

Hamter: Adistration of 0.24, 0.16, 0.08, 0.04 or 0% niridazole

in the diet of Syrian golden haters either infected with 30 Schistosoma
mansoni cercariae by Yarinsky 1 s method (Yarinsky, 1975) 56 days before or
non-infected led to a significant increase in the incidence of tururs of
the forestomach, mainly papillomas, at all doses (15-90% versus 0-3%)
except the lowest; and a total of 13 transitional-cell papillomas of the
b1adder were observed aIng animls treated with the 3 highest dose levels.
The miim mmibrs of animls i:r group ~re 30 males and 29 femles.
Infection did not api:ar to influence turur yield (Bulay et aL., 1976).

3.2 oter relevant biological data

(a) [Link] system

Oral admistration of O. 2 or O. 4 % niridazole in the diet to SWiss
mice led to reduction in boy weight and to severe testicular atrophy.
Sirlar findings were observed in Syian golden hamters gi ven 0.32 or
0.64% in the diet (Bulay et ai., 1976).

Niridazole inhibi ts granulam forntion and suppresses delayed hypr-

sensitivity in mice (Maud & Warren, 1974). It pro longs the surival of
lIuse skin allografts; its inunosuppressive effects have be shaw to be
associated wi th long-acting suppression of cellular imi ty but not wi th
inhibition of antiby production (Pelley et aL., 1975). In mice, niridazole
inhibits the grow but enances the rate of metastasis of transplanted
tururs from bath sygeneic and a1logeneic donors (Dear et al., 1976).

In rats, rabbits and dogs, orally admnistered ii.C-imdázinane

ring-lablled niridazole is slowly but ~ll absorbe and excreted in the

126
urine and faeces wi thin a few days. In exrirts invol ving rat tissue
hOrrenates, i t was degaded rrst rapidl y by li ver and kidney and to a
lesser extent by testis, spleen, heart, lung, brain, mucle and thyms -
this order corresfXnds to the levels of ni troreductase in these tissues.
Simlarly, it is rrtalized IISt rapidly in vitro by lIuse liver and then
in descending order by the liver of rats, rabbits, sheep, pigs and bovines.
In bloo, the rrtablites attain a higher level th the parent drg
(Faigle & Kebrle, 1969).

The principal rrtablite of niridazole fome by rat liver microsors

was the hydroxylamne (N-hydroxyamnothiamdazol), fome by reduction of
the nitro group of niridazole (Fel 1er et aL., 1971). This reduction was
also rriated by rat liver xathne oxidase (Morita et al., 1971).

Mutagenicity was reported in Salmonella typhimurium TAOO and TAl538,

in the absence of rrtablic activation by li ver hOIenates (McC et al.,
1975), and the induction of tadem duplications in Escherichia coli has
been described (Straus, 1974). The mutagenic activity of niridazole for
Salmonella TA1538 has also been detected in bloo and urine from mice
treated with 200 mg!kg hw and in the host-meiated assay after admnistra-
tion of 10 mg!kg hw by an unspecified route (Leator et aL., 1975). Mitotic
recomination in Saccharomyces cerevisiae (D5 strain) given 100-200 ~g/rn
(Shahin, 1975) and specific locus mutations in the ad-3 region in Neurospora
crassa (Ong & de Serres, 1975) have also been reported to occur. Mutagenic
activity in Salmonella typhimurium TAl538 was detected follawing exosure
of the bacterial test system to the urine of patients treated with 250 mg
niridazole orally thice daily for ten days (Leator et al., 1975).

In the dominant lethal test in mice, after either a single i.p.

injection (530-650 mg!kg hw) or 5 oral doses of 125 mg!kg hw, a significant
decrease in the numr of pregnant anirls and total imlants per pregnancy,
but no difference froID controls in pOst-imlantation los ses , were observed
(Epstein ct; al., 1972). There lS no indication of an ability to cause
chrolIsorr abnonnlities after an i.p. injection of 450 mg/kg li in bone-
rnow cells of rats (Sauro & Green, 1973) or mice (Holden et al., 1975).
Absence of an effect on micronuclei in lIuse bone-rnrow cells after single
i.p. injections of 10-200 mq!kq hw or multiple iniections of 100 mg!kq hw
for 5 days was also refXrted (Webr et al., 1975).
127
 (b) Ma
Niridazole suppresses delayed hyrsensi ti vi ty in hum (wester
et al., 1975). Orally adrnistered niridazole is well absorbe (Faigle &
Kebrle, 1969); it is rrtablized in the liver and elimnated largely
and alist eqll y in the urine and faeces (Rollo, 1975).

3.3 Case reports and epidemological studies

No data were available te the oorking Group (see also 'Geeral Remks
on Substaces Considered', pp. 24-25).

4. Conts on Data Reported and Evaluation 1

4. 1 Animl data
Niridazole is carcinogenic in rnce and haters after i ts oral adm-
stration: in nuce it induced lymhonas and tururs of the lung, stomch,
ma gland, ovar and bladder; in haters it proouced tururs of the
forestomach and papilloms of the urinar bladder. Infection of treated
rnce and haters with Schistosoma mansoni cercariae did not affect the
carcinogenici ty of the compund.

4 . 2 Hll data
No case reports or epidemological studies were available to the
Working Group.

1 Se also the section 'Anl Data in Relation to the Evaluation of

Risk to Ma' in the introouction to ths volur, p. 13.

128
 5. References

Antaki, H. & Tewik, J. (1968) A study on the rrtablisr and excretion of

1- (5-nitro-2-thazolyl)-2-irdazolidinone (nitrothiamdazole).
J. Egt. me. Ass., 51, 991 -996

Archer, S. & Yarinsky, A. (1972) Recent developets in the cheitherapy

of schistosomiasis. In: Jucker, E., ed., Progress in Drug Research,
VoL. 16, Basel, Stuttgart, Birkhäuser Verlag, pp. 14-23

Blacow, N.W., ed. (1972) Matindale, The Exra Pharcopoia, 26th ed.,
London, Pharmceutical Press, pp. 1618-1621

Bulay, O., Urm, H., Clayson, D.B. & Shubik, P. (1976) Carcinogenic
effects of niridazole on rodents infected with Schistosoma mansoni.
J. nat. Cancer Inst. (in press)

Ciba Ltd (1963) Ne 2-oxotetrahydroirdazoles. Belgian Patent, 632,989,

29 Novemr
Dear, S.D., Le, V.W., Chang, T., Maud, A.F. & Warren, K.S. (1976)
Effects of the imosuppressi ve drg niridazole in isogeneic and
allogeneic mouse tumr system in vivo. Cancer Res., 36, 3147-3150

Epstein, S.S., Arold, E., Andrea, J., Bass, W. & Bishop, Y. (1972)
Detection of chemcal mutagens by the domiant lethal assay in the
mouse. Toxicol. appl. Pharncol., 23, 288-325

Faigle, J.W. & Kebrle, H. (1969) Metalisr of niridazole in various

speies, including ma. An. N.Y. Acad. Sci., 160, 544-557

Feller, D.R., Morita, M. & Gillette, J.R. (1971) Enzymtic reduction of

niridazole by rat liver microsorns. Biocher. PhacoL., 20, 203-215

Holden, H.E., Ray, V.A., Wahenburg, M.G., Ellis, J.H., Jr & Florio, J.R.
(1975) A comparative study of schistosorncides in cyenetic and
point mutation assays. Mutation Res., 31, 309-310

Jacobs, J .B., Cohen, S.M., Arai, M., Friedell, G.H., Bulay, O. & Urm, H.K.
(1976) Chemcally induced Slth mucle tumrs of the lIuse urinary
bladder. Cacer Res., 36, 2396-2398

Legator,. M.S., Connor, T.H. & Stoekel, M. (1975) Detection of rntagenic

activity of -metronidazole and niridazole in boy fluids of hums and
rnce. Science, 188, 1118-1119

.Mud, A.A.F. & Warren, K.S. (1974) Anti-inflartory effects of tata

ertic and niridazole: suppression of schistosor egg granuloma.
J. Immol., 112, 222-228

129
McCan, J., Choi, E., Yarsaki, E. & Ars, B.N. (1975) Detection of
cacinogens as rntagens in the Sa lmone L ia/mlcrosaæ test: assay of
300 cherals. Proc. Nat. Acad. Sei. (Wash.), 72, 5135-5139

rbrita, M., Feller, D.R. & Gillette, J.R. (1971) Reuction of niridazole
by rat liver xathine oxidase. Biochem. Phanncol., 20, 217-226

Ong, T-M. & de Serres, F. J. (1975) Mutagenic evaluation of antischistosomal

drgs and their derivatives in Neurospora crassa. J. 'lxicol. environr.
- -
Hlth, l, 271-279

Pelley, R.P., Pelley, R.J., Stavitsky, A.B., Maud, A.A.F. & Warren, K.S.
(1975) Niridazole, a fXtent long-acting suppressant of cellular hypr-
sensi ti vi ty . III. Miiml suppression of antiJy resfXnses.
J. Imol., 115, 1477-1482

Rollo, LM. (1975) Drugs used in the cheitherapy of helmnthasis. In:

Gc, L.S. & Gilm, A., eds, The Pharncological Basis of Thera-
peutics, 5th ed., Ne York, Macrllan, pp. 1026-1027

Sauro, F.M. & Gree, S. (1973) In vivo cyogenetic evaluation of chloro-

indazole thoxanthene IA-4 (a hycathone anog) and niridazole in rat
bone marow. J. Phacol. ex. Ther., 186, 399-401

Shan, M.M. (1975) Geetic activity of niridazole in yeast. Mutation

Res., 30, 191-198

Stecher, P.G., ed. (1968) The Meck Index, 8th ed., Raway, ID, M=rck &
Co., pp. 733-734

Straus, D.S. (1974) Induction by mutagen of tadem gene duplications in

the glyS region of the Escherichia coii chomso. C~netics, 78,
823-830

Urn, H.K., Bulay, O., Clayson, D.B. & Shubik, P. (1975) Cacinogenic
effects of niridazole. Cacer Lett., l, 69-74

Webr, E., Bic1ll, K. & lBator, M.S. (1975) An evaluation of the

micronuclei test using triethylenenlame, trinthylphosphate,
hycatlne an niidaole. Mutation Re., 28, 101-106

Webster, L.T., Jr, Buttervrt, A.E., Maud, A.A.F., Mngola, E.N. &
Warren, K.S. (1975) Suppression of delayed hyprsensitivity in
schistosaæ-infected patients by niridazole. Ne Engl. J. Me., 292,
1144-1147

WHO (1973) Schistosomiasis control. wid Hlth Orge techn. Rep. Ser.,
No. 515, p. 18

Yarinsky, A. (1975) Evluation of schistosorcides againt exri1tally

estalished Schistosoma mansoni infections in rnce and hamters.
J. Toxicol. environr. Hlth, l, 229-242

130
 OXYHETHOIDNE

1. Chercal and Physical Data

1. 1 Synonyr and trade nas

Cher. Abstr. Re. Serial No.: 434-07-1

Cher. Abstr. Nar: 17 - Hydroxy- 2- (hydroxythy lene) - 1 7 -rrthy 1 -Sa, 17 ß-

androsta- 3-one

4, 5-Dihydro-2-hydroxyethylene- l7a-methyltestosterone; HM; l7ß-

hydroxy- 2-hydroxythylene- 1 7 a-rnthyl - 3-androstaone; 17 ß-hydroxy-
2- (hydroxythylene) -1 7a-methyl -5a-androstan-3-one; l7ß-hydroxy-2-
(hydroxythy lene) -17 -rnthy 1 -5a-androsta- 3-one ; 2-hydroxythy lene-
1 7a-methy 1 - 5a-androstan- 17 ß-ol - 3-one; 2-hydroxythylene- 1 7a-methy 1-
dihydrotestosterone; 2- (hydroxythylene) -1 7-rrthy ldihydrotesto-
sterone; 2- (hydroxythylene) - 1 7a-methyldihydrotestosterone; 2-
hydroxythy lene- 1 7 a-rnthy 1 - 1 7 ß-hydroxy- 3-androstaone; 17 a-rnthy 1-
2-hydroxythylene- l7-hvdroxy-5a-androstan-3-one; oxitholonum;
oximtolona; oxvthenolone

Adroidin; Adroyd; Anadrol; Anadroyd; Anapolon; Anasteron;

Anasteronal; Ansterone; Beorel; CI-406; Dyasten; Methabl;
Nastenon; NSC-26 198; Oxtosona-50; Pavisoid; Plenastril;
Protanabl; Roboral; Synasteron; Zenalosyn

1. 2 Chemcal formla and 1I1ecular weight

C2l H3203 MoL. wt: 332.5

131
1. 3 Chemcal and physical properties of the pure substace
From Weat (1975), unless oth:iise spfied

(a) Description: Crystals (Stecher, 1968)

(b) Mel ting-point: 178- 1800C; l820C

(c) Optical rotation: (cd~5 = + 360 in dioxane

(d) Spetroscopy data: À 285 and 315 mu (El -- 294 and 547) in
m: 1

0.01 N rrthanolic sooium hydroxide

(e) Solubility: Practically insoluble in water; soluble in ethanol,

dioxae and ether¡ very soluble in chloroform (Blaco, 1972)

1. 4 Technical proucts and iruri ties

Oxtholone is available in the US as a National Formlar grade
containing 97-103% active ingredient on a dried basis with a ni of
3% foreign steroids or other imurities. Tablets are available in 2.5,
5, 10 and 50 mg doses that contain 90-110% of the stated amunt of
o:xtholone (Kastru, 1974; National Formlar Board, 1970). In Japan,
oxytholone is also available in poer forme

2. Proouction, Use, O:currence and Anal ysis

For imrtant background inforntion on this section, see prearle,

p. 15.

2. 1 Proouction and use

(a) Proouction

A rrthod of prepaing oxytholone was reported in 1959 and involved

the following steps: l7a-rnthylandrostan-17ß-ol-3-one in anydrous
thiophene-free benzene is reacted wi th ethy 1 formte and soium hydride by
stirring the mixture under ni trogen for several hours. The resul ting
sooium deri vati ve is washed and then treated wi th cold dilute hydrochloric
acid to librate crude oxytholone, which is purified by recrystallization
fror ethyl acetate (Rigold et aL., 1959).

No evidence has been found that oxytholone was ever ~rcially

produced in th US. In 1975, two Japanese coanies proouccd a combined
total of 480 kg oxytholone.
132
 (b) Use

Oxtholone iS a sythetic androgenic-anablic steroid hOrIne

pririly intended for use in clinical therapy ta maintain a pJsitive
nitrogen balance. It ca be used to reverse excess excretion of calcium
and nitrogen resulting froID corticosteroid therapy, prolonged imbilization
and other diseases characterized by catablism and tissue depletion
(Amrican Soiety of Hospital Phacists, 1968). Oxtholone is used
mainly to prorrte weight gain to counteract weaess and emciation
resulting froID debilitating diseases an after serious infections, burs,
traum or surgery. The usual adult dosage of 5 ta 10 nq pèr day is
admistered orall y for abut 3 weeks, but not exceeing 13 wees, for a
single course of therapy. Occasionall y, 30 nq pèr day is gi ven (Harvey,
1975) .

Oxytholone can also be used in the treaTInt of anaemas caused by

deficient red-cell production and in acquired and congenital aplastic
anemas, rnelof ibrosis and hypplastic anaemas due to rnelotoxic drgs.
It can be used as an adjunct in the treatrt of senile and :ost-nenopausal
osteoporosis (Kastrup, 1974).

'Ital US sales of oxytholone for use in hum nedicine are estirted

to be less than 20 kg anuall y. In Japan, 360 kg are estirted to have
ben used in 1975.

Oxtholone has reportedly ben used in veterinar rricine as an

anablic steroid for smll animls (Stecher, 1968).

2. 2 Occurrence
Oxtholone iS not know to occur in nature.
2. 3 Anal ysis
A general surey of steroid analysis includes nethods for anablic
steroids (Forist & Johnson, 1961). An ultraviolet spectral assay for
oxytholone in bulk and talet forr (National Fonnary Board, 1970) and
thin-layer chromtographic analysis (Hara & Mi, 1967) have been
describ .

133
 3. Biological Data Relevant to the Evaluation
of Cacinogenic Risk ta Ma
3.1 Cacinogenicity and related studies in animls

No data were available to the Working Group.

3. 2 Oter relevant biological data

(a) Exrimntal system
No data were available to the Working Group.

(b) Ma
Abut 5% of an oral dose of 10 ID oxytholone was recovered from the
urine as tw unidentified rrtablites, which were present in roughly eqal
proportions (Adhikary & Harkness, 1971). Adnistration of oxytholone
and other androgenic anablic agents can give rise to various alterations
of liver IIrpholog , peliosis hepatis (Bagheri & Boyer, 1974; Bernstein
et al., 1971; Medows et al., 1974), cholestasis and haemsiderosis
(Johnson et al., 1972), haemrrhagic necrosis (Bruguera, 1975), haem-
siderosis (Lesna et al., 1976) and altered liver function (Anon., 1973;
Holder et al., 1975; Johnson et al., 1972; Mulvihill et al., 1975).

3. 3 Case reports and epiderological studies

Ten cases of li ver-cel 1 tUlurs have ben reported in young patients
treated with oxytholone alone or in combinat ion with other androgenic
anablic . steroids or drgs for aplastic anaera (5 cases), Fanconi' s anaema
(3 cases) or paoxysmal noctural haeilobinuria (2 cases) (see Table I) .
In all the cases reported, patients were treated for extended periods. In
five of these patients bloo cysts (peliosis hepatis), haemrrhagic les ions
or haeisiderosis were found in the neoplastic as well as in the nornl
tissue. The tUlurS have ben variously describ and diagnosed owing to
the difficulty of distinguishing betwen benign and malignant liver-cell
neoplasr. However, no rrtastases were deinstrated in any of the patients.
ln two patients the li ver lesions were reported to regress after cessation

- --
of the drg (Farell et al., 1975; Johnson et al., 1972).

A single case of hepatocellular carcinOI was reported to have occured

in a 22-year old girl with Fanconi 's anaera diagnosed at 8 years of age

134
 TABLE l

Cases of liver-cell tururs associate with oxyth1one therapy

!
Case Aqe9 Sex Disease Dose ¡:r Duration Oter drgs Li ver cx-F'eto- Oriqina1 Other Reference
no. (yrs) day (m: ) of function protein pathoicx:Tcal hepatic
treatrnt tests diagnosis les ions

(irnths)
1 20/21 M Fanconi 1 s 100 10 - not re- not well-diff. peliosis Bernstein
anaema ported tested hepa tOff hepatis et aL., 1971

2£ 2V6~ F aplastic 30- 100 46 predisone norm1 negative hepatocellu- - Johnson

anaema 1ar carcinoff et al., 1972

3 17/19~ F aplastic 150-250 28 predisone abnorm1 negative well-diff. choles- Johnson

anaema hepatocell u- tasis; et al., 1972
1ar carcinor haei-
sidero-
sis
4£ 4/15~ M ap1astic 100 36 prednisone; slightly high con- ? ? Henderson
anaema irthy 1 testo- abnorml centra- et aL., 1973
sterone; tion
norethdro-
lone;
stanozolo1
5 2/6 F ap1astic 40-60 41 predisone; norm1 not well -dif f. peliosis ~adows
anaema nandrolone tested hepator hepabs et al., 1974
decanoate
6 5/11 M Fanconi 1 s not 13 predisone; abnorml negati ve well --if f. - Ho1cìr
anaema reported irthandro- hepator et al., 1975
stenolone; 1

fluxoxy- !

sterone;
nandro1one
decanoate

~
W
ui
1-
w
C\

Case
a
Age- Se Disease J)se per Duation Oter drqs Li ver o.-Peto- Original Oter Reference
no. (yrs) cly (mg) of function protein pathological hepatic
treatrnt tests diagnosis lesion::
(rrnths)
7 16/19 M 1 paoxysrl not 36 - not re- not re- liver-cell haeirr- Bruguera, 1975
noctural reported ported oorted adenom hagic
haer 10- necrosis;
binuria bl00
cysts in
turur
8.0 28/34 M paoxysr1 100-150 65 predisone norml negative weii-aiff. - Farrell
noctura1 hepatocellu- et aL., 1975
haer1o- lar carcinom
binuria
9 8V12 M Fanconi 1 s 20-200 37 predisone abnorml negative well-diff. - Mul vihill
anaema hepatic et al., 1975
adenom
10 2/17 M aplastic 50 68 testosterone; not re- not 1iver haemsi - Lesna
anaema predisone; ported tested adenom derosis et aL., 1976
azathoprine

~ Age at diagnosis of disease/age when 1iver turur was diagnosed or age at death

e. Regression of hepatic lesion shaw by liver scan; death by gastrointestinal haemrrhage. No autopsy perforrd
~ Diagnosis of liver turur mde on clinical basis on1y; oxytho10ne given only during the 1ast 3 years
.0 Decrease of 1iver size and regression of neop1astic lesion shaw by 1iver scan
and not treated wi th androgen therapy. H~ver, :Jst-necrotic cirrhosis
was found, VJch could have predisposed to developt of the cancer
(Catta et al., 1974).

Eight other cases of hepatic tururs have recentl y be reported in

patients recei ving long-terr therapy wi th other androgenc anablic steroids
for Fanconi' s anema (5 cases), hyppituitaisr (1 case), crytorchidisr
(1 case) or imtence (1 case) (Corberand et al., 1975; Farrell et ai.,
1975; Guy & Auslander, 1973; Johnson et al., 1972; Sweney & Evan,
1975; Ziegenfuss & Carabsi, 1973).

It has ben proposed that prolonged use of such drgs may cause liver-
cell dage or hyprplasia, thus predposing ta subseqent malignant
transforntion (Johnson et aL., 1972). On the other had, the underlying
disease itself may have hepatic neplasia as a complication, which becoræs
appaent when surival is extended by drgs (Corttee on Neoplastic
Diseases, 1974).

4. Catts on Data Reported and Evluation

4 . 1 Animl data
No data were available to the Working Group.

4 . 2 Hur data
Al though ten cases of li ver-cell tururs have been reporte in patients
wi th aplastic anema, Fanconi' s anaema or paoxysml noctural haer
globinuria treated for long periods with oxytholone alone or in corination
with other androgenic drgs, a causal relationship caot be estalished.

The increased risk of developing li ver-cell tururs could be related to

hepatic dage know ta be caused by oxytholone. On the other hand,
patients with congenital anaemas may have an intrinsically higher risk of
developing tUIurS; ths risk may becor rrifest during the extended
suri val resul ting from admnistration of the drg.

137
 5. References

Adik, P.M. & Hakness, R.A. (1971) Th use of caton skeleton chomato-
graphy for the detection of steroid drg rrtali tes: the rrtalisr
of analx:Üic steroids in mm. Act endocrinol., 67, 721-732
AIrica Soiety of Hospital Phacists (1968) AIrican Hospital Fonnar
Seice, Setion 68:08, Washington OC

Anon. (1973) Liver tunurs and steroid horrnes. Lacet, ii, 1481

Bagheri, S.A. & Boer, J.L. (1974) Peliosis hepatis associated with andro-
genc-anlic steroid therapy. A severe forr of hepatic injur.
An. int. Me., 81, 610-618

Berntein, M.S., Hlter, R.L. & Yachnin, s. (1971) Hepatc: and peliosis
hepatis developing in a patient with Fanconi 's anera. Ne :Egl.
J. Me., 284, 1135-1136

Blaco, N. W., ed. (1972 ) Maindale, The Exa Phacopoia, wndon,

Phaceutical Press, pp. 1680-1681

Bruguera, M. (1975) Hepatoma associated with androgenc steroids.

Iacet., i, 1295

Catta, D., Kalifat, R., Wautier, J-L., ~ign, S., Vesin, P. & Piet, R.
(1974) Maladie de Fanconi et cancer du foie. Arch. franç. MaL. Ap.
dig., 63, 41-48

Co ttee on Neplastic Diseases (1974) Is li ver cacer induced by

treating aplastic anema with androgenc agents? Pedatrics, 53, 764
Corberand, J., Pris, J., Dutau, G., Ruau, J-L. & Régner, C. (1975)
Association d'une maladie de Fanconi et d'une tuiur hépatique chez
une malade soumse à un traitet androgénique au long cours.
Arch. franç. Pédiat., 32, 275-283

Farell, G.C., Joshua, D.E., Uren, R.F., Bad, P.J., Perkins, K.W. &
Kronenrg, H. (1975) Androgen-induced hepato. Lacet, i, 430-432

Forist, A.A. & Johnson, J.L. (1961) Steroids. In: Higuchi, T. & Brochm-
Hassen, E., eds, Phaceutical Analysis, New York, Interscience,
pp. 69-136

Guy, J. T. & Auslander, M. O. (1973) Androgenic steroids and hepatocellular

cacinom. Iacet, i, 148

Haa, S. & Mi, K. (1967) Systetic analysis of steroids. VII. Thin-

1ayer [Link] of steroidal pharceuticals. Chem. phar. Bull.
(Tokyo), 15, 1036-1040

138
Harvey, S.C. (1975) Horrnes. In: Osol, A. et aL., ed, Regton's
Pharmceutical Sciences, l5th ed., Eaton, Pa, Mack, pp. 931-932

Henderson, J.T., Richrnd, J. & Smnrling, M.D. (1973) Androgenic-anlic

steroid therapy and hepatocellular carcinoma. Lacet, i, 934
Holder, L.E., Gnra, D.J., Lakin, B.C., Nishiyam, H. & Perkins, P.
(1975) Hepato associated with anablic steoid therapy.
Amr. J. Roetgenol., 124, 638-642

Johnson, F.L., Feagler, J.R., Leer, K.G., Majeru, P.W., Siegel, M.,
Har, J.R. & Thmas, E.D. (1972) Association of androgenc-
anlic steoid therapy with developiæt of hepatocellular carcinan.
Lacet, ii, 1273-1276

Kastrup, E.K., ed. (1974) Facts and Caarisons, St umis, Missouri,

Facts and Compisons, Inc., p. lllc

Lesna, M., Spencer, 1. & Walker, W. (1976) Liver noules and androgen.
Lacet, i, 1124

~adow, A.T., Nai, J.L. & Valdes-Dapea, M. (1974) Hepatoo associated

with androgen therapy for aplastic anera. J. Pediat., 84, 109-110

Muvihill, J.J., Ridolfi, R.L., Schultz, F.R., Barzy, M.S. & Haughton, P.B.T.
(1975) Hepatic adenoma in Fanconi anena trated with oxtholane.
J. Pediat., 87, 122-124

National Forrar Bod (1970) National Fonnular XIII, Washigton OC,

Arrica Phaceutical Association, pp. 506-508

Ringold, H.J., Batres, E., Halpern, O. & Necchea, E. (1959) Steroids.

OJ. 2-~thy 1 and 2-hydroxythy lene-androstae de ri vati ves.
J. Amr. Cheie So., 81, 427-432

Stecher, P.G., ed. (1968) Th ~rck Index, 8th ed., Raay, ID, rlrck &
Co., p. 775

Swney, E.C. & Evs, D.J. (1975) Liver lesions and androgenc steroid
therapy. Lacet, ii, 1042

Weast, R.C., ed. (1975) CRC HadJk of Cherstr and Physics, 56th ed.,
Cleveland, Ohio, Chemcal Ruber Co., p. C-752

Ziegenfuss, J. & Caabsi, R. (1973) Androgens and hepatocllular

carcinoma. Lacet, i, 262

139
 PHEIN
1. Chcal and Physical Data

1. 1 Synonyr and trade :rs l

Cher. Abstr. Reg. Serial No.: 62-44-2

Cher. Abstr. Nar: N- ( 4- Ethoxyheny 1) acetamde

para-Acetophenetide; para-acetophenetidide; acetophenetidin;

acetophenetidine; para-acetophenetidine; aceto-4-phenetidine;
acetophenetin; acetparaphenalide; acetphenetidin; para-acetphene-
tidin; acet-para-phenetidin; N-acety 1 -para-phenetidine; 4 '-ethoxy-
acetanilide; 4-ethoxyacetanilide; para-ethoxyacetanilide; para-
phenacetin; phenacetine; phenaci tin; phenazetin

Fenacetina; Fenedina; Fenidina; Fenina; Pertonal; Phenacet;

Phenacetinum; Phenazetin; Phenedina; Phenidin; Phenin

1.2 Chemcal fonrla and 1I1ecular weight

o
Il
~C -CH,

o -CH2CH3

C10H13N)2 MoL. wt: 179 . 2

1. 3 Chemcal and physical properties of the pure substance
From Stecher (1968), unless otherwise speified

(a) Description: Crystalline scales or JJder

(b) Melting-point: l34-l350C

lOnly trade nas of those products that contain phenacetin alone are
gi ven; there are rny other products in which phenacetin is comined wi th
other drgs.
141
 (c) Spetroscopy data: Àni 250 nr in ethol (US Phacoial
Convention, Inc., 1970); 285 nm in chlorofonn and iSCXtane
(National Foi:ar Bod, 1970)

(d) Solubility: Soluble in cold water (0.076 g/lOO ml), in boiling

water (1. 2 g/lOO ml), in ethl (6.7 g/lOO ml), in chlorofonn
(7.1 g/lOO ml), in ether (1. 1 g/lOO ml) and in glycerol

1.4 Technical products and imurities

Various national and interntional phacopoias gi ve specifications

for the purity of phencetin in phaceutical products. For exle,
phenacetin is available in the US as a USP grade containg 98-101%
active ingredient on a dried basis and O. 03% im para-chloroacetalide;
it is also obtainable as talets in 300 mg doses oontaining 94-106% of
the stated arunt (US Phacopeial Convention, Inc., 1970). Phenacetin is
also available in the US in talet fonn oontaing 150 mg phencetin in
combination with 230 mg aspirin and 15 or 30 mg caffeine, or with 230 mg
aspirin, 30 mg caffeine and 8, 15, 30 or 60 mg axeine phosphate and
containing 90-110 % of the state arunt of pheceti (National Fonnar
Bord, 1970).

2. Production, Use, Occurence and Analysis

For imrtant background inforntion on ths section, se preale,

p. 15.

2.1 Production and us

(a) Production

A rnthod of prepaing phenacetin was first reported in 1894 and

invol ved reaction of 4-acetamophenol wi th JXtassium ethy 1 sulphate in an
aqueous alcoholic alkaline solution under 'pressure at l500C (Prager &
Jacobson, 1930). Phecetin can aiso be prepa by cadesing para-
nitrophenol, dissolved in a sodum hydroxde solution, with ethyl brande
to give para-nitrphetole, which is then reduce with sodum sulphide.
The resultig para-phenetidine is acetylated by refluxg with acetic
anydide to give phacetin (SWinyard, 1975).

142
 In Japa, phencetin is produced corcially by the phenol sythesis
procss, which uses the following rrterials: para-ni trophenol , sodium
nitrate, sulphuric acid and ethyl iodide, or by the nitration-fractional
crystallization process, whch uses para-chloroni trobezene and mixed acid.

Phenacetin has been produced ccrciall y in the US for over 50 years

(US Tariff Commssion, 1927); only one US company reported production
(see preale, p. 15) in 1974 (US Interntional '!ade Cossion, 1976a).
The camined production of the acetanilide deri vati ves, phenacetin and
acetaophen, was reported to be 3,500 thousand kg in 1974 (US International
Trade Corssion, 1976a). US imrts of phenacetin though the principal
cutam districts were 67,000 kg in 1972 (US Tariff Comssion, 1973),
94,000 kg in 1973 (US Tariff Cossion, 1974) and 192,000 kg in 1974 (US
Interntional Trade Comssion, 1976b).

Phenacetin was first produced in Japa in 1935. 'l coies mau-

factured a coined total of 276,000 kg in 1975; in that year, 40,000 kg
were irrted fror France an less than 1,000 kg exrted.

Total anual production of pheacetin in western Euope is estimted

to be 1-5 million kg; the Federal Republic of Gerry and France are
believed to be major producing countries.

(b) Use

Phenacetin is used as an anlgesic and antipyretic agent but has

little anti-inflaitory activity. It is us rrinly to counteract mild
to m:erate pain of the rncu0skeletal system and is freqently corrined
with aspirin and caffeine. Th usual dosage is 300 mg taen 4 to 6 tirs
per day, not exceeing 2 g pe day (SWinyard, 1975). Prepaations containing
phenacetin coined with oter drgs usually contain 150 ta 200 mg phenacetin
(Arrican Soiety of Hospital Phacists, 1959).

'Itål US sales of phenacetin for use in hum rrcine are estimted

to be less th 640,000 kg anuall y.

Phenacetin is also us as a stailizer for hydrogen peroxide in

hair bleachig prepaations (Makland, 1966). In veterinar rrcine, it
is used as an anlgesic and antipyretic agent (Stecher, 1968).

143
2.2 Occuence
Phencetin 1S not know ta occu in nature.
2. 3 Anl ysis
Methods of assay for phenacetin tht rrt reguatory reqrernts for
phaceutical products include non-aqueous titration, gravimtric IIthods
and UV sptrophotoitry. A selective lN spctrophotoitric IIthod based
on :rsurert of i ts oxidation products by cobal tic oxide ca deterrne
phenacetin in biological rrterials in the range of 5-50 llg/rn (Wallace
et al., 1973).

Agas chromatoaphic IIthod ca determne phenacetin in mixtures wi th

other drgs (Ryabtseva et al., 1970), and another deterrnes phenacetin in
urine and plasra. (after its conversion ta a silyl derivative) with a limt
of detection of 0.05 llg/rn (Prescott, 1971).

Colorimtric IIthods include a determation of phenacetin in fonn-

;-
lations with aspirin and cxeine phosphate (Wosiak & Krzernska, 1975),
an assay for phenacetin based on its reaction with 1,2-naphthoqone-4-
sulphonate (Noma et al., 1966) and a procedure baed on oxidation of
phenacetin with cerium (St. Ajer et aL., 1969).

3. Biological Data Relevat to the Evluation

of Cacinogenic Risk ta Ma
3.1 Cacinogenicity and related studies in anls
Oral admistration
Rat: A group of 30 BD rats, 100 days of age (sex unspeified),
received 40-50 rn/anirl phenacetin daily in the diet (average total dose,
22 g). One rat died after a total dose of 10 g and was found to have an
osteohondrom. The IIan age at death was 770 days, coed with 750 days
in an unspecified numr of controls. NO tlTurs related to treatrt
were observed (Scli & Reiter, 1954).

Four groups of 15, 20, 20 and 24 rrle alino rats (150-180 g) were
fed diets containing 0 (control), 0.05, 0.1 or 0.5% N-hydroxyhenacetin
(a putative IItablite of phenacetin) for up ta 73 ~eks. Of treated
144
animls 11, 13 and 15 rats were still alive at the ti of the appeance
of the first tuur after 45, 45 and 38 wee, and 8/11, 13/13 and 15/15
develope liver tuiurs (describd as hepatocellular cacinomas), coed
with 0/15 controls; one animl fed 0.1% in th diet develope a transitional-
cell carcinoma of the renal pelvis (Calder et aL., 1976).

3.2 Oter relevant biological data

(a) Exrimtal system
The single oral ID of phenacetin in male wistar rats is abut 4 gl
~ 0

kg lM (Boyd & Hottenoth, 1968) and in guinea-pigs 2.6 g/kg lM (Boyd &
Caro-iami, 1970).

Papillar necrosis of the kidney was produced in Wista rats fed a

mixture of aspirin (210 rn/kg l:/day), phenacetin (210 rn/kg l:/day) and
caffeine (80 rn/kg lM/day) but not in rats receiving 500 rr/kg l:/day
phenacetin alone (Saker & Kicaid-Smth, 1969).
There are thee knOt iætalic pathays for phencetin: de-ethylation,
N-deacetylation an ring hydroxylation. The main route is oxidative de-
ethylation, giving rise ta N-acetyl-para-amophenol, which is excreted in
the urine as the sulphate or as the glucuronide (Dubch & Raaflaub, 1969).
ln rats, rabbits, guea-pigs and ferrets given 125 rr/kg l: by oral intu-
bation or rned with foo, 63, 57, 81 and 47% of the dose, respetively,
were excreted as N-acetyl-para-amnophenol (free or conjugated). Metalisr
by the second pathay, N-deacetylation, was greatest in rats (21% of the
dose) and least in gunea-pigs and rabbits (7 and 4% of the dose) (Smth
& Timrell, 1974). The para-phenetidine resulting from N-deacetylation
can be converted to 2-hydroxy-para-phenetidine, whch in rats is excreted
in increasing anunts with increasing doses of phenacetin (Duch &
Raaflaub, 1969). Other rntalites that have ben found in the urine of
rats, guea-pigs and rabbits are 2-hydroxyhenacetin and 3( (5-acetado-2-
hydroxyhenyl)thoJaniline (Smth & Timrell, 1974); and the 2-hydroxy-
acetophenetidine-glucuonide conjugate has been found in the urine of dogs
and cats admistered phenacetin (route not given) (Klutch et aL., 1966).
The possible role of N-hydroxy lation in the iætalisr of phenacetin
ha ben discussed by Nery (1971). N-Hydroxyhenacetin, a sythetic
145
cound, uner acid conditions or after N-ester foi:tion, will react with
rrthonie to give 4-hydroxy-3-rrthylthoacetailide (Cader et al., 1974),
a urin rrtalite of phenacetin in dos (Foclla et al., 1972).

In rats treated wi th 3-rnthy lcholanthene or exsed to cigarette

srke (which caused the induction of bezo(alpyene hydroxylase in lung
tissue), there was an increased ability of lung and intestine to rrtalize
phecetin to N-acetyl-para-amnophenol (Welch et aL., 1972).

A coison of the rrtablisr of phenacetin wi th that of other strc-

turally-related caunds bas be rrde in rats (Smth & Griffiths, 1976).
N-Hydrxy deri vati ves of phencetin were found to be nephrotoxic in
ferle Wista rats following single i. v. injections of abut 1 !T/kg li
(Calder et al., 1973).

In a limted study, an intragastric dose of 2 g/kg li/we (eqvalent

to a hur dose of abut 20 g/day) was given in five divided doses/wee to
25 male rats for up to 220 days; at 176 days 80% of the rats were sterile.
No significant difference in the numr of stillbrns was seen caared to
tht in controls (Boyd, 1971).

Phencetin ca be N-nitrosated i-n vitro ta forr an untale N-nitroso

carund, N-nitroso-2-nitro-4-ethoxyacetanilide (Eisenrand & Preussm,
1975) .

(b) Ma
The ma toxic effec of chronic phencetin ingestion is papillar
necrosis of the kidney (se section 3.3). Several acute cases of poisonig
with paacetal (a major rætalite of phenacetin) have been reported, in
whch hepatic dage occued (Clark et al., 1973; Prescott et aL., 1971;
Proudfoot & Wright, 1970).
A group of 623 warr knaw regarly to ingest phenacetin-containng
analgesics wee coed over a 4-yea period with a group of 621 controls.
A high intae of phenacetin analgesics was associated wi th increased seru
creatinine ievels an a lCM urine speific gravity (Duch et al., 1975).

lætherlobinaera and baerlytic anaera bave occued in subjects

ingesting phencetin; ræthaerlobinaera bas be associated with the

146
excretion of increase anunts of 2-hydroxyhenetidie sulphate and the
glucuonide or its oxidation products in the urine of the se subjects

(Shahdi, 1967; Shahdi & Hemdan, 1969; V\ur & Fingl, 1975).
Nornl subjects ingesting phenacetin excrete the major pat of the dose
in the urine as conjugated N-acetyl-para-amophenol (abut 70-80%), soiæ
as free N-acetyl-para-amnophenol (3-5%), a smll pecetage (0.2%) as
unchaged phenacetin and a smler percetage (0.1%) as para-phenetidine
(Brodie & Axlrod, 1949). In 3 male volunteers given 2 g phenacetin orally,
abut 2% of the dose was found ta be excreted as S- (1 -acetado-4-hydroxy-
phenyl)cysteine in the urine (Jagenurg & Tozko, 1964). In cigarette
sikers, there is a higher ratio of N-acetyl-para-amophenol :phenacetin
in the plasm than in corresponding controls (Pantuck et al., 1974).

The urinar excretion of 2-hydroxyhenetidin and of N-acety 1 -para-

amophenol, and of their conjugates, was signficantl y decreased when
phenacetin was ingested in coination with aspirin, caffeine and coeine
(Gault et al., 1972).

3.3 Case reports and epiderological studies

(a) Case reports

Hultengren et aL. (1965) in Stokholm, Svden, reported 6 cases of

renal papillar necrosis associated wi th transi tional -cell cacinoma of the
renal pel vis; 5 of the cases were reported to be heavy users of phenacetin-
containg anlgesics (IIre than 1 g anlgesic per day for IIre th one
year). Since tht ti there have been several case reports of carcinomas
of the renal pelvis (RP) or of the bladder (B) in heavy users of phenceti-
containing analgesics (Ad et al., 1970, 1 case RP; Beley et al., 1970,
1 case B; Grob, 1971, 2 cases RP; Güller & Duch, 1973, 1 case RP, 1
case B;' Liu et aL., 1972, 1 case RP; Maion & Suso, 1971, 1 case B;
Rathert et al., 1975, 1 case RP, 1 case B).

(b) Epiderological studies

Of 242 patients in GÖterg, Swen, with chronic non-obstrctive

pyelonephritis, 142 were considered ta be heavy users of phenacetin-containg
anlgesics; 104 of these cases were follow for 1-11 years (average 5.3
years) and comped with 88 control cases not considered to be heavy users
147
of analgesics. Eight patients in the heavy usrs' group develope
tranitional -cell cacinoms of the renl pelvis; in seven, a renal
papillar necrosis was present. ln addition, one male and one ferle
develope s:ilar tUIurs of the bladder. ì' such tUIurs ~re observed in
the control group (Begtson et aL., 1968). A nith patient later develope
renal pelvic cacin() (Angervll et al., 1969).

In another report fran Jönköping County Hospital, ~eden, 15 cases of

tranitional-cell cacino:r of the renal pelvis ~re observed during a 9-
year period: 10 cases cxcured arng the inabi tats of Huskvara (popula-
tion 13,000), and 9 of these were arng the WJrkers of a smll-an factory
emloying 1800 peple. Ths ooes wi th an incidence of renl pel vic
carcinoma or papill() in Swen as a whole of only 1 case :¡r 183,000 per
year during 1960-1963. In 10 (or possibly 12) cases, heavy use of Hjorton's
poer (each dose containing 0.5 g phencetin, 0.5 g phenazone and 0.15 g
caffeine) was recorded, and renal papillar necrosis was present in 9 (or

possibl y 10) cases. 'I of the heavy users also had a carcinom of the
bladder. The study did not include a caeful search for other possible
carcinogenic agents, but, accordig to the authors, an occupational carci-
nogen seer unlikel y in a factory of ths ty (rnufacturing smll -an,
sewing machines, bicycles, garden tex:Üs and simlar products) (Angervall
et al., 1969).

In a furer report of 62 cases of carcino:r of the renal pelvis in

knaw phenacetin users (which included 19 of the cases describd in the tw
previous reports), detailed data an the use of phenacetin-containg
an1gesics were available for 38 cases reported. The average total dose
was 9.1 kg, average exsure tim, 17 years and average induction tim,
22 years. Papillar necrosis was a prornent feature in 59 patients; 8
patients also had urinary bladder tUIurs (Begtson et al., 1975;
Johansson et aL., 1974).

Bok & Hogrefe (1972) describd 31 cases in the Federal Republic of

Genny of cacinoma of th renal pelvis, 5 cacinoms of the ureter and
106 carcinoms of the bladder; onl Y 1 case of papillar cacin() of the
renl pelvis assoiated wi th a benign paillom of the ureter and 2 cases
of carcinom of the bladder ~re found in heavy users of phenacetin.

148
 Taylor (1972) reported that in the OK in a series of 189 prir tururs
of the adult urinar tract, 2/30 cacinorns of the renl lxy, 7/13 carcinor
of the renal pelvis, 0/2 carcinoms of the ureter and 2/144 cacinomas of the
bladder occued in peple niing heavy use of analgesics, including
phenacetin-containing mitures.

ln a surey in Zurich, Swtzerland, of 24,683 autopsies, 5/269 hypr-

nephroid carcinomas (1.9%), 4/15 cacinomas of the renl pelvis (26.6%)
and 11/218 carcinoms of the bladder (5%) \\re found in heavy users of
phenacetin-containing analgesics. Papillar necrosis wa present in 9 of
these 20 subjects. Only the cacinoms of the renal pelvis were considered
to be related to heavy use of phenacetin (Kfug, 1976).

Of 320 patients in Dek with a diagnosis of chronic pyelonephritis,

101 had a history of heavy use of phenacetin-containing anlgesics; 2 of
these presented papillary necrosis and a trani tional -cell tuiur of the
renal pelvis (Hj2ybye & Nielsen, 1971). Leistenschneider & Eh (1973)
in Basel, Swi tzer land, reported tht of 17 cacers of the renl pelvis (out
of 21,291 autopsies), 8 occured in heavy users of phenacetin-containing
anlgesics (2 presented pyelonephritis) .
ln a case-control study in the OK, in whch subjects with urinar-
tract cancer were comped wi th age and se-matched controls, heavy
consurtion of anlgesics was not found to be associated with cancers of
the renal pelvis but was associated wi th cacers of the renal paenchym.
ln 106 patients with adenocarcinoms of the renal paenchym there were 15
heavy users of analgesics, and only 2 in 106 controls. It rnt be note
tht only 4 of the 15 heavy users with a renl paenchynl tuiur were
users of analgesic miures containng phecetin (Artrong et al., 1976).

149
 4. Cots on Data Reported and Evluation

4.1 Animl data

ln one lim ted study in whch phencetin wa admistered orall y ta

rats, no carcinogenic effects were observed l . One putative iætali te
of phenacetin, N-hydroxyhenacetin, is carcinogenic in rats after its oral
admistration: it produced hepatocellular carcinOIs.

4.2 Hum data

Available data indicate that heavy use of anlgesic mixtures contaig
phencetin is associated wi th papillar necrosis of the kidney and suggest
a relationship betw such use and the developint of transi tional -cell
carcinor of the renl pelvis.

ITh Working Group wa aware that several studies on the cacino-

genicity of phenaceti are under (IA, 1976).
150
 5. References

Adam, W.R., Dawbrn, J .K., Price, C.G., Riddell, J. & Story, H. (1970)
Anaplastic tranitional -cell cacinorr of the renal pelvis in
association with anlgesic abuse. ME. J. Austr., l, 1108-1109

Amrica Soiety of Hospital Phacists (1959) Amrican Hospital

Formar Service, Setion 28:08; Washington OC

Angervall, L., Begtson, U., Zetterlund, C.G. & Zsigind, M. (1969)

Renal pelvic carcinoma in a ~dish district with abuse of a
phencetin-containing drg. Brit. J. Urol., 41, 401-405

Artrong, B., Gacx, A. & I):Ül, R. (1976) A retrospective study of renal

cancer with speial reference to coffee and anl protein consurtion.
Brit. J. Cacer, 33, 127-136

Beley, M., Chdwick, J.M. & Jepson, R.P. (1970) A pJssible case of anal-
gesic abuse associated wi th transi tional -cell carcinoma of the bladder.
Me. J. Austr., 2, 1133-1134

:Bngtsson, U., Angervall, L., Ek, H. & Le, L. (1968) Tranitional

cell tuirs of the renal pelvis in anlgesic abusers. Scad. J. Urol.
Nephrol., ~, 145-150

Bengtsson, U., Angervall, L., Johasson, S. & Wahlgvist, L. (1975) Phen-

acetin abuse and renal pel vic carcinom. Int. J. clin. Pharcol.,
12, 290- 294

Bok, K.D. & Hogefe, J. (1972) Anlgetika-Abusus und maligne 'lren der
ablei tenden HaW2ge - Eine retrospeti ve Studie. Mfch. rr. Wschr.,
114, 645-652

Boyd, E.M. (1971) Sterility fror phenacetin. J. clin. PhacoL., 11,

96- 102

Boyd, E.M. & Caro-Ciami, G. (1970) Th oral 100-dy IDso index of

phencetin in guea pigs. ToxicoL. appL. Phacol., 16, 232-238

Boyd, E.M. & Hottenoth, S.M.H. (1968) Th toxicity of phenacetin at the

range of the oral ID ~ 0 (100 days) in albino rats. ToxicoL. appL.
PharncoL., 12, 80-93

Brcxie, B. B. & Axelrcx, J. (1949) The fate of acetophenetidin (phenacetin)

58-67 -
in ma and rrthcxs for the estimtion of acetophenetidin and its
iætali tes in biological rrterial. J. Phacol. ex. Ther., 97,

Calder, LC., Creek, M.J., William, P.J., Fuder, C.C., Green, C.R.,
Ha, K.N. & Tange, J.D. (1973) N-Hydroxylation of p-acetophenetidide
as a factor in nephrotoxicity. J. rr. Cher., 16, 499-502

151
Calder, LC., Cree, M.J. & William, P.J. (1974) N-Hydroxyhecetin as
a precsor of 3-substituted 4-hydroxyacetanilide rntalites of
phenacetin. Cher. -bioL. Interact., 8, 87-90

Calder, I.C., Gass, D.E., William, P.J., Fuer, C.C., Gree, C.R., Ha,
K.N. & Tange, J .D. (1976) Neoplasia in the rat induce by N-hydroxy-
phenacetin, a netalite of phenacetin. Patholog,~, 1-6

Clark, R., Thanson, R.P.H., Borirakchayavat, V., Ttiiddop, B., Davidson, A.R.,
Gaulding, R. & William, R. (1973) Heptic dage and death from over-
dose of paacetal. Lacet, i, 66-70

Duch, U.C. & Raaflaub, J. (1969) Neue Aste zur Frage der Nephro-
toxizität von Phenacetin. Exrientia, 25, 956-958

Duch, U.C., Ievy, P.S., Rosner, B., Baumler, H.R., l1:ller, A., Peier, A.
& Ehenspeger, T. (1975) Relation betwen reglar intae of phena-
cetin-containing anlgesics and labratory evidence for urorenal dis-
orders in a \'rking feile population of Swtzerland. Lacet, i,
539-543
Eisenrand, G. & Preussm, R. (1975) Nitrosation of phenacetin. Formtion
of N-nitroso-2-nitro-4-ethoxyacetalide as an unstale product of the
nitrosation in dilute aqueous-acidic solution. Arzneimttel-Forsch.,
25, 1472-1475

Focella, A., Heslin, P. & Teitel, S. (1972) Th sythesis of tw phenacetin

metali tes. Cad. J. Chen., 50, 2025-2030

Gault, M.H., Shadi, N.T. & Ga, A. (1972) Th effect of acetylsalicylic

acid, caffeine, and coeine on the excretion of phenacetin netablites.
Caad. J. Physiol. Phacol., 50, 809-816

Grob, H. U . (1971) Phenazetinabusus und Nierenckenkarzinom. Hel v. chir.

acta, 38, 537-539

Güller, R. & Duch, U.C. (1973) 'lren der Haege nach [Link]
Einnah phenazetinhltiger Anlgetik? Hel v. ræ. acta, 36, 247-250
HØybye, G. & Nielsen, O.E. (1971) Rel pelvic cacinam in ohencetin
abusers. Scan. J. Urol. Nephrol., 5, 190-192

Hultengren, N., Lagergren, C. & Ljungqvist, A. (1965) Cacinam of the

renal pelvis in renal papillar necrosis. Acta chir. scand., 130,
314-320

IA (1976) IA Inonntion Bulletin on the Surey of Chcals Being

Tested for Cacinogenicity, No. 6, Lyon, pp. 71, 91, 279

Jagenurg, O.R. & Tozko, K. (1964) Th rntalisr of acetophenetidine.

Isolation and chaacterization of S- (1 -acetado-4-hydroxyheny 1) -
cysteine, a rntalite of acetophenetidine. Biocher. J., 92, 639-643

152
Johasson, S., Angervll, L., Begtsson, U. & Wahqvist, L. (1974)
Uroepithelial turrs of the renl pelvis associate with abuse of
phenacetin-containing anlgesics. Cacer, 33, 743-753

Klutch, A., Harfenist, M. & Conney, A.H. (1966) 2-Hydroxyacetophenetidine,

a new rntalite of acetophenetidine. J. rr. Ch., 9, 63-66

KÜIg, L.G. (1976) Hyrnephroides Kazinor un Kazinan der ableitenden

Hamwee nach Phenacetinusus. Schweiz. rr. Wschr., 106, 47-51
Leistenschneider, W. & Et, R. (1973) Nierenkenarzinor nach Phen-
azetinabusus. Schweiz. rn. Wschr., 103, 433-439

Liu, T., Smth, G. W. & Rain, J. T. (1972) Renal pelvic turur associated
with anlgesic abuse. Cad. rn. Ass. J., 107, 768, 771

Maon, R.A. & Suso, D. (1971) Phenacetin abuse causing bladder tur.
J. Urol., 106, 692

Makland, \"'.R. (1966) Hair prePaations. ln: Kik, R.E. & Otr, D.F.,
eds, Encyclopea of Chemcal Tehnolog, 2nd ed., Vol. 10, Ne York,
John Wiley and Sons, pp. 798-799

National Formlar Board (1970) National Formlar XIII, v.7ashigton OC,

Arrica Phaceutical Assoiation, pp. 66-68 & 178-180

Nery, R. (1971) The possible role of N-hydroxylation in the biological

effecs of phecetin. Xenobiotica, l, 339-343

Nora, N., Ito, T. & Shiho, D. (1966) Determnation of mied m:cines

I. Quantitative colorimtric rrthod for phenacetin with scium 1,2-
naphthoqinone-4-sulfonate. Yakugak Zasshi, 86, 331-335

Pantuck, E.J., Hsiao, K-C., Maggio, A., Naka, K., Kuntzr, R. &
Conney, A.H. (1974) Effect of cigarette siking on phenacetin rnta-
bolism. Clin. Phacol. Thr., 15, 9- 17

Prager, B. & Jacobson, P., eds (1930) Beilsteins Handbuch der Organische
Chere, 4th ed., VoL. 13, Syst. 1847, Berlin, Springer-Verlag, p. 461

Prescott, L.F. (1971) 'Te gas-liquid chrantographic estimtion of phen-

acetin and paacetal in plasm and urine. J. Pha. Phacol.,
23, 111-115

Prescott, L.F., í"'right, N., Rosco, P. & BrOt, S.S. (1971) Plasm-para-
cetal half- life and hepatic necrosis in patients with paacetal
overdosage. Lacet, l, 519-522

--
Proudfoot, A.T. & Wright, N. (1970) Acute paacetal poisoning. Brit. rr
J., iii, 557-558

153
Rathert, P., Melchior, H. & Lutzeyer, W. (1975) Phencetin: a carcinen
for the urin tract? J. Urol., 133, 653-657

Ryabtseva, LM., Kuleshova, H.L, Ruenko, B.A. & Kucherov, V.F. (1970)
Gas- liqud chrtography of drgs. Izv. Akd. Nauk SSR, Se. Kh.,
12, 2676-2680

Saker, B.M. & Kicaid-Smth, P. (1969) Papillar necrosis in eximtal

analgesic nephropathy. Brit. rn. J., i, 161-162

Scl:, D. & Reiter, A. (1954) Fehen einer cacerogenen lAlirkug beir

Phenacetin. Arzneimttel-forsch., 4, 404-405

Shadi, N.T. (1967) Acetophenetidin sesitivity. Arr. J. Dis. Chld.,

113, 81 -82

Shadi, N.T. & Hemida, A. (1969) Acetopheetidin-induce rætharlo-

binera and its relation to the excretion of diazotizable amnes.
J. La. clin. i~., 74, 581-585

Smth, G.E. & Griffith, L.A. (1976) Coative metalic studes of

phenacetin an strcturally-related ccunds in th rat.
Xenobiotica, 6, 217-236

Smth, R.L. & Timrell, J.A. (1974) Factors affecting the rætalism of
phencetin. 1. Influence of dose, chronic dosage, route of adm-
stration and speies on the metalism of (1-14C-acetyl)phenacetin.
Xenobiotica, 4, 489-501

Ste Ajer, G., Laszl, O.V. & Szab, O.A.E. (1969) Cerimtric detetion
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Stecher, P .G., ed. (1968) The Meck Inex, 8th ed., Ray, ID, l'Eck &
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SWinyard, E.A. (1975) Anlgesics and antipyretics. In: Osl, A. et aL.,

ed, Rergton's Phaceutical SCiences, 15th ed., Eaton, Pa, Hack,
p. 1051

Taylor, J.S. (1972) Cacin of th urin tract and anlgesic abse.

Me. J. Austr., l, 407-409

US Interntional Trade Cassion (1976a) Synthetic Organc Chcals, US

Production and Sales, 1974, USITC Pulication 776, l"lashington OC,
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US International Trade Ccssion (1976b) Imrts of Bezenoid Chcals
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154
us Phacopeial Convention, Inc. (1970) Th us Phacopeia, 18th rev.,
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US Tariff Cossion (1927) Cesus of Oyes and Oter Synthetic Organic

Chemcals, 1926, Tariff Inormtion Series :N. 35, Washington OC,
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1972, 'I Pulication 601, Washington OC, us Cbvenit Printing
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us Tariff Ccmssion (1974) Imrts of Bezenoid Chemcals and Prcxucts,

1973, 'I Pulication 688, Washington OC, us Goenit Printing
Office, p. 83

Wallace, J.E., Biggs, J.D., Halton, H.E., Foster, L.L. & Blum, K. (1973)
lN spectrophotomtric rrthcx for determtion of phenacetin in bio-
logical speims. J. pha. Sei., 62, 599-601

Welch, R.M., Cavallito, J. & Loh, A. (1972) Effect of exsure to cigarette

srke on the rætalisr of bezo (a Jpyene and acetophenetidi by lung
and intestine of rats. ToxicoL. appL. Phacol., 23, 749-758

Vbur, D.M. & Fingl, E. (1975) Analgesic-antipyetics, anti-inflartory

agents, and drgs emloyed in the therapy of gout. In: Go, L.S.
& Gilm, A., eds, Th Phacological Bais of Therapeutics, 5th ed. ,
Ne York, Macrllan, pp. 343-347

Vbsiak, B. & Krzerka, A. (1975) Colorimtric determation of phenacetin

in poers also containing aspirin and coeine phosphate. Far. pol.,
31, 771-773

155
 PHEBAITAL AN PHEBAITA SODIUM

1. Chercal and Physical Data

Phenobabi tal

1.1 Synonym and trade nas

Cher. Abstr. Reg. Serial No.: 50-06-6

Cher. Abstr. Nar: 5-Ethyl-5-phenyl-2,4,6- (lH ,3H ,5H)pyrirnidinetrione

5-Ethyl-5-phenylbbituric acid; phenobabitone; phenobabituric
acid; pheny lbrbi tal; phenylethylbbi turate; pheny lethy lbbi turic
acid; 5-phenyl-5-ethylbbituric acid; phenylethy1rlonylurea

Monal; Aephenal; Agrnal; AI1lofene; Aphenylbbit¡ Aphenyletten;

Austrornal; Babapil; Babellen; Babellon; Babey 1; Babilettae;
Babinal; Babiphen; Babiphenyl¡ Babipil; Babita; Babivis;
Babonal; Babophen; Badorr; Batol; Bialmal; Blu-phen;
Caronal; Calrtten; Canal; Cadenal; Celonal; Coibbital;
Coronaletta; Dairal; Dezibbitur; Dormna; Dormral; Doscalun;
Dueryl; Ensobab; Ensodorr; Epal; Epidorr; Eoilol¡ Episedal;
Epsylone; Eskabb; Etilfen; Eueryl; Fenital; Fenerl; Feno-
babital; Fenosed; Fenylettae; ('"adenal; Gadepayl; Glysoletten;
Haplopan; Haplos; Helional; Henoletten; Hynaletten; Hypnogen;
Hypnolone; Hypno-Tablinetten; Hynotal; Hysteps; Lefeb; [Link] ¡
Lepheb; Lepinal; Liasen; Liqutal; Liophen; Lubrgal;
Lubrokol; Lll; Lursettes; Lusyn; Lumal; Lurfridetten;
Luphenil; Luramn; Molinl; Neurobab ¡ Nirvonal; Noptil; Nova-
Pheno; Nurl; Nunol¡ Parkotal; PEB; [Link]; Phenaerl;
Phen-Ba; Pheneml; Phenobal; Pheobabyl; Phenoluric; Phenonyl;
Phenoturic; Phenyletten; Phenyral; Phob; Polcominal; Promptonal;
Sedab; Seda-Tablinen; Sedicat; Sedzorin¡ Sedlyn; Sedofen;
Sedonal; Sedonettes; Sevenal; Sautol; Sornolens; Sooletten;
Sornosan; Sonal¡ Spasepilin; Staifen; Stailettae; Stental
Extentabs; Telax; Thnobabi tal; Triabb; Tridezibbi tur;
Triphenatol; Versoml; Zadoletten; Zadonal

157
1.2 Chemcal formla and 1I1ecular ~ight

C12H12N203 rbl. wt: 232.2

1. 3 Chercal and physical properties of the pure substace

From Stecher (1968), unless otherwise speified

(a) Description: Crystals

(b) Melting-point: l74-l780C

(c) Spetroscopy data: Àrr 257 nr in rrthanol; nuclear magnetic

resonace an mass sotra are also given (Grasselli, 1973)
(d) Solubility: Soluble in water (0.1 g/lOO ml), in ethanol (12.5
g/lOO ml), in chloroforr (2.5 g/lOO ml), in ether (7.7 g/lOO ml) ,
in benzene (0.14 g/lOO ml) and in alkaline hydroxides and
carbonates
1.4 Technical products and Ì1uri ties

Various national and international pharcapias give spcifications

for the puri ty of phenobabital in Pharceutical prcxucts. For exle,
phenobabital is available in the US as a USP grade containing 98-101%
active ingredent on a dried basis. It is availab1e in 7.5 and 20 rr
doses per 5 ml of an elixir which contains 92.5-107.5% of the stated arunt
of phenobarbitaL. Th alcoholic cxmtent of the elixir is 12 to 15% as
ethol. Phenobarbital talets in doses of 8, 15, 25, 30, 60 and 100 mg
are available containing 94-106% of the stated arunt of phenobabitaL.
It is also available in poer forr (US Phacopeial Convention, Inc.,
1975). Cominations of 15 and 30 rr Phenobabital with 25 and 50 mg
ephedine sulphate in capsules, and of 8 rr phenbabital with 130 rr
theophylline and 24 mg ephedine hydrochloride in talet forr are available
(National Fonmlar Bod, 1970). Phenobarbital is also forrated in a

158
variety of combinations with other babituratcs and other drgs, such as
scopolamne hydrobrorde and phenyltoloxae, for mutiple rrication
therapy (Kastrp, 1976).

In Euope, phenobarbital prepaations contain 99-101% active ingredient

on a dried basis (Council of Euope, 1969). It is available in the OK in
tablets, containing 92.5-107.5% active ingredient, and for injection
(Bri tish Pharcopoia Comssion, 1973).
In the USSR, phenobabital is also available in talets containing
20 rn phenobabital, 250 rn theobromine an 20 rn papaverine hydrochloride
(Mashkovski, 1972).
Phenobabi tal sodium

1. 1 Synnyr and trade nas

Cher. Abstr. Re. Serial No.: 57-30-7

Cher. Abstr. Nar: 5-Ethyl -5-phenyl -2,4,6- (lH, 3H , 5H) pyirdinetrione,

IInosodium salt

- -
5-Etyl-5-phenylbbituric acid soium; 5-ethyl-5-phenylbbituric
acid sodium salt; phenemlum; phenobabital; phenobabital elixir;
phenobabital-sodium; phenobabitone sodium; phenobabitone sodium
salt; phenylethylbabituric acid, sodium salt; sodium 5-ethyl-5-
phenylbbiturate; soium phenobabital; sodium phenobarbitone;
sodium phenylethylbbiturate; soluble phenobabital; soluble
phenobarbi tone

Gardenal sodium; Lumnal soium; PBS; Pheobal soium; Soium

lumnal
1.2 Chemcal formla and 1I1ecular ~ight

C12Hii N2Na03 l-l. wt: 254. 2

159
1. 3 ChemC?l and physical properties of the pure substance
From Stecher (1968)

(a) Description: Slightly hygroscopic crystals or white paer

(b) Solubility: Soluble in water (100 g/lOO ml) and in ethanol
(10 g/lOO ml); insoluble in ether and chloroforr

(c) Staility: Aqueous solutions are stale for a few days at

100C

1.4 Technical products and imuri ties

Various national and international phacapias gi ve SPcifications

for the puri ty of phenobabital soium in pharceutical products. For
exale, phenobabital soium is available in the US as a USP grade con-
taining 98.5-101% active ingredient on a dried basis with a m: of
0.003% heavy rrtals. Phenobabital soium injections are available in
doses of 60, 130 and 160 mg per ml as sterile solutions in a suitable
solvent (such as water, ethanol or propylene glycol) and contain 90-105%
of the stated anunt. Sterile phenobabital soium sui tale for parenteral
use is available in 60, 130, 200 and 320 mg doses. Tablets containing
35 mg phenobarbital sodium are also available. Phenobabital sodium can
be obtained in poder forr (US Phacopeial Convention, Inc., 1975).
In the OK, it is available on a dried basis containg no less than 98%
active ingredient and in talets containing 92.5-107.5% of the stated
anunt (British PhacolXia Conssion, 1973).

2. Prouction, Use, Ocurrence and Anal ysis

For imrtant background inforntion on this section, see preamle,

p. 15 .

2. 1 Production and use

(a) Production

A rrthod of preparing phenobabital by ethylating pheny1mlonic ester

followed by condensation wi th urea in the presence of sodium ethoxide was
first patented in Germy in 1911 (Stecher, 1968). Phenobabital is

160
produce corcially in the follCMing way: benZ'.Il chloride is treated
with sodium cyanide in rrthanol; the resulting bezyl cyanide is treated
with a rnthanol-sulphuric acid mixture to yield rrthyl-phenylacetate; ths
is condensed with diethyl oxalate in soium rnthoxide, and the resulting
..
dirthyl-phenyloxalacetate is converted to dithyl-pheny1mlonate by
heating in vacuo; this is ethylated with ethyl bromide in sodium rnthoxide,
and the resul ting ethy lated ester is condensed wi th dicyanodiamde to gi ve
the imobarbituric acid; this is hydrolysed with sulphuric acid, and
crude phenobarbital is recrystallized froID ethanol (Meyer & Rollet, 1964).

Phenobarbi tal sodium can be prepared by dissol ving phenobabital in

an eqi valent arunt of soium hydroxide solution in ethanol, follCMed by
evaJ?ration at low terrature (Swinyard, 1975).

Phenobarbital and phenobarbital sodium have be produced corrcially

in the US for over fifty yeas (US Tariff Camssion, 1927). Duing the
period 1950-1964, an average of 140,000 kg phenobabital were produced
anually in the US; in 1974, only two us ccies re:prted production
(see preale, p. 15) (US International Trade Ccmssion, 1976a). Duing
the period 1955-1968, an average of 4700 kg phenobarbital sodium \Vre
produced anually in the US. In 1968, the last year in which separate US
production data were reJ?rted, 5450 kg phenobabital sodium were produced;
only two US cornies re:prted production (see prearle, p. 15) in 1974
(US International Trade Coimssion, 1976a). Curent production of pheno--
barbital in the US is abut 140,000 kg anually. US imrts of phenobarbital
though the principal cutorn districts \Vre reported to be 14,400 kg in
1972 (US Tariff Commssion, 1973), 500 kg in 1973 (US Tariff Commssion,
1974) and the sam aiunt in 1974 (US International Trade Comssion, 1976b).

No evidence was found that phenobabital or phenobarbital sodium is

produced corncially in Japa; in 1975, less than 7,000 kg phenobabital
and less than 100 kg phenobarbital sodium \Vre [Link].

Anual production of phenobabital in Euope is estimted to be in the

range of 100-500 thousand kg: less th one thousand kg in Austria and in
Benelux; and between 1-100 thousand kg in the Federal Republic of Germy,
in France, in Hungar, in l tay, in Poland, in Scandinavia, in Spain, in
Swi tzer land and in the OK.

161
 Anual production of phenobabital soum in Euope is estirted to
be in the range of 1-100 thousand kg: less th one thousand kg in
Austria, in Beelux, in Scadinvia and in Spain; and 1-100 thousand ka
in the Federal Republic of Gey, ln France, in Hungary, in Ital y, in
Poland, in Swi tzer land and in th OK.
(b) Use

Phenobabital and phenobabital soium are long-acting barbiturates

used in hum iæicine as hypnotics, sedatives and in the treatrnt of
epilepsy. When the se coI1unds are used as hypnotics, they are admistered
orally in an average adult dose of 100 ta 200 mg on retiring; the sodum
salt may also be admnistered intravenously at a dose of 130-200 mg
(Swinyard, 1975).

Phenobabital or phenobabital scxium ca be used to prcxuce mild,

prolonged sedation for the treatrnt of conditions such as hyprtension,
functional gastrointestinal disorders, aniety neuroses, coronar heart
disease and preoperati ve apprehension. Th average adul t sedative dose
is 15 to 30 mg orally tw or thee tins daily; the scxium salt can be
given by injection at a dose of 100-130 mg, repeated after 6 hours if
necessar (Swiyard, 1975).

Phenobabi tal and phenobabital sodium are use in the treatrnt of

epilepsy, espeially for grand mal seizures, and are often enloyed in
cominat ion with a ketogenic diet, phenytoin or other therapeutic treatrnt.
An average oral adult dose of either cOlund is 50 to 100 mg admnistered
2 or 3 tims dail y; the sodum salt ca be admistered paenterall y in a
dose of 200-320 mg, repeated after 6 hours if necessary (Swinyard, 1975).

Because of potential abuse leading to a lim ted physical or psycho-

logical depedence, phenobarbital and phenobabital scxium are classified
in the US as controlled substaces in Schedule IV of the Controlled
Substaces Act, whch reqres all maufacturers and distributors of
these cOI1unds to register and report to the US Drug Enorcent Agency
any chages in the quti ty maufactured or distributed and reares
physicians to review their patients' status pericxicall y (US Drug
Enforcet Adistration, 1976).

162
 Total US sales of phenobabital for use in hin iæicine are estirted
te be less th 78,000 kg anually, and those of phenobarbital scum,
less than 2000 kg anua1ly.
Phenobabi tal is used in veterinar iæicine as a seative and anti-
convusant and in canination wi th dicyclome hydroc10ride to control
nausea and vomiting (Siegmd, 1973). It is also reJ?rtedly used in the
treatrt of eclamsia, neuri tis , :orui tis , strchine poisoning, as a
preanaesthetic and as an anesthetic (Stecher, 1968). Phenobabital scium
is reportedl y used in the sa: ways and, in addition, for the treat.t
of epileptiform convulsions (Stecher, 1968).

2. 2 Occuence
Nei ther phenobabital nor phenobabital scum is know te occur ln
nature.
2 . 3 Anal ysis
A general review covers anal ytical Ilthod for babi turic acid
derivatives (Connors, 1961). Methods of assay for phenobarbital that rrt
regulatory reqrerts for phaceutical products include aqueous and
non-aqueous titrations; those for phenobabital scium include graviItr
and spetrophotortry.

Spectro:ohotoIltric anl ysis has æen used to determne phenobarbital

in a miure with caffeine and amdopyrine (Brutko & Sapin, 1972).
Phenobarbital sodium has be detected in miures containing brordes and
a valerian tincture by UV sperophotartry (Mosiniak, 1974). Another
method, based on the corined use of lN sptrophotartr, vohnætr and
complexantry, ca determe phenobabital scum in talets containing
ephedine hydrochloride and calcium camhorsulphonate (Dobrecky, 1969).
Radioimoassays of phenobabital have be reJ?rted (CCDk et al.,
1975; Satoh et al., 1974). l3ker & Darcey (1975) rrde a caison
of enzyrtic imoassay and gas- liqud chrantography for the deteration
of phenobabital. Polarogaphic Ilthods cao be used to determe pheno-
babital in babiturate miures (Zum, 1974) and in b100 (Broks et al.,'
1973). Determnation of phenobabital, whch has be sepaated fran

163
biological materials by fibre-glass intat th- layer chra:togaphy, has
be accomlished by the use of a [Link] ræthod (Hsiung
et al., 1974).

For IIst gas chra:tographic procedures phenobabital is alklated

prior to its sepaation an dete:ition. An exactive ræthylation
technique is claim to elimte may of the cclications associated
with gas-chromtogaphic determations (Ehsson, 1974). lbre recent
alklation rnthods invol ve flash heating wi th th appropriate quternar
arrm:)Dium hydroxide (Friel & Troupin, 1975; Heope et al., 1975).

Quanti tati ve thn- layer chra:toaphic ræthods have be develope

for the dete:ition of phenobabital in the presence of other pha-
ceutical counds (Szasz & Dessouk, 1973) and for the detertion of
phenobabital in biological fluids (Garceau et al., 1973).

High-pressure liquid chra:togaphic ræthods are used to detere

phenobabital and phenytin in biological materials (Atwll et al., 1975;
Evans, 1973) and phenobabital in antispasric drg miures (Honigbeg
et al., 1975).

A potentiCJtric titration assay for pheobabital soium uses per-

chloric acid as the titrant (Sell, 1968).

The rnthods of anl ysis used to determe phenobabital in biological

system VXuld, in principle, be applicable ta pheobabital soium also.

3. Biological Data Relevant ta the Evluation

of Cacinogenic Risk to lm
3.1 Cacinogenci ty an related studies in anls
(a) Oral admistration

Mouse: Phenobabital soium (purity not less than 98%) was adrnisteed
for up to 109 wees at a level of 500 rr pe kg of diet to 30 male and 30
femle 4-wee öld CFl mice. Wh the exprimt was termated at 109 wes,
liver tuiurs wee found in 24/30 maes and 21/28 femles, coed with
11/45 and 10/44, respetively, in the controls. Th IIrtality rate in the
treated group was no different fra tht of the controls, an tUlur inci-

164
dences at other sites were of the sa order in ooth treated and control
groups. Histologically, the liver tuurs W2re classified as ty A (in
which parenchyml strcture is basicall y retained) and ty B (in whch
parenchyml strcture is distort). In the treated mice, there W2re 16
typ A and 8 ty B tUIurS in males and 12 ty A and 9 ty B tUIurS
in ferles. In the controls there W2re 2 ty B tuurs in rrles, all
other tUIurS being typ A. No rntastases W2re observed in ths study;
hawever, in a study reported in a footnote ta the paper, the authors stated
that when 1000 or 3000 ID phenobabital soiur/kg of diet wee adrnistered
in life-tim tests, lung rntastases were observed in mice wi th hepatocellu1ar
tUIurs of typ B but not in those with ty A (Thorp & Waler, 1973).

The incidence of liver tUIurS in untreate 4 or l2-wee old C3H mice

(C3Hf/Anl) (Anl 70) caged in groups of five for 12 IInths was 7/17 in rrles
and 1/16 in ferles. Addi tion of 500 ID phenobabi tal/kg of diet raised
these incidences to 16/17 and 10/16, respetively. In anls caged
singly for 12 rnnths, the incidences of hepatic tUIurs were 25/37 in
males and 5139 in ferles. The addition of 500 ID phenobabital/kg of diet
increased the incidence in anls caged singly ta 35/36 and 29/29,
respetively (Peraino et al., 1973a) (No evaluation of foo intake was
made in ths study).

A group of 112 male and 74 ferle 4-wek old CFl mice received 0.05%
phenobabital sodium in the drinking-water for life (im duration,
120 wees), and this group was coed with a group of 49 rrle and 47
ferle untreate controls. Surival at 80 W2s was abut 50% in ooth
groups and was reduced to abut 5% in treated animIs, coed with 15% in
controls, at 120 W2ekS. The incidence of liver tUIurS in treated rrles
was 77/98 and tht in treated ferles 45/73, coed with 12/44 in rrle
and 0/47 in ferle controls (the reference numr is the effective num
of mice suriving at the appearance of the first tUIur). Th incidences
of other tUIurS W2re not increased in treated anirls. Of the 122 paren-
chyml li ver tUIurs observed in treated mice, 58 were classified as li ver-
ceIl carcinors; no rntastases W2re observed (Ponankov et al., 1976).

Rat: In a study in whch the effect of phenobabital on the incidence

of acetylamofluorene-induced liver cacinogenesis was obsered, a control

165
group of 48 3-week old male Sprague-Daley rats wee fed a diet containing
500 mgjkg of diet phenobabital alone. No tuurs were obsered in ths
group withn 64 wees (Peraino et aL, 1975) (Th l"lrkig G:rup noted the
short duration of this study).

Phenobabital sodium was admistered at a concentration of 500 m:/l

in the drinking-water to 36 male and 34 femle 7-wee old wista rats for
lifespa. Twenty-tw males and 28 femles were still alive when the first
hepatic turur appeared at abut 99 weeks. At the termnation of the
exprirt at 152 wes of age, 13 males and 9 femles had develope benign
hepatic neoplasr. No hepatic tuurs were obsered in a coable group
of 36 male and 35 femle control rats. Th incidences of other neoplasm
were comarable in test and control groups of rats (Rossi et al., 1976).

(b) Intraperi toneal admistration

Rat: A group of 36 male and 36 ferle l2-day old Sprague-Dawley rats
received weely i.p. injections of 2 m:jkg b. phenobabital for life; rr
surival tims were 80 wes in males and 85 we in ferles (significatly
reduced in coarison wi th controls, in which they were 96 and 94 wees,
respectively). Am:mg 32 males and 30 femles suriving 200 or irre days,
2 tlJurs were observed (a malignant turur of the adrenl gland and 1
carcinor of the nasal cavi ty). Arng 36 male and 33 ferle cxmtrols, 1
haemgioendothelirn of the li ver and 3 rr adenoccinors were
observed (Schml & Habs, 1976).
3.2 Oter relevant biological data
(a) Exrimntal system
The Lp. ID of pheobabital sodium in mice is 340 mgjkg (Collins &
50
Horlington, 1969); the oral ID in rats is 660 m:jkg b. (Stecher, 1968).
50

After an 1. v. injection to rats of 50 m:jkg b. ll¡C-phenobabital, the

concentration in the liver is higher than that in other internal organs,
and elimnation in the urine reaches a pe after 6-8 hours (Glasson
et al., 1959). Of a 75 rnjkg rn i. v. dose of pheobabital scium to male
Sprague-Dawley rats, 18% was excreted in the bile in 6 hours as undentified
[Link] (Klaassen, 1971). Hetalites of pheobabital scium produced

166
in rats and gunea-pigs are 5- (3, 4-dihydroxy- l, 5-cyclohexdien- 1 -y 1) -5-
ethylbbituric acid, 5- (1-hydroxyethyl)-5-phenylbbituric acid, 5- (3,4-
dihydroxyhenyl) -5-ethylbbituric acid and 5- (4-hydroxyhenyl) -5-ethyl-
barbituric acid (Harey et al., 1972).

Since the observation that babiturates stirate drg rrtalisr

(Rer, 1958), several reviews have dealt with the exensively docte
effect of phenobabital on li ver enlargemt and wi th the induction of
microsoml enzys in various speies, including ma (Barka & Popper, 1967;
Conney, 1967; Conney & Gelbin, 1972; Kunz et al., 1966; l'1aering, 1971¡
Rerr, 1965; Schulte-Hern, 1974). Phenobabital-induced microsal
enzyis are chaacterized by a low substrate spificity. Conseently,
they are invol ved in the rntalisr of a large variety of endogenous and
exogenous substaces, e. g., in the activation and detoxification of chercal
carcinogens. Therefore, phenobabital treatrt often leads to al terations
in the biological acti vi ty of nurrous counds, including toxici ty and
carcinogenicity (Mitchell & Jollows, 1975; í'Jattenrg, 1975).

The sirultaeous adrnistration to rats of phenobabital soium with

N-nitrosoiethylame (NDEA), 2-acetylamofluorene (AA), 4-diithylarno-
azobezene or aflatoxin reduced the prcxuction of hepatic tUlurS (Ishidate
et al., 1967; Kunz et al., 1969; ~~Le & Mashall, 1971; Peraino et al.,
1971; Takarya et aL., 1973; Weisburger et al., 1975). Havever, when
phenobabital was admistered after treatrnt for a few ~s with AA or
NDEA, the hepatic tUlur incidence was enced (Peraino et al., 1971,
1973b¡ Weisburger et al., 1975).

Phenobabital admistration does not al ter the renl turrigenic

resp:mse to a single dose of N..nitroscxirthylarne in rats on a protein-
free diet (McLean & Magee, 1970). It parially counteracted the carcino-
genic effect of N-nitrosodutylame on the lIuse bladder epithelium but
failed to affect tht of N-nitrosobutyl (4-hydroxybutyl)arne (Betram &
Craig, 1972).

The incidence of lung adenans in mice aftt a single high dose of

urethane was reduced when the mice were pretreated wi th several successive
injections of phenobabital (Adenis et al., 1970); but when the pheno-

167
babi tal treatrt was gi ven after that of urethe, opposi te resul ts were
obtained (De Azevedo e Silva, 1972).

No clear effects on skin carcinogenesis by polycyclic arortic hydro-

carbons were obtained, whether phenobabital was admistered simltaeously
or sor days or ~eks after the carcinogens (Gni et al., 1975).

ici pathogen-free mice were treated wi th phenobabital soium in the

diet on days 6 to 16 of pregcy: with 50 rrjkg of diet phenobabital
sodium, 0.6% of foetuses had cleft palates, and wi th 150 rrjkg, 3.9%,
whereas there were very few cleft palates in untreated controls (0.3%)
(Sullivan & McElhattan, 1975). An increased incidence of cleft palate
was also reported in mice given s.c. injections of 175 rrjkg b. phenobabital
sodium between the llth and l4th day after conception (Walker & Patterson,
1974) .

Phenobabital bins to rat chrantin in vivo (Alam & Steele, 1973).

It has ben reported to be weakly mutagenic in Drosophila melanogaster
after addition of 2-3 mg (the ID ) ta the nutrient rrium (Filippova
50
et aL., 1975); it did not induce point mutations in Salmonella typhimurium
TAlOO, TAl535, TAl537 or TA98 (.l1cC & Ams, 1976; ~~ et al., 1975).

(b) Ma
Phenobabi tal is readil y absorbe fran the gastrointestinal tract of
ma (lDus, 1954). Its rntalism see to bear a close simlarity to tht
reported in rats by Glasson & Beakis (1961). The major rntalite is the
para-hydroxy derivative, whch is excreted in the urine patly as the
sulphate conjugate (Butler, 1956). For reviews of the action of pheno-
babi tal as an enzy inducer, see 3. 2 (a) .
A possible association be~n maternl epilepsy, anticonvlsat drgs
(mainly phenobabital and phenytoin) and congenital malforntions in off-
spring was rePJrted by M2dow (1968). In tv reports, a total of 38
children born to IIthers who took anticonvulsants, including phenobabital,
thoughout pregcy revealed a simlar constellation of rnlfonntions
suggesting a syndr invol ving 1ip and palate, cadiovascular system and
ske1etal abnormlities (lim, face, skull) (Meadow, 1968, 1970). Subseqent

168
case reports of epileptic wa treated thoughout pregcy wi th phen-
babi tal and phenytoin have describ affected inants wi th sim1ar abor-
iilities (Aase, 1974; Anerson, 1976; Ba et aL., 1974; Da et aL.,
1975; Loughn et al., 1973; Seip, 1976).
ln 51 pregcies identified from US hospital recrds over th peioo
1960-1970 occuing in 42 epileptic patients receiving phenobabital and
phenytoin in 44 cases, an phenytin alone or with rnso1ine in the othr
cases, thoughout pregancy, 3 congenital abnormlities occued (5.8%).
Al though no anarlies were found in 50 rntche pregcies, th effect was
not considered to be statistica11y signficant (Watson & Spellacy, 1971).

In the OK, 168 epileptic wa treated with pheobabital, pheytin

or other anticonvulsant drgs wee identified from the neurolog depa-
rnts of tw general hospitals. These w: had exience 365 pregcies,
during whch phenobabital alone or in caination was taen in 240 cass
and phenytoin alone, or in coination, in 192 caes. Seventee of the
365 infants had malforntions, caed with 7 of 483 controls and 0 of 62 ,
offspring of epileptic w: Ilt taing iæication. Th calculated risk
was tv to thee tims greater th nornl (Speidel & HeadOW, 1972).

Duing 1969 and 1970, 7,896 w: gave birt at a single IDdon

hospital, and 192 of the infants rad malformtions, to give a rate of
2.44%. In ths group, 22 epileptic IIthers had taen anticonvulsant drgs
throughout pregcy. 'I of the children born ta rrthers who had taen
phenobabi tal and phenytoin in one case, and phenytoin and other drgs in
the other case, had cleft palate and/or hae lip, an incidence of 9%,
whereas only 0.13% of the inants bom to non-epileptic rrthers væe sa
affected (South, 1972).

In Cadiff, 31,877 inants born be~ 1965 and 1971 ~re studied;
245 rnthers in this series had a history of epilepsy. Arng the 111 bir
to epileptic IIthers not on anticonvulsant therapy, the malformtion
rate was 2.7%, caed to 2.8% of infants ln the total poulation. ln
contrast, 6. 7% of 134 inants born to IIthers recei ving anticonvulsant
therapy had malformtions. One child had a hae lip but no cleft
palate arng 53 born to rnthers treated with phenobabital alone; 'Vereas

169
6 children wee rom wi th malformtions arng 60 birt te Ilthers treate
with phenobabital an phenytoin (UM, 1973).

In a surey in Norter Ire1and, the risk to inants oorn to epi1eptic

rrthers taking anticonvusant drgs, mainl y phenobabital or phenytoin and
other drgs, was 6.4% caed wi th a maformtion rate of 3.8% for all
live and stil1birth in the province. 'Tre was a 7-fold increase in the
risk of cleft lip with or withut cleft palate (1.8% oomped with 0.22%)
(Millar & Nevin, 1973).

In th OXord Reord Linkge Study, of li ve baies oorn betwee 1966

and 1970 te epileptic rrthers, 17/217 (7.8%) had congental abnormlities
at birt comed with 21/649 (3.2%) of th controls matched for civil
status, social class, mat~l age, paity, hospital an year of delivery.
Subseently, the rates rose to 13.8% (30/217) for baies of epileptic
rrthers and 5.6% (36/649) for thse in the control group (Tls may be due
to a longer observtion tim and to the inclusion of different tys of
malformtions, which may have be considered te be congental before).
Maternl phenobabital ingestion alone did not accunt for an increased
incidence of defects (2/41, 4.9%) over tht in inants of rrthers not
taing drgs (2/19, 10.5%), but when phenobabital was given in caination
wi th phenytin the proportion of infants wi th defects was rrre th doubled
(11/50, 22%). The incidence of defects am::mg inants of rrthers taing
phenytin alone (5/33, 15.2%), while higher th tht am:mg inants of
rrthers taing phenobabital alone, was 10wr th tht arng inants of
rrthers taing phenytoin in caination with phenobabital (FErick, 1973).

A study of pregancies fo11ow in a single US c1inic included 284

birt te 138 epileptic rrthers betw 1939 and 1972. Of 141 birt
to rnthers tang anticonvulsants during the first trimste, 10 inants
had malformtions; of 56 birt te epileptics not tang irication, 1
infant had a malformtion; and of 26 birts te epileptic IIthers in
remssion no malformtions v;re se. Th assoiation of c1eft palate
and hea malformtions with anticonvusant usge was simlar in 28
patients treated wi th phenobabital alone an in 24 vo treate wi th
phenytin alone (Anegers et al., 1974).

170
 Bary & Das (1974) re¡:rted that in Australia betwee 1967 and 1972,
10/39 infants bom to nothers who ~re treated with phenytoin and babit-
urates during pregcy had congenital abnonn1ities. tb congenital
abnornlities were found in children of IIthers treated with babiturates
alone, whereas thy occured in 2/8 children of those treate with phenytin
(see also IInogaph on phenytoin, p. 201).

After treatrnt with 22.4-388 llg/rn phenobarbital 1-n vitro, a ~ak

increase in the mmibr of chrorsoiæ gaps and breas was observed in culti-
vated peripheral hum leucoes (Foerst, 1972). tb ChrorsOO abnonnli-
ties were reported in hum fibroblasts exsed in vi tro to concentrations
of 0.13-130 llg/rn for 5 days (Stenchever & Jaris, 1970). Very high doses
of phenobabital (100-400 llg/rn) induced accuation of irtaphases in
cultured hum leucocyes; h~ver, ths study did not indicate if chror
so abnornlities wee noted (Caratzali & Rcm, 1969).

3. 3 Case reports and epiderological studies

(a) Case reports

Of 7 patients originally describ by Saltzstein & Acke:i (1959) as

developing pseudolymhomas follawing phenobabital and/or phenytoin treatrt,
tw died, one fror rmltiple Ilelam with an unCM surival ti after
diagnosis and another fror nalignt 1 ymhan, wi thin 4 years (Harington
et aL., 1962).

Of 4 patients recei ving phenobabital and phenytoin, 2 were reported

to have develope Hodgkin' s disease, 1 a 1 ymhosarco and 1 a reticu1ur-
cell sarcaI (Hy & Sors, 1966) (see also nonogaph on phenytoin, p. 201).

Two cases of neuroblastona, a 3-year old girl and a 7-day old boy,
have ben reported in the foetal hydantoin sydror. Both nothers had taen
anticonvulsants (phenobabital and :penytoin) since girlhoo because of
epilepsy (Pendergrass & Hanson, 1976; Sherm & Roizen, 1976).
(b) Epidemological studies

The carcinogenicity of anticonvusant drgs, including phenobabital,

in ma was studied in a retrospetive in"':stigation conducted on 9136 epi-
leptic patients adrtted to the neuropsychiatric hospital of Filadelfia

171
in Dek betwee 1933 an 1962. The patients \\re treate with pheno-
babital (100-300 mg), phenytin (100-400 mg) or primdone (500-1500 mg).
In those treated for up to 10 yeas, the incidence of cacer at all sites
except the li ver was the sa as or lowr th tht exted when caed
with the incidence in the general population. In patients treate for rrre
th 10 yeas, 3 cases of li ver cacer \\re observed in males whereas 1.1
were exted, and 1 li ver cacer was obsered in a ferle where O. 7 was
exted; and in males treated for 1ess th 10 years, 1 liver cacer was
observed where 0.4 was excted. Th author reported tht one ma with
liver cance had be treated with thorotrast 18 years before death. In
patients treated for irre th 10 yeas, tururs of brain and neous system
were obsered in 10 males (exted 3. 5) and in 6 ferles (exted 2.9)
(see also 'General Raks on Substances Considered', pp. 24-25) (Clersen
et al., 1974). Schneidenn (1974) reconsidered the se results with respet
to li ver tururs and suggested tht the cases of li ver cacer rnght represet
an increase incidence; but Clensen (1975) report that 3 out of the 4
liver cancers see in male patients had previously be treated with
thorotrast, whch is know to induce liver tuurs in ma (Kiely et al.,
1973; MacMon et al., 1947; Ma et al., 1976; da Silva Hort et al.,
1965; Smron & Bettifora, 1972).

4. O:ts on Data Rerted

~ and Evaluation 1

4 .1 Animl data
Phenobabital soium is cacinogenic in mice and rats afte its oral
admnistration for lifetii. In mice, it produce begn an malignant
li ver-cell tUIurs; in rats, beign li ver-cell tUfurs wee observed
ver late in life.

1 See a1so the section 'Anl Data in Relation ta the Evluation of

Risk to ~1a' in the introduction to ths volui, p. 13.

172
4.2 Hum data
A possible relationship ætwee anticovusant thrapy in which
phenobarbital was know to æ included and the occurence of cacer in
ma has be investigated in one large epiderological study and reported
in several case studies. ln IIst intaces, phenobabi tal was gi ven in
conjunction with other drgs, in paicular phenytoin. The available
evidence is insufficient to alla. an evaluation of the cacinogenicity
of phenobabital to æ made (se also IInogaph on phenytoin, p. 201).

173
 5. References

Aase, J .M. (1974) Anticonvusant drgs and congenital abnornlities.

Arr. J. Dis. Chld., 127, 758

Adenis, L., VlaeInck, M.N. & Driessens, J. (1970) L'adénoiæ puJnaire

de la souris Swiss recevant de l'uréthane. VIII. Acion du phéno-
babitaL. C.R. So. BioL. (Paris), 164, 560-562

Alar, S.N. & Steele, W.J. (1973) In vivo binding of i:enobabita1 (PB)
and actinorncin D (AM) to rat liver chomtin. Fed. Proc., 32, 684

Anderson, R.C. (1976) Cadiac defects in chi1dren of rrthers receiving

anticonvusant therapy during pregncy. J. Pediat., 89, 318-319

Anegers, J.F., Elvebck, L.R., Hauser, W.A. & Kurland, L. T. (1974) 1)

anticonvusants have a teratogenic effect? Arch. Neurol., 31, 364-373

Atwell, S.H., Gree, V.A. & Haey, W.G. (1975) Developlt and evaluation
of iæthod for slltaeous determtion of phenobabital and diphenyl-
hydatoin in plasm by high-pressure liqud chonatogaphy.
J. phar. Sci., 64, 806-809

Baka, T. & Popper, H. (1967) Liver enlargert and drg tocity.

Meicine, 46, 103-117

Bar, M.,Jr, Poznanski, A.K. & Schmckel, R.D. (1974) Digital hypplasia
and anticonvusants during gestation: a teatogenc sydraæ?
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Bary, J.E. & Das, D.M. (1974) Anticonvulsants and congenital abnorni-
ties. Lacet, ii, 48-49

Bertram, J.S. & Craig, A.W. (1972) Spific induction of bladder cacer in
mice by butyl- (4-hydroxybutyl)nitrosame and the effects of honmna1
m:fications on the sex difference in response. Euop. J. Cacer,
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Boker, H. E. & Darcey, B. A. (1975) Enymtic iioassays vs. gas/liqud

chromtoaphy for detertion of phenobabital and diphenylhydatoin
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British PharcoJ;ia Cæssion (1973) British Phaco~ia, lDndon,

HMO, pp. 358-359
Brooks, M.A., De Silva, J .A.F. & Hackm, M.R. (1973) Determation of
phenobabital and diphenylhydatoin in bloo by differential pulse
polarogaphy. Anlyt. chi. acta., 64, 165-175
Brutko, L.I. & Sapein, L.P. (1972) Polybuffer-sptrophotoitric
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Butler, T.C. (1956) Th iætalic hydrxylation of pheobabita.
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Caatzali, A. & Ran, I.C. (1969) Action stathinétique de quelques

dérivés babituriques sur le lymhoce htm en cuture.
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Cleise, J. (1975) Phenobabitone, liver tururs and thorotrast.

Lacet, i, 37-38

Cleisen, J., Fuglsang-Frederiksen, V. & Plum, C.M. (1974) Are anti-

convusants oncoenc? Lacet, i, 705-707

Collins, A.J. & Horlington, M. (1969) A seqential screeg test ba on

the rug cannent of audiogenc seizures in rnce, includg
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Conney, A. H. (1967) Phacological iilications of rncrosan enyi

induction. PhacoL. Rev., 19, 317-366

Conney, A.H. & Gelbin, H.V. (1972) Drug-induced Diseases, !, 179

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(lk, C.E., AIrson, E., Poole, W.K., Ieser, P. & O'Tu, L. (1975)
Phenytoin and phenobabital concentrations in saliva an plasm
rnsured by radioinoassay. Clin. Phacol. Thr., 18, 742-747

Council of Euope (1969) Euop PhaC(ia, Vol. l, 57-Sainte-Ruffine,

France, Maisonneuve, pp. 346-348

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175
Fedick, J. (1973) Epilepsy an pregcy: a report from the Oxord Rerd
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181
 [Link] AN OXYHEUTAZONE

1. Chemcal and Physical Data

Pheny lbutazone

1. 1 Synonym and trade nars

Cher. Abstr. Reg. Serial No.: 50-33-9

Cher. Abstr. Nar: 4-Butyl - l, 2-diphenyl - 3, 5-pyrazolidinedione

4-Buty 1- i, 2-dipheny 1 - 3, 5-dioxopyrazolidine; 4-butyl - l, 2-dipheny 1-
pyrazolidine- 3, 5-dione; 3, 5-dioxo- l, 2-dipheny 1 -4-n-buty lpyrazo-
lidine; diphebuzol; diphenylbutazone; 1,2-diphenyl-4-butyl-3,5-
pyrazolidinedione; l, 2-diphenyl-3, 5-dioxo-4-butylpyrazolidine;
l, 2-diphenyl-3, 5-dioxo-4-n-butylpyrazolidine; l, 2-diphenyl-2, 3-
dioxo- 4-n-buty lpyrazol idine; fenibutazona; fenylbutazon
Alindor; Alkabutazona; Alqoverin; Anerval; Anpuzone; Antadol;
Anuspiramn; Arthizin; Arrizin; Arrizone; Arropan; Azdid;
Azobutil; Benzone; Betazed; Bizolin 200; B. T . Z .; Butacote;
Butacompren; Butadion; Butadiona; Butadione; Butagesic; Butal-
gina; Butalan; Butalidon; Butaluy; Butaphen; Buta-Phen; Buta-
pirazol; Butapyrazole; Butarecbon; Butartril; Butartrina; Buta-
zina; Butazolidin; Butazolidine; Butazona; Butazone; Butidionà;
Butiwas-simle; Butone; Butoz; Butylpyrin; Buvetzone; Buzon;
Chemutazone; Digibutina; Diossidone; Diozol; Diphebuzol; Eco-
buta zone ; Elmal; £qi Bute; Eributazone; 'Esteve' ; Febuzina;
Fenartil; Fenibutasan; FenibutDl; Fenilbutazona; Fenilbutina;
Fenilbutine; Fenilidina; Fenotone; Flexazone; C; 13,871; IA- But;
Intalbut; Intrabutazone; Ipsoflar; Kadol; Ligel; Malgesic;
Mephabutazone; Merizone; Nadazone; Nadozone; Neo-Zoline; I\Tovo-.
phenyl; Phebuzin; Phebuzine; Phen-Buta-Vet; Phenbutazol; Pheno-
pyrine; Phenylbtazone; Phenylbutaz; Phenylbutazonum; Phenyl-
M:buzon; Pirarreurl' B' ; Praecirheurn; Pyabutol; Pyazolidin;
Rectofasa; Reudo; Reudox; Remnasyl; Reumzin; Eeumzol; Remmme;
Reupolar; Robizon-V; Rubatone; Scanbutazone; Scherrgin; Shigrodin;
Tazone; Tetnor; Tevcod~me; Therazone; Ticinil; Toalgil; Uzone;
VAC-10; Wescozone; Zolaphen; Zolidinum
183
1.2 Chemcal formla and rrlpc:ular wpight:

~
\ 0 ~Ù~H'_CH,_CH'_CH,
C19H20N202 Mol. vrt: 308.4

1. 3 Chercal and physical prooerties of the pure substance

From Stecher (1968), unless otherwise specified

(a) Description: Crystals

(b) Melting-point: 1050C

(c)
. max 1
Spectroscopy data: À 239.5 nr (El = 516.2) in acidic rrthanol;
264 nm in sodium hydroxide solution (US Pharmcopeial Convention,
Inc., 1975)

(d) Solubility: Soluble in water, 70 mg/lOO ml at 22.50C (also

re¡:rted as 220 mg/100 ml)

1. 4 Technica1 products and imuri ties

Various national and international pharrcoooias gi ve specifications
for the puri ty of pheny lbutazone in pharnceutical products. For exale,
phenylbutazone is available in the US as a USP grade containing 98-l00.5~5
active ingredient on a dried basis wi th a maimum of 0.001% heavy metals.
Tablets are available in 100 mg doses containing 93-107% of the stated
arunt of phenylbutazone (US Phacopeial Convention, Inc., 1975). Capsules
are also available in which 100 mg phenylbutazone (or 50 mg phenylbutazone
and 1. 25 mg prednisone) is combined with 100 mg dried alumnium hvdroxide
gel and 150 mg magnesium trisilicate (antacids added to rnnirze gastric
upset) (Kastrup, 1976).

In the UK, phenylbutazone is available as suppositories (250 mg)

containing 92.5-107.5% of the stated arunt and as talets (100 mg)
containing 95-105% of the stated arunt (British Pharnco¡x)eia Commssion,
1973) .

184
Oxyhenbutazone
1. 1 Synonyrs and trade nars
Chem. Abstr. Reg. Serial No.: L~9-20-4

Chem. Abstr. Nar: 4-Buty1-l- (4--hydroxyhenyl)-2-phenyl-3,S-

pyrazolidinedione
4-Butyl -2- (4-hydroxyhenyl) - l-pheny1- 3, S-dioxopyrazo1idine; 4-butyl-
1- (paY'a-hydroxyhenyl) -2-phenyl -3, S-pyrazolidinedione; 4-butyl -2-

(para-hydroxyheny 1) - 1 -pheny 1- 3 , ~)-pyrazo1idinedione; hydroxyheny 1-

butazone; pcn'a-hydroxyhenylbutazone; 1- (para-hydroxyhenyl) -2-
phenyl-4-butyl-3,S-pyrazolidinedione; 1 -para-hydroxyheny1 -2-pheny1-
3,5-dioxo-4-n-butylpyrazo1idine; rrtablite I; oxazolidin; oxiphen-
buta zone ; oxyhenobutazone; oxypheny lbuta
z one ; para-oxyheny lbuta-

zone; 1 -pheny 1- 2- (para-hydroxyheny 1) - 3, S-dioxo-4-buty 1Dyrazolidine;

1 -pheny 1- 2- (para-hydroxyheny 1) - 3, S-dioxo-4-n-buty lpyrazolidine
Artroflog; Butaflogin; Butanova; Butapirone; Butilene; Crovaril;
Def1ogin; Etrozolidina; Flamil; Flanaril; Floga1; F1oghene;
Flogistin; Flogito1o; Flogodin; F1ogori1; Flogostol); Flopirina;
Frabl; G 27202; Idrobutazina; Infar1; Inf1am1; Ipabutona;
Iridil; Isobutazina; Isobutil; Neo-Fardol; Neofen; Offitril;
Oxlid; Oxzolidin-C'-iigy; Oxiutol; Oxi-"penibuto1; Pirabutina;
Piraflogin; Poliflogil; Remzin; ReUIx; Rumpax; Tandacote;
Tandalgesic; Tandearil; Tanderal; Tanderil; Telidal; Tendearil;
Validil; Vi subutina
1. 2 Chercal formula and molecular weight

IQN/
~
~i-~H2 N F
_CH2 _CH2 -CH3

C19H2üN203 lvl. wt: 324.4

185
 1. 3 Chemcal and physical ~roporties of L~e pure substance

From Stecher (1968), un1ess otherwise specified

(a) Description: Crystals

(b) Me1ting-point: l24-l250C

(c) Spectroscopy data: Àmax 254 nr in sodium hydroxide solution

(National Forrulary Board, 1970)

(d) Solubility: Soluble in benzene, ch10roforr, ethanol, ether and

rnthno 1

1. 4 Technical products and imuri ties

. - --
Various national and international pharmacopoias give specifications
for the purity of oxyhenbutazone in )Jharmceutical products. For examle,
oxyhenbutazone is available in the US as a National F'orrular grade
containing 98-100. 5% active ingredient on an anydrous basis. Tablets are
available in 100 mg doses which contain 94-106% of the stated aiunt of
oxyhenbutazone (National Forrulary Board, 1970).
In the OK, oxyhenbutazone is available on a dried basis containing no
less than 99% of the active ingredient and as talets (dose 100 mg) con-
taining 95-105% of the stated aiunt (British Pharmcopoia Commssion,
1973) .

2. Production, Use, Occurrence and Analysis

For imrtant background informtion on this section, see prearle,

p. 15 .

2. 1 Production and use

(a) Production

A rnthod of preparing phenylbutazone was patented in the US in 1951,

in which butylmlonyl chloride is condensed with hydrazobenzene in ether
solution with pyridine. Aqueous hydrochloric acid is used to extract the
pyridine, and phenylbutazone is extracted with aqueous sodium carbonate.
The alkaline solution is acidif ied wi th hydrochloric acid to gi ve phenv 1-
butazone (Stenz1, 1951).

186
 A rrthod of preparing oxyhcnbutazonc v.'aS oatcntcd in thc us in 1956
and invol ves the following steps: diethy 1 but Y lmlonate is condensed wi th
para-benzy loxyhydrazobenzene in a solution of sodium ethoxide in anydrous
ether; the addition of xy lene forr 1- (para-benzv loxy) - 2-pheny 1 -4-buty 1 - 3,5-

pyrazolidinedione; this is debenzy lated by Raey nickel hydrogenation ta

give oxyhenbutazone (H~fliger, 1956).
Commrcial production of phenylbutazone was first re~rted in the us
ln 1965 (US Tariff COmmssion, 1967); only one us company re~rted
production (see preamle, p. 15) in 1974 (US International Trade Commssion,
1976a) . US imrts of phenylbutazone though the principal custorn
districts were 2700 kg in 1972 (US Tariff Commssion, 1973), 15,300 kg in
1973 (qs Tariff commssion, 1974) and 10,300 kg in 1974 (US International
Trade Commssion, 1976b).
Corrrcial production of oxyhenbutazone was first re~rted in the US
in 1960 (US Tariff COmmssion, 1961); only one US company reported
production (see preamle, p. 15) in 1974 (US International Trade Commssion,
1976a). US imrts of oxyhenbutazone through the principal custorn
districts were 10 kg in 1972 (US Tariff Comssion, 1973) and 175 kg in
1973 (US Tariff Commssion, 1974).

Production of phenylbutazone was first reported in Japan in 1957; i

major compy and 10 smaller firr maufactured Dhenylbutazone in 1975.
Over 50,000 kg phenylbutazone were used in Japan during that year. No
evidence was found that oxyhenbutazone has ever been produced corrrcially
in Japa; abut 800 kg were imrted in 1975.

Production of phenylbutazone in India in 1972 was reoorted to be at

least 8900 kg. Imrts for the period 1972-1973 were reoorted to be
24,180 kg (Anon., 1974).
Estimted anual production of phenylbutazone in Europe is estimted
to be in the range of 100-500 thousand kg. Anual production is believed
to be less than 1 thousand kg in Austria and in Benelux, and 1-100 thousclnd
kg in the Federal Republic of Germy, in France, in Hungar, in Italy, in
Poland, in Scandinavia, in Swi tzer land and in the OK. Anual production
of oxyhenutazone in Italy and in Spain is estimted ta be in the range of

187
1-100 thousand kg; Scandiavia prcxuces abut 200 kg oxyenutazone
anua11y for captive use on1y as a chenca1 intei:iate.

(b) Use

Phenylbutazone and oxyhenbutazone are anti-inflamtory agents

possessing analgesic, antipyretic, sodium retention and mild uricosuric
properties (Amrican Society of Hospital Pharrcists, 1961).

Both comounds are used in the symptomatic treatrt of acute gout

and to relieve the sytorn of rheur toid arthi tis, rheur toid si;::ndy li tis ,
os teoari tis and psoriatic arthi tis; they are used in the sytomatic
treatint of acute sui::~rficial thoniphlebitis and for severe forr of a
variety of acute local inflamtory conditions (Kastrp, 1976).

When phenylbutazone is used to treat gout, a disease which afflicts

abut 800,000 peple in the US (Cavallito, 1968), an initial oral dosage
of 600 to 800 mg is adrnistered on the first day of an acute attack, and
the dose is reduced as the inflamtion subsides (Knott, 1974).

Total US sales of pheny lbutazone for use in hUI rædicine are esti-
mated to be less than 35,000 kg anuall y; those of oxyhenbutazone are
less than 10,000 kg anually.
Pheny lbutazone is also used in veterinary rædicine as an analgesic,
antipyretic and anti - inflamtory agent (Stecher, 1968).
2. 2 Occurrence
Phenylbutazone and oxyhenbutazone are not known to occur in nature.

2.3 Analysis

~thods of assay for pheny lbutazone that rnt regulatory reqrernts

for pharceutical products include aqueous and non-aqueous ti tration and
spectrophotometry; those for oxyhenbutazone include aqueous titration
and UV spectrophotometry.

Spectrophotorntric ræthods develope for the assay of phenylbutazone

(Magalhaes & Piros, 1973) include one based on the detection of the per-
magante oxidation product of phenylbutazone following its extraction from
plasm (Jähchen & Levy, 1972).
188
 Phenylbutazone can be anlysed in plasm by high-speed liqud
chromatography (Pound et aL., 1974), and levels of 0.25 llg/fi oxyhen-
buta zone can be determned in plasn by this rrthod (Pound & Sears, 1975).

Gas- liquid chromatographic rnthod for deterng phenylbutazone and

oxyhenutazone as their silyl derivatives in plasm have been describd
(Midha et aL., 1974; Tanirura et aL., 1975). Comparisons have been made
of agas chromatographic rrthod and a W-spectrophotorrtric ræthod for the
deterrnation of phenylbutazone (McGilveray et aL., 1974) and of oxyhen-
buta zone in biological fluids (Bruce et al., 1974). A gas chromtographic
ræthod for the determation of bulk and dosage forr of phenylbutazone has
be reported (Watson et aL., 1973).
Pheny lbutazone ca also be anal ysed by thn- layer chromatographic
rnthods (Krechniak et al., 1972; Pomazánska-Kolodziejska, 1972; šar~~ov~
& Kaá~, 1972) or electrochemcally by [Link] (Proksa et al., 1974) or
alternating-current oscillopolarography (Szyszko & Weglowska, 1974).

3. Biological Data Relevant to the Evluation

of Cacinogenic Risk to Ma
3.1 Cacinogenicity and related studies in animls

No data ~re available to the Working Group.

3. 2 oter relevant biological data

(a) Exrimtal system

The oral ID50of phenylbutazone in rnce is 680 ng/kg 00, that in rats
1000 ng/kg bw and that in rabbits 146 rr/kg 00; the i. v. ID
50 in mice is
120 rn/kg bw and that in rats 150 rr/kg 00. The oral ID 50 of oxyhen-
butazone in dogs is 575 rr/kg bN; the i. v. ID50
in rats is 105rn/kg bw
(Sunshine, 1969).

Gastric ulcerations were produced in dogs after prolonged i.m. admi-

stration of doses of 3.6-35.2 g phenylbutazone over 10-103 days (Kirsner &
Ford, 1955).

The biological half- life of pheny lbutazone in plasn was abut 6 hours
in dogs, 5 hours in guinea-pigs and 3 hours in rabbits (Burs et al., 1953).

189
 Negative results have been reported with phenylbutazone in Salmonella
typhimurium TAlOO, TA98, TA535 and TA537 in the presence or absence of
rat liver hOTIenate (McCa & Ars, 1976; McC et al., 1975). It did
not induce non-disjunction or crossing-over in Aspergillus nidulans in the
absence of rntalic activation, whereas i ts rntali te, oxyhenbutazone,
had weak but significant activity (Bignar et aL., 1974). Negative results
were also obtained with phenylbutazone in the domiant lethal test folla...ing
admistration of the drg either to male mice at 50-100 rrjkg bN by i.p.
injection (Macherr & Hess, 1971) or to ferle mice as a single oral dose
of 400 mg/kg hw (Macherr & Hess, 1973).
NO increase in the numr of chrolIsorn abnornli ties has been observed
in bone-marrow cells of Chnese haters after oral admistration (Müller
& Strasser, 1971) or of rats after i.p. admnistration of phenylbutazone
(Gehat & Wissrüller, 1973) or in geral cells of male rnce after oral
admnistration (Rathenbrg & Müller, 1972).

(b) Ma
Adnistration of pheny lbutazone to ma is associated wi th peptic
ulcer, hyprsensi ti vi ty reactions, hepati tis, nephri tis and bone-marraw
suppression. The toxicity of oxyhenbutazone ls simlar to that of the
parent cOITund (Mauer, 1955; Vbur & Fingl, 1975).
The biological half-life of phenylbutazone in plasm is 72 hours
(Burs et al., 1953). Phenylbutazone given at 2-3 tirs the therapeutic
dose is rntablized slowly, with the acculation of oxyhenbutazone in
the plasm. Oxhenbutazone and y-hydroxyhenylbutazone were excreted in
the urine; no glucuronic acid conjugates of phenylbutazone were detected.
In ma, oxyhenutazone has a biological half- life of abut 2 days (Burs
et al., 1955).

Significant increases in the numr of chrolIsorn abnornlities

resulting froID chrOIsan breakage events W2re reported in cultured hur
peripheral leucoes from :ptients treated with phenylbutazone for rhetmtic
disorders for at least 3 ITnth in doses ranging frar 100-500 mg/day. In
the sar study, a few of the :ptients W2re examed for chrolIsan damge
in bone-rnCJ cells, with rigative results (Stevenson et aL., 1971).

190
 J ~ J
Neaative results have also been retXrted for chroIlsorn damae in bone-
marow cells from hi.s receiving 400 mg/day phenylbutazone for 1 week
(Jensen, 1972). In vitro treatrnt of hum leucoctes with 0.62-2.16 x
10-3 Mil phenylbutazone caused an increased incidence of ChrolIsorn
abnormlities (wis srül 1er , 1971; Wissmtller & Gehart, 1970).
3. 3 Case reports and epiderological studies
(a) Case reports

Six cases of leukaema in patients who had recei ved pheny lbutazone
were reported by Bean (1960). In case l, a male aged 69 years was treated
with 600 mg then 400 mg phenylbutazone daily for 3 weeks, when he developed
a mild anaema, leucocosis and 1 ymhadenopathy in the neck and axillae.
He died 20 IInths after the begining of treatrt and post mortem shoved
myeloid leukema. Case 2 was a male aged 67 years who had suffered froID
lu: spondylitis for may years and who had been treated with phenyl-
buta zone intermttently over 4 years prior to a termnal lymhatic leukaera.
Case 3 was a male aged 70 years 'Wo had been treated with 200-600 mg phenyl-
buta zone , then wi th an increased dose dail y for 5 IInths prior to admssion
to hospitaL. Investigations revealed that he was suffering froID lymhatic
leukaema, and he died 3 W2eks later. In case 4, the period of treatInt
with phenylbutazone was approximtely 1 year before death froID myeloid
leukaema. The patient was a male agE-,¿ 80 years and had suffered froID
osteoari tis and spondy l:L tis for many years. There was a history of sorr
intolerance to phenylbutazone. Case 5, a male aged 66 years, had ben
treated with 600 then 100 ID::/day phenylbutazone for 4 years before admssion
to hospital for acute diarrhoea and had tolerated the drug welL. During
investigation it was found that he had generalized lymhadenopathy and
splenorral y. Histological exarnation of the 1 ymh nodes revealed the
presence of mlignant lyrhoproliferative disease. Case 6, a 63-year old
male, had received a course of fifty 100-rn phenylbutazone tablets approxi-
mate 1 y 18 IInths before admssion to hospi tal for severe angina. Thee
weeks after admssion his bloo picture was suggestive of myeloid leukaera,
confinn at necropsy six days later.
Lawrence (1960) reported a leukaeiid reaction in a worn aged 69
1 ITnth after treatrnt wi th 8.4 g pheny lbutazone spread over 4 weeks, while

191
Garett (1961) reported a simlar reaction in a 64-year old ~ who had
taen 18 g pheny lbutazone spread over 3 IInths. The bloo dyscrasia was
discovered a few wees after cessation of treatmnt.

Cast (1961) rnntioned sorn degree of intolerance to phenylbutazone in

a femle aged 59 after the admnistration of 300 mg daily for tw months.
Drug therapy was resurd after a respite of 14 IInths, when she was
admtted for an operative procedure for the relief of osteoarthritis.
Duing routine bloo investigations, myeloid leukaera was diagnosed and
she died 14 days after operation.

Cadm & LÌ1nt (1962) reported a case of acute leukaera in a 71 -year

old ma who 3 IInths earlier had been treated with 20 200 mg phenylbutazone
tablets over 6 days.

Chatterjea (1964) drew attention to a case of acute myeloid leukaera

ln a 46-year olò male wÌio had takei-i phenyThuL:azone intermttently fer a
significa~t pcriod prior to the onset of leukaema.

A 56-year old femle was treated with 300 mg phenylbutazone daily for
17 days. After 2 weeks she becam seriousl y ill and was found to have an
aplastic anaema which was accompanied by extensi ve bruising; she died a
week later. Autopsy showed massive cerebral haeirrhage, and diagnosis was
of an acute leukaera (Thorp, 1964).

Chalmrs & McCarthy (1964) describd the onset of a fatal myelomono-

cyic leukaera in a 44-year old won treated wi th dail y doses of 100-300
mg phenylbutazone for consecutive periods of 12, 8 and 19 IInths for
rheumtoid arthi tis .
Hart (1964) re:¡rted on a 58-year old ma who was treated with 400 mg
phenylbutazone daily for 7 days on two occasions separated by an interval
of approxitely 16 IInths. 'I \teks after the second episode he developed
acute myeloid leukaera.

Golding et al. (1965) reported a case of fatal monocytic leukaema

after treatmnt for crippling rheumtoid arthitis with phenylbutazone ln
doses of 300 mg daily for one year and thereafter at 200 mg daily for
abut 4 years more. The patient' s sister had died of the sam disease at

192
the age of 61 without ever having received phenylbutazone.

Five cases of acute leukema followng phenylbutazone therapy, from

a total of 55 cases of acute leukaema obsered in Australia in the years
1959- 1963 were reported by Wooliff & Dougan (1964); 3 further cases
associated with phenylbutazone treatmnt were reported by Dougan & Wooliff
(1965). Of the total of 8 cases, 5 were in rnn and 3 in WJiæn; the doses
ranged froID approximtely 3-100 9 and the duration of treatrnt lasted fror
1 week to 4 years continuously, or 7 years intermttently. The first
syrptom related to leukaema were observed from a few weeks to 1 year after
cessation of phenylbutazone admnistration. Four other cases were excluded
because of short duration of treatrt or prior radiotherapy. Dougan &
Wooliff (1965) also reported 4 cases of chronic myleoproliferative dis-
orders (2 of Hodgkin' s disease, 1 chronic lymhatic leukaema and 1 poly-
cythaera vera) follawing admnistration of phenylbutazone for periods
ranging from a few vveks to 2 years. The doses in two of these cases were
abut 6 and 45 g; in the other two they were unow.
Thee cases of acute leukaema following pheny lbutazone therapy, out
of 50 cases of acute leukaema, were recorded in Scadinavia between 1959
and 1964 (Jensen & Roll, 1965). A 78-year old male was given 600 mg
phenylbutazone daily for 4 TInths. In the follawing IInth he was admtted
to hospital with anema, and haemtological investigations revealed the
early stages of myeloctic leukaema, froID which the patient died approxi-
ITtely 13 IInths later. The second case concerned a 67-year old ma who
received 300 ir daily for a few TInths for rheumtoid arthitis. Shortly
afterwds he had an acute episode of gastrointestinal haeirrhage fror
which he recovered, but 2 years later he died of acute myeloblastic leukaema.
The third case was a comparatively young femle, 31 years of age, given
repeated courses of phenylbutazone in doses of 400-600 ir daily for 10 days.
Abut 14 nDnths later she was adrtted to hospital with severe anaema,
and a haemtological investigation revealed 1 yrphoblastic leukaema; she
died one year later.

Acute leukaema, or a leukaeiid reaction, has also ben observed

after treatrent with oxyhenbutazone. In one case, a 30-year old wom
suffering froID rnld myasthenia gravis was given approxitely 5 9 oxyhen-
193
buta zone over a IInth. Shortl y afterwards, she develope an anaenua, a
leucocyosis; this was followed by leucocyopeia, which becar steadil y
worse over a reriod of 26 days of observation but showed signs of imrove-
rrt after 11 IInths. In a second case, a 40-year old ma recei ved a total
dose of 1 9 oxyhenutazone over approximtel y 5 days, 2 IInths before
admssion to hospital with a severe anaema and leucoctosis; he died on
the third day after developing ths condition. The third case concerned a
32-year old vvma who was treated with 2.5 9 oxyhenutazone over 7 days;
she also develored severe leucoctosis follCMed by leucopeia and anaera,
but there was a subseqent recovery. The last case of white-bloo cell
disorder concerned a 47-year old ma who, 13 IInth before admssion with a
tenrature, had been given 1. 9 g oxyhenbutazone over a 2-week periode
On admssion, he was given 0.6 9 of the drg; bloo exarnation showed
that he had a leukaema, of which he died approximtely 4 IInths later
(Perers & Sjöberg, 1966).

(b) Epidemological studies

Fraumni (1967) conducted a follow-up study of 25 patients who develope

bone-rnrrow depression following phenylbutazone therapy. Eighteen patients
recvered within 2 [Link] ta 4 years, 4 were still under care at the tir
of the report and 3 died wi thin 2 weeks to 2 years, 2 of aplastic anaema
and 1 of a myeloproliferati ve disorder, very probal y a leukaema.

4. Cots on Data Reported and Evaluation

4. 1 Animl data
:N data were available to the Working Group.

4 . 2 Hur data
A numr of cases of leukaera were reported between 1960 and 1966 in
subjects treated with phenylbutazone or oxyhenbutazone. The available
evidence is, hovver, insufficient to substantiate the suggestion that use
of phenylbutazone or oxyhenbutazone is related to subseqent developet
of leukaera.

194
 c:
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tored by gas-liquid chromatography. J. Chomt., 76, 141-147

Wissrüller, H.F. (1971) Untersuchungen ilr die cytogenetische i;virkung

von Pheny lbutazon an rrnschlichen Lymhocten in vi tro .
Arzneirttel-Forsch., 21, 1738-1750

Wissmüller, H. & Gehat, E. (1970) Cytogenetic effects of Butazolidin

in hum leukoces in vi tro . EM Newslett., 3, 28-29

Woour, D.M. & Fingl, E. (1975) Analgesic-antipyretics, anti-inflamtory

agents, and drgs emloyed in the therapy of gout. In: Goodm, L. S .
& Gilm, A., eds, Th Pharmcological Basis of Therapeutics, 5th ed.,
New York, l'acrllan, pp. 339-341

Wooliff, H.J. & Dougan, L. (1964) Acute leukaera associated with phenyl--
buta zone treatrnt. Brit. rrd. J., 1,744-746

199
 PHEIN AN PHEIN SODIUM

1. Chemcal and Physical Data

Phenytoin
1. 1 Synonym and trade nas
Chem. Abstr. Reg. Serial No.: 57-41-0

Chem. Abstr. Nai: 5, 5-Diphenyl-2, 4-irdazolidinedione

Diphenylhydatoin; 5, 5-diphenylhydantoin¡ 5, 5-diphenylirdazolidin-
2 , 4-dione; DPH ¡ phaantine; Dhenantoine
Aleviatin; lmtisacer; Deyl; Didan-TD-250; Difhydan; Dihycon;
Di-Hydan¡ Dihydantoin¡ Di-La; Dilantin¡ Dilantine¡ Dillantin;
Dintoina ¡ Diphantoin; Diphedan¡ Diphenin¡ Diphenine; Diphentoine¡
Diphentyn; Enefal¡ EDutin¡ Epilan; Epinat¡ Eotoin; r-irot-
Epilan-D; Hidantal¡ Hydantin¡ lepitoin; Soanton¡ Tacosal¡
Zentropil
1.2 Chemcal formla and molecular weight

N'f0
1

N-H

C15H12N202 lvb1. wt: 252.3

1.3 Chemcal and physical properties of the pure substance
From Stecher (1968), unless otherwise specified

(a) Descr iption: PCMder

(b) Melting-point: 295-2980C

(c) rr 1
Spectroscopy data:;\ 258 nr (El = 28.1) in water (Weast,
1975) ¡ 264 and 257 nm (El - 18.5 and 29.1) in methanol
1

(Grasselli, 1973)

(d) Solubility: Practically insoluble in water; soluble in ethanol

(1. 7 g/lOO rrl) and in acetone (3.3 g/lOO rri)
201
 - ~-
1.4 Technical products and imuri ties

Various national and international pharncoooias gi ve specifications

for the purity of phenytoin in pharceutical products. For examle,
phenytoin is available in the US as a USP grade containing 98.5-100.5%
active ingredient on a dried basis with a maum of 0.002% heavy metals,
and as 50 mg talets that contain 93-107% of the stated amunt of phenytoin
(US Pharncopeial Convention, Inc., 1975). Oral suspensions of 6, 20 or
25 mg phenytoin per ml of a suitable irdium are also available and contain
90-110% of the stated amunt of phenytoin (National Fonnlary Bod, 1970).
Sustained release capsules of 50, 100, 125 or 250 mg phenytoin are also
obtainable (Kastrup, 1975).

Phenytoin sodium

1. 1 Synonyr and trade nams

Cher. Abstr. Reg. Serial No.: 630-93-3

Cher. Abstr. Nar: 5,5-Diphenyl-2,4-imdazolidinedione, TInosodium salt

Dipheny lhydantoin sodium; 5, 5-dipheny lhydantoin sodium; dipheny 1-

hydantoin sodum salt; 5, 5-diphenylhydantoin sodium salt; phenytoinum
sodium; sodium diphenylhydantoin; sodium 5, 5-diphenylhydantoin;
sodium 5,5' -diphenylhydantoin; sodium diphenylhydantoinate; sodium
5,5-diphenylhydantoinate; sodium 5, 5-diphenyl -2, 4-imdazolidinedione;
sodium phenytoin; soluble phenytoin

Alepsin; Antilepsin; Antisacer; Auranile; Citrullamn; Dantoin;

Denyl; Difenin; Difetoin; Difhydan; Di -Hydan; Dihydantoin;
Dilantin; Dilantin Soium; Dintoina; Diphantoine; Diphedan;
Diphenate; Diphenin; Diphentoin; Di-Phetine; Ditoin; Divulsan;
Enefal; Eparn; Eputin; Etlin; Epifenyl; Epihydan; Epilan-D;
Epilantin; Epinat; Eptoin; F'enantoin; Fenitoin; Fenytoine;
Hidantal; Hydantin; Hydantoin; Hydantoinal; Idantoin; Idantoinal;
Lepitoin; Mietoin; Novantoina; Novodiphenyl; OnHydantoine;
Phenhyda; Saceril; SDPH; Soanton; Solantoin; Solantyl;
Sylantoic; Tacosal; Thilophenyt; Zentropil

202
1.2 Chemcal formla an rrlecular weiaht

~
N yONa
N

C15Hii N2Na02 MoL. wt: 274.2

1.3 Chemcal and physical properties of the pure substance

From Stecher (1968)

(a) Description: Wh te 'pder

(b) Solubility: Soluble in water at pH :; 11. 7 (1. 7 g/lOO ml) and

in ethanol (10 g/lOO ml); insoluble in ether and chloroforr

(c) Staility: Gradually absorbs carbon dioxiòe on exsnre to air,

librating phenytoin

1. 4 Technical products and imuri ties

Various national and international pharcoooias gi ve specifications
for the puri ty of phenytoin soium in pharnceutical products. For examle,
phenytoin sodium is available in the US as a USP grade containing 98.5-
100.5% active ingredient on a dried basis with a mxir of 0.002% heavy
rntals. Capsules are available in 30 or 100 mg doses and contain 93-107%
of the stated aiunt of phenytoin soium. Sterile ohenytoin sodium
sui table for parenteral use is avail~Jle in 100 or 250 mg doses in solution
containing 90-115% of the stated aiunt (US Pharcopeial Convention, Inc.,
1975). Phenytoin sodium is available in POder forr in l, 4 or 16 oz doses
(Kastrup, 1975).

In the OK, prepaations contain 98.5-101% active ingredient on a

dried basis and 92.5-107.5% of the stated aiunt of active ingredient in
tablets (British Pharcopoia Commssion, 1973).

203
 2. ProductJ-_on, U~t:!_Occurrence_~~d _~ìal y~js

For imrtant background informtion on this section, see preamle,

p. 15 .

2 . 1 Production and Use

(a) Production

A rrthod of preparing phenytoin was oatented in the us in 1946 (Henze,

1946). In one corrrcially used nethod, benzaldehyde is converted to
benzoin with sodium cyanide followed by oxidation with nitric acid to benzil;
this is heated with urea in aqueous ethanolic lXtassium hydroxide and
acidified to give phenytoin. In another nethod, phenytoin is obtained by
heating urea with benzilic acid in acetic anydride (Smth, 1966).

In a rnthod of preparing phenytoin sodium, re:orted by Biltz in 1911,

benzil was condensed with urea followed by treatrnt with sodium hydroxide
(Stecher, 1968). Ths nethod is still used currently, in addition to a
sirlar nethod which invol ves the following steps: benzaldehyde is converted
to benzoin using sodium cyanide, followed by oxidation with nitric acid to
give benzil; ths is heated with urea in the presence of ethanolic sodium
ethoxide or isopropoxide to give phenytoin sodium (Smith, 1966; Swinyard,
1975) .

Commrcial production of Dhenytoin was first relXrted in the us in

1946 (US Tariff Comssion, 1948); only one US compy reported production
(see preamle, p. 15) in 1974' (US International Trade Commssion, 1976a).
us imrts of phenytoin though the principal cutom districts were 900 kçr
in 1972 (US Tariff Comssion, 1973), 550 kg in 1973 (US Tariff Commssion,
1974) and 2,400 kg in 1974 (US International Trade Commssion, 1976b).

Corrrcial production of phenytoin sodum was first reoorted in the US

ln 1938 (US Tariff Commssion, 1939); only one US company reported
production (see preamle, P. 15) in 1974 (US International Trade Commssion,
1976a). US imrts of phenytoin sodium were 1,500 kg in 1972 and in 1973
(US Tariff Comssion, 1973, 1974) and 6,700 kg in 1974 (US International
Trade Commssion, 1976b).

204
 No evidence was found tliat phenytoin is prcxuced comrærcially in
Japa. In 1975, 1,250 kg of this drg were imrted; approximtely 95%
was converted into the scxium salt, and only 5% was used directly as an
antiepileptic agent. Comrrcial prcxuction of phenytoin scxium was begu
in 1951. In 1975, one company prcxuced abut 1300 kg, of which abut
1100 kg were used within the country.

Anual production of phenytoin and of phenytoin sodum in western

Euope is estimted to be less than 1000 kg. Beelux, the Federal
Republic of Gerny and Italy are believed to be the major producing
countries.
(b) Use

Phenytoin and phenytoin scxium are used largel y in the treatrnt of

grand mal and psycholItor seizures, often in cornination wi th phenobarbital
or other anticonvulsants. The oral dosage for adul ts and children over
six years of age 1S ini tial1 y 100 mg 3 tirs oer day and can gradual1 y be
increased by 100 mg every tw to four wees until the desired response 1S
obtained. Maintenance dosages usually range from 300 to 600 mg daily.
Maintenance dosages for children under six are usually 3 to 10 mg!kg bt

per day (Amrican Society of Hospital Pharrcists, 1969).

Phenytoin sodium is also used for the control of status epilepticus.

It is preferably adrnistered intravenously, but can be adrnistered
intramscular 1 y if necessar. l t can also be used for the prophy lactic
control of seizures in neurosurgery. It is adrnistered intramuscularly
during surgery and is continued during the pJst-operati ve oeriod (Kastrup,
1975) .

Phenytoin and phenytoin scxium have been proposed for use as cardiac
depressants (anti-arrhythc). The oral dose 1S usually 100 mg 2 to 4
tires per day. An intravenous dose 0:: 50 to 100 mg of the sodium salt 1S
repeated every ten to fifteen minutes as necessar up to a total maum
dose of 10 to 15 mg!kg bt (US Pharncopeial Convention, Inc., 1975).

Phenytoin has been used in the treatrnt of chorea or Parkinson 1 s

syndrome to control involuntary Ilverents. Its use has been investigated
for the treatrnt of trigemnal neuralgia, migraine, oolyneuritis of

205
 pregcy, acute alcoholisr an certain psychoses (Arrican Soiety of
Hospital Pharncists, 1969). Phenytoin sodiUl has also be suggested for
use in the treatrt of migraine, trigemal neuralqia ëid certin psychoses.
However, these uses are not yet approved by the US Foo and Drug Admnis-
tration (Swinyard, 1975).

Total US sales of phenytoin for use ln hum rnicine are estimted

to be less th 78,000 kg anually.

Phenytoin is used in veterinary rrdicine to control epileptiforr con-

vulsions in dogs (Swinyard, 1975). Phenytoin sodium has also been reconm-
ded as an anticonvulsant for dogs (Stecher, 1968).

2. 2 Occurrence
Phenytoin and phenytoin sodium are not known to occur in nature.

2. 3 Anal ysis

.Mthods of assay of phenytoin tht rnt regulatory reqrernts for

pharceutical products are usuall y lx1Sed on non-aqueous ti trations; those
for phenytoin sodium are usually based on gravirtric rnthods.

A collabrative study investigated a colum chromatographic IDthod to

separate phenytoin sodium in capsules followed by direct ultraviolet

quantitation (Cuningham et al., 1972).

Agas chromatographic rrthod can de

termne phenytoin in seru in the
range of 0.5-8.00 lJg/rn (Brien & Ina, 1974; Chin et al., 1972). Another
gas chromatographic rrthod has l:en develope to determne phenytoin as i ts
alkyl derivatives after flash-heater alkylation with tetraalkl (C -C )
1 7
amnium hydroxides (Giovaniello & Pecci, 1976; Pecci & Giovaniello,
1975). Comparisons have l:en made of gas chromatographic rrthods for the
deterrnation of phenytoin in seru wi th a spectrophotorntric rrthod
(Janz & Schmdt, 1974) and with an enzymtic imunoassay IDthod (Boker &
Darcey, 1975). A spin imunoassay det~errnation of phenvtoin in seru has
.. -
been reoorted to have a minimum sensi ti vi ty of 20- 30 nml (Montgorrry
et aL., 1975).

Thin- layer chromatographic analyses of Dhenytoin can be made in

pharnceutical preparations (Prelini & ('.,rosa, 1975) and in bloo and [Link]
(Pippnger et al., 1969).

206
 High-pressure liqud chromtographic rrthod use lJV spectrophotorntric
detection to determe phenytoin and phenobabital in plasm (Atwell et aL.,
1975), phenytoin and its para-hydroxylated rntablites in plasm (Alrt
et al., 1973), phenytoin in bloo (Gauchel et al., 1973) and phenytoin,
phenobabital and primdone in bloo (Kabra et aL., 1976). The limt of
detection of these methods is in the order of 1 ~g/rn.

A mass fragrtographic determnation of phenytoin and its metablite

5- (4-hydroxyhenyl)-5-phenylhydantoin in hum plasm has a limt of detec-
tion of 0.01 ~g/rn (Hoppel et al., 1976).

An irroved lN spectrophotorntric rrthod for the microdetermnation of

phenytoin in bloo (Saitoh et al., 1973) and a simlified fluorimtric
method for its determation in plasm (Dill et al., 1976) have ben
re:ported. A phosphorescence spctrorntry rrthod for determation of
phenytoin in bloo has a detection lim t of O. 05 ~g/rn (l-brrison & 0' Donne
1 1,

1974) .

A conilexometric determation of phenytoin has also ben reported

(Hentrich & Pfeifer, 1967).

The rnthods of anl ysis for phenytoin in biological samles would, in

principle, also be applicable to phenytoin sodium.

3. Biological Data Relevant to the Evaluation

of Cacinogenic Risk to Ma
3.1 Carcinogenicity and related studies in animls

(a) Oral admnistration

Mouse: Groups of 2- 3-lInth old ferle CS 7BL, C3H/F or SJ/J mice,

48 animls per group, were fed phenytoin sodium at a dose level of 60 mg/kg
bw/day in a liquid diet for 168 days. Thee of 24 C57BL rnce suriving 10
IInth develope thymc lyrpho:rs, whereas no pathologic lesions were found
in 48 controls. Thee of 24 C3H/F femle rnce suri ving 10 IInths develooed
loclized thyrc lymhomas, whereas no pathologic lesions were found in 48
controls (P=0.03). Six of 42 SJ/J mice autopsied after the 4th IInth had
generalized lymhars. After 8 or nore IInths, 48 SJ/J controls showed
proliferative atypical reticulurcells but no lyrhoms (Krger et al., 1972).

207
 Rat: No tUlurS were observed in femle or male Sprague-Daw1ey rats
treated wi th high doses of phenytoin by gavage or in the diet and observed
for periods of 6 to 13 IInths . Neative resul ts were also obtained in
femle Holtzr rats given phenytoin in the diet and observed for 60 weeks
(Griswld et al., 1966, 1968; Morris et al., 1969; Peraino et al., 1975).
No vesicuar tUlurS were observed in rn1e Iog-Evans rats given phenytoin
for one year, despi te the presence of a surgicall y imlanted foreign boy
in the vesicle. No tUlurS were seen in non-operated controls (McDonald,
1969) (These negative studies were inadeqate in duration to be of value
in assessing cacinogenic acti vi ty ) .
(b) Intraperitoneal admnistration

Molie: Fifty random bred albino rnce of bath sexes (18-20 g) received
daily i.p. injections of 0.6 ng/aninl phenytoin suspeded in water or in
saline over 66 days (total, 57 injections or 34.2 mg/animl). No weight
gain was observed, and 10 aninls died during ths periode The remining
40 anirls were observed for 9 IInths; 50 untreated controls were observed
for 11 IInths. Ten treated rnce develope tUlurS, comprising 4 thymc and
2 rrsenteric 1 yrphomas and 4 leukaemas. The leukaemas developed between
60 and 142 days and the lymhomas between 100 and 255 days. In controls,
1 thyrc lymhoma and 1 lung adenoma were observed (Juhász et al., 1968).

3.2 oter relevant biological data

(a) Exrimntal system

The Lv. ID of phenytoin sodium in rabbits is 125 mgjkg bw (Stecher,
5 a

1968). Oral admnistration of phenytoin sodium to dogs produced higher

plasm peak levels at earlier tim periods than phenytoin (Glazko, 1972).

Phenytoin admnistered intraveneously to male Sprague-Dawley rats was

found to accumulate to a considerable degree, :Iiately after admis-
tration, in the Iiver, kidney and salivar gland and at a slower rate and
to a smller degree in fat, muscle and brain (Noach et aí:., 1958). With
continuous admistration to ferrets, high concentrations were found in
the oral mucosa as weIl as in the salivar gland (Steinrg et al., 1973).
Breakdow of 14C-phenytoin in the li ver of Sprague-Dawley rats first
suggested that li ver was the principal si te of rrtablisr (Noach et al. ,

208
1958), and its hepatic rrtablism has been deInstrated in isolated
perfused livers of rats of that strain (Gerber et aL., 1971). Metablites
are excreted in bile (Gerber et al., 1973) and in urine (Chang et al., 1972).

The principal rrtablites ~re forr by hydroxylation of one of the

phenyl rings (Butler, 1957) in the para )Jsition in rats (Chang et al.,
1972) and in the meta position in dogs (Atkinson et al., 1970) and were
excreted in the urine as glucuronides (Atkinson et aL., 1970; Chang et aL.,
1972; Maynert, 1960).

In addition, 5- (3, 4-dihydroxy- l, 5-cyclohexadien- 1 -y 1) - 5-pheny lhydantoin

has been identified in rat and IInkey urine (Chg et aL., 1970). In rat
urine, the diphenolic rrtablite 5- (3,4-dihydroxyhenyl)-5-phenylhydantoin
(Borga et al., 1972) and its rrthoxylated derivative, 5- (4-hydroxy-3-rrthoxy-
phenyl)-5-phenylhydantoin, are present as glucuonide conjugates (Chang
et al., 1972). The glucuronic acid conjugates of 5-(3,4-diydroxyhenyl)-5-
phenylhydantoin and 5- (3-rrthoxy-4-hydroxyhenyl) -5-phenylhydantoin were
identified in rat bile (Gerber et al., 1973) as well as 5,5-bis(4-hydroxy-
phenyl)hydantoin (Thompson et aL., 1976).

A single intragastric dose of phenytoin to pregnant Sprague-Dawley rats

was transferred to the foetuses and was concentrated rrre in the kidney thn
in the liver, in contrast ta the situation in adult rats (Gabler & Falace,
1970). Transplacental transport has been docurted in mice (Stevens &
Habison, 1974; Waddell & Mirkin, 1972), rats (Mirkin, 1971; Stevens &
Harbison, 1974), hamters (Stevens & Habison, 1974), goats (Shoe et al.,
1972) and monkeys (Gabler & Hubbard, 1972).

A/Jax rnce were injected subutaneously with 12.5, 25 or 50 rr/kg l:

phenytoin sodium at various tims from days 9-15 of pregnancy; the 50 rr/kg
bw dose produced cleft palates in 26-43% of the offspring (Massey, 1966).

Pregnant Swiss-Webster and A/J mice ~re injected subutaneously with

50 rr/kg l: phenytoin sodium on days 7-9 or 11-13 of gestation. Treatrnt
during late gestation induced significant increases in the incidence of
cleft palates in the offspring of Swss-Wester (15%) and A/J mice (31%)
(Gibson & Beker, 1968). Th teratogencity of phenytoin has ben confinæd
in other exrimnts in rnce (Elshove, 1969; Harbison & Becker, 1969;

209
l-1ash & Fraser, 1973; Sullivan & McElhatton, 1975), rats (Harbison &
Becker, 1972; Mercier-Parot & Tuchm-Dulessis, 1974) and monkeys
(Wilson, 1973).

After oral treatrt of rats with 250 Il/kg l: daily for the first 5
days of gestation, bane-rnow cells had 26% abnornl rætaphases coed
with 8% in controls (and accuation of rrtaphases was also observed)
(Caratzali & Roma, 1971; Ram & Caatzali, 1971).
Negative results have be reported in the danant lethal test ln
mice after 115 or 145 Il/kg l: phenytoin sodium were adrnistered by i.p.
injection (Epstein et al., 1972).

(b) Ma
A single oral dose of phenytoin soium produced higher bloo Ievels
of the soium salt than did phenytoin (DilI et al., 1956).

The principal adverse reaction to phenytin is gingival hyprplasia

(Hassell et al., 1976; Kapur et al., 1973). In addition, hepatic necrosis
(Le et aL., 1976) and a reduction in the numr of peripheral blcx lymho-
cyes (Brandt & Nilsson, 1976) have ben reported; a-tyical lymh node
hyprplasia (' pseudol ymhoma') rry also occu (see section 3. 3) .
Follawing a single oral dose of phenytoin soium, 5- (para-hydroxy-
phenyl)-5-phenylhydantoin and a 10-fold greater level of its glucuonide
were present in plasm (Albrt et al., 1974). Urine sanles from individuals
recei ving phenytoin contain 5-13 % of th~ hydroxy lated rntabli te as 5-

(meta-hydroxyhenyl) -5-phenylhydatoin (Atkinson et aL., 1970). Oter

urin rntalites are 5- (3, 4-dihydroxy- l, 5-cyclohexadien- 1 -yl) -5-phenyl-
hydantoin (Horning et aL., 1971), 5- (3, 4-dydroxyhenyl) -5-phenylhydantoin
(Borga et aL., 1972) and 5,5-bis-(4-hydroxyhenyl)hydatoin (Thamson et
aL., 1976).

A possible association between maternal epilepsy, anticonvusant drgs

(mainly phenytoin and phenobabital) and congenital malforntions in
offspring was reported by Medow (1968). In two reports, a tota of 38
children barn to rrthers who took anticonvusants, including phenytoin,
thoughout pregcy revealed a simlar constellation of rrlforntions

210
suggesting a sydroræ invol ving the lip and palate, cadiovascuar system
and skeletal abnormlities (lim, skull, face) (Meadow, 1968, 1970).
Subseqent case reports of epileptic v;iæ treated during pregnancy with
phenytoin and phenobarbital have describd affected infants wi th sirlar
abnormlities (Aase, 1974; Anderson, 1976; Bar et al., 1974; Dabe
et aL., 1975; Go et aL., 1976; Loughnan et al., 1973; Pettifor &
Benson, 1975).
In a retrospeti ve study of epileptic imthers in the Federal Republic
of Gerny, 262 pregancies W2re accorranied by continuous treatrt with
phenytoin and babiturates; five malforntions W2re found aIng the
offspring, to give a rate of 2.2%, whch was not considered to be increased
over that of the general :ppulation (Janz & Fuchs, 1964).

ln 51 pregcies identified from us hospital records over the period

1960- 1970 occuring in 42 epi1eptic patients, phenytoin was admistered
alone or with mysoline in 7 pregncies and wi th phenobarbital in 44
pregancies, thoughout pregnancy; 3 congenital abnorrli ties occured
(5.8%). Although no anomalies W2re found in 50 matched pregnancies, the
effect was not considered ta be statistically significant (Watson &
Spellacy, 1971).

In th OK, 168 epileptic v;rn treated with phenytoin, phenobabital

or other anticonvulsant drgs were identified from the neurolog depants
of tw general hospi tals. These v;ræn had exrienced 365 pregnancies,
during which phenytoin alone or in combinat ion was given during 192
pregnancies and phenobabital alone or in combination during 240 pregnancies.
Seventeen of the 365 infants had malformtions, comred with 7 of 483
controls and 0 of 62 offspring of epileptic waen not taking rædication.
The calculated risk was tw to thee tis greater than nornl (Speidel &
Meadow, 1972).

During 1969 and 1970, 7,896 v;ræn gave birt at a single London
hospi tal, and 192 of the infants had ma 1 formt ions , to gi ve a rate of
2.44%. In ths group, 22 epileptic IIthers had taen anticonvusant drgs
thoughout pregcy. 'I of the children oorn to IIthers who had taken
phenytoin and phenobarbital in one case and phenytoin and other drgs in

211
the other case had cleft palate and/or hae lip, an incidence of 9%,
whereas only 0.13% of the infants torn ta non-epileptic rrthers were so
affected (South, 1972).

In the US, the outcor of 50,897 pregnancies selected frar patients

attending 12 hospitals betw 1959 and 1965 were exed. The congenital
malformtion rate for a wide range of defects, including cleft lip and cleft
palate, was 6.12% am:mg birt to 98 epileptic rrthers who took phenytoin
reguarly during the first four IInth of pregnancy. The rate was signifi-
cantly higher th that of 2.45% aIng children tom to ~ with no con-
vulsive disorder. The abnormlity rate aIng baies torn to 101 wa with
convulsive disorders but who did not receive phenytoin was 2.97%, which is
not significantly different from nornl (Monson et al., 1973).

In Cadiff, 31,877 infants torn betwee 1965 and 1971 were studied;
245 IIthers in this series had a history of epilepsy. Aing the 111 birt
to epileptic IIthers not on anticonvulsant therapy, the rnlforntion rate
was 2.7%, compared to 2.8% of infants in the total population. In contrast,
6. 7% of 134 infants torn to rrthers recei ving anticonvusant therapy had
malforntions: 1/53 children whose IIthers had received phenobabital
alone, 2/9 children whose IIthers had received phenytoin alone and 6/60
children whose IIthers had recei ved phenobabital and phenytoin during
pregnancy (Ioe, 1973)

In US Ai Force hospitals between 1965 and 1971, aIng the children

barn to 410 epileptic rrthers alst certainl y exsed to anticonvulsant
therapy during pregncy, from a population of 347,097 livebm infants,
there was a twfold increase in the risk for all malforntions and a
five- to six-fold increase for cleft lip and/or palate (Niswander &
Wertelecki, 1973). In a surey in tbrtern Ireland, the risk to infants
barn to epileptic IIthers taking anticonvulsant drgs, mainly phenytoin
and phenobarbital and other drgs, was 6.4 % comped wi th a malforntion
rate of 3.8% for all live and stillbirts in the province. There was a
7-fold increase in the risk of cleft lip with or without cleft palate
(1. 8% compared with 0.22%) (Millar & Nevin, 1973).

ln the Oxord Reord Linage Study, of li ve baies barn between 1966

and 1970 to epileptic IIthers, 17/217 (7.8%) had congenital abnormlities

212
at birt corared with 21/649 (3.2%) of the controls matched for civil
status, social class, maternal age, :ii ty, hospi tal and year of deli very .
Subseqently, the rates rose to 13.8% (30/217) for baies of epileptic
irthers and 5.6% (36/649) for those in the control group (This may be due
to the longer observation period and to the inclusion of different tys
of rrlforntions, which could have been considered to be congenital beforeJ.
Maternl phenobarbital ingestion alone did not account for an increased
incidence of defects (2/41, 4.9%) over that in inants of mothers not
taking drgs (2/19, 10.5%), but when given in combinat ion with phenytoin
the proportion of infants with defects is IIre than doubled (11/50, 21%).
The incidence of defects anng infants of IIthers taking phenytoin alone
(5/33, 15.2%), whle higher than that anng infants of IIthers taking
phenobabi tal alone, is lower than that aIng infants of IIthers taking
phenytoin in combinat ion with phenobabital (Fedick, 1973).

In Scotland between 1965 and 1967, a prospetive study of 15,181

pregancy outcomes, including abrtions and includig 48 epileptic IIthers,
revealed 452 wi th malforntions, of which 30 had hae lip and/or cleft

palate. None of these were associated with epilepsy or anticonvusant

therapy in the nother. It was concluded tht any causative relationship
between anticonvulsant therapy and teratogenic effects would reqire a
larger study (Kuenssberg & Knox, 1973).

A study of pregnancies follawed in a single US clinic included 284

birts to 138 epileptic nothers betwee 1939 and 1972. Of 141 birts to
IIthers taing anticonvusants during the first trimster, 10 infants had
malforntions; of 56 birts ta epileptics not taing rnication, 1 infant
had a malformtion; and in 26 birt to epileptic IIthers in remssion
no malformtions were sen. The association of cleft palate and hear
malformtions with anticonvulsant usage wa simlar in 28 patients treated
with phenobabital alone and in 24 patients treated with phenytoin alone
(Anegers et al., 1974).

Bary & Dans (1974) reported that in Australia betwee 1967 and 1972,
10/39 infants barn to IIthers treated with phenytoin and babiturates
during pregncy had congenital abnormli ties . No congenital abnormli ties

213
occurred in children of irthers treated wi th babi turates alone, whereas
they occued in 2/8 chldren of rrthers trated wi th phenytoin.

The teratogenicity of phenytoin has also been designated as the foetal

hydantoin syndrorn (Hanson, 1976; Hanson & Smth, 1975) (see also IIno-
graph on phenobabital and phenobarbital sodum, p. 157).
In vitro treatrnt of hum lJripheral leucotes with concentrations
of 50-100 llg/rn phenytoin produced chrolIsorr abnornlities (Muiz et aL.,
1969). Negative results have also been obtained with sirlar concentrations
(Alving et aL., 1976; Bishun et al., 1975; Stenchever & Jaris, 1971).

A weak colchicine- like effect on rntaphase arrest in cul tured hum

lymhoces has ben observed in vitro with concentrations of 3.6 x 10-4 M.
In the sai study, the rrtablite 5- (4-hydroxyhenyl) -5-phenylhydantoin was
3 tims IIre patent (MacKiney et al., 1975). A prelirnar repart indi-
cated tht bath phenytoin and the rrtabli te interfere wi th rncrotubule
formtion in vitro (MacKinney & Vyas, 1975).

CholIsorrs in cul tured leucoctes from 7 epileptic patients recei ving

phenytoin sodium shCMed no abnormlities (Muñiz et al., 1969). CholIsomal
abnornlities were, however, reported by Neuhauser et al. (1970). In a study
of leucocte cultures from 32 IIthers taing anticonvusants, the average
increase in chroIIsoml abnormli ties was 17% in those of all 5 womn taking
only phenytoin, whereas only 5% was seen in cultures froID 32 controls;
leucocyes of 4 children barn ta rrthers recei ving phenytoin onl y also had
an increased numr of mitotic abnormlities (Grosse et al., 1972).
3.3 Case reports and epidemological studies

(a) Case reports

Of 7 patients originally describd by Saltzstein & Ackerm (1959) as

developing pseudolymhomas following ohenvtoin and/or phenobabital treat-
rnnt, two died, one from multiple myeloma wi th an unow suri val tim
after diagnosis, and another froID malignant 1 ymhoma wi thin 4 years
(Harrington et al., 1962).

Of 6 patients recei ving phenytoin alone or wi th phenobarbital, 3 were

reported to have develope Hodgkin' s disease, 2, 1 ymhosarcoms and l, a
reticulumcell sarcoma (Hyr & Sommrs, 1966).

214
 Nurrous case reports have describ lyrhOIs (Bichel, 1975; Brow,
1971; Gar et al., 1968; Jungi et al., 1975; Rausing & Trell, 1971;
Sorrell & Forbes, 1975; Tashim & de los Satos, 1974) and leukaemas
(wildhck, 1973) occuring in patients treated with phenytoin.

Two cases of neuroblastom, a 3-year old girl and a 7-day old boy,
have been reported in the foetal hydantoin sydrar .Both nothers had taken
anticonvulsants (phenytoin and phenobarbital) since gir lhoo because of
epilepsy (Pendergrass & Hanson, 1976; Sherm & Roizen, 1976).
(b) Epidemological studies

The records of 85 persons dying with malignant lyrhoms during the

period 1958 to 1968 at a single us hospital were reviewed for a history of
phenytoin therapy. Four of the 85 were epileptics who recei ved phenytoin
sodium alone or phenobarbital. The occurrence of epilepsy was estimted
to be 1 in 200 in the general J:pulation and in the necropsy group in this
hospital, 1 in 400. Therefore, four lìmihoms in 85 autopsies was 10 tims
the excted incidence (Anthony, 1970).

In 300 patients fror a single US rrical centre wi th malignant 1 yrhomas,

7 were found to have a history of antiepileptic phenytoin therapy. Since
onl y 1 in 300 indi viduals in the general population takes anticonvulsant
drugs, this was considered to be a significant association (Charlton &
Lunsford, 1971).

In a Danish hospi tal, 9,136 cases of epilepsy treated wi th phenytoin,

phenobarbital or prirdone durÌlg the peiod 1933 to 1962 were reviewed
for the incidence of neoplasrn. There was no increase in the incidence of
neoplasr of the 1 ymh and bloo system in the group of epileptics as
corned wi th that in th general l:pulation (For a discussion on the
incidence of li ver tururs, see IInograph on phenobabital and phenobarbital
sodium, p. 157). In patients treated for IIre than 10 years, tururs of
brain and nervous system were observed in 10 males (expcted 3.5) and in 6
femles (expted 2.9) (Clersen et al., 1974) (see also 'r.,neral Rerks
on Substaces Considered', pp. 24-25).
A review of 516 cases of malignant lymhoms treated in a radiation
centre during the period 1968 to 1972 revealed that 8 patients (1.6%) had

215
 had prolonged treatrt with phenytoin. Only 2 of 516 tuiur-free indi-
viduals (0.4%) interviewed in connection wi th other epidemologic studies
had had phenytoin therapy. Thee of 516 patients (0.6%), rntched to within
5 years of age at diagnosis of cacers other than 1 ymhorn, had recei ved
phenytoin therapy. Therefore, the excess risk of malignt 1 ymha: was
concluded to be tw- to thee-fold for patients recei ving phenytoin
(Li et al., 1975).

4. Corrts on Data Reported and Evluation

4. 1 Animl data
Phenytoin and its soium salt are cacinogenic in rnce following their
intraperitoneal injection and oral adnistration, respectively: they
produced lymhomas and leukaemas. Studies in which rats were given
phenytoin by oral adnistration were of too short duration to a11CM an
evaluation to be made.

4. 2 Himian data

An association has be observed in epileptic patients betwen the

occurence of 1 ymhomas and long-term anticonvulsant therapy in which
phenytoin alone or in camination with other anticonvulsants such as
phenobabi tal were gi ven (see also IInogaph on phenobarbi tal and pheno-
barbital sodium, p. 157).

216
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Pecci, J. & Giovaniello, T.J. (1975) Gas chromatographic studies of

phenobabital and diphenylhydantoin after flash-heater alklation.
J. Chomt., 109, 163-167

Pendergrass, T. W. & Hason, J. W. (1976) Fetal hydatoin syndrorr and

neuroblastoma. Lacet, ii, 150

Peraino, C., Fry, R.J.M., Staffeldt, E. & Chistopher, J.P. (1975)

Comative encing effects of phenobarbital, arbabital, diphenyl-
hydantoin and dichlorodipheny 1 trichloroethane on 2-acety lamofluorene-
induced hepatic tmorigenesis in the rat. Cancer Res., 35, 2884-2890

Pettifor, J .M. & Beson, R. (1975) Congenital malforntions associated

with the admistration of oral anticoaguants during pregnancy.
J. Pediat., 86, 459-462

Pippeger, C.E., Scott, J.E. & Gillen, H.W. (1969) Thin-layer chromatogaphy
of anticonvusant drgs. Clin. Chem., 15, 255-260

Prelini, R. & Gerosa, A.Z. (1975) Applicazione della densitorrtria

all 'analisi routinaria di spcialita farceutiche. BolL. Chir. Fam.,
114, 355-360

Rausing, A. & Trell, E. (1971) Maignant lyrhogranulomatosis and anticon-

vulsant therapy. Acta ir. scand., 189, 131-136

Ro, I.C. & Caratzali, A. (1971) Effects of anticonvulsant drgs on

chrarsorrs. Bri t. ir. J., iv, 234

223
Saitoh, Y., Nishiara, K., Naagawa, F. & Suzuki, T. (1973) Imraved
rncrodeterrnation for diphenylhydantoin in b100 by W spetrophoto-
iætr . J. pha. sei., 62, 206-210

Saltzstein, S.L. & Ackerm, L.V. (1959) Lymhadenopathy induce by anti-

convusant drgs and rncking clinicall y and path010gicall y malignant
lymhoms. Cacer, 12, 164-182

Sherm, S. & Roizen, N. (1976) Fetal hydatoin sydroiæ and neuroblastoma.

Lacet, ii, 517

Shoer, D.W., Kauffr, R.E., Azamoff, D.L. & Boulos, B.M. (1972)
Placental transfer of dipheny lhydantoin in the goat. Biod1e. Phancol.,
21, 1237-1243

Sr th, E. (1966) Hydantoin. In: Kirk, R. E. & Otr, D. F ., eds,

Encyclopa of Chemcal Tehnolog, 2nd ed., Vol. 11, Ne York, John
Wiley and Sons, p. 156

Sorrell, T.C. & Forbes, I.J. (1975) Phenytoin sensitivity in a case of

phenytoin-associated Hodgkin 1 s disease. Austr. N. Z. J. ~d., ~, 144-147

South, J. (1972) Teratogenic effect of anticonvusants. Lacet, ii, 1154

Spidel, B.D. & Meadow, S.R. (1972) M3temal epi1epsy and abnoi:1ities of
the fetus and newbrn. Lacet, ii, 839-843

Stecher, P.G., ed. (1968) Th Meck Index, 8th ed., Raay, ID, M=rck &
Co., p. 388

Steinrg, A.D., Allen, P.M. & Jeffay, H. (1973) Distribution and iætalism
of dipheny lhydatoin in oral and nonoral tissues of ferrets.
J. dent. Res., 52, 267-270

Stenchever, M.A. & Jaris, J.A. (1971) Diphenylhydantoin: effect on

the chrorsors of hum leukoctes. .Ar. J. Obstet. Gyec., 109,
961-962

Stevens, M.W. & Habison, R.D. (1974) Placetal transfer of diphenyl-

hydatoin: effects of spies, gestational age, and route of
admistration. Teratolog, 2., 317-326

Sullivan, F.M. & McElhatton, P.R. (1975) Teratogenic activity of the anti-
epileptic drgs phenobabital, phenytoin, and primdone in mice.
ToxicoL. appL. Phai:col., 34, 271-282

Swinyard, E.A. (1975) Antiepileptics. In: Osol, A. et al., eds,

Regton 1 s Phaceutical Sciences, 15th ed., Eaton, Pa, Mack,
p. 1017-1018

Tashi, C.K. & de los Satos, R. (1974) Lymhoma and anticonvusive therapy.
J. Amr. me. Ass., 228, 286-287

224
Thorson, R.M., Behin, J., Fife, W.K. & Geber, N. (1976) 5,5-Bis(4-
hydroxyhenyl)hydatoin, a mior rrtalite of diphenylhydatoin
(dilantin) in the rat and hi.. Drug M2ta. Disposition, i, 349-356

US Interntional Trade Cossion (1976a) Sythetic Organic Chercals, US

Production and Saes, 1974, USITC Pulication 776, Washington DC, US
Govert Printing Office, p. 106
US Interntional Trade Cossion (1976b) I:rts of Bezenoid Chercals
and Products, 1974, USITC Pulication 762, Washigton DC, US
Governt Printing Office, p. 83
US Phacopeial Convention, Inc. (1975) The US Phacopeia, 19th rev.,
Rokville, Md, pp. 379-381

US Tariff Ccssion (1939) Sythetic Organc Chemcals, US Production and

Sales, 1938, Rert No. 136, Seond Series, Washington DC, US
Govert Printing Office, p. 36
US Tariff Carssion (1948) Sythetic Organic Chercals, us Production and
Sales, 1946, Report l'. 159, Send Seies, Washington DC, US
Govermt Printing Office, p. 103
US Tariff Corssion (1973) Inrts of Bezenoid Chcals and Products,
1972, TC Pulication 601, Wahigton De, US Goverrt Printing Office,
p. 85

US Tariff Carssion (1974) IIrts of Bezenoid Chcals and Products,

1973, TC Pulication 688, Washington De, US (':iverrt Printing Office,
p. 81

Waddell, W.J. & Mikin, B.L. (1972) Distribution and rrtalisr of diphenyl-
hydatoin-l '+c in feta and rrtemal tissues of the pregnt lIuse.
Bioche. PhacoL., 21, 547-552

Watson, J.D. & Spellacy, W.N. (1971) Nenatal effects of mateal treabæt
with the anticonvusant drg diphenylhydantoin. Obstet. Gyec., 37,
881-885

ÝÈst, R.C., ed. (1975) CR Hadbk of Cherstr and Physics, 56th ed.,
Clevelan, Oho, Chercal Ruber Co., p. C- 336

Wildhck, R. (1973) Leukfue nach Hyantoin-Bedlung. Mfch. rrd. Wschr.,

115, 1275-1279

Wilson, J.G. (1973) Present status of drgs as teratogens in ma.

Terato1og, 2, 3-16

225
 PRONAIL HYRIE
1. Chemcal and Physical Data

1.1 Synonym and trade nas

Chem. Abstr. Re. Serial t'.: 51-02-5

Chem. Abstr. Nar: a-H (1-Methylethyl)amnoJ:rthyl)--2-naphthalene-

:rthanol, hydrochloride

Alder lin hydrochloride; a- ( (isopropy lamo) rnthyl J - 2-naphthalene-

:rthol, hydrochloride; 2-isopropy lamno- 1 - (2-naphthyl) ethanol
hydrochloride; naphthylisoproterenol hydrochloride; pronethalol
hydrochloride
ici 38174; Inetol; Nethalide hydrochloride

1. 2 Chemcal formla and 1I1ecular w=ight

1 CHi
OH

~ .HCI
~O CH-CH2-NH-CH(CHi

C15H19OO' ILl r.l. wt: 265.8

1. 3 Chencal and physical properties of the pure substace
From Stecher (1968)

(a) Description: Crystals

(b) Melting-point: l840C

1.4 Technical products and imurities

No data were available to the Working Group.

2. Prcxuction, Use, O:currence and Analysis

For imrtat background informtion on ths section, see prearle,

p. 15.

227
2.1 Production an us
(a) Production

A irthod of sythesizing th free bae, pronetalol, by the reduction of

2-naphthcy 1 brorde wi th soium borohydride, follCM by reaction wi th
isopropylame, wa patented in the OK in 1962 by Stephenson (Stecher, 1968).

No evidence was found that pronetalol hydrochloride has be produced

corrcially in the US or Japa. It has been produced by one coy in
Euope in the past but is no longer available.
(b) Use

Pronetalol hydrochloride is the salt of pronetalol, a i:tent ß-

adrenergic blocking agent wi th li ttle sythomtic acti vity . It was
withawn fror clinical us prior to 1970 (Grollr & Grollr, 1970)
because it was found to produce tunurs in rnce after prolonged admstra-
tion (Howe et al., 1968).

2. 2 Occurence
Pronetalol hydrochloride 1S not know ta occur in nature.

2. 3 Anal ysis
Several quti tati ve anal ytical rrthods, including thn- layer and gas
chromtogaphy, have ben develope to determe the free base, pronetalol
(Cerri, 1970).

A sensi ti ve assay for ß-adrenergic blocking drgs, includig pronetalol,

in biological fluids is based on the use of gas chromatogaphy with electron-
capture detection of th trifluoroacetyl derivatives. The limt of detection
is in the order of 0.1 ng/rn (Walle, 1974).

228
 3. Biological Data Relevant ta the Evaluation
of Cacinogenic Risk to Ma
3.1 Carcinenicity and related studies in anls
Oral admistration
Mouse: In an inadeqtely reported study, doses of up to 200 rr/kg
hw prcruced lymhoms in various organs (Paget, 1963).
Groups of 25 male and 25 ferle mice (strain unspecified) ~re fed
pronetalol Hel in the diet at concentrations of 0.2, 0.1, 0.05 and 0%
for up to 454 days, at which tim the exrirt was termated. Four
thymc tUlurs occured after 141, 240, 358 and 353 days in feres fed
a diet containing 0.1%. Thee thymc tUlurS occured in nales fed 0.1%
in the diet after 115, 329 and 450 days. Anng mice fed 0.2%, 1 nale and
2 femles develope thymc tUlurs after 283, 171 and 257 days, respectively.
Arng mice fed 0.05%, no nales and 3 femles develope thyrc tururs after
172, 220 and 428 days, respectivelYi one thyrc turur was observed in
control males after 338 days (Have, 1965).

Rat: In an adeqtely reported study, no tururs occured in rats

treated with doses up to 200 rr/kg l: for 2 years (Paget, 1963).

3.2 Oter relevant biological data

(a) Exrimntal system
The i. v. LD
50
of pronetalol HCl in mice is abut 46 ff/kg hw and the
oral LD abut 550 ff/kg l: (Black et aL., 1965).
50
Pronetalol Hel is absorbe rapidl y fror the intestine of anesthetized
dogs. In conscious dogs peak bloo levels were reached abut one hour after
oral admistration. The highest average tissue concentrations were found
in the spleen, follow by kidney, brain, hear and uteru (Black &
Stephenon, 1962).
Five rntalites of the drg have been identified in the urine of
various speies. Four of these, 2-amo-l- (2-naphthyl)ethanol, 2-naphthyl-
glycolic acid, 2-naphthylglyoxylic acid and 2-naphthoic acid, are fonæ by
degadation of the isopropylamoethanol side-chain. The fifth, the 7-
hydroxy anloge, is present pal y in the free forr but predorantl y as

229
its glucuonide. ln mice, rats, guinea-pigs, rabits, dogs, cats and
rnnkeys, the differences in the rrjor iætalites are of a quantitative
rather than of a quli tati ve character. In rrst of the hi therto tested
species, ring hydroxylation and conjugation is the rrjor pathway; in
guea-pigs, side-chain oxidation is rrre freqent than ring hydroxylation
and conjugation (Bond & Howe, 1967).

A dihydrodiol iætalite and the glycol, 1- (2-naphthyl) ethane-l, 2-

diol, were identified in the urine of rats, whle 4 phenolic iætalites,
a dihydrodiol and the glycol ~re detected in rruse urine. The major
rrnohydroxylated iætal:Üite excreted by rnce was the 7-hydroxy analoge of
the drg. In guea-pig urine the glycol was the rrjor iætablite
(Stillwell & Harning, 1974).

(b) Ma
In thee patients treated orally with 200 mg 14C-pronetalol HC1,
[Link] all of the radioactivity was recovered from the urine within 72 hours.
The excreted iætabli tes ~re simlar ta those found in exrirtal animls
(Bond & Howe, 1967).
3. 3 Case reports and epidemological studies
No data were available to the Working ('~up.

4. Cots on Data Reported and Evluation 1

4.1 Animl data

Pronetalol hydrochloride is carcinenic in rnce following i ts

oral admistration: it produced thyrc lymhons.

4. 2 Hun data
No case reJ?rts or epiderological studies were available to the
Vbrking Group.

1 See also the section 'Animl Data in Relation to the Evaluation of

Risk to Ma' in th introduction to ths voltme, p. 13.

230
 5. References

Black, J.W. & Stephenson, J.S. (1962) Phcolog of a ne adrenergic

beta-rector-blocking caund (nethalide). Iat, ii, 311-314
Black, J.W., Duan, W.A.M. & Shs, R.G. (1965) Caison of SC
properties of prothlol and propranlol. Brit. J. Phcol., 25,
577-591
Bond, P.A. & Haw, R. (1967) 'l rrtablisr of pronethalol. Biocer.
Pharmcol., 16, 1261-1280

Cerri, O. (1970) Detenntion of ß-blocking drgs. fulL. Ch. Far.,

109, 338-343

Grollm, A. & Grollm, E.F. (1970) Phacolog an Thrapeutics,

7th ed., Philadelphia, I. & Febiger, p. 311

Hawe, R. (1965) Cacinogenicityof 'alderlin' (pronethlol) in mice.

Nature (Lond.), 207, 594-595

How, R., Crower, A.F., Stephenson, J.S., Rao, B.S. & Smth, L.H. (1968)
ß-Adenergic blocking agents. i. Pronethal01 and related N-alkl
and N-aralkyl derivatives of 2-amo-l- (2-naphthyl)ethol. J. rr.
Cher., 11, 1000-1008

Paget, G.E. (1963) Cacinenic action of pronethlol. Brit. rrd. J.,

iv, 1266-1267

Stecher, P .G., ed. (1968) The rvrck Index, 8th ed., Raay, ID, rvrck &
Co., p. 871

Stillwell, W.G. & Horning, M.G. (1974) rvtalisr of proneth10l in th

rat, the gunea pig and the rruse. Res. Cæm. chem. Path. Phacol.,
9, 601-619

1885- 1891 --
Wallß, T. (1974) GI detennation of propranlol, othr ß-blocking drgs,
and rrtalites in bi010gical fluids and tissues. J. phar. Sei., 63,

231
 PYR
1. Cherca and Physical Data

1.1 Synanym and tract nas

Cher. Abstr. Reg. Serial No.: 58-14-0

Cher. Abtr. Nar: 5- (4-Chlorophenyl) -6-ethyl -2, 4-pyrimdiediarne

5- (4' -Chlorophenyl) -2, 4-diamo-6-ethylpyrimdie; 2,4-diamo-5-

( 4-chloropheny 1) -6-ethy Ipyrimdine; 2, 4-diamo-5- (para-chloro-
phenyl) -6-ethylpyrimdie; pyrerthame
BW 50-63; Chloridi; Chloridie; Daaclor; Darapram; Daaprir;
Daapr.i ; Diamopyri ta; Erbaprelina; Erboprelina; Fansida ;
Malocide; Maloprir; NSC 3061; [Link]; 4753 R.P.; Tindurin
1.2 Chercal forma and nolecular weight

C12H13ClN 4 MoL. wt: 248.7

1.3 Chercal and physical propees of the pure substace

Fran Stecher (1968), unless othei:se specified

(a) Description: Cistals

(b) ~ltig-point: 238-2420C (US Phamcopeial Convention, Inc.,

1975 )

(c) Solubility: Practically insoluble in water; very spaingly

soluble in propylene glycol at 700C and in [Link] at
700C; slightly soluble in ethol (0.9 g/lOO ml) and in dilute
hydrochloric acid (0.5 g/lOO ml); soluble i. boiling ethol
(2.5 g/lOO ml)

233
1. 4 Technical products and :Iuri ties

Various national and interntional phccpias gi ve spifications

for the puri ty of pyirthame in phceutical products. For exle,
pyiithame is available in th US as a USP grade containg 99-101%
active ingredient on a dried basis and as 25 ri talets containing 93-107%
of the stated anunt (US Phacopeial Convention, Inc., 1975).
In th OK, pyirthamne is available on a dried basis cotaining
99-101% of th active ingredient and as 25 ri talets containing 92.5-107.5%
of the stated anunt (British Pharncopoia Cassion, 1973). Caninations
of pyriitham with other antimarial drgs, e. g., chloroqe, are
available in so countries.

2. Production, Use, Occuence and Anal ysis

For imrtt background informtion on ths section, se preale,
p. 15.

2.1 Production and use

(a) Proucion
A rrthod of sythesizing pyirthame was first patented in th US in
1951 (Hitchigs et al., 1951). Pyimthne ca also be prepared by the
condensation of ethyl propionate with para-chlorophenylacetonitrile in the
presence of soium rrthylate, ta give a-propionyl-para-chlorophenylaceto-
nitrile, which is then reacted wi th isoa 1 alcohol to produce a hemacetal.
Ths is dehydated to a- (para-chlorophenyl) -ß-ethyl-ß-isoloxyacrylo-
nitrile, whch is reacted wi th guanidine to yield pyrirtharne (Haey,
1975) .

Corcial production of pyrirthe in the US was first reported

in 1953 (US Tariff Commssion, 1954); only one US compy reported
production in 1974 (see preamle, p. 15) (US International Trade Corrssion,
1976) .

It has be produced in Japan since 1967, and production in 1975 was

reported to be 7500 kg; 4500 kg of this were exrted to south-east Asia.

234
 Anua production of pyiithame in the UK 1S estirte to be in
the range of 1-100 thousand kg.

(b) Use

Pyirthe is used as an antirarial agent in hur ræcine. It

is active agaist asexl bloo forr in all tys of nalaria, producing
clinical cue in all and radical cue in most cases of Plasmodium falciparu
infection. Its action is, hawver, slOW, and it is not, therefore,
recded for treatrt of acute attacks. [Link] is a p:erful
suppressive agent; suppressive cue is achieved againt P. falciparu
infection and [Link] against P. vivax (Cavell et aL., 1955). It is a
rrr of a group of diydrofolate reductase initors which bind selec-
ti vel y ta the essential enzyi of the malaria parasite, dihydrofolate
reductase. Folic acid antagonism may arise with high dosages but can be
prevented by concantat adnistration of folinic acid (WHO, 1973).

Combinations of [Link] wi th other drgs, such as chlorogine,

qunine, sulphadoxine, sulphafurazole and sulphadiazine have be us
against chloroqe-resistat falcipa nalaria (WO, 1973).

Pyiitharne may also be used to treat toxoplasmosis, in conjunction

with sulphonarde drgs such as sulphadiazine or triplesulpha (Kastrp,
1975). When used for the prophylactic treatrt of toxoplasrsis in
pregnancy, several courses of treatrt of 25 ID twice a day are gi ven
fror the 9th to the 40th week of pregnancy (Mahkovski, 1972).

Pyiitharne has also ben reported to be usful as an irosuppressi ve

agent (Haey, 1975).

Use of pyrimthne as an antirlarial agent in veterinar ræicine

has also been reported (Stecher, 1968). In Japan, pyrirtharne is added
to dorrstic li vestock animl fee ta prevent and to suppress nalarial
attacks and toxoplasisis.

2. 2 O:curence
Pyiitharne is not know to occur in nature.

235
2. 3 Anal ysis
Methods of as say for pyrimthamne tht rrt reguatory reqirernts
for phaceutical products include non-aqueous ti tration and UV spectro-
photoiætry .
Its determnation in boy fluids and tissues dow to levels of 0.01 lJgI
ml can be accomlished by thin- layer chromatogaphic sepaation and UV
absorption iæasurerts on the thin- layer chromtograi::hic plates (Degelis
et al., 1975). A sirlar technique, using fluorirtr, has ben emloyed
for its detection in urine, with a lirt of detection of 0.1 IT/ml (Jones
& King, 1968).

Pyrirthne has been deterrned in anl fee by gas chromtography

with electron capture detection (Royere et al., 1972). Gas chromtogaphy
wi th bath electron capture and mass spetroiætr detection have ben used
for i ts determnation in tissues, wi th a lir t of detection of O. 1 m:/kg
(Cala et al., 1972).

It can be determed in pharceutical products by nuclear rrgnetic

resonace spectroscopy (Girgis & Askam, 1974).

3. Biological Data Relevant to the Evluation

of Cacinogenic Risk to Ma
3.1 Cacinogenici ty and related studies in animls

Intraperi toneal admnistration

J'use: In a large-scale screening study, groups of 10 male and 10
femle A/He rnce, 6-8 weeks old, received 5 i.p. injections over 8 weeks
of pyrirthame dissolved in 0.1 ml redistilled tricaprylin (total doses
pyrimthamne, 0.125, 0.062 and 0.025 g/kg l:). Animls were killed 24
weeks after the first injection; the numrs of surivors were 9/20, 9/20
and 18/20, respectively. Control groups received 24 i.p. injections of
0.1 ml tricaprylin or [Link] untreated; the numrs of surivors of bath
sexes were 154/160 and 94/100, respectively. Urethane-treated animls
served as posi ti ve controls. Th numr of lung tumurs per lIuse in
an:ils of bath sexes treated with the highest dose was 0.78, which was

236
signficantly higher (P":0.05) th that in untreated (0.22 in males, 0.17
in fenles) or vehicle-treated (0.24 in maes, 0.20 in femes) controls.
At lower doses the increase in the nurr of lung tururs per lIus wa not
statistically significat. The nurrs of rnce with lung tururs (calcuated
on the basis of surivors of bath sexes) were 5/9 (56%), 4/9 (44%) and 4/18
(22%) in the treated groups, respetively. In control groups, the results,
exressed as a pecentage of hmour incidence, ~re: 22% in males and 17%
in ferles (untreated group) and 28% in males and 20% in fenles (tricaprylin-
treated group). The JXsitive control group treated with urethane had a
100% turur incidence (Stoner et al., 1973).

3.2 oter relevat biological data

(a) Exprimtal system

In rats, daily oral doses ln the diet of approxitely 190 rn/kg hw
were lethl wi thin 142 days. ln rhesus IInkeys, dail Y doses of 5 rn/kg hw
were alist uniforry fatal withn 9-18 days: 1/4 anirls died withn 36
days after daily doses of 2.5 ff/kg l:. Pone-rnrow and renal toxicity
were observed in bath spcies (Schmdt et al., 1953).

In rhesus nonkeys, 1 4 c- lablled pyimthame admistered orall y,

intravenously or intramcularly, is completely but slowly absorbe from
the gastrointestinal tract and undergoes rætalisr; 82-84.5% of the
radioacti vi ty was found ln the urine and 3. 5- 5. 2 % in the faeces (Rollo,
1975; Smth & Schmdt, 1963).
Doses of 25-50 ff/kg admistered ta rats on the 9- l3th day of
pregncy induced malformtions in the erryo skeletons (Akimva, 1972 i
Kotin & Repin, 1973). Injection of 0.1-1.5 ff into the yolk sac before
or up to 4 days after cormcing incubtion induced a high incidence of
malformtions in chick erryos (Stazhevskaya, 1966).

Bailyak & Kharchenko (1973) found that concentrations of 1.5 x 10-'+

g/rn in the fee caused a 3-4 fold increase in the numr of X- linked
recessive lethls in Drosophila melanogaster. Chomosome abnormlities
have be observed in nouse bane-rnrow cells (Matveeva et al., 1973) and
in blastamrs from pregnt rats (Dyban & Udalova, 1967) following treat-
met with pyrirtharne in vivo.
237
 (b) Nan

Following admistration of single oral doses of 100 mg pyrimthame

to hur sujects, the average concentration in the seru fell slCMly,
reaching 0.12 ff/l 9 days after dosing. Urinar excretion, whch accounted
for 20-30% of the drg adistered, oontinued for at least 30 days before
falling below detectale lirts (Smth & Ihig, 1959).

Pyimthamne crosses the placental baier and is also excreted in

the IIther' s milk (Rollo, 1975; Sadoff, 1972).

Chorsorr abnormlities were observed in rrtaphases of bone-rnrow

cells exed in 3 of 5 patients receiving total doses of 200-300 ff
pyrimthame (Bottura & Coutino, 1965). Chorrsoi abnormi ties were
observed in vitro in hum peripheral lymhoes treated with 2.5 x 10-5
g/rn (Bailyak & Khcheno, 1973).

3.3 Cae reports and epidemological studies

It has been suggeste (Sadoff, 1972) tht pyrirthame might be
incrimated in the etiolog of Bukitt' s lymhom, since the drg was
introduced in tropical Africa for the suppression and treabæt of malaria
in 1949, and the first cases of Bukitt' s lymhom ~re reported in the
area 4 years later. However, available evidence does not support the
hypthesis tht Bukitt' s 1 ymhoma is a 'new disease' or that the children
affecte by ths malignancy, or their IIthers, ~re treated with the drg
(Hutt & Bukitt, 1973; William, 1973). An apparent connection exsts,
hCMever, betwn rrlarial infection and cacer in chldren in Africa (see
'General Reks on the Substaces Considered', pp. 24- 25) .

4. Conts on Data Reported and Evluation 1

4. 1 Animl data
In the only study available, pyrirthame prouced a significant
increase in the numr of lung tliurs per lIuse when it was given intra-

1 Se also the section 'Anl Data in Relation to the Evaluation of

Risk to Ma' in the introduction to ths volui, p. 13.

238
peritoneally at high doses. rlhs lirted evidece of cacinogencity
awaits confirmtion 1.
4 . 2 Hl. data
"N case reports or epidemological studies were available to the
Vbrking Group.

IThe WJrkig Group wa also aware of ongoing cacinoenici ty tests in

mice and rats (IA, 1976).

239
 5. References

Akiiva, LM. (1972) Anomlies in the developrt of the rat emryo

skeleton after ch10ridine (pyrimthame). Arkh. Anat. Gistol.
Emriol., 62, 77-88

Bailyak, I. R. & Khrchenko, T. I. (1973) Investigation of the mutagenic

activity of sorr drgs. TsitoL. Geet., 7, 455-458

Bottura, C. & Coutinho, V. (1965) The effect of pyrimthame on the hum

chromosorrs. Rev. Brasil. Biol., 25, 145-147

British Pharncopoia Conrssion (1973) British Phacopoia, London,

HM, p. 405
Cala, P.C., Trenner, N.R. & Boos, R.P., lXing, G.V., Jr, Snth, J.L. &
VandenHeuv1, W.J.A., III (1972) Gas chomtogaphie detennation
of pyrimtharne in tissue. J. agric. Fc: Cher., 20, 337-340

Cavell, G., Coatney, G.R., Field, J.W. & Singh, J. (1955) Cheitherapy of
malaria. wid Hlth Org. M::mogr. Ser., No. 27, pp. 51, 87

Degelis, R.L., Sirns, W.S. & Nichol, C.A. (1975) Quantitative thin-
layer chromatogaphy of pyrimthame and related diarnopyrirdines
in boy fluids and tissues. J. Chomat., 106, 41-49

Dyban, A.P. & Udalova, L.D. (1967) A study of chrolIsorn abrrations at the
ear 1 y stages of m:lian emryogenesis. I. The exprirts wi th the
effect of 2,4-diamo-5-p-chlorophenyl-6-ethyl-pyrirdine (pyrimtha-
mine) on rats. Genetika, 4, 52-65

Girgis, P. & Askam, V. (1974) The deterration of pyrimtharne by

nuclear magnetic resonance spctroscopy. J. Ass. publ. Anal., 12,
55-59

Harvey, S.C. (1975) Antircrobial drugs. In: Osol, A. et aL., ed,

Rengton' s Pharnceutical Sciences, l5th ed., Easton, Pa, Mack,
p. 1159

Hitchings, G.H., Russell, P.B. & Falco, E.A. (1951) 2,4-Diamo-5-phenyl-6-

alkylpyrirdines. US Patent 2,576,939, to Burroughs Wellcorr and Co.
(USA) Inc.

Hutt, M.S.R. & Burkitt, D.P. (1973) Aetiolog of Burkitt's lymhom.

Lacet, i, 439

IAe (1976) IAe Informtion Bulletin on the Surey of Chemcals Being

Tested for Carcinogenicity, No. 6, Lyon, p. 166

Jones, e.R. & King, L.A. (1968) Detection and fluorescent ræasuremnt of
pyrimtharne in urine. Biocher. rnd., 2, 251-259

240
Kastr, E.K., ed. (1975) Facts and Corisons, St Louis, [Link],
Facts and Coisons, Inc., p. 391
Kotin, A.M. & Repin, V.S. (1973) Effect of pyrirthe on nucleic acid
rrtablism in white rat emryos. Ontogenez,!, 128-138

Mashkovski, M.D. (1972) Drug COTIunds, rbsCCM, Mezina, pp. 384-385

Matveeva, V.G., Kerkis, Y.Y. & Osipova, L.I. (1973) Mutagenc effect of
DDB pesticide and chloridine in bone maraw and gerr cells of mice.
Geetika, ~, 67-76

Rollo, LM. (1975) Drugs used in the cheitherapy of malaria. In:

Go, L.S. & Gilm, A., eds, The Pharcological Basis of
Therapeutics, 5th ed., New York, Macmllan, pp. 1053-1058

Royere, G., Duoir, J. & Faure, J. (1972) Dosage de la pydn"éthe dans

les alimts pour animux pa chromtogaphie en phase gazeuse.
Analusis, l, 362-364

Sadoff, L. (1972) Aetiolog of Bukitt's lymhom. Lacet, ii, 1414

Schmdt, L.H., Hughes, H.B. & Schmdt, I.G. (1953) The pharcological
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Smth, c.C. & Ihig, J. (1959) Persistent excretion of pyrirtharne

follawing oral admnistration. Amr. J. trop. Me. Hyg., ~, 60-62

Smth, C.C. & Schmdt, L.H. (1963) Observations on the absorption of

pyrimtharne from the gastrointestinal tract. Ex. Parasitol., 13,
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Stanzhevskaya, T. I. (1966) The influence of chloridine on chick emryo-

genesis. Bjull. eksp. BioL. M:., 61, 85-88

Stecher, P. G., ed. (1968) The Merck Index, 8th ed., Raay, ID, Merck &
Co., p. 892

Stoner, G.D., Shiin, M.B., Kniazeff, A.J., Weisburger, J.H., Weisburger,

E.K. & Gari, G.B. (1973) Test for carcinogenicity of foo additives
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US International Trade Commssion (1976) Synthetic Organic Chemcals, US

Production and Sales, 1974, USI'I Pulication 776, Washington OC,
US Gavernnt Printing Office, p. 101
US Phacopeial Convention, Inc. (1975) The US Pharrcopeia, 19th rev.,
Rokville, Md, p. 430

241
US Tariff Comssion (1954) Sythetic Organic Chemcals, us Production and
Sales, 1953, Report tb. 194, Second Series, Washington OC, US
Goverrt Printing Office, p. 106
WHO (1973) Chtherapy of nalaria and resistace 1:0 antimlarials.
Wld Hlth Org. techn. Rep. Ser., No. 529, pp. 16, 20-21

William, E.H. (1973) Aetiolog of Bukitt' s lymhor. Lacet, i, 1123

242
 SUPPLEARY CDRRGE TO VOLUM 1 - 12

Corrigenda covering Volurs 1 - 6 appeared in Volur 7, others

appeared in Volurs 8, 10, IL "and 12.

V01ui 4

p. 231 1.1 replace Chem. Abstr. No.: 432-88-1 by 542-88-1

p. 253 1.1 replace Chem. Abstr. No.: 1633-83-6 by 1120-7l~4

Volui 9

p. 183 3.1 (a) replace 100 am3 by 0.01 ml

2nd line

Volur 10

p. 24 Reference replace (1975) by (1976)

No. 32

V01ur 11

p. 26 Reference replace (1975) by (1976)

No. 33

p. 222 References replace 576-567 by 576-577

last line

V01ur 12

p. 22 Reference replace (1975) by (1976)

No. 34

p. 123 2.2 delete ¡rom "For informtion " to "see IA (1974). Il

lst paa.

243
 CUMUIATIV INEX 'I IA HONORAHS ON TH EVATION
OF CACINENIC RISK OF CHCA TG MA
Ninrs under lined indicate volur, and ninrs in i talics indicate
page. References to corrigenda are gi ven in paentheses.
Acetarde 7,197
Acriflavinium chloride 13,31
Actinancins 10,29
Adriarcin 10 , 43

Aflatoxins 1,145 (corr. 7,319)

(corr. 8,349)
10,51
Aldrin 5,25
Amanth 8,41
para-Amnoazobenzene 8,53
ortho-Amnoazotoluene 8,61 (corr. 11,295)

4-Amnobipheny 1 1,74 (corr. 10,343)

2-Amno-5- (5-nitro-2-furl) - 1,3, 4-thiadiazole 7 , 143

Am trole 7,31
Anaesthetics, volatile 11 , 285

Aniline 4,27 (corr. 7,320)

Apholate 9,31
Arar te (R) 5,39
Arsenic and inorganic arsenic compunds 2,48
Arsenic (inorganic)
Arsenic pentoxide
Arsenic trioxide
Calcium arsenate
Calcium arsenite
Potassium arsenate
Potassium arsenite
Sodium arsenate
Sodium arseni te
Asbestos 2,17 (corr. 7,319)
Aisite
245
 Anthophyllite
Chsotile
Crocidoli te
Aurame 1,69 (corr . 7,319)
Aurothoglucose 13,39
Azaserine 10 , 73
Aziridine 9,37
2- (1 -Aziridinyl) ethanol 9,47
Aziridy 1 benzoqinone 9,51
Azobenzene 8,75
Bez (c lacridine 3,241
Bez (a L anthacene 3,45
Bezene 7,203 (corr . 11,295)
Bezidine 1,80
Bezo(b lfluoranthene 3,69
Bezo (j L fluoranthene 3,82
Bezo (a L pyrene 3,91
Bezo (e L pyrene 3,137
Bezy 1 chloride 11 , 21 7 (corr. 13,243)
Beryllium and berylliur comunds 1,17
Beryl ore
Beryllium oxide
Beryllium phosphate
Beryllium sulphate
BHC (technical grades) 5,47
Bis (1 -aziridiny 1) morpholinophosphine sulphide 9,55
Bis (2-chloroethy 1) ether 9,117
N~ N-Bis (2-chloroethyl) -2-naphthylame 4,119
Bis (chloromthy 1) ether 4,231 (corr . 13,243)
1,4-Butanediol dimthanesulphonate 4,247
ß-Butyrolactone 11 , 225
y-Butyrolactone 11 , 231
Cadmum and cadmum carunds 2,74
11 , 39

246
 Cadmum acetate
Cadmum carbonate
Cadmum chloride
Cadmum oxide
Cadr um :Jder
Cadmum sulphate
Cadmum sulphide
Catharidin 10 , 79

Cabary 1 12 , 37

Cabon tetrachloride l,53

eaisine ~,83
Chlorarucil 9,125
Chloramhenicol 10 ,85

Chlormdinone acetate ~, 149

Chlorobezilate ~,75
Chloroforr l,61
Chloroithy 1 rnthy 1 ether 4,239
Chloropropham 12 , 55

Chloroqine 13 , 4 ?

Cholesterol 10,99
Chomium an inorganic chromium cauunds 2,100
Barium chromte
Calcium chromate
Chomic chromte
Choic oxide
Chorum acetate
ChOIum carbonate
ChOIum dioxide
Chornum phosphate
Chrorum trioxide
Led chrorrte
Potasium chromte
Potassium dichromte
Soium chromte
Sodium dichrorrte

247
 Strontium chrte
Zinc chromate hydroxide

Chsene 3,159
Chsoidine 8,91
C.I. Disperse Yellaw 3 8,97
Ci trus Red No. 2 8,101
[Link] 10 ,113

Cycasin 1,157 (corr . 7,319)

10,121
Cyclochlorotine 10,139
Cyclophosphamde 9,135
Daunomcin 10,145
D & C Red No. 9 8,107
DDT and associated substaces 5,83 (corr . 7-,320)
DDD (IDE)
DDE

Diacety lamoazotoluene 8,113

Diallate 12 , 69

2, 6-Diamno- 3- (phenylazo) pyridine (hydrochloride) 8,117

Diazepa 13,5 ?

Diazoræthane 7,223
Dibz(a~ hJacridine 3,247
Dibnz (a ~ j J acridine 3,254
Dibz (a~ hJanthacene 3,178
7H-Dibzo (c ~ g J cabazole 3,260
Dibzo(h~ rst Jpetaphene 3,197
Dibnzo(a~ e Jpyrene 3,201
Dibzo(a~ hJpyrene 3,207
Dibzo (a ~ i J pyrene 3,215
Dibnzo(a~ L Jpyrene 3,224
ortho-Dichlorobezene 7,231
para-Dichlorobenzene 7,231
3, 3 ' -Dichlorobenzidine 4,49
Dieldrin 5,125
Diepoxybutane 11 , 115

248
l, 2-Diethy lhydrazine !,153
Diethylstilbstrol ~,55
Diethyl sulphate 4,277
Dig 1 ycidy 1 resorcinol ether 11 , 125
Dihydrosafrole 1,170
10,233
Dimthsterone 6,167
3,3' -Dimthoxybenzidine (o-Dianisidine) 4,41
para-Dimthy lamoazobezene 8,125
para-Dimthy lamobezenediazo sodium sulphonate 8,147
trans-2 ( (Dimthylarno)methylimo)-5-(2- (5-nitro-
2-furl)vinyl)- 1,3, 4-oxadiazole 7,147
3, 3' -Dimthy 1bnzidine (o-Tolidine) 1,87
Dirthy lcarbay 1 chloride 12 , 77
l, 1 -Dirthy lhydrazine 4,137
1,2-Dirthylhydrazine !,145 (corr. 7,320)
Dimthyl sulphate !,271
Dini trosopetarthy lenetetrarne 11 , 241

l, 4-Dioxane 11,247
Disulfiram 12 , 85

Di thanol 13 , 75
Dulcin 12 , 9 7

Endrin 5,157
Epichlorohydrin 11 , 131

1 -Epxyethy 1-3, 4-epoxycyclohexe 11 , 141

3, 4-Epxy-6-methy lcyclohexy lmthy 1 - 3, 4-epoxy-

6-rnthy lcyclohexane carboxy late 11 , 1 47

cis-9,10-Epxysteaic acid 11 , 153

Etiny loestradiol 6,77

Ethionarde 13,83
Ethy lene oxide 11 , 157

Ethy lene sulphide 11 , 257

Ety lenethourea 7,45

Etyl methesulphonate 7,245
Ethy 1 selenc 12 , 107

249
Etyl tellurac 12 , 11 5
Ethynodiol diacetate 6,173
Evans blue 8,151
Ferba 12,121 (corr . 13,243)
2- (2-Formlhydrazino) -4- (5-nitro-2-furl) thiazole 7,151 (corr . 11,295)
Fuarenon-X 11 , 1 69
Gl ycidaldehyde 11 , 1 75
Glycidyl oleate 11 , 1 83
Gl ycidy 1 steaate 11 , 187
Griseoful vin 10,153
Haemti te 1,29
Heptachlor and i ts epoxide 5,173
Hycanthone 13 , 91

Hycanthone llsylate 13,92

Hydrazine 4,127
4-Hydxyazobezene 8,157
8-Hydroxyquinoline 13,101
Hydroxysenkirkine 10,265
Indeno(l, 2, 3-cdJpyrene 3,229
Iron-dextran comlex 2,161
Iron-dextrin comlex 2,161 (corr. 7,319)
Iron oxide 1,29
Iron sorbitol-citric acid complex 2,161
Isatidine 10,269
Isonicotinic acid hydrazide 4,159
Isosafrole 1,169
10,232
Jacobine 10,275
Lasiocarine 10,281
Lead sal ts 1,40 (corr . 7,319)
(corr. 8,349)
Led acetate
Led arsenate
Lead carbonate
Lead phosphate

250
 Led subcetate
Lete 12 , 131
Lindane 5,47
Luteoskyrin 10,163
Magenta 4 , 57 (corr. 7, 320)
Maleic hydrazide 4,173
Maeb 12 , 137
Maornstine (dihydrochloride) 9,157
Mephalan 9,167
Meoxyrogesterone acetate 6,157
Melphalan 9,167
Merphalan 9,167
Mestranol 6,87
Methoxychlor 5,193
2-Methylaziridine 9,61
Methy lazoxythanol acetate 1,164
10,131
Methyl carbate 12,151
N-Methy 1 -N, 4-dinitrosoaniline 1,141
4,4' -Methylene bis (2-chloroailine) 4,65
4,4' -Methylene bis (2-methylaniline) 4,73
4,4' -Methylenedianiline 4 , 79 (corr. 7, 320)
Methyl rnthanesulphonate 7,253
N-Methyl -N' -ni tro-N-ni trosoganidine 4,183
Methy 1 red 8,161
Methy 1 selenc 12 , 1 61
Methy 1 thiouracil 7,53
Metronidazole 13,113
Mirex 5,203
Mitorncin C 10 , 1 71
Monocrotaline 10,291
~nuron 12 , 167
5- (Morpholinathy 1) - 3- ( (5-ni trofurfur lidee) -
amno J - 2-oxazolidinone 7,161
Mutad gas 9,181 (corr. 13,243)

251
1 -Naphthy lame 4,87 (corr . 8,349)
2-Naphthylame 4,97
Native carageeans 10,181 (corr . 11,295)
Nickel and nickel canunds 2,126 (corr. 7,319)
11, 75

Nickel acetate
Nickel carbonate
Nickel carbony 1
Nickelocene
Nickel oxide
Nickel poer
Nickel subsulphide
Nickel sulphate
Niridazole 13,123
4-Nitrobiphenyl 4,113
5-Nitro- 2-furaldehyde semcarbazone 7,171
1 ( (5-Ni trofurfur lidene) amo ) - 2- imdazolidinone 7,181
N- ( 4- (5-Ni tro- 2-fur 1) - 2-thazol y 1) acetamde 1,181
7,185
Nitrogen rntad (hydrochloride) 9,193
Nitrogen rntad N-oxide (hydrochloride) 9,209
N-Ni trsodi -n-butylame 4,197
N-Ni trosoiethylame 1,107 (corr. 11,295)
N-Nitrosoimthylame 1,95
N-Nitrosothylurea 1,135
N-Nitrosothy lurea 1,125
N-Nitroso-N-rnthy lurethane 4,211
Norethsterone 6,179
Norethsterone acetate 6,179
Norethynodel 6,191
Norgestrel 6,201
üchratoxi A 10,191
Oestradiol - 17 ß 6,99
Oestradìol rntad 9,217
Oestriol 6,117

252
Oestrone 6,123
Oil Orange SS 8,165
Orange i 8,173
Orange G 8,181
OXazepa 13 , 58

OXtholone 13,131
Oxhenutazone 13,185
Parasorbic acid 10 , 199
Patulin 10,205
Penicillic acid 10 , 211
Phenacetin 13 , 1 41
Phenicarbazide 12,177
Phenobabi tal 13,157
Phenobabital sooium 13,159
Phenxybezame (hydrochloride) 9,223
Phenylbutazone 13 , 183
Phenytin 13 , 201
Phenytin soium 13,202
Pol ychlorinated bipheny ls 7,261
Ponceau MX 8,189
Ponceau 3R 8,199
Ponceau SX 8,207
Potassium bis (2-hydoxyethyl) dithocarbate 12 , 183
Progesterone 6,135
Pronetalol hydrochloride 13,227
1,3-Propae sultone 4,253 (carr. 13,243)
Propha 12,189
ß-Propíolactone 4,259
n-Propyl cabate 12 , 201

Propylene oxide 11, 191

Propy lthouracil 7,67

Pyiithe 13,233
Qutozene (Pentachloroni trobezene) 5,211
Reserin 10,217
Retrorsine 10,303
Ridde11iine 10,313
253
Saccharated iron axide 2,161
Safrole l,169
10,231
Scar let red 8,217
Selenium and selenium cCIuns 9,245
Sercarbazide (hydrochlaride) 12 , 209

Seeciphylline 10,319
Senirkine 10,327
Soium diethyldithocarbate 12,217
Sot, tas and shale ails 3,22
Sterigntocstin 1,175
10,245
Streptozotocin 4,221
Styrene axide 11 , 201

Sudan r 8,225
Sudan ir 8,233
Suda III ~, 241

Sudan brCM RR 8,249

Sudan red 7B 8,253
Sun set yellow FC 8,257
Tanic acid 10,253
Tanins 10,254
Terpne pol ychlorinates (Strobae (R) ) 5,219
Testosterone 6,209
Tetraethy llead 2,150
Tetrarthy llead 2,150
Thoacetade 7,77
Thiouracil 7,85
Thiourea 7,95
Thiram 12 , 225

Trichloroethylene 11,263
Trichlorotriethylame hydrochloride 9,229
Triethylene glycol diglycidyl ether 11,209
Tris (aziridinyl) -para-benzoqinone 9,67
Tris (l-aziridinyl)phosphine oxide 9,7.5

254
Tris (l-aziridinyl)phosphine sulphide 9,85
2,4,6-Tris (1 -aziridinyl) -s-triazine 9,95
Tris (2-methyl-l-aziridinyl)phosphine oxide 9,107
Try blue 8,267
Uracil mustard 9,235
Urethan 7,111
Viryl chloride 7,291
4-Viny lcyclohexene 11 , 277
Yellow AB 8,279
YellOW OB 8,287
Zectran 12,237
Zineb 12 , 245
Ziram 12,259

255
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