Vol.3/Issue1/Jan.-Feb. 2021 Inter. J.

Pharma O2 ISSN: 2582-4708

International Journal of PharmaO2

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(IJPO: A Peer-reviewed Bi-monthly online journal)

Review Article
Genotoxicity of Drugs: Mechanisms, Testing Guidelines and Methods
for Evaluation
Leena T. Shinde*, Snehal B. Gaikwad, Riyaa R. Patel and Dr. M.R. Kumbhare
S.M.B.T. College of Pharmacy, Nandi Hills, Dhamngaon, Nashik-422401, India

ARTICLE INFO ABSTRACT

Article history: It is estimated that 80% of world population rely on traditional herbal
Received: 25/12/2020; medicine for primary health care. With the rising utilization of herbal
Revised: 09/01/2021 products, safety and efficacy of herbal medicine have become a public
Accepted: 12/01/2021; health concern. Adverse health effects associated with herbal products
Available online:
could be attributed to both inherent toxic effects of herbal medicine and
23/01/2021.
toxicities induced by adulterants.Although often perceived as innocuous
Key Words: by the general public, many herbs phytochemicals that are either directly
Genotoxicity, reactive towards DNA or likely to disturb cellular homeostasis, cell cycle,
Cancer, andr genome maintenance mechanisms; this may translate into
Herbal, genotoxicity, carcinogenicity, or co-carcinogenicity. Genotoxicity refers
Carcinogenicity, to the deleterious effect of a chemical compound or a physical event on
DNA. the genetic material; such genotoxic events are considered hallmarks of
cancer risk. The numerous genome maintenance mechanisms of the cell
Please cite this article and may not lead to cancer. The long-term safety evaluation is probably
as: Shinde, L.T. et al.,
(2021). Genotoxicity of better investigated through carcinogenicity, which denotes the capacity of
Drugs: Mechanisms, a chemical substance or a mixture of chemical substances to induce
Testing Guidelines and cancer or increase its incidence.
Methods for Evaluation.
3(1), 021-028.
©2021 Published by International Journal of PharmaO2. This is an open access article.
*
Corresponding author: Ms. Leena T. Shinde, Final Year B. Pharmacy; S.M.B.T. College of Pharmacy, Nandi Hills,
Dhamangaon, Nashik-422401, India. Contact-+91 9623195719, e- mail: [email protected]

Introduction to mutations. The permanent, hereditary changes

In genetics, genotoxicity describes the property can affect either somatic cells of the organism or
of chemical agents that damages the genetic germ cells to be passed on to future generations.
information within a cell causing mutations, Cells prevent expression of the genotoxic
which may lead to cancer. While genotoxicity is mutation by either DNA repair or apoptosis;
often confused with mutagenicity, it is important however, the damage may not always be fixed
to note that all mutagens are genotoxic, however, leading to mutagenesis. Genotoxicity is a word in
not all genotoxic substances are mutagenic. The genetics defined as a destructive effect on a cell's
alteration can have direct or indirect effects on genetic material (DNA, RNA) affecting its
the DNA: the induction of mutations, mistimed integrity. Genotoxins are mutagens; they can
event activation, and direct DNA damage leading cause mutations. Genotoxins include both
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radiation and chemical genotoxins. A substance lesions leading to gene mutation that are as
that has the property of genotoxicity is known as follows, (EMEA, 2006)
a genotoxin. (IV) 1. Direct pair
Toxic substances which directly shows their 2. Base excision pair
impact on cell viability is for the most part 3. Nucleotide excision pair
mainly referred to as cytotoxins. All the 4. Mismatch repair
chemicals which produce genetic knock out
leading to mutation are known as genotoxic. Direct Repair
Further, some classes of substances which are Direct repair acts by removing or reversing the
capable of damaging and interacting the genome DNA lesions. These lesions can occur due to
within chemical genotoxins. Genotoxins include alkylating agents. Direct repair is carried out by
both radiation and are genotoxic (NCBI, 2020). specific enzymes called alkyl guanine-DNA
Genotoxins can be of the following category methyltransferases (AGMT), which remove the
depending on its effects; alkyl group from the guanine residue of DNA
1. Carcinogens or cancer-causing agents. and transfers it to one of its cysteine residues.
2. Mutagens or mutation is causing agents. Alkylating agents are reactive compounds that
3. Teratogens or congenital disability is causing can transfer methyl or ethyl groups to a DNA
agents. base, thereby chemically modifying the base. A
Hence, genotoxicity can be described as the particularly important type of damage is
capacity of a substance to cause damage to the methylation of the O6 position of guanine,
genetic information inside the cell. This DNA because the product, O6-methylguanine, forms
damage could result in mutations, thus promoting complementary base pairs with thymine instead
carcinogenesis or establishing the framework for of cytosine. This lesion can be repaired by an
congenital disorders. The damage which is enzyme (called O6-methylguanine
caused by agents of genotoxins may also involve methyltransferase) that transfers the methyl
in direct interaction with the DNA, and resulting group from O6-methylguanine to a cysteine
either in the base substitutions, frame-shift residue in its active site (OECD, 1981).
mutations, and also even double-stranded breaks. Only a few types of DNA damage are repaired in
In some other cases, the substances which are this way, particularly pyrimidine dimers resulting
genotoxic may also interact with various types of from exposure to ultraviolet (UV) light and
proteins are either engaged with replication or alkylated guanine residues that have been
maintaining chromo-somal stability. modified by the addition of methyl or ethyl
Toxicological studies have encountered a groups at the O6 position of the purine ring. UV
compelling evolution during the previous decade, light is one of the major sources of damage to
with enough outstanding emphasis being placed DNA and is also the most thoroughly studied
on chronic toxicity, teratogenicity, form of DNA damage in terms of repair
carcinogenicity, mutagenicity, etc. (V) mechanisms. The major type of damage induced
by UV light is the formation of pyrimidine
Mechanism dimers, in which adjacent pyrimidines on the
One of the endpoints of genotoxicity is gene same strand of DNA are joined by the formation
mutations. Mutagenic chemicals cause of a cyclobutane ring resulting from saturation of
predominantly gene mutations, which are the double bonds between carbons 5 and 6
generally not lethal but can form a major threat (Mohamad, S. et al., 2017).
to the integrity of chromosomes and viability of
cells. Depending on the specific classes of DNA Base excision repair
lesions, one or more DNA repair pathways In excision repair, the damaged DNA is
become active. Four of the 4 major DNA repair recognized and removed, either as free bases or
pathways are involved in the repair of DNA as nucleotides. The resulting gap is then filled in
by synthesis of a new DNA strand, using the

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undamaged complementary strand as a template. relationship between HNPCC and defects in

The repair of uracil-containing DNA is a good mismatch repair was discovered in 1993, when
example of base-excision repair, in which single two groups of researchers cloned the human
damaged bases are recognized and removed from homolog of MutS and found that mutations in
the DNA molecule. Uracil can arise in DNA by this gene were responsible for about half of all
two mechanisms: (1) Uracil (as dUTP HNPCC cases. Subsequent studies have shown
[deoxyuridine triphosphate]) is occasionally that most of the remaining cases of HNPCC are
incorporated in place of thymine during DNA caused by mutations in one of three human genes
synthesis, and (2) uracil can be formed in DNA that are homologs of MutL (EMEA, 2006;
by the deamination of cytosine. The excision of Mohamad, S. et al., 2017).
uracil in DNA is catalyzed by DNA glycosylase, Guidelines
an enzyme that cleaves the bond linking the base OECD Guidelines
(uracil) to the deoxyribose of the DNA For the Testing of Chemicals (OECD TG) are a
backbone. This reaction yields free uracil and an set of internationally accepted specifications for
apyrimidinic site—a sugar with no base attached. the testing of chemicals decided on by the
(EMEA, 2006) Organisation for Economic Co-operation and
Development (OECD). They were first published
Nucleotide-excision repair in 1981. They are split into five sections:
The result of DNA glycosylase action is the Section 1: Physical Chemical Properties
formation of an apyridiminic or apurinic site Section 2: Effects on Biotic Systems.
(generally called an AP site) in DNA. Similar AP Section 3: Environmental Fate and Behaviour.
sites are formed as the result of the spontaneous Section 4: Health Effects
loss of purine bases, which occurs at a significant Section 5: Other Test Guidelines.
rate under normal cellular conditions. For ICH Guidelines
example, each cell in the human body is This guidance replaces and combines the ICH
estimated to lose several thousand purine bases S2A and S2B Guidelines. The revised guidance
daily. These sites are repaired by AP describes internationally agreed upon standards
endonuclease, which cleaves adjacent to the AP for follow-up testing and interpretation of
site. The remaining deoxyribose moiety is then positive results in vitro and in vivo in the
removed, and the resulting single-base gap is standard genetic toxicology battery, including
filled by DNA polymerase and ligase. Whereas assessment of no relevant findings. This
DNA glycosylases recognize only specific forms guidance is intended to apply only to products
of damaged bases, other excision repair systems being developed as human pharmaceuticals
recognize a wide variety of damaged bases that (Kamath, G.H. et al., 2013; Gallowa, S.M.,
distort the DNA molecule, including UV-induced 2017).
pyrimidine dimers and bulky groups added to
DNA bases as a result of the reaction of many SCHEDULE Y
carcinogens with DNA. (Savale, S.K., 2018) In the Drugs and Cosmetics Rules, 1945
(hereinafter referred to as said rules), (1) in Part
Mismatch repair X-A, after rule 122-DA, the following shall be
The importance of this repair system is inserted, namel,
dramatically illustrated by the fact that mutations 122-DAA.: Definition of Clinical trial.- For the
in the human homologs of MutS and MutL are purpose of this Part, ―Clinical trial‖ means a
responsible for a common type of inherited colon systematic study of new drug(s) in human
cancer (hereditary nonpolyposis colorectal subject(s) to generate data for discovering and /
cancer, or HNPCC). HNPCC is one of the most or verifying the clinical, pharmacological
common inherited diseases; it affects as many as (including pharmacodynamic and
one in 200 people and is responsible for about pharmacokinetic) and /or adverse effects with the
15% of all colorectal cancers in this country. The

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objective of determining safety and / or efficacy battery.In general, the three standard
of the new drug. genotoxicity test battery is adequate for
In the said rules for Schedule Y, the following evaluation of genotoxicity of NCE (New
Schedule shall be substituted, namely; Chemical Entities).
SCHEDULE- ‗Y‘ ( 122A, 122B, 122D, 122DA, S2A: Genotoxicity Guidance on specific aspects
122DAA and 122E) (Shah, S.U., 2016; Gallowa, of regulatory genotoxicity tests for
S.M., 2017) pharmaceuticals.
Standard Test Battery for Genotoxicity S2B: Genotoxicity: A standard battery for
There are two fundamental areas in which genotoxicity testing of pharmaceuticals.
harmonization of genotoxicity testing is S2A Guideline: The S2A guidelines cover the
considered necessary is strategic issues and protocol design for in-
1. Identification of a standard set of tests to be vitro and in-vivo genotoxicity test.
conducted for registration. The standard test battery for genotoxicity
2. The extent of confirmatory experimentation recommends the following for genotoxicity
in in-vitro genotoxicity tests in standard Evaluation (Shah, S.U., 2016; EMEA, 2006).
Table 1: Standard Test for Genotoxicity Evaluation
TG 471 Bacterial Reverse mutation test ( Ames test)
TG 472 Genetic toxicology; Escherichia coli, reverse assay
TG 473 In-Vitro Mammalian Chromosome Aberration
TG 474 Mammalian Erythrocyte Micronucleus
TG 475 Mammalian Bone Marrow Chromosome Aberration
TG 476 In-Vitro Mammalian Cell Gene Mutation
TG 477 Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila
melanogaster.
TG 478 Genetic Toxicology: Rodent Dominant Lethal Test
TG 479 Genetic Toxicology: In-Vitro Sister Chromatid Exchange Assay in
Mammalian cells.
TG 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene
TG 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic recombination
Assay.
TG 482 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA
synthesis in mammalian cells In-Vitro.
TG 483 Mammalian Spermatogonial Chromosome Aberration Test
TG 484 Genetic Toxicology: Mouse Spot Test
TG 485 Genetic Toxicology: Mouse Heritable Translocation Assay
TG 486 Unscheduled DNA Synthesis (UDS) Test with Mouse Liver cells In-
Vitro.
TG 487 In-Vitro Mammalian Cell Micronucleus Test.
Testing for gene mutation in bacteria: In-vitro: Procedure- There are two methods; 1. Plate
cytogenetic evaluation of chromosomal damage incorporation method 2. Pre incubation method
with mammalian cells or mouse lymphoma Suspensions of bacterial cells are exposed to the
assay. test substance in the presence and in the absence
In-vivo: test for chromosomal damage using of an exogenous metabolic activation system.
rodent hematopoietic cells. Treatment mixture is incubated and then mixed
In-Vitro testing methods with an overlay agar plating onto minimal
Ames test (Bacterial reverse mutation test) medium. After two or three days of incubation,
Bacteria: 1. Salmonella typhimurium: TA1535; revertant colonies are counted and compared
TA1537 or TA97a or TA97; TA98 and TA100. (Fig. 1).

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Principle- After identifying the mutation it strain to grow in the absence of amino acids. The
reverts it back and restores the functional bacterial reverse mutation test being rapid,
capability of the mutant cell to synthesize inexpensive and easy to perform is commonly
Histidine. In this test the reverent bacteria cells used as an initial screening test for genotoxicity
are identified by the ability of the parent test or mutagenicity (OECD, 1981).

Fig. 1: Ames Test

TG487: INVITRO MAMALIAN CELL chemical.. Harvested and stained interphase cells
MICRONUCLEUS TEST-2010 are analysed.Treated with a cytokinesis blocker;
Principle- detection of the frequency of this is easily achieved by scoring only binucleate
micronuclei. cells. Assay detects the activity of clastogenic
Procedure- Cell cultures of human or other and an eugenic chemicals (Fig. 2) (NCBI, 2020).
mammalian origin are exposed to the test

Fig. 2: In-Vitro Mammalian Cell Micronucleus Test 2010

TG 474: Mammalian Erythrocyte treatment and smear preparations are made and
Micronucleus Test stained.
Principle- Detection of damage induced by the Take number and sex of animals: 1.treated and
test substance to the chromosomes or the mitotic 2.control group must include at least 5 analysable
apparatus of erythroblasts, by analysis of animals per sex and then animals are exposed to
erythrocytes as sampled in bone marrow and/or the test substance by an appropriate route, after
peripheral blood cells of animals. 1, 2, or more treatments at 24 h intervals.
Procedure- Bone marrow = the animals are The limit dose has been used, and dosing
sacrificed, bone marrow extracted, and continued until the time of sampling also be
preparations made and stained. Peripheral blood administered as a split dose. It can be given by 2
= the blood is collected at appropriate times after ways
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1) Treated with once. 2) 2 or more daily Blood: tail vein or other appropriate blood
treatments vessel, smear preparations are made and then
1st: Samples of bone marrow =24hr, peripheral stained
blood twice =36hr DNA specific stain e.g. acridine orange or
2nd: bone marrow samples collected once Hoechst 33258 plus pyronin-Y and at the end
between 18 and 24 hours, peripheral blood: observe the presence of micronuclei (Fig.3).
sampling between 36 and 48 hours. (NCBI, 2020; OECD, 1981)
Bone marrow cells are usually obtained from the
femurs or tibias and stained using established
methods.

Fig. 3: Mammalian Erythrocytes Micronucleus Test

TG 473: In-Vitro mammalian chromosomal And Treatment of results we observe that % of
abberation test cells with structural chromosome aberration.
Principle: metaphase cells are analysed (OCED, 1981)
microscopically for the presence of chromosome In-vivo genotoxicity testing methods: The in-
aberrations. And identify the agents which can vivo genotoxicity test or assays are done
cause structural mutations in chromosomes or supplemental to in-vitro assay if an in-vitro
chromatids, chromatid mutation being the positive result is obtained. Some of the in-vivo
common. Other type of chromosomal changes tests done are as follows,
like polyploidy and duplication can also be found In-vivo comet assay: It is one of the commonly
using this test. used in-vivo test used for hazard assessment of
Procedure: Treatment of test with lymphocytes agents which have potential for genotoxicity or
started at about 48 hours after mutagenicity. It helps in detecting the DNA
mitogenicstimulation. Cells should exposed to damage and detects a broad variety of primary
the test substance both with and without DNA lesions which cannot be identified by any
metabolic activation for 3-6 hours.sampled at a other tests. This test can be applied to a wide
time equivalent to about 1.5 normal cell cycle variety of tissues or any special cell types. Being
length after the beginning of treatment. sensitivity to even low level of DNA damage it
Chromosome preparation: culture treated with requires only small amount of cells per sample
Colcemid or colchicine 3 hr prior to harvesting, and it can be completed in a short period of time.
process involves hypotonic treatment of the cells, In-vivo micronuclei test/In-vivo chromosome
fixation and staining. aberration test: It is a test done to identify the
Analysis: should be independently coded before damage done chromosome or spindles. On
microscopic analysis. At least 200 well-spread exposure to the mutagen the cell may undergo
metaphases should be scored per concentration damage and on division it will form smaller
and control micronucleus additional to the main nucleus.
it is important to record polyploidy and (EMEA, 2006)
endoreduplication when these events are seen.
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Conclusion 6. The Central Drugs Standard Control

Genotoxins are agents that can interact with the Organisation (CDSCO). (2020).
DNA thus causing mutations and damaging its https://rgcb.res.in/documents/Schedule-Y.pdf
structure and may lead to cancer. They act by 7. International conference on harmonization
changing the chromosomal structure by addition, (ICH). (2011). ICH of technical requirements for
deletion, duplication, forming rings etc. The registration of pharmaceuticals, for human use
mutations may lead to a wide variety of diseases ICH harmonized tripartite guidline guidance on
to cancer. It is very important to do genotoxicity genotoxicity testing and data interpretention for
studies so as to avoid the potential damage that pharmaceuticals intended for human use S2(R1)
can be caused by it. These genotoxicity tests are Current Step 4 version dated 9 November 2011.
done to identify if a drug or other substance have 8. Kamath, G.H. and Rao, K.S. (2013).
the potential to cause mutation and genotoxicity. Genotoxicity guidelines recommended by
By doing so they help us identifying the hazards International Conference of Harmonization
in the early stage of drug development itself. (ICH),
Identification of the genotoxic agents helps us https://link.springer.com/protocol/10.1007%2F97
understand the mechanism of the mutation and 8-1-62703-529-3_24#citeas
genotoxicity thereby paving us way to better 9. Gallowa, S.M. (2017). International
prevent the frequency of such mutation and regulatory requirements for genotoxicity testing
genotoxicity. The development of broad range of for pharmaceuticals used in human medicine, and
short-term assays for genotoxicity serves to their impurities and metabolites. Applied Genetic
identify many mutagens and their relationship Toxicology: From Principles to Practice, 58(5),
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