DARU Vol. 17, No.

1 2009
13

Evaluation of antimicrobial effectiveness of ophthalmic drops

according to the pharmacopeial tests criteria
1
Samadi N., 2Tarighi P., 1Fazeli M.R., 3Mehrgan H
1
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of
Medical Sciences, 2Department of Microbiology, School of Basic Sciences, Islamic Azad
University (North Tehran Branch), 3Department of Pharmaceutics, Faculty of
Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

Received 16 July 2006; Revised 26 Oct 2008; Accepted 28 Oct 2008

ABSTRACT
Background: In this study antimicrobial effectiveness test was performed on eye-drops
which had high microbial contaminations in hospital practice to find out whether their
antimicrobial efficacies affect the magnitude of microbial contamination during their uses.
Materials and Methods: Artificial tear, atropine sulfate, betamethasone, homatropine
hydrobromide, phenylephrine hydrochloride, phenylephrine zinc, pilocarpine
hydrochloride, tetracaine hydrochloride and tropicamide eye-drops were subjected to the
United States Pharmacopeia (USP) and British Pharmacopeia (BP) antimicrobial
preservative effectiveness tests.
Results: The results of this study showed that eight out of the nine products met the BP 'B'
and USP criteria. The preservative employed in phenylephrine zinc eye-drop did not
possess adequate antimicrobial activity against P. aeruginosa. Other eye-drops showed
appropriate reductions in bacterial viability after 6 hrs, 24 hrs and 7 days, but showed a
very low bacterial recovery after 28 days which didn’t comply with the no recovery (NR)
term of BP 'A' criteria. Since viable microbial counts were usually determined by plate
count method, it seems that the term of NR should define an acceptable range.
Conclusion: The results indicated that there is not a clear correlation between antimicrobial
efficacy testing of eye-drops and the rate of their microbial contamination while are being
used. Other factors such as hygienic practices of eye-drops, proper bottle design and
training of patients could influence their microbial contaminations. Regulation of in-use
efficacy testing of eye-drops which is influenced by the environment, the frequency and
technique of use, might be essential.
Keywords: Antimicrobial effectiveness test, challenge test, preservative, eye-drop,
ophthalmic drop

INTRODUCTION States Pharmacopeia (USP) (11), but sampling

Ophthalmic drops are sterile preparations which times and logarithmic (log) reduction performance
are usually packed in multi-dose containers. In criteria of the BP are more stringent than those in
their uses, microbial contamination may lead to the USP. It has been reported that there is a
product degradation or result in ocular infection correlation between the performance of eye-drops
(1-4). Protection of these multiple dose products according to the BP antimicrobial efficacy test
against microbial contamination is usually and magnitude of microbial contamination during
achieved by addition of a suitable preservative their uses (12), suggesting other investigators to
extend similar studies on other multi-dose
system (5-7). The antimicrobial effectiveness test
products. The aim of this study was to determine
is designed to provide a laboratory test that the antimicrobial efficacy of eye-drops produced
gauges the level of antimicrobial activity by a by Iranian manufacturers according to the both
pharmaceutical product and to evaluate how well United States and British Pharmacopeia to assess
a product withstands microbial contamination the correlation of antimicrobial performance of
while being used (8, 9). The method is similar in the eye-drops with magnitude of microbial
both British Pharmacopeia (BP) (10) and United contamination during their uses.

Correspondence: [email protected]
 Antimicrobial effectiveness of ophthalmic drops 14

MATERIALS AND METHODS in P. aeruginosa count appeared well preserved

against all the challenging organisms (3 logs
Test samples reduction).
The tested eye-drops were artificial tear, 1% After 28 days, there was no bacterial recovery
atropine sulfate, betamethasone, 2%homatropine from betamethasone eye-drop, while the number
hydrobromide, 5% phenylephrine hydrochloride, of P. aeruginosa in phenylephrine zinc increased.
phenylephrine zinc, 2% pilocarpine hydro- Other eye-drops showed no increase in bacterial
chloride, tetracaine hydrochloride and 1% tropic- counts after 28 days which were about 10-50 CFU
amide which showed high microbial ml-1 of the products.
contamination during hospital uses (13). All In all cases the number of fungi after 7 and 14
products except phenylephrine zinc which had days were acceptable and those after 28 days were
only benzalkanium chloride, contained at least 2 logs lower than the initial counts (Table
benzalkanium chloride and ethylene diamine 2).
tetra-acetic acid (EDTA) as antimicrobial As shown in Table 3, more than 2 logs reduction
preservative system. All samples were produced in bacterial counts (after 30 min) and more than 3
by Iranian manufacturers. logs reduction in fungal counts (after 24 hrs) were
observed for all eye-drops except phenylephrin
Antimicrobial effectiveness testing zinc.
Possible antimicrobial effects of all samples were The results of this study showed that eight out of
eliminated and validated following the method the nine products met the BP 'B' criteria and USP
proposed by the USP under validation of while all of them except artificial tear were highly
microbial recovery from pharmacopeial articles contaminated during hospital uses (Table 4). The
using Fluid Casein Digest-Soy Lecithin- preservative employed in phenylephrine zinc eye-
Polysorbate 20 medium (Merck) (14, 15). The drop did not possess adequate antimicrobial
unused eye-drops were subjected to the BP (10) activity against P. aeruginosa to be able to bring
and USP (11) preservative challenge tests using about acceptable low levels of microbial
Escherichia coli ATCC 8739, Pseudomonas contamination as demanded by regulatory bodies.
aeruginosa ATCC 9027, Staphylococcus aureus Therefore another effective antimicrobial
ATCC 6538, Candida albicans ATCC 10231 and preservative system for this formulation should be
Aspergillus niger ATCC 16404 as test organisms. employed.
To determine the microbial killing rate, the eye- Other eye-drops showed appropriate reductions in
drops were inoculated with challenging bacterial viability after 6 hrs, 24 hrs and 7 days,
microorganisms at a final concentration of 105-106 except a very low bacterial recovery after 28 days
CFU ml-1 and the viable organisms were (10-50 CFU ml-1) which didn’t comply with the
determined 30, 90 and 180 min after inoculation no recovery (NR) term of BP 'A' criteria. Viable
for bacteria and 24 hrs for fungi. microbial count, as recommended in both
Aerobic viable cell count of 1:10 dilution of Pharmacopeia, was determined by plate count
product in neutralizer was determined by plate method using 1 ml of 1:10 dilution of product in
count method and 0.5 log increase in colony neutralizer and no bacterial growth means that the
forming units was accounted for variability. number of challenging bacteria was reduced to
lower than 10 CFU per ml of the product instead
RESULTS AND DISCUSSION of NR. Therefore, it seems that the term of NR
The antimicrobial preservative efficacy of the should define an acceptable range.
eye-drops challenged with E. coli, S. aureus and
P. aeruginosa is shown in Table 1. After a contact CONCLUSION
time of 6 hrs all the eye-drops except All the eye-drops except artificial tear were
phenylephrine zinc which showed only 1.7 logs contaminated after 1, 2, 4 and 7 days of hospital
reduction in P. aeruginosa initial count, reduced uses with different rate of contamination from
at least 2 logs of all bacterial counts. 23.5% for atropine sulfate to 84.4% for tetracaine
Most of the eye-drops eradicated the inoculated hydrochloride (Table 4) while similar results were
microorganisms more than 3 logs in 24 hrs and obtained for all the eye-drops except phenylehrine
also 7 days, except phenylephrine zinc. The zinc when subjected to antimicrobial effectiveness
number of P. aeruginosa in phenylephrine zinc testing according to the both Pharmacopeia. These
was reduced 2 logs after 24 hrs of inoculation and comparisons indicate that there is not a clear
was increased to about the initial count after 7 correlation between antimicrobial efficacy
days. testing of eye-drops and the rate of their
After 14 days, all the eye-drops except microbial contaminations during their
phenylephrine zinc which showed 1 log reduction usage. Therefore in addition to preservatives,
 Samadi et al / DARU 2009 17 (1) 13-18 15

Table 1. Antimicrobial preservative efficacy of the eye-drops challenged with E. coli, S. aureus and P. aeruginosa
Microorganism Eye-drop Sampling time/Viable count (CFU ml-1)
0 6 hours 24 hours 7 days 14 days 28 days
Artificial tear 3.8105 102 101 101 <10 <10
Atropine sulfate 6.0105 <10 101 101 <10 101
Betamethasone 3.8105 101 <10 101 101 <10
Homatropine HBr 6.0105 101 <10 101 <10 5101
1
E. coli Phenylephrine HCl 3.510 5
<10 10 <10 310 1
101
5 2
ATCC 8739 Phyenylephrine zinc 3.510 <10 <10 <10 8.110 3101
Pilocarpine HCl 3.8105 <10 101 4101 <10 2101
1
Tetracaine HCl 1.010 5
<10 10 210 1
710 1
101
5 1 1
Tropicamide 3.810 <10 210 510 <10 2101

Artificial tear 7.1105 <10 <10 <10 <10 101

Atropine sulfate 4.9105 <10 <10 <10 101 101
Betamethasone 7.1105 <10 <10 101 <10 <10
Homatropine HBr 4.9105 2101 <10 <10 <10 3101
S. aureus Phenylephrine HCl 2.0105 5101 <10 <10 <10 <10
ATCC 6538 Phyenylephrine zinc 3.5105 <10 <10 <10 <10 <10
Pilocarpine HCl 7.1105 <10 101 <10 <10 <10
Tetracaine HCl 4.9105 <10 2101 101 <10 <10
Tropicamide 7.1105 <10 3101 101 <10 101

Artificial tear 3.3105 <10 101 <10 <10 2101

Atropine sulfate 4.7105 <10 <10 2101 <10 2101
Betamethasone 3.3105 2101 <10 <10 <10 <10
Homatropine HBr 4.7105 <10 <10 3101 <10 101
P. aeruginosa Phenylephrine HCl 7.0105 <10 <10 <10 4101 <10
ATCC 9027 Phyenylephrine zinc 1.0105 2103 1103 1.1105 1.5104 2.2105
Pilocarpine HCl 3.3105 <10 1.6101 101 <10 <10
Tetracaine HCl 4.7105 <10 <10 101 3101 101
Tropicamide 3.3105 <10 5101 <10 <10 <10

Table 2. Antimicrobial preservative efficacy of the eye-drops challenged with C. albicans and A. niger
Microorganism Eye-drop Sampling time/Viable count (CFU ml-1)
0 7 days 14 days 28 days
Artificial tear 2105 101 101 <10
Atropine sulfate 1.1105 <10 <10 <10
Betamethasone 2105 <10 <10 <10
Homatropine HBr 1.1105 <10 <10 <10
C. albicans Phenylephrine HCl 1.1105 <10 <10 <10
ATCC 10231 Phyenylephrine zinc 105 <10 <10 2101
5
Pilocarpine HCl 210 <10 <10 <10
Tetracaine HCl 1.1105 <10 <10 5101
5 1 1
Tropicamide 210 510 10 <10

Artificial tear 2.5105 <10 <10 <10

Atropine sulfate 1.1105 <10 <10 <10
Betamethasone 2.5105 <10 <10 <10
Homatropine HBr 1.1105 <10 <10 <10
A. niger Phenylephrine HCl 1.1105 <10 <10 <10
ATCC 16404 Phyenylephrine zinc 1.1105 2.5101 <10 <10
Pilocarpine HCl 2.5105 101 <10 <10
Tetracaine HCl 1.1105 6101 <10 <10
Tropicamide 2.5105 <10 <10 <10
 Antimicrobial effectiveness of ophthalmic drops 16

Table 3. Logarithmic reductions in challenging microorganisms viable counts after 30, 90 and 180 min for bacteria and
1440 min (24 hrs) for fungi
Log reducations
Time
Eye-drop
(min) E. coli S. aureus P. aeruginosa C. albicans A. niger
ATCC 8739 ATCC 6538 ATCC 9027 ATCC 10231 ATCC 16404

30 4 4 4
90 >5 >5 >5
Artificial tear >5
180 4 >5
1440 >5 3

30 2 4 3
90 4 4 4
Atropine sulfate 180 >5 >5 >5
1440 >5 4

30 4 >5 >5
90 >5 >5 >5
Betamethasone 180 >5 >5 >5
1440 4 4

30 4 >5 >5
90 4 4 >5
Homatropine HBr 180 4 >5 >5
1440 >4 5

30 >5 >5 4
90 >5 >5 >5
Phenylephrine HCl 180 >5 4 4
1440 >4 5

30 >5 2 <1
90 >5 4 2
Phyenylephrine zinc 180 >5 >5 4
1440 >4 1

30 >5 >5 >5

90 >5 >5 >5
Pilocarpine HCl 180 4 >5 >5
1440 >5 3

30 5 >5 >5
90 5 4 4
Tetracaine HCl 180 5 4 >5
1440 4 3

30 4 3 >5
90 >5 >5 >5
Tropicamide >5
180 4 >5
1440 >5 5
 Samadi et al / DARU 2009 17 (1) 13-18 17

Table 4. Antimicrobial preservative efficacy of the tested eye-drops according to the BP1 and USP2 criteria and their in-
use microbial contaminations
Overall contamination after 1, 2,
Eye-drop BP 'B' criteria USP
4 and 7 days use (%), (13)
Artificial tear Pass Pass 0
Atropine sulfate Pass Pass 23.5
Betamethasone Pass Pass 80
Homatropine HBr Pass Pass 29.4
Phenylephrine HCl Pass Pass 43.58
Phyenylephrine zinc Fail Fail 50
Pilocarpine HCl Pass Pass 58.3
Tetracaine HCl Pass Pass 84.4
Tropicamide Pass Pass 40
1
BP, British Pharmacopeia, A criteria for bacteria requires not less than 2 and 3 log reduction from the initial count after 6 and 24 hrs
respectively and no recovery of viable cells after 28 days. B Criteria for bacteria requires not less than 1 and 3 log reduction from
the initial count after 24 hrs and 7 days respectively and no increase from the 7 days count after 28 days. A criteria for yeast and
molds requires at least 2 log reduction after 7 days and no increase from the 7 days count after 28 days. B criteria for yeast and
molds require at least 1 log reduction after 14 days and no increase from 14 days count after 28 days.
2
USP, United States Pharmacopeia, for bacteria requires not less than 1 log reduction from the initial count after 7 days, not less than
3 log reduction from the initial count after 14 days and no increase from the 14 days count after 28 days. For yeast and molds
requires no increase from the initial count after 7, 14 and 28 days.

other factors could be responsible for microbial environment, the frequency and technique
contamination of eye-drops. Hygienic practices of of use (18).
eye-drops especially in the hospitals, proper bottle
design and training of patients could influence ACKNOWLEDGEMENTS
their microbial contaminations (16-17). It is This work was supported by a research grant from
essential to maintain and regulate the in-use the Research Council of Tehran University of
efficacy testing which is influenced by the Medical Sciences.

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