Aim: Eye drop products must be properly packaged and must be sterile throughout the period of use. For this reason, we evaluated eye drops offered for sale and used for the treatment of eye diseases in Nigeria for their pharmaceutical quality and antimicrobial effectiveness. Materials and Methods: Eleven different sterile eye-drop product samples containing gentamicin, chloramphenicol, timolol, tropicamide, tetrahydrozoline, pilocarpine, antazoline-tetryzoline, diclofenac, dexamethasone, and flubiprofen were opened and tested for bacterial contamination after exposure to air over 56 days. To assess the possibility of contaminations by pathogens due to repeated handling during use about 10 7 -10 8 cells of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans were added to other newly opened eye drops and incubated at room temperature. Samples were collected and the number of viable organisms was estimated. Eye-drop product samples were also investigated for packaging, pH, and clarity. Results: No viable microorganisms were detected in the eye drops during and at the end of the 56 days exposure to air. All the samples exhibited rapid bactericidal and fungicidal activities at a rate which depended on the species of pathogens and the content of the sample. The anti-infective samples demonstrated the highest rapidity in microbicidal activities. There were no microbial survivors in any of the samples after 4 h of inoculation by pathogens. Conclusions: The study concluded that generally multi-dose eye drop products offered for sale in Nigeria were of acceptable microbial quality and possessed good antimicrobial effectiveness.
Keywords: Antimicrobial preservation, drug quality, eye drop products, Nigeria
Reports of poor microbial quality in some commercially available pharmaceutical preparations in Nigeria are frequent. Most of the reports have focused non-sterile preparations such as creams and lotions, , tablets,  creams,  and other oral and topical liquid preparations.  In this study, commercially available eye drop products, which are sterile pharmaceutical preparations, were investigated for their microbial quality.
When non-sterile preparations are allowed to contain some micro-organisms within specified limits, sterile preparations are expected to be totally free from all microbial contaminants. For eye drop products, the requirement for sterility should be maintained throughout the period of their use.  This is because, while in use, microbial contamination may lead to product degradation or result in ocular infection. , Therefore, it is essential to protect the product against the effects of opportunistic contamination by the use of appropriate preservatives. 
To assess the suitability of the added preservative in the eye drops, the antimicrobial effectiveness tests such as the microbial challenge tests were carried out.  Other properties of the eye drop products were also investigated.
Procurement of eye drops
The eye drops were purchased from registered pharmacy premises in Ile-Ife, Nigeria. A total of 11 different brands of eye drops were used in the study. These included various preparations of gentamicin sulphate, chloramphenicol, timolol, tropicamide, tetrahydrozoline, pilocarpine HCl, antazoline HCl, tetryzoline HCl, diclofenac sodium, dexamethasone, and flubiprofen sodium from different manufacturers (local and foreign). Each container label was noted for the following: contents, manufacturing and expiry dates, preservative used, and batch number.
Physical and organoleptic examinations and pH test
The solutions in the eye drop were examined visually under bright light for color, presence of particles, and clarity. The pH of the eye drops was investigated by placing several drops of the solution on red and blue litmus paper.
Evaluation of microbiological quality of eye drops
Each freshly opened eye drop of 1.00 ml was serially diluted to 10 3 , 10 4 , and 10 5 with sterile distilled water. Each dilution of 1.00ml and each eye drop of 1.00ml was subcultured on Nutrient Agar (Oxoid, England) and on Sabouraud Dextrose Agar (Oxoid, England) in duplicate and incubated for 48 h at 37°C for bacteria and for 3 days at 25°C for fungi to determine the colony forming units in the original eye drop.
Investigation of contamination by the air
The ability of the eye drops to withstand contaminations when exposed to air for a specific period of time was investigated.  Eye drops were opened and exposed to the atmosphere by placing them on the open shelves in the laboratory. Samples were taken at 0, 2, 4, 8, 24, 48, and 72 h, then 7, 14, 28, and 56 days. Approximately 0.5 ml of each solution was cultured in 10 ml of thioglycollate (Brewer) broth as described. 
After 24 h incubation at 37°C the broth was examined for bacterial growth. Each broth was then subcultured on blood agar plates and incubated overnight at 37°C. All thioglycollate broth cultures were subsequently examined for 7 days. The thioglycollate broth not only acted as an enrichment medium to support the growth of Pseudomonas as well as sporing and non-sporing anaerobes, but also helped to dilute out any antibacterial effect of the preservative in the eye-drop bottles.  The inclusion of the blood agar plates was to allow for the culturing of a very wide spectrum of organisms.
Preparation of inoculums for the challenge test
Reference strains of organisms used were from culture collections maintained in our laboratory. For the bacteria: Pseudomonas aeruginosa NCIB 950, Escherichia More Details coli NCIB 86, and Staphylococcus aureus NCIB 8586; inoculums of the organisms were taken from Nutrient Agar slope culture and subcultured in Nutrient Broth and then on Nutrient Agar plates. Inoculum size was determined by viable colony counts on Nutrient Agar plates after appropriate serial dilutions.
For the fungi: Inoculum of the Candida albicans NCYC 6 was taken from Sabouraud Dextrose Agar slope and subcultured in Sabouraud Dextrose Broth and then on Sabouraud Dextrose Agar plates and incubated at 25°C for 3 days. Inoculum size was determined by viable colony counts on Sabouraud Dextrose Agar plates after appropriate serial dilutions. Each eye drop was challenged with these organisms.
Inoculation of eye drops with organisms
Challenging organisms of 0.1 and 0.05 ml (equivalent to 1% of the volume of the 10 ml and 5 ml of each eye-drop, respectively) each at a final concentration of 10 6 -10 8 cfu/ml was inoculated into each eye drop and mixed thoroughly to determine the rate of microbial kill. Each organism was tested separately and the inoculated product was maintained at 20-25°C throughout the test period. At 30 min, 1, 2, 4, 6, and 24 h time-points, 1.00 ml samples were taken and inoculated in 9 ml of thioglycolate medium to neutralize the preservative before plating on Nutrient Agar or Sabouraud Dextrose Agar for bacteria or fungi, respectively, to determine the viable organisms after serial dilutions as appropriate.
The plates for bacteria were incubated at 35°C for 48-72 h, and the fungal plates were incubated at 25°C for 5-7 days. Following incubation, the colonies were counted.
Properties of the eye drops
All the eye drops have their date of manufacture, expiry date, and batch number clearly stated on the package of each of them [Table 1]. They were all within their expiry date which ranged from 2 to 5 years and were all labeled to be used within a month after first opening. The volume of the eye drops ranged from 5 to 15 ml. With regards to the National Agency for Food and Drug Administration and Control (NAFDAC) numbers, one company did not provide the NAFDAC number of her product on the label.
The label disclosures indicated that most of the eye drops were preserved with 0.01% benzalkonium chloride. The three exceptions to this were products B, F, and ‘I’. In products B and F, the specific preservative agent used was not stated. On the other hand, product ‘I’ was labeled as having 0.05% benzalkonium chloride. The cost of each brand of eye drop ranged from NGN100.00 to NGN1500.00 (i.e., approximately $0.62-$9.33). The anti-infectives were found to be of lower cost whereas the analgesics, anti-inflammatory, and anti-glaucoma eye drops were of higher cost. All the eye drops were clear and colorless. Eye drop product C was found to be alkaline in pH whereas the rest were neutral.
Ability of the eye drop to withstand contamination when exposed to air
Over the period of 56 days for which the eye drops were exposed, no growth was detected on the blood agar plates. There was also no surface growth, deposit, or cloudiness in all thioglycolate broth medium throughout the 7 days of exposure.
Results of the challenge test
The initial microbial load for the reference organisms in each product was 5.0 × 10 6 cfu/ml for P. aeruginosa, 3.5 × 10 5 cfu/ml for S. aureus, and E. coli and 2.5 × 10 4 cfu/ml for C. albicans. All the eye drops exhibited rapid bactericidal and fungicidal activities at a rate which depended on the species of pathogens and the content of the eye drop [Table 2]. The anti-infective eye drops demonstrated the highest rapidity in microbicidal activities. All the eye drops reduced the load of P. aeruginosa to 0 within 2 h of inoculation. The microbial load of S. aureus and E. coli was reduced to 0 within 1 h of inoculation of gentamicin eye drop, whereas the microbial load of C. albicans was similarly reduced to 0 within 2 h for all the eye drops [Table 2].
The single dominant factor characteristic of all ophthalmic products is specification on sterility not only after preparation but throughout the period of use. This is a legal requirement that dates back to 1955  and is still in force today. Despite this requirement, extensive researches have not been done on the quality and antimicrobial effectiveness of commercially available eye drops purchased and used by patients. Most reported studies have focussed patient-used preparations and high prevalence of microbial contaminations in these preparations have been reported. , Although most of these studies have attributed these contaminations to the use of the eye drops, it is doubtful if these assertions have any scientific basis since the microbial quality of the original eye drop products are largely assumed.
The eye-drop products used for evaluation in this study were purchased in Ile-Ife, a town in south-western Nigeria. It is to be noted, however, that the same eye drop brands are available and on sale throughout the country and are among the ones being prescribed, dispensed, and administered in hospitals and in community pharmacies in other countries beyond Nigeria because they were found to be manufactured and distributed by local and foreign companies.
Ophthalmic products are required to be packaged in such a way that they will retard contamination. This is because multi-dose containers may be opened, closed, and used many times by the consumer. The dates of manufacture and expiry are also important and should be specified which would give an idea about the timeframe for which the wholesomeness of the product can be reasonably assured. In these respects, all the eye drops used in this investigation were properly packaged and labeled.
There were however, a few inadequacies and inconsistencies observed in the label disclosures. These should be viewed with serious concern particularly from the standpoint of non-disclosure of NAFDAC number for one of the product samples which was from a foreign manufacturer. This could mean that the product did not undergo appropriate regulatory screening before being released into the Nigerian market.
The design of the containers might also influence contamination. All the eye drops used in this study have their tips attached to the bottle. This is of a great advantage over the old type of eye drops container that comprised a dropper separately packaged from the main container. These old container types have been reported to encourage contamination during use leading to serious ocular infections. 
According to the label disclosure, benzalkonium chloride was the preservative in all the eye drops except in product B and F in which the preservatives were not stated. Chloramphenicol is an anti-infective and probably does not need a preservative due to its intrinsic antimicrobial action. Pilocarpine on the other hand does not possess antimicrobial activity and is therefore expected to contain a preservative.
The results obtained from this study, however, indicated that the pilocarpine eye drop probably contained a preservative since it was able to withstand microbial contamination. Another possibility of this observation was that the product was able to withstand contamination probably because of another novel preservation system. A novel preservative system that consists of a balanced composition of common buffers and ions has been described.  The effectiveness of this preservative system has been reported to be a result of the unique combination of the ingredients in the eye drop preparation. 
The results of challenge tests indicated that gentamicin eye drops demonstrated the highest antimicrobial effectiveness followed by chloramphenicol. Timolol eye drops were next and were closely followed by Antazoline-Tetryzoline and pilocarpine. These results are not unexpected since gentamicin and chloramphenicol are antimicrobial agents with broad spectrum activities against a wide range of bacteria including S. aureus, species of Pseudomonas, Proteus, and many coliform bacilli.  This produces a synergistic effect with the preservative against any invading micro-organisms.
Results also indicated that all the eye drops demonstrated comparable fungicidal activities against the Candida strain tested. For example, both gentamicin and timolol sterilized an inoculum of 2.5 × 10 6 cells of C. albicans within 2h of inoculation at a rate different from that obtained for all the bacteria strains. Since none of the eye drops that are anti-infective has been reported to possess intrinsic antifungal activity, this result is not unexpected. Therefore the anti-candidal activity observed in this study could be attributed mainly to the effect of the benzalkonium chloride preservative.
The requirement of the British Pharmacopiea (BP 2007) on the efficacy of antimicrobial preservation is that, for ophthalmic preparations intended for use on more than one occasion, the number of bacteria recovered per ml or per g is reduced by a factor of not less than 10 3 within 6 h of challenge, and no organism is recovered from 1 ml or 1 g at 24 h and thereafter. Also with respect to moulds and yeasts, the number of organisms recovered per ml or per g is reduced by a factor of not less than 10 2 within 7 days of challenge, and there is no increase thereafter. 
Our results showed that no organism was recovered from 1 ml of all the eye drops investigated after 2 h of challenge indicating that official requirements as specified by the BP was satisfied by all the eye drops. These results suggest that the sample eye drops have a high probability of being safe during the period of use. ,
The antimicrobial action of the eye drops supports the previously described self-sterilizing effect of some eye medications.  It can therefore be concluded that the antimicrobial effectiveness of the eye drops depends on the efficacy of the preservatives and intrinsic antimicrobial activity of the eye drop solution itself especially in antibiotic drops.
In conclusion, the investigated eye drop products sold in Nigeria are of the appropriate microbial quality since they were shown to have complied with official requirements with respect to sterility and demonstrated ability to effectively kill microbes as required. However, it is obvious that this singular study cannot be a permanent solution to the issue of quality of eye drop products in Nigeria. There is a need for continuous and regular monitoring of other aspects of the quality such as biological and content quality. Ongoing studies in our laboratory are currently geared towards these issues.
For quality to be maintained throughout the use of the products it is recommended that patients should adhere to standard guidelines of using eye drops. Each eye drop product should not be used by more than one patient and should be used for only one eye of the patient. In addition to this, the eye drop tip should not touch an infected eye or any other object during application. It is also important that eye drop products should be stored as recommended. In this way, the assurance of safety of the eye throughout the period of use of the eye drop products could be guaranteed more satisfactorily.
The technical assistance of Adeoluwa Adediran is appreciated. Also, the valuable comments and suggestions of Dr. G Alebiowu of the Department of Pharmaceutics, Obafemi Awolowo University, on the manuscript are appreciated.
Source of Support: None, Conflict of Interest: None