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A guidance document on microbiological control of cosmetic products 4 Analytical Methods
Through its technical committee for Cosmetics ISO/TC 217, the international organization for standardization (ISO) has presented a number of new international standards for microbiological examination of cosmetic products. These standards are detailed and cover the needs of a large part of available cosmetic products. It is strongly recommended to incorporate the use of ISO standards in microbiological testing of cosmetic products. In the following some of these standards are presented. In genera, the efficacy of antimicrobial preservation in cosmetics can be tested by the Challenge test. The test shows the ability of the cosmetic product to reduce the count of micro-organisms after a contamination. Challenge testing is mandatory for all cosmetic products that under normal conditions of storage and use may deteriorate or form a risk to the consumer. As neither a legal nor a universal challenge test method is available; it is up to the manufacturer to decide on the details of the test to be used. 4.1 Standards under developmentThe technical committee for Cosmetics ISO/TC 217 is developing two new guidelines. The laboratories should be updated on the status of the standards. ISO/CD 29621 is a guideline for the risk assessment and identification of microbiologically low-risk products. In the committee draft stage the guideline has been approved for registration as a draft international standard. ISO/NP 11930 is general information on evaluation of the antimicrobial protection and has been approved as a new project. 4.2 Neutralization and preparation of water-immiscible samples.Microbial examination of cosmetics has at least two inherent problems, namely the toxic properties of the conservation systems and water-immiscibility of some products. These problems are very important to deal with in order to achieve a correct result. Cosmetic products are usually conserved to maintain a hygienically good quality during storage and to prevent growth of microorganisms during use of the product. The conservation system is likely to inhibit growth on agar plates or in enrichment broths and thereby likely to give rise to false negative results. Therefore the conservation system must be inactivated or neutralized before analysis. The initial steps of microbial examinations involve preparation of an initial suspension of the microorganisms in the sample. This suspension is subsequently diluted to achieve sub-samples with appropriate concentrations of microorganisms. If the cosmetic product to be tested is water-immiscible, the diluents should contain a suitable amount solubilising agents such as Polysorbat 80. Samples with antimicrobial properties must be neutralized before analysis. This is done by adding neutralizers to the diluents. Relevant neutralizers are suggested in the specific standards (see section 4.3) and in ASTM E 1054 (10). In all cases and whatever methodology, the neutralization of the antimicrobial properties of the product must be checked and validated. The validation procedures are described in each specific standard. The principle of the validation procedure is to add a known amount of the relevant test strain(s) to the initial sample suspension and compare the number of micro-organisms with a control without the sample. In case of qualitative or presence/absence tests, growth and characteristics of the colonies are examined. 4.3 Examination of microbial quality of productsCosmetic products must be subjected to microbiological control as described in Chapter 2. A number of ISO standards have been developed to give guidelines for the manufacturers. It is recommended that all laboratories use the ISO standards described in this section. 4.3.1 ISO 21149 Cosmetics – Microbiology – Enumeration and detection of aerobic mesophilic bacteriaThe standard contains guidelines for enumeration and detection of mesophilic aerobic bacteria in cosmetics by counting colonies on agar medium after aerobic incubation or by checking absence of bacterial growth after enrichment. 4.3.2 ISO 18415 Cosmetics – Microbiology – Detection of specified and non-specified micro-organisms.The standard contains guidelines for the detection and identification of specified microorganisms in cosmetic products as well as for the detection and identification of other kinds of aerobic mesophilic non-specified microorganisms in cosmetic products. The standard contains guidelines for the detection of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans. The detection is carried out by enrichment in a non-selective broth followed by isolation and identification. The identification consists of gram staining, catalase- and oxidase test followed by the use of an identification test kit. 4.3.3 ISO- methods for the detection of specific microorganisms: E. coli (ISO 21150), Pseudomonas aeruginosa (ISO 22717), Staphylococcus aureus (ISO22718) and Candida albicans (ISO 18416).In all four standards, the first step is enrichment in a non-selective broth to increase the number of microorganisms without the risk of inhibition by the selective ingredients present in the growth media. The second step is isolation on selective media followed by identification tests. 4.3.4 ISO/FDIS 16212 Cosmetics – Microbiology – Enumeration of yeast and mould.This standard is currently available as a draft. The method involves enumeration of colonies on Sabouraud dextrose chloramphenicol agar medium. Enumeration may be carried out as a pour plate, surface spread or membrane filtration method. 4.4 Efficacy of preservation – Proposal for a Challenge test-not validatedA challenge test is a procedure in which a product is challenged by exposure to specified types of bacteria and fungi. The product is then incubated at a given temperature, samples are taken at specified intervals and the number of microorganisms is determined. Normally the product is challenged to the microorganisms Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, but in-house microorganisms, found as contaminations in the products, may be used for additional specific purposes of challenge testing. The antimicrobial properties of the product are acceptable, if a significant decrease or no increase in viable count of micro-organisms is seen, when the product is tested under consideration to storage and use. The challenge test should be performed both during development of the preservative system and as an evaluation of the protection efficacy in intact, in-use or in ending cosmetic products. There are no approved ISO standards available for challenge tests. Until the new ISO standard is available, the procedure below in section 4.4.1 is recommended. The procedure has not been validated why it is suggested to perform an in-house validation before use. Similar challenge tests are described in the European Pharmacopoeia (11), the US Pharmacopoeia (12) and in ASTM (the American Society for Testing and Materials) (13). The methods are similar but differ in the detailed procedures, test organisms, criteria for passing the test and requirements for validation. Inexperienced laboratories should send the samples to an accredited laboratory. Another alternative is to contact the producer/deliverer of the preservative; they can usually provide laboratory capacity. 4.4.1 Proposed procedure for challenge testingThe product is challenged with cell/spore suspensions (108 cells/spores pr. ml) at a concentration of 105 – 106 cells/spores pr. ml. The challenged product is incubated at 22 °C ± 1 °C in the dark. Samples for determination of plate counts are taken after 0, 7, 14, 21 and 28 days. Challenge the product with
Other relevant organisms, such as commonly observed contaminating organisms, should also be used as test organisms. Use Letheen agar which contains the neutralisers polysorbat 80 and lecithin, or Casein soya bean digest agar for bacteria, and Saboraud-glucose agar without antibiotics for fungi. The initial plate counts are determined immediately after addition of the test organisms. The concentration of bacteria should reach a log 3 reduction after 14 days, and there should be no increase in the concentration after day 14. The concentration of fungi should reach a log 2 reduction after 14 days and there should be no increase in concentration after day 14. The efficacy of the neutralisers shall be validated according to the specific ISO standards (se section 4.3) or according to ASTM E 1054.
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