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A guidance document on microbiological control of cosmetic products

Appendix 2: Challenge test of water miscible cosmetic products

Claus Jørgensen, DHI
Ann Detmer, DHI

1.1 Purpose

The purpose of this procedure is to determine the effectiveness of antimicrobial preservatives used to stop proliferation or to prevent microbial contamination in cosmetic products.

The procedure is particularly useful during development of new products.

1.2 Principle

The cosmetic product is challenged by adding 105-106 CFU/ml or g of a single strain of test microorganism via cell suspensions of approximately 108 cells/spores pr. ml and is incubated at 20-25° C protected from light. After 0, 7, 14, 21 and 28 days of incubation, samples are taken to determine the number of microorganisms by plate count. The product will pass the test, if the analyses are valid and results are in compliance with the acceptance criteria.

1.3 Scope

This procedure can be used for water miscible products.

1.4 References

European Pharmacopoeia (6.0). Method 5.1.3 Efficacy of antimicrobial preservation. 01/2008:50103.

ASTM E 640 – 78 Standard test method for preservatives in water containing cosmetics. American Society for Testing and Materials, 1991 (reapproved 1998). Annual Book of ASTM Standards. ASTM, Philadelphia , Pa.

ISO 16212:2008 Cosmetics – Microbiology – Enumeration of yeast and mold. First edition 2008-10-07.

ISO 22149:2006 Cosmetics – Microbiology – Enumeration and detection of aerobic mesophilic bacteria. First edition 2006-03-01.

ISO 22717:2007 Cosmetics – Microbiology – Detection of Candida albican. First edition 2007-07-15.

ISO 22717:2006 Cosmetics – Microbiology – Detection of Pseudomonas aeruginosa. First edition 2006-02-01.

ISO 22718:2006 Cosmetics – Microbiology – Detection of Staphylococcus aureus. First edition 2006-02-01.

1.5 Equipment

Balance (± 0.01g)

Equipment for homogenisation (e.g. stomacher)

pH meter (± 0.1 pH units)

One autoclave to clean equipment and media, and another autoclave to treat contaminated waste

Incubators: 20-25 °C and 30-35 °C.

Other standard equipment for microbiological laboratories necessary to perform sterile work, sample handling and work with cultures.

1.6 Test organisms

Pseudomonas aeruginosa CCUG 22801 (ATCC 9027; NCIMB 8626; CIP 82.118)

Staphylococcus aureus CCUG 10778 (ATCC 6538; NCTC 10788; NCIMB 9518; CIP 4.83)

Candida albicans CCUG 19915 (ATCC 10231; NCPF 3179; IP 48.72)

Aspergillus niger CCUG 18919 (ATCC 16404; IMI 149007; IP 1431.83)

The organisms can be obtained from type culture collections.

1.7 Chemicals and substrates

For P. aeruginosa, S. aureus and C. albicans a tryptone (1,0 g/l) sodium chloride (8,5 g/l) diluent is used.

For A. niger a tryptone (1,0 g/l) sodium chloride (8,5 g/l) solution with Polysorbat 80 (0.5 g/l) is used.

Agar for cultivation and quantification of P. aeruginosa, S. aureus and C. albicans: Letheen agar

Agar for cultivation and quantification of A. niger: Saboroud-glucose (Saboroud-dextrose) agar without antimicrobials.

Letheen agar contains polysorbat 80 and lecithin, which inactivates many antimicrobial preservatives.

1.8 Procedure

1.8.1 Preparation of inocula

Prepare stock and working cultures of the test organisms as described by the supplier.

Before the test, inoculate the surface of the agar with recently grown stock or working cultures of each of the specified microorganisms. Incubate the agar plates with P. aeruginosa and S. aureus at 30 °C to 35 °C in 18 to 24 hours, agar plates with C. albicans at 20 °C to 25 °C in 48 hours, and agar plates with A. niger at 20 °C to 25 °C in 1 week or until good sporulation is obtained. Examine the agar plates for contamination before use.

To produce challenge suspensions, harvest the bacterial and fungal cultures by transferring colonies from the agar plates to diluents to a concentration of approximately 108 P. aeruginos or S. aureus pr. ml or approximately 107 C. albicans or A. niger pr. ml. Determine the number of colony forming units (CFU) pr. ml immediately after resuspension by plate count on the specified agar.

In order to make quick assessments of the concentration of organisms in the challenge suspensions, it is advised that the laboratory compares different concentrations of the specific test organisms (determined by plate count) in diluent against rapid detection methods, i.e. absorbance at 620 nm (typical absorbance of 108 bact/ml is 0.15 to 0.46 at 1 cm), turbidometry or visual comparison to McFarland standards.

1.8.2 Preparation of challenge test samples

Transfer 10 times 100 g or 10 times 100 ml of the product to be tested into sterile double Stomacher bags (one bag inside the other). Of these, two are used for each of the four test organisms and two are used as uninoculated controls.

Inoculate the test samples by adding no more than 1 ml of challenge suspension pr. 100 ml (or g) test product. Homogenise the test samples in the Stomacher for 30 seconds.

1.8.3 Sampling and incubation

Take a zero sample for each microorganism immediately after homogenisation (see 8.4) and incubate as described in 8.1. Incubate the Stomacher bags with the challenged test samples at 20 – 25 °C. It is important to seal the bags to avoid evaporation. Leave approximately 10 % headspace in the Stomacher bags.

Take further samples after 7, 14, 21 and 28 days. Homogenise before each sampling.

1.8.4 Analysis of samples

Transfer 10 g or 10 ml to 90 ml diluents (or 1 g/1ml to 9 ml). The density of the product must be known if the transfer is based on weight. Homogenise the dilutions and spread 100 µl of dilutions in duplicate on the surface of the agar. Use the 10-1, 10-2, 10-3, 10-4 and 10-5 dilutions for the bacterial challenges and 10-1, 10-2, 10-3, 10-4 for mold and yeast challenges at time zero. For subsequent sampling select the dilutions to be analysed on the basis of the results of the initial analyses. It is suggested to use the 10-1, 10-2 and 10-3 on day 7 for P. aeruginos and S. aureus and 10-1 and 10-2 for C. albicans and A. niger. Incubate as described in 1.8.1, count the number of colonies and calculate the plate count according to ISO 21149:2006, and plot the log10 transformed results versus time.

1.9 Validity criteria

1.9.1 Validity of analyses

If the organisms grow readily on the plates, and if there is a 1:10 relation between the dilutions used for calculation of each analysis, the result of the analysis is valid. The relation between dilutions can be tested by a χ² test with 1 degree of freedom:

Formula

where C1 and C2 are the number of colonies counted in dilutions V1 and V2. If χ² ≥ 3.84 then the dilution is significantly different from 1:10 at the 5% level. If a low dilution (more sample) has a relatively low count compared to a higher dilution (less sample) it may be caused by inhibition by the antimicrobial preservative in the tested product, and the test should be repeated with an alternative neutralizer. See for instance ISO 22717 annex B for information on alternative neutralizers.

1.9.2 Validity of test organisms and neutralising agent

If no growth is observed on the agar plates it may be caused either by poor viability of the test organisms or infectivity of the neutralising agent.

If no growth is observed, test organisms are spread in duplicate on the 10-1 and 10-2 plates and on clean control plates. Poor viability of the test organisms is shown, if no growth is observed on the control plates. In this case the entire test should be repeated with new and viable test organisms.

If no growth is observed on the 10-1 or 10-2 plates, and growth is observed on the control plates, then the neutralizing agent has been ineffective. The lab should try to wash away/neutralize the antimicrobial preservative with a sterile diluent to which an alternative neutralizer has been added. Again, the test organisms are spread on the plates. If no growth is observed on the washed plates and growth is observed on control plates treated in a similar way, then the antimicrobial preservative is accepted. If growth is observed, the test is repeated with an alternative neutralizer.

1.10 Acceptance criteria

The antimicrobial preservation system is accepted if:

the performed analyses were valid according to section 1.9.1, and

  • bacteria are reduced by 3 log10 units after 14 days, and no increase in plate counts after day 14.
  • mold and yeast are reduced by 2 log10 units after 14 days, and no increase in plate counts after day 14.

or if

no growth is observed after washing with alternative neutralizers according to section 1.9.2.

 

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Version 1.0 January 2010, © Danish Environmental Protection Agency