MICROBIOLOGICAL STABILITY TESTING COSMETIC FORMULATIONS

When it comes to cosmetic products, the key requirements are stability, effectiveness and safety. Microbiological stability plays a major role here, especially with regard to consumer safety. If, for example, a product that has not been adequately preserved contains enough water and nutrients for microorganisms, these can multiply accordingly. This is particularly likely to happen in warm and sometimes humid bathrooms, where most cosmetics are used and stored. To guarantee product quality and consumer safety, cosmetics should not become contaminated with microorganisms, either when unopened or when freshly opened.

ORIGIN OF MICROBIOLOGICAL CONTAMINATION

Experience shows that microbiological contamination of cosmetic products can be traced back to two sources. Firstly, cosmetics can become contaminated with microbes during the manufacturing and filling process. However, unopened cosmetic products are usually free of microorganisms. The quality of the raw materials, operational hygiene and regular microbiological checks generally ensure that cosmetics do not carry any pre-contamination. The second possible source of microbiological contamination is the consumer themselves. Between the time the packaging is opened and the product is completely used up, the consumer continuously introduces microbiological contaminants, for example through their own hands. Cosmetics in jars are particularly susceptible to this. In order to counteract contamination of the product, a wide variety of preservatives are used in the manufacturing process.

MICROBIAL CHALLENGE TEST FOR COSMETIC PRODUCTS

When assessing the safety of cosmetic products, cosmetics legislation requires a microbial challenge test (alt. Preservative Efficacy Test – PET) to be carried out to ensure sufficiently effective preservation. There are various recognised methods for this, which can differ with respect to the microorganisms used, the amounts inoculated in the samples, and the acceptance criteria.

One of the best-known methods comes from the field of pharmacy and is described in the European Pharmacopoeia (Eu. Ph.) 5.1.3. under „Testing for adequate preservation“. This method is designed to test the microbiological stability of non-sterile pharmaceutical products, but can also be used for cosmetic products.

In April 2012, the first version of the ISO 11930 standard „Cosmetic products – Microbiology – Evaluation of the antimicrobial protection of a cosmetic product“ was published, which is specifically tailored to cosmetics. The challenge test according to ISO 11930 is now widely used and recognised as a common test for checking the microbiological stability of a cosmetic product.

Although both methods are recognised and similar in many test parameters, they differ in some respects. Both challenge test methods use the same test organisms. These include three representatives of bacteria, two of which are Gram-negative and one Gram-positive, as well as one representative of yeasts and one mould. The inoculation of the germs is identical for bacteria in both methods, but differs for yeasts and moulds. The challenge test according to Ph. Eu. 5.1.3. specifies the same inoculation quantity of between 100,000 and 1,000,000 germs of all 5 microorganisms per gram of sample. The challenge test according to ISO 11930, on the other hand, specifies a slightly lower inoculation of yeasts and moulds between 10,000 and 100,000 microorganisms per gram of sample.

KBT TEST ORGANISMS

Ph. Eu. 5.1.3. / ISO 11930
		test organisms	trunk
bacteria	Gram-negative	Escherichia coli	ATCC 8739
	Gram-negative	Pseudomonas aeruginosa	ATCC 9027
	Gram-positive	golden staph	ATCC 6538
yeasts		Candida albicans	ATCC 10231
mould		Aspergillus brasiliensis	ATCC 16404

INOCULATION

MICROBIAL CONCENTRATION PER GRAM OF SAMPLE
	Ph. Eu. 5.1.3.	ISO 11930
bacteria	10⁵ – 10⁶	10⁵ – 10⁶
yeasts	10⁵ – 10⁶	10⁴ – 10⁵
moulds	10⁵ – 10⁶	10⁴ – 10⁵

Differences between the methods presented

The main difference between the two methods presented here lies in the test times and the acceptance criteria for the evaluation stages. According to Ph. Eu. 5.1.3., the bacteria are tested at four test times, whereas according to ISO 11930, the earliest of the test times is omitted. According to Ph. Eu. 5.1.3., both fungal species are tested only two weeks after inoculation, while according to ISO 11930 the yeast is tested already on day 7 after inoculation.

TEST TIMES – Ph. Eu. 5.1.3. vs ISO 11930

In general, there are two assessment levels for an challenge test, namely A and B. These are based on the log level reduction in viable cell count compared to the start of the test. If the test does not meet the criteria for either level A or level B, the test is rated as failed. While according to Ph. Eu. 5.1.3. the bacteria are assessed already on day 2, according to ISO 11930 this is not done before day 7. However, the criteria for assessment are then comparable from day 7 onwards. With an challenge test according to ISO 11930, the yeasts are assessed already from day 7.

However, the criteria for assessment are then comparable from day 7 onwards. With an challenge test according to ISO 11930, the yeasts are assessed already from day 7. In order to achieve an A rating, however, they have to be reduced by one log level less in the comparison.

The moulds are assessed using both methods from day 14 onwards. However, the same applies to the assessment as for yeasts. One log reduction less is sufficient for an A rating.

ACCEPTANCE CRITERIA

	REQUIRED LOGARITHMIC REDUCTION VALUES
	Ph. Eu. 5.1.3.						British Standard 11930^a
	bacteria		yeasts		mushrooms		bacteria		yeasts		mushrooms
criterion	A	B	A	B	A	B	A	B	A	B	A	B
T2	≥2	-	-	-	-	-	-	-	-	-	-	-
T7	≥3	-	-	-	-	-	≥3	-	≥1	-	-	-
T14	-	≥3	≥2	≥1	≥2	≥1	≥3^b	≥3	≥1^b	≥1	≥0^c	≥0^c
T28	≥3^b	≥3^b	≥2^b	≥1^b	≥2^b	≥1^b	≥3^b	≥3	≥1^b	≥1^b	≥1	≥0^b
^a A deviation of 0.5 log is permissible in this test in accordance with ISO 11930, Chapter 5.7. ^b No increase since the last count ^c No increase since first count

Comparing the cosmetics challenge test according to ISO standard 11930 with the specifications of the European Pharmacopoeia, the latter provides for a somewhat stricter assessment of the microbial reduction through the inclusion of an additional test time point for the bacteria. In addition, the European Pharmacopoeia specifies higher reduction in terms of log levels with a likewise higher inoculation quantity for the fungal species. This may be necessary for pharmaceutical products, but for cosmetic products the test time points, inoculation levels and acceptance criteria described in ISO 11930 are sufficient. In addition, the challenge test of ISO standard 11930 takes into account a measurement uncertainty with regard to microbiological fluctuation ranges of up to 0.5 log levels. The challenge test of ISO standard 11930 has proven an appropriate alternative to the European pharmacopoeia since its publication in 2012.

CHALLENGE TEST FROM COSPHATEC

Cosphatec offers challenge tests in accordance with both ISO 11930 and Ph. Eu. 5.1.3. The choice of test depends on individual factors. A method is selected according to the customer's requirements and the preservation system is tested with our Multifunctionals adjusted and optimised accordingly.

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General information about challenge tests

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