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Special Newsletter Cosmetics Part 21 / 03.2019
Frequent errors in the microbiological examination of cosmetics
 
Dear Mr. Mustermann,
ISO standards for the microbiological examination of cosmetic products do exist for many years.

In this special edition, frequent errors in the microbiological testing of cosmetics are to be pointed out and explained.

We wish you informative reading and would be happy to answer any questions you may have.

Best regards


Dr. Bernhard Fellenberg 
Customer consulting Cosmetics / Pharmaceuticals
 
 
Frequent errors in the microbiological examination of cosmetics
 
For some years now, ISO standards have been in existence for cosmetic products regarding microbiological testing including specifications and quantitative limits. The microbiological methods are described in detail in the respective test standards. DIN EN ISO 17516 specifies the maximum permitted content of microorganisms.

The implementation and performance of these methods is associated with high financial and personnel costs for many companies. As a result, the degree to which these methods are implemented varies widely across enterprises.

This document is intended to identify and explain common errors in the microbiological testing of cosmetics.
 
Question 1: Must the cosmetic product always be diluted before the microbiological examination?

ANSWER: YES

The basic principle of classical microbiological examination methods is the application of a certain amount of sample onto or into an agar medium. After incubation of the medium, microorganisms can multiply on or in the medium and become visible and thus countable.

The sample matrices in the analysis are very diverse and include cosmetic end products, bulk or semi-finished goods as well as raw materials used. Dry, solid, powdery, liquid, fatty and other matrices are examined. Theoretically, all these product categories can be examined by applying them directly to the medium (or by dipping media into the product (dip slide), rubbing with swabs, etc.). This is a simple methodology, but it is NOT suitable for the safe and reliable detection of microorganisms. Meaningful results are NOT achieved with this type of investigation. The sample MUST be diluted before examination.

Why is a simple application or immersion not sufficient?
  • The vast majority of cosmetic products contain preservative substances to be microbiologically stable. These substances interfere (= inhibit) with the growth of microorganisms on the test medium. It is therefore not ensured that possible contaminations in the product are reliably detected (especially within a short incubation period of e.g. 2-3 days).
  • For a simple application or immersion of the sample, the exact test quantity is usually not known. Depending on the consistency of the sample, the amount can vary considerably. Similarly, due to the surface activity of a product, only a small amount of product may remain on the medium. The result cannot therefore be determined and reported quantitatively. With this methodology it is not possible to check compliance with quantitative limit values.

    DIN EN ISO 17516 (microbiological limit values) is relevant for cosmetic products:

    Table 1: Requirements for the microbiological quality of cosmetic products (from DIN EN ISO 17516)


    It is important to note that in addition to a quantitative determination (bacteria, yeasts and moulds) the absence of so-called specified microorganisms in 1g of product is required. This requirement can only be met if 1g of product is actually tested. The result is then given as the presence or absence of specified microorganisms per g of product.

    Further information on the limit values and specifications of DIN EN ISO 17516 can be found in the special edition Cosmetics Part 10.
  • Striations or particles of a sample can mask the growth of microorganisms.
 
Question 2: How is the sample diluted?

The first dilution is usually carried out 1:10 with a dilution liquid containing de-inhibiting or neutralizing substances.

Certain ingredients in this dilution solution may remove a possible inhibition (see question 1). The substances required for this are called inhibitors (e.g. lecithin, polysorbate 80 and other substances). Only in this way it can be assumed that microorganisms can multiply under the test conditions. (see special edition Cosmetics part 17).
 
Question 3: Growth of microorganisms is detectable. What to do?
 
  • Even the detection of a small number of microorganisms in products can lead to serious problems. Product contamination can impair the safety of the product and lead to product recalls. In individual cases, the so-called "Phoenix effect" is observed (microorganisms are initially only detectable in small numbers, but after a certain time strong growth occurs). Therefore, even if there is only ONE colony on the agar, it must NOT be classified as a contamination caused by the laboratory.

    Any growth of microorganisms (whether quantitative and/or qualitative) therefore requires identification. It is initially irrelevant whether a limit value is exceeded or not. DIN EN ISO 17516 provides guidelines on how to evaluate positive findings. The following factors in particular are taken into account when assessing risk:
    - Number of microorganisms
    - Possible detection of specified microorganisms or health-relevant germs
    - Type of product (low microbiological risk yes/no?)
    - User groups: also use on children under 3 years of age, in the eye and mucous membrane area, immunosuppressed persons, elderly persons?
    - Is it possible to multiply the microorganisms in the product?
  • The performance and interpretation of identifications requires sufficient laboratory equipment and appropriate microbiological expertise. When microorganisms grow on a nutrient medium, some optically visible unambiguous properties are recognizable. However, this is not always the case, as the germs can be damaged by preservative substances. Growth is then altered or atypical.

    A too rapid or insufficiently substantiated classification of a bacterium (e.g. solely on the basis of the type of growth on an agar plate) can result in a false hazard classification.
     
  • There is already sufficient empirical data on the type and extent of contaminants that are particularly problematic in the cosmetic environment. Mostly gram-negative bacteria such as Enterobacteria e.g. Pluralibacter gergoviae (formerly Enterobacter gergoviae) or typical water germs e.g. Pseudomonas aeruginosa, Pseudomonas putida, Burkholderia cepacia are in focus. The type of microorganism usually allows conclusions to be drawn about possible sources of input (e.g. Pseudmonas species mostly water as cause).
     
The scope of the follow-up examinations and the handling of the product concerned will not be discussed in detail here (see special edition Cosmetics Part 14, 15 and 16).
 
Conclusion
 
The following points are of central importance in the microbiological examination of cosmetic end products or cosmetic raw materials:
  1. Investigation using appropriate methods
    The test must be carried out using validated and appropriate methods. As a rule, the examination is carried out in accordance with ISO standards for cosmetics.
    The use of so-called "rapid methods" usually does not provide the same analytical reliability, the microbiological product safety cannot be proven beyond doubt.
  2. Avoidance of false negative results. As a rule, a dilution as well as a disinhibition or neutralisation of the sample is necessary within the scope of the examination of cosmetic samples. This is the only way to prevent false negative test results from being obtained (microorganisms possibly present in the product are NOT detected).
  3. Identification of detected microorganisms. In the case of detection of micro-organisms in a sample (regardless of the number), it is important to perform identification. The possible entry source can thus be identified much more easily and quickly, and a risk assessment is thus possible.

Further comprehensive information as well as frequently asked questions on the topic "Requirements for microbiological tests" can be found in a summary article of the DGK e.V. (German Society for Applied Cosmetics). The article is available at the following link: https://web.dgk-ev.de/publikationen-fg2/


Your customer consult will be happy to answer any questions you may have about microbiological testing of cosmetics. Contact us at any time, we are looking forward to hearing from you!
 
 
BAV INSTITUT
Hygiene und Qualitätssicherung GmbH
Hanns-Martin-Schleyer-Str. 25
77656 Offenburg

T +49 781 9 69 47 0
F +49 781 9 69 47 20

info@bav-institut.de
bav-institut.de

   

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Dipl.-Ing. Paul Andrei

Registergericht: Amtsgericht Freiburg i. Br.
Registernummer: HRB 471864

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Inhaltlich Verantwortlicher gem. §/10 Absatz 3 MDStV: Dipl.-Ing. Paul Andrei
 
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