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Challenge Test – Antimicrobial Protective Effectiveness Test
Antimicrobial protective efficacy test (Challenge Test) is a laboratory test that measures the level of biological activity possessed by the preservative system of a cosmetic product. This test is designed to evaluate the performance of the cosmetic product with preservatives added to inhibit the growth of microorganisms during or after the manufacturing process.

The screening test is based on assessing the risk of microbial contamination by exposing the finished product formula to artificial contamination. The basis of all methods used is to determine the number of microorganisms remaining alive in test samples taken at different times, after combining the test sample with different microorganisms. The methods applied vary in terms of the test microorganisms, the media used, the storage conditions of the samples and the application of the test.
According to the ISO 29621 standard, Challenge Test is not required for products with low microbiological risk product groups.
Physico -Chemical Factor Limit Example
pH ≤3.0 Skin peelers ( glyclic acid )
pH ≥10.0
hair straighteners
water removed -
body oils
Ethanol or other alcohols ≥ 20% Hair sprays, tonics, perfumes
filling temperature ≥65.0°C Lip balm, lipsticks , cream blushes
water activity ≤0.75 Lip balm, lipsticks , cream blushes
Organic solvents:
Ethyl acetate
Butyl acetate

> 10%
Solvent-based products:
Nail polishes
Alkaline compounds:

≥ 0.5%
Oxidizing products:
Hair dyes, perms
aluminum chlorohydrate and salt related substances ≥ 25%
Antiperspirant products
hydrogen peroxide ≥ 3% Hair lighteners, bleaches , perms
NOTE: Soaps, synthetics and solid cleaners are considered low risk because they have low water activity and high pH .

This test, which checks the effectiveness of the protective system using the ISO 11930:2019 standard, is based on the inoculation of the cosmetic product with 5 different microorganism strains. The remaining concentration of microorganisms is then determined 7, 14 and 28 days. The methods applied vary in terms of the test microorganisms, the media used, the storage conditions of the samples and the application of the test. Microorganism strains to be used
  • Pseudomonas aeruginosa (ATCC 9027)
  • staphylococcus aureus (ATCC6538)
  • Candida albicans (ATCC 10231)
  • Escherichia coli (ATCC 8739)
  • aspergillus brasiliensis (ATCC 16404)
The test involves challenging a clean product with a prescribed appropriate microorganism inoculum and storing the inoculated product at a prescribed temperature. Using serial dilution and plate counting, the number of organisms remaining in test products is determined at regular intervals. Products that meet the specified criteria are deemed to be adequately protected for manufacturing and consumer use. Products that do not meet the criteria are considered inadequately protected. While

the product is in the R& D stage, a Challenge test should be applied to determine the compatibility of the preservative and the formulation and the effectiveness of the added preservative in the finished product. Important note: If a formulation change has been made in the existing product, a new challenge test must be applied for that product.
First of all, the cleanliness and microbial flora of each cosmetic product should be checked at the beginning of the test. According to the ISO 11930 Standard used, before starting the protective effectiveness test, the total aerobic mesophilic organism (ISO 21149) and mold-yeast (ISO 16212) count of the incoming sample must be made. After these tests are completed, protective effectiveness testing begins .