🧴 Challenge test — preservative protection assessment

Pharma and Cosmetics PN-EN ISO 11930

Overview

STATUS (as of 02.11.2026). PN-EN ISO 11930:2019-03 — English version (published 07.03.2019), implements EN ISO 11930:2019 and ISO 11930:2019 [IDT], replaced PN-EN ISO 11930:2012 with corrigendum Ap1:2017-05; amendment PN-EN ISO 11930:2019-03/A1:2023-02 (published 23.02.2023) implements ISO 11930:2019/Amd 1:2022. The catalogue cards carry no withdrawal notice. In the ISO catalogue the standard is at stage 90.92 (“to be revised”). The second ISO edition (15.01.2019) added two diluents for bacteria and Candida albicans (composition 1 and 2) and changed how results are recorded at low colony counts (fewer than 30 for bacteria and C. albicans, fewer than 15 for A. brasiliensis — the number of colonies multiplied by the dilution factor, and with no colonies “< 1” multiplied by that factor). Amendment Amd 1:2022 added modified Eugon LT broth as an alternative neutralizer.

SCOPE (Clause 1). The standard specifies a procedure for the interpretation of data from the preservation efficacy test and from the microbiological risk assessment (or both) when evaluating the overall antimicrobial protection of a product. It comprises the preservation efficacy test — a reference method for assessing products on the market — and a procedure for evaluating products that are not low-risk products according to ISO 29621. The test does not apply to products whose microbiological risk has been determined to be low (Annex A and ISO 29621). It is primarily designed for water-soluble or water-miscible products and, with modification, also for products in which water is the internal phase. During product development the test may serve as a guideline for an in-house method (modified strains, media, conditions), and the criteria may be adapted to the objective.

PRINCIPLE (Clauses 4 and 5.1). Antimicrobial protection results from chemical preservation, the characteristics of the formulation, the packaging and the manufacturing process. The evaluation combines the characteristics of the formulation (ISO 29621) or the results of the preservation efficacy test with the characteristics of the product, the production conditions (ISO 22716, ISO 29621), the packaging, the recommendations for use and the area of application and target users (Annex D). In the test the formulation is inoculated with calibrated inocula of relevant strains, the number of surviving microorganisms is determined at defined intervals over 28 days, and the log reduction for each time and strain is compared with the minimum values of criteria A or B (Annex B). Before the test, the efficacy of the neutralizer must be demonstrated (5.5) and the microbiological quality of the product determined (ISO 21149 and ISO 16212 or ISO 18415), so that microorganisms present in the sample do not interfere with the recovery of the test strains. When used as a reference method, the test is carried out strictly according to the procedure.

MATERIALS (5.2). Distilled or purified water according to ISO 21148:2017, 8.2; glass beads 3–4 mm, a sintered glass filter of porosity 2 (40–100 µm), sterile glass containers with closures, a centrifuge up to 2000 g. Diluent for bacteria and C. albicans: sodium chloride 8,5 g in 1000 ml of water (composition 1) or with the addition of 1,0 g of tryptone (composition 2, pH 7,0 ± 0,2); for A. brasiliensis — a polysorbate 80 solution (0,5 g/l); all sterilised at 121 °C for 15 min. Neutralizer: Eugon LT 100 broth (egg lecithin and polysorbate 80 neutralize inhibitory substances, octoxynol 9 acts as a dispersing agent; pH 7,0 ± 0,2) or, since Amd 1, modified Eugon LT broth (polysorbate 80 15 g/l, sodium lauryl ether sulfate 1,56 g/l); the suitability of the neutralizer for the strains and the formulation is always demonstrated (5.5), other examples are given in Annex C. Media: tryptic soy agar (TSA, pH 7,3 ± 0,2) for bacteria, Sabouraud dextrose agar (SDA, pH 5,6 ± 0,2) for C. albicans and potato dextrose agar (PDA, agar 20 g/l, pH 5,6 ± 0,2) for A. brasiliensis.

STRAINS (5.3). Pseudomonas aeruginosa ATCC 9027 (equivalents: CIP 82.118, NCIMB 8626, NBRC 13275, KCTC 2513 or an equivalent national collection strain); the available text of the standard ends at this strain — the other four according to descriptions by testing laboratories: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404.

CRITERIA (Annex B, according to descriptions by two testing laboratories). Criteria A — bacteria: reduction of at least 3 orders of magnitude after 7 days, after 14 and 28 days at least 3 with no increase from the previous reading; C. albicans: at least 1 after 7 days, after 14 and 28 days at least 1 with no increase; A. brasiliensis: no increase from the initial count after 14 days, at least 1 after 28 days. Criteria B — bacteria: at least 3 after 14 days and no increase after 28 days; C. albicans: at least 1 after 14 days and no increase after 28 days; A. brasiliensis: no increase after 14 and 28 days. A formulation meeting criteria A satisfies the requirements of the test; one meeting only criteria B requires additional justification that the product is sufficiently protected (e.g. by packaging, process); one meeting neither — further evaluation and usually reformulation.

LEGISLATION. Regulation (EC) No 1223/2009 requires the cosmetic product safety report to contain the results of the preservation challenge test (Annex I Part A point 3). According to the Commission guidelines 2013/674/EU, the challenge test is not necessary for products with a low microbiological risk (with a scientific justification) or for single-use and non-openable products, and is necessary for all other products.

WHAT WE DO NOT GIVE HERE. The concentrations of the inocula and their volume relative to the sample mass, the temperature and storage conditions of the inoculated samples, the procedure for sampling, dilution and counting, the calculations and the overall evaluation procedure of Clause 6 — the available text of the standard ends at the list of strains (5.3), and we give the criteria of Annex B from descriptions by testing laboratories.

Method principle

Five portions of the formulation are inoculated separately with calibrated suspensions of test microorganisms — three bacteria, the yeast Candida albicans and the mould Aspergillus brasiliensis. At defined intervals over 28 days portions are taken, the action of the preservatives is neutralized with a neutralizer of demonstrated efficacy and the number of viable microorganisms is determined by the plate count method. For each strain and time the log reduction relative to the initial count is calculated and compared with criteria A or B of Annex B; the result is combined with a risk assessment according to ISO 29621.

Applications

• Assessment of the preservation efficacy of a cosmetic formulation — reference method for products on the market and in disputes
• Results of the challenge test for the cosmetic product safety report (Regulation (EC) No 1223/2009, Annex I Part A point 3)
• Evaluation of the overall antimicrobial protection of a product that is not a low-risk product according to ISO 29621 — together with packaging and manufacturing process
• Guideline for an in-house method during formulation development (modified strains, media, conditions and criteria adapted to the objective)
• Water-soluble or water-miscible products; with modification — products with water as the internal phase

Key parameters

Standard

Standard number

PN-EN ISO 11930:2019-03 + A1:2023-02

Title (PL)

Kosmetyki — Mikrobiologia — Ocena skuteczności ochrony przeciwdrobnoustrojowej produktu kosmetycznego

Title (EN)

Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product

Step-by-step procedure

1. Is the test needed

   Determine according to Annex A and ISO 29621 whether the product is a low-microbiological-risk product — if so, the preservation efficacy test is not performed.

2. Microbiological quality of the product

   Determine the microbiological quality of the sample according to ISO 21149 and ISO 16212 or ISO 18415, so that microorganisms present in the product do not interfere with the recovery of the test strains (5.1).

3. Neutralizer efficacy

   Demonstrate the efficacy of the neutralizer against the strains and the formulation (5.5); without it the result for a given strain is assessed according to 5.7.3.

4. Inocula

   Prepare calibrated suspensions of bacteria, C. albicans and A. brasiliensis spores (5.4) and inoculate separate portions of the formulation with them.

5. Readings

   At the intervals of the standard over 28 days, take portions, neutralize them and determine the number of viable microorganisms by the plate count method (5.6).

6. Calculations and criteria

   Calculate the log reduction for each strain and time relative to the initial count and compare it with criteria A or B (5.7, Annex B).

7. Overall evaluation

   Combine the test result with the risk assessment (formulation, production, packaging, users) according to Clause 6 and Annex A; prepare the test report according to 5.8.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The available text of the standard contains no safety requirements — the rules for working with the test microorganisms are set by the laboratory on the basis of a risk assessment

Frequently asked questions