🧴 Challenge test — preservative protection assessment
In short
Cosmetic preservative efficacy test (challenge test / PET). Product inoculation with test strains and monitoring microbial reduction over 28 days according to ISO 11930. The test is performed according to PN-EN ISO 11930:2019/A1:2022; Test strains: S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231, A. brasiliensis ATCC 16404. The procedure comprises 11 steps (about 31 days 6 h 45 min–33 days 8 h 45 min in total); it is used for: Assessment of preservative system effectiveness in new cosmetic formulations, Verification of preservative protection after formula changes (preservative replacement, pH change), Testing microbiological stability of products for registration.
At a glance
- Standard: PN-EN ISO 11930:2019/A1:2022
- Category: Pharma and Cosmetics
- Procedure steps: 11
- Total time of stages: 31 days 6 h 45 min–33 days 8 h 45 min
- Test strains: S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231, A. brasiliensis ATCC 16404
- Inoculum concentration: 10⁵–10⁶ CFU/g product
- Incubation temperature: 20–25°C
Overview
Challenge test (provocation test, Preservation Efficacy Test — PET) is a key microbiological test assessing the ability of a cosmetic product to protect against secondary microbiological contamination during production, storage, and use. The PN-EN ISO 11930 standard describes the reference method for assessing antimicrobial protection of cosmetics.
The test involves controlled introduction (inoculation) of known quantities of test microorganisms into a cosmetic product sample, then monitoring changes in numbers of these microorganisms at strictly defined time intervals over 28 days. Log reduction is calculated for each strain and each interval, and results are compared with standard acceptance criteria.
The test is required as part of Safety Assessment of cosmetic products according to Regulation (EC) No 1223/2009. Challenge test is one of the basic elements of cosmetic product documentation (PIF — Product Information File) and is required by the person responsible for product safety (safety assessor).
The standard defines two acceptance profiles: profile A (preferred — strong protection) and profile B (acceptable — minimal protection). Challenge test result confirms that the product preservative system (preservatives, pH, water activity, composition) is sufficient to protect against realistic contamination under use conditions. Minimum required sample quantity is 250 g for full test (5 strains × duplicates × 4 time points).
Method principle
Cosmetic product sample is inoculated separately with five test strains: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus brasiliensis ATCC 16404. Each strain is introduced at concentration approx. 10⁵–10⁶ CFU/g product (inoculation must not change product composition by more than 1%). Samples are incubated at temperature 20–25°C. On days 7, 14, and 28 (bacteria and yeasts) and 14 and 28 (molds), portions of sample are taken, preservative system is neutralized (by dilution method or chemical neutralizers), and number of viable microorganisms is determined by plate method. Log reduction is calculated: log R = log N₀ − log Nₜ, where N₀ is CFU count after inoculation, Nₜ — on day t. Results are compared with profile A or B criteria.
Applications
- Assessment of preservative system effectiveness in new cosmetic formulations
- Verification of preservative protection after formula changes (preservative replacement, pH change)
- Testing microbiological stability of products for registration
- Safety Assessment documentation according to Regulation 1223/2009 (PIF)
- Optimization of preservative concentration (minimization while maintaining effectiveness)
- Comparative testing of preservative systems (traditional vs. "preservative-free")
- Assessment of products with "natural" / "without parabens" claims — protection confirmation
- Validation of production technologies (hot fill, aseptic filling)
Key parameters
| Parameter | Value |
|---|---|
| Test strains | S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231, A. brasiliensis ATCC 16404 |
| Inoculum concentration | 10⁵–10⁶ CFU/g product |
| Incubation temperature | 20–25°C |
| Measurement intervals (bacteria/yeasts) | Day 7, 14, 28 |
| Measurement intervals (molds) | Day 14, 28 |
| Profile A (preferred) — bacteria | ≥3 log reduction (day 7), ≥3 log (day 14), no growth (day 28) |
Standard
- Standard number
- PN-EN ISO 11930:2019/A1:2022
- Title (PL)
- Kosmetyki — Mikrobiologia — Ocena ochrony przeciwdrobnoustrojowej produktu kosmetycznego
- Title (EN)
- Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product
Step-by-step procedure
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Test strain preparation
Activate lyophilized strains on appropriate media (TSA for bacteria, SDA for fungi). Perform 2–3 passages. Prepare suspension with density 10⁷–10⁸ CFU/mL.
-
Inoculum standardization
Adjust suspension density to McFarland 0.5 (approx. 10⁸ CFU/mL) or measure with densitometer. Dilute to obtain 10⁶–10⁷ CFU/mL.
-
Product sample inoculation
Weigh 20–30 g product into sterile containers (separately for each strain). Add inoculum in volume <1% of sample mass. Mix thoroughly. Obtain concentration 10⁵–10⁶ CFU/g.
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Initial concentration confirmation (T₀)
Immediately after inoculation, take 1 g sample. Dilute in neutralizer. Plate on TSA/SDA. Count colonies after incubation — confirm N₀.
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Sample incubation
Store inoculated samples at temperature 20–25°C, in darkness, for 28 days. Do not open without necessity.
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Day 7 sampling
Collect 1 g sample (bacteria + yeasts). Dilute in neutralizer (Dey-Engley broth). Perform dilution series (10⁻¹ to 10⁻⁵). Plate on TSA/SDA. Incubate.
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Day 14 sampling
Repeat day 7 procedure for all five strains (bacteria, yeasts, and molds). Plating, incubation, reading.
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Day 28 sampling
Last measurement point. Collect, dilute, plate, incubate. Read and count colonies for all strains.
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Log reduction calculation
For each strain and each time point, calculate: log R = log₁₀(N₀) − log₁₀(Nₜ). Compare results with ISO 11930 profile A and B criteria.
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Result assessment
Profile A (preferred): bacteria ≥3 log reduction by day 7, no growth by day 28; yeasts ≥1 log (day 7), no growth (day 28); molds — no growth (day 14, 28). Profile B (acceptable): milder criteria.
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Report and documentation
Prepare report with: product identification, test strains, results (CFU/g and log R tables), profile A/B assessment, conclusion on preservative protection effectiveness. Archive as PIF element.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Class II laminar cabinet | Thermo Fisher MSC-Advantage, Esco Labculture, FASTER BH-EN | 25 000–60 000 PLN |
| Incubator 20–25°C (with cooling) | Memmert IPP110, Binder KB240, POL-EKO ST 500 | 10 000–25 000 PLN |
| Incubator 30–35°C | Memmert IN110, Binder BD115 | 8 000–20 000 PLN |
| Autoclave | Tuttnauer 3870ELV, Systec VX-75 | 20 000–60 000 PLN |
| Water bath 45–50°C | Memmert WNB 22, GFL 1012, for tempering media | 3 000–8 000 PLN |
| Vortex mixer | IKA Vortex 3, Heidolph Reax top | 1 500–4 000 PLN |
| Colony counter | Interscience Scan 300, IUL Flash & Go | 5 000–20 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| S. aureus strain ATCC 6538 | — | Lyophilized reference strain, storage -20°C / -80°C, max 5 passages from reference |
| E. coli strain ATCC 8739 | — | Lyophilized reference strain, storage in cryobeads -80°C |
| P. aeruginosa strain ATCC 9027 | — | Reference strain, storage in cryobeads -80°C |
| C. albicans strain ATCC 10231 | — | Yeast — reference strain, culture on SDA, 30–35°C |
| A. brasiliensis strain ATCC 16404 | — | Mold — reference strain, culture on PDA, 20–25°C, spore collection after 7–14 days |
| Casein-soy agar (TSA) | 91079-40-2 | For bacteria culture and TAMC determination, 500 g |
| Sabouraud Dextrose agar (SDA) | — | For yeast and mold culture, with chloramphenicol 50 mg/L |
| Neutralizing solution (Dey-Engley broth) | — | Neutralizing broth for broad spectrum of preservatives (parabens, formaldehyde, QAC, isothiazolinones) |
Health and safety (OHS)
- Work with pathogenic microorganisms (S. aureus, P. aeruginosa, E. coli) — BSL-2 laminar cabinet mandatory
- Nitrile gloves, safety glasses, laboratory coat — mandatory
- A. brasiliensis fungus — allergenic spores, avoid inhalation, work in laminar cabinet
- Autoclaving all biological waste before disposal (121°C, 15 min, 1 atm)
- Workstation disinfection with 70% alcohol before and after work
- Strain cryobeads — store in -80°C freezer, maintain passage register
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 11930:2019/A1:2022 — “Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product”.
How does this method work?
Cosmetic product sample is inoculated separately with five test strains: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus brasiliensis ATCC 16404. Each strain is introduced at concentration approx.
What is the measuring range and accuracy?
Test strains: S. aureus ATCC 6538, E. coli ATCC 8739, P. aeruginosa ATCC 9027, C. albicans ATCC 10231, A. brasiliensis ATCC 16404; Inoculum concentration: 10⁵–10⁶ CFU/g product; Incubation temperature: 20–25°C; Measurement intervals (bacteria/yeasts): Day 7, 14, 28.
How long does the test take?
The times given for the individual stages add up to approximately 31 days 6 h 45 min–33 days 8 h 45 min. The procedure comprises 11 steps.
What equipment is required?
Class II laminar cabinet, Incubator 20–25°C (with cooling), Incubator 30–35°C, Autoclave, Water bath 45–50°C, Vortex mixer, Colony counter. Indicative cost of the core instrument (Class II laminar cabinet): 25 000–60 000 PLN.
Where is this test used?
Assessment of preservative system effectiveness in new cosmetic formulations; Verification of preservative protection after formula changes (preservative replacement, pH change); Testing microbiological stability of products for registration; Safety Assessment documentation according to Regulation 1223/2009 (PIF); Optimization of preservative concentration (minimization while maintaining effectiveness); Comparative testing of preservative systems (traditional vs. "preservative-free"); Assessment of products with "natural" / "without parabens" claims — protection confirmation; Validation of production technologies (hot fill, aseptic filling).
What safety precautions apply?
Work with pathogenic microorganisms (S. aureus, P. aeruginosa, E. coli) — BSL-2 laminar cabinet mandatory; Nitrile gloves, safety glasses, laboratory coat — mandatory; A. brasiliensis fungus — allergenic spores, avoid inhalation, work in laminar cabinet; Autoclaving all biological waste before disposal (121°C, 15 min, 1 atm); Workstation disinfection with 70% alcohol before and after work; Strain cryobeads — store in -80°C freezer, maintain passage register.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 11930.