CERTIFICATE OF ANALYSIS OF CHALLENGE TEST (ISO 11930:2012)
Results – Microbiological Stability Test – EPSAN® C
RESULTS OF THE CHALLENGE TEST (ISO 11930:2012)
The UFC/g are expressed on a log scale. UFG/g is estimated at the time of initial inoculum (t0) and after 7, 14 and 28 days.
INTERPRETATION AND CONCLUSIONS OF TEST RESULTS
The tested product must present an inhibiting activity against the microbiological strain used, according to the acceptability criteria of the ISO 11930:2012 method.
To evaluate the efficacy of the storage system, the method must fulfill two criteria:
• CRITERION A
It is assigned if the obtained results satisfy all the conditions reported below:
– after 7 days (t7): reduction of at least 3 logs for bacteria and 1 log for C. albicans;
– after 14 days (t14): reduction of at least 3 logs for bacteria (without any increase compared to the previous time), at least 1 log for C. albicans (without any increase with respect to the previous time), and no increase compared to t0 for A. brasiliensis;
– after 28 days (t28): reduction of at least 3 logs for bacteria (without any increase compared to t14), at least 1 log for C. albicans (without any increase compared to t14), and at least 1 log for A. brasiliensis.
If the cosmetic product is in accordance with the criterion A:
– the microbiological risk is acceptable: the tested product is to be considered safe against the microbial proliferation and it is not necessary to ponder on further factors which result to be independent from the formulation.
• CRITERION B
It is assigned if the obtained results satisfy all the conditions reported below:
– after 14 days (t14): reduction of at least 3 logs for bacteria, at least 1 logs for C. albicans and no increase compared to time zero (t0) for A. brasiliensis.
– after 28 days (t28): reduction of at least 3 logs for the bacteria (without any increase compared to t14), at least 1 log for C. albicans (without any increase compared to t14), and no increase for A. brasiliensis.
If the cosmetic product is in accordance with the criterion B:
– the microbiological risk is acceptable, but its true evaluation will have to take into account also other control factors independent from formulation (i.e. packaging features) to reduce the microbiological risk.
REFERENCES
– Regulation (EC) No 1223/2009 (The “Cosmetic products regulation”), replaced the European Union (EU) Cosmetics Directive 76/768/EEC, which harmonizes and simplifies the cosmetics regulations across the EU member states by Product Information File (PIF).
– COLIPA Guidelines “Product Information File (PIF)” – December 15th 2011.
– ISO (International Organization for Standardization) 11930:2012.
The data reported in the table below express the Colony Forming Unit (CFU) relative to 1 g of product.
T: analytical time of the analysis.
The data below express the reduction of microbial load in log scale as a function of time
(NI: no increase-ND: no decrease).
TABLE OF LOG REDUCTION OF MICROBIAL LOAD
| Microrganisms | t7 | t14 | t28 | Criterion A |
| S. aureus ATCC 6538 | ≥ 3 Log | NI | NI | Acceptable |
| P. aeruginosa ATCC 9027 | ≥ 5 Log | NI | NI | Acceptable |
| E. coli ATCC 8739 | ≥ 3 Log | NI | NI | Acceptable |
| C. albicans ATCC 10231 | ≥ 4 Log | NI | NI | Acceptable |
| A. brasilienisis ATCC 16404 | Not performed | ≥ 2 Log | NI | Acceptable |
The charts above show the variation of the microbial concentration, evaluated at each time studied, relative to the initial inoculum load (t0) of the product.
LOG CFU/g: Colony-Forming Unit related to 1 g of product expressed in log scale.
Time of analysis:
t0: microbial load in the inoculated sample;
t7: microbial titer 7 days after the inoculum;
t14: microbial titer 14 days after the inoculum;
t28: microbial titer 28 days after the inoculum.
