AIM

The Challenge test is a procedure that verifies the microbiological stability of a cosmetic product.

The test implies the contamination of the products with different species of microorganisms and the following evaluation of bacterial load variation by count in plates at regular intervals for a period of 28 days.

The defensive capacity of the cosmetic products from the aggression by the microbes is verified by observing the reduction of the bacterial load (of each species) in a given time interval, according to the acceptability criteria issued by the ISO 11930:2012 Regulation.

The procedure consists of:
– microorganism preparation for the inoculum;
– preliminary bacterial load measurement of the sample to be tested;
– inoculum of the samples;
– check of microorganism’s survival at given time intervals;
– analysis of results.

MATERIALS AND METHODS

MICRORGANISMS USED FOR ANTIBACTERIAL TESTING
As stated by the ISO 11930:2012 regulation:

• GRAM POSITIVE COCCI
Staphylococcus aureus (ATCC 6538), being a common saprophyte of the skin and a potential pathogen;

• GRAM NEGATIVE RODS
among fermenters, Escherichia coli (ATCC 8739), as its presence is an index of fecal contamination; among non fermenters Pseudomonas aeruginosa (ATCC 9027) being ubiquitous and well-known pathogen.

• YEASTS
Candida albicans (ATCC 10231), a widespread yeast in nature and often involved in pathological processes

• MOULDS
Aspergillus niger (ATCC 16404), a microorganism often responsible for pollution of badly stored products.
The initial inoculum concentration (t0) has to range from 105 to 106 Colony-Forming Units (UFCs/g) of products.

GROWTH MEDIA
The growth media used are:
– Tryptone Soya Agar (TSA) for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus;
– Sabouraud Dextrose Agar (SDA) for Candida albicans;
– Potato Dextrose Agar (PDA) for Aspergillus niger.

LAB-IMPLEMENTED REAGENTS
The reagents used are:
– Diluting solution;
– Neutralizer;
– Diluting solution (Polysorbate) to prepare Aspergillus niger suspension.

DETERMINATION OF THE NEUTRALIZER EFFICACY
As indicated at paragraph 5.5 of the ISO 11930:2012 method, the determination of the antimicrobial protection of a neutralizer was performed.

DETERMINATION OF THE ANTI-MICROBIAL PROTECTION OF A COSMETIC PRODUCT
As indicated at paragraph 5.6 of the I SO 11930:2012 method, the determination of the antimicrobial protection of a cosmetic product was performed by inoculating the formulation with calibrated inocula.

EVALUATION OF THE TEST TIME
The checking time for each sample and for each standard strain corresponds to the inoculum of the microorganisms at time zero (t0) and after 7, 14, 28 days (t7, t14, t28) to verify the lack of resistant strains, eventually till the complete exhaustion of the initial inoculum.

ANALYTICAL TECHNIQUE
Petri dish seeding is the technique used in the test. The inoculated product was stored at room temperature and repaired from light still the end of the test.