According to ISO 11930 (2019) Challenge test, also known as “Preservation efficacy test”, is a procedure specially developed for evaluating the antimicrobial protection of a cosmetic product.
Why is Challenge Test so important for cosmetics?
In the cosmetic industry, all manufactures should ensure that their products are free of (specific) microorganisms that may affect their own quality or even the consumer health. In addition, it should be ensured that any microorganism introduced into the cosmetic will not affect its quality and, essentially, its safety. Antimicrobial preservatives are ingredients added to the cosmetic formulations for protection from microorganisms that could contaminate the product during its production and/or normal use. However, is that enough? To answer this question, challenge test emerges as the leading protocol. Evaluating how effective is the preservative system and its ability of withstanding some types of contamination, challenge test offers a guarantee of safety and efficacy to a product during its shelf life.
How is Challenge Test performed?
The challenge method is based on the inoculation of a formulation with a known concentration of five relevant strains of microorganisms; Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis. At the same time, the neutralization of the possible antimicrobial activity of the formulation(s) shall be checked and demonstrated.
In this stage, in the presence of each microorganism (without its inhibition), the ability of the neutralizer to neutralize the antimicrobial activity of the formulation is verified. A calibrated suspension is inoculated in the neutralizer with and without the formulation(s) under study and the different counts are compared. If the obtained results do not comply with the requirements, additional tests must be carried out (such as, for instance, the selection of a different neutralizer or a further sample dilution). If the results still do not comply, the formulation is not susceptible to contamination by the strain concerned and the result should be "Not susceptible to contamination".
Preservation efficacy of the formulation:
The evaluation of the preservation efficacy is based on inoculation of the cosmetic formulation with a calibrated inocula previously prepared from each one of the five strains of microorganisms. The surviving microorganisms are enumerated at defined intervals during a period of 28 days, specifically after 7 (T7), 14 (T14) and 28 (T28) days. With the obtained results, it is determined the number of microorganisms present at T0 and the number of survivors for the remaining times. Finally, for each time and strain, the log reduction is should be calculated and compared to the values required for evaluation criteria.
Results - Understanding the acceptance criteria
As previously mentioned, for each time (T0, T7, T14 and T28) and each strain is determined the number of surviving microorganisms and calculated the log reduction. It should be considered the inherent variability in microbial counts used to calculate the reduction values. Therefore, a deviation of 0.5 log units is acceptable in the present method. Then, for each specific time, the obtained values are compared to the acceptance criteria required by the method for acceptable preservation. In challenge test two different levels of acceptability criteria are available, representing the protection capacity of cosmetic formulation.
For bacteria (S. aureus, E. coli, P. aeruginosa), at T7, there should be at least a 3-log reduction from the initial count and, from this time onward there should be no increase in the count from the previous contact time (at T14 and T28).
For C. albicans, at T7, there should be at least a 1 log reduction from the initial count and, from this time onward there should be no increase in the count from the previous contact time (at T14 and T28).
For A. brasiliensis there should be no increase from the initial count, at T14, and no less than a 1 log reduction at T28.
With this criterion, the formulation is protected against microbial proliferation which can present a potential risk for its use, meeting the requirements of the ISO. No additional factors need to be considered.
For bacteria, at T14, there should be at least a 3-log reduction from the initial count and, at T28, there should be no increase in the microbial population.
For C. albicans, at T14, there should be at least a 1 log reduction from the initial count and, at T28, there should be no increase in the microbial population.
For A. brasiliensis, at T14 and also at T28, there should be no increase from the initial count.
With this criterion, the level of protection is acceptable. However, the risk analysis shall demonstrate the presence of control factors (not related to the formulation itself) indicating that the microbiological risk is tolerable. For instance, the package design plays a major role in the microbiological risk assessment and different factors can be considered (and adjusted) such as packaging configuration, its size and the quantity used per application.
If the obtained values do not comply with criterion A or B, the formulation under study does not satisfy the requirements of the challenge test. However, the overall evaluation of the antimicrobial protection of a cosmetic takes into account the challenge test combined with the microbiological risk assessment. Therefore, in the presence of these results, the status of the product shall be evaluated solely according the last one. Parameters such as characteristics and composition of the formulation, the production conditions, characteristics of the packaging, specific recommendations for the use and the area of application play an important role in this assessment. Thus, if the risk analysis demonstrates the existence of strengthened control factors, the cosmetic product still meets the requirements of the ISO.
Challenge test is required for all of cosmetic products?
Despite its impact in the cosmetic industry, challenge test is not required for all products. Microbiologically low-risk cosmetic products can be exempt from this test because, due to their intrinsic characteristics, the possibility of microbiological contamination is very low or even nonexistent.
Water, specifically the available water in a formulation, is one of the most important factor controlling the rate of growth of an organism. As the amount of free water is reduced, slower will be the microbial growth, eventually leading to the cell death. In a formulation, water activity is a measure of the available water (aw). Since the possibility of microbial proliferation is non-existent in formulations with a aw lower than 0.75, there is no need to carry out the challenge test in these types of formulations.
pH of formulation
An acidic or alkaline pH may create a hostile environment and may, in some products, be used as part of their preservative system. In the presence of an extreme pH, either acidic or alkaline, the microorganisms need to expend energy on maintenance of intracellular pH rather than growth. Thus, products with pH above 10.0 and below 3.0 do not require microbiological testing, including challenge test.
Raw materials that can create a hostile environment
The use of certain raw materials will help to create an environment hostile to microbial growth such as strong oxidizing agents or strong reducing agents, polar organic solvents, ammonia and monoethanolamine, oxidizing dyes, aluminium chlorohydrate (and related salts) and propellant gases. In addition, microbial growth is also prevented in aqueous systems containing higher than 20 % (v/m) of absolute ethyl alcohol. For levels below, other physico-chemical factors need to be evaluated.
As the temperature rises, growth is inhibited and microorganisms are killed. Indeed, temperatures above 65°C can cause thermal inactivation of the microbial activity. Thus, for formulations that are filled at a temperature above this value, challenge test is not required.
In a cosmetic, the type of packaging has a direct influence on the risk of its contamination. Certain packaging components (e.g. a pump dispenser, single dose units) contribute to the protection and preservation of a formulation. On the other hand, certain presentations, such as pressurized delivery or unit-dose, also provide full protection of the product from contamination during its use. Other factors (e.g. indication of short duration of use) also contribute to the protection of formulation.
Combinations of some of mentioned factors can help to create an environment that is hostile to microbial growth or survival.