58 TESTING
TABLE 2: EUROPEAN PHARMACOPOEIA (EP) PASS CRITERIA FOR TOPICAL PRODUCTS
Log Reduction 2 Day Bacteria Fungi 2 - 7 Day 3 - 14 Day
- 2
28 Day NI NI
needed to be able to offer a vegan alternative for challenge testing, not only to keep up with changing cosmetic trends and also to ensure the entirety of the testing process from start to finish. In October 2022, ISCA partnered up with
The Vegan Society to certify our challenge test methods. We needed to ensure that replacing TSA with VeganSure (VSA), and SDA with Potato Dextrose Agar (PDA) would not affect the standard test methods and that we would get the same performance results.
Preservative efficacy testing (PET) Microbiological assessments, alternately referred to as ‘challenge tests’ or ‘preservative efficacy tests’ (PET), exhibit slight variations depending on the intended global market for the product. The quality of the test method employed varies in accordance with the specific standard procedure being executed. At ISCA, we perform the following preservative efficacy tests: ■ European Pharmacopeia (EP) ■ United States Pharmacopeia (USP) ■ ISO 11930 The European Pharmacopeia (EP) test method is the most stringent method within our testing portfolio. This traditional test method is recognised and accepted globally. Alternatively, our United States Pharmacopeia (USP) and ISO 11930:2019 test methods are recognised standards of ‘Evaluation of the antimicrobial protection of a cosmetic product’. All three test methods employ the same microbiological test species (Table 1).
Testing process Testing was initiated using samples A and B. By conducting both a traditional challenge test (EP) against a Vegan EP method running simultaneously to avoid any other contributing factors interfering with the test. Aseptic techniques were used throughout the entirety of the test procedure to ensure accurate results and eliminate any chance of contamination.
TABLE 4: SAMPLE A RESULTS FROM TRADITIONAL TEST METHOD
Test Species P. aeruginosa S. aureus
C.albicans A. brasilliensis
Colony forming units per g after inoculation 2 Day
7 Day <10
8.60 x 101
- -
<10 -<10
- -
14 Day
- -
<10 <10 PERSONAL CARE November 2023 28 Day <10 <10 <10 <10 P. aeruginosa S. aureus
C.albicans A. brasilliensis
Pseudomonas aeruginosa Staphylococcus aureus Candia albicans
Aspergillus brasiliensis
NCTC 12924 NCTC 10788 NCPF 3179 NCPF 2275
TABLE 3: INOCULATION COUNTS FOR THE TRADITIONAL EUROPEAN PHARMACOPEIA (EP) AND VEGAN ALTERNATIVE
Test Species Initial Inoculum Level
Traditional Test Method (cfu/g) 8.20 x 105 2.12 x 106 1.96 x 106 3.00 x 105
Initial Inoculum Level Vegan Test Method (cfu/g)
2.34 x 106 2.80 x 106 1.66 x 105 2.50 x 105
Initially, the focus was on ensuring that the
vegan-friendly media, PDA and VSA, could support the growth of our microbial test species, thus facilitating the creation of inoculations to purposely contaminate our sample cosmetic products for testing. All media was prepped and sterilised by autoclaving at 121°C. PDA was employed to cultivate the test
species Candida albicans and Aspergillus brasiliensis, and they were incubated at 25°C for five to seven days. For the bacterial test species, Pseudomonas aeruginosa and Staphylococcus aureus, incubation took place at 35°C for two days. We achieved successful growth of all test species, resulting in inoculation counts ranging from 105
to 106 . Four 20g portions of the samples were transferred to sterile containers and inoculated
TABLE 5: SAMPLE A RESULTS FROM VEGAN ALTERNATIVE TEST METHOD
Test Species
Colony forming units per g after inoculation 2 Day
7 Day <10 <10
- -
<10 -<10
- -
14 Day
- -
<10 <10 28 Day <10 <10 <10 <10
separately with 0.2 ml culture of the test species as detailed below. The inoculated sample portions were mixed using sterile implements to evenly distribute the inoculum and stored at room temperature. The challenge test protocol of the European Pharmacopoeia (EP) 10.0 was followed in exact detail.
Results Inoculation counts tend to exhibit slight variations and are not consistently replicable. This variation is elucidated when comparing the inoculation counts between the traditional test method and the vegan alternative test method (Table 3). All inoculation counts are above the desired threshold for the challenge testing. For both the traditional and vegan alternative test methods, Sample A meets the criteria set
TABLE 6: SAMPLE B RESULTS FROM TRADITIONAL TEST METHOD
Test Species
Colony forming units per g after inoculation 2 Day
7 Day P. aeruginosa 3.0x105 S. aureus
C.albicans A. brasilliensis
2.30x 102
- -
3.0x105
1.50x 102
14 Day
- -
- 3.0x105 - 3.0x105
28 Day / / / /
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