TESTING ET50
140 120 100 80 60 40 20 0
1 Time (h) ET50
140 120 100 80 60 40 20 0
1 10 Time (h) 100
determination of B 0.3% CAPB
160 140 120 100 80 60 40 20 0
1 10 Time (h)
Figure 5: The results of four surfactants tested for mildness using the non-animal XtraMild test. The ET50 values produced by the test provided the same rank order of irritation as clinical scoring (CS) from human patch testing
testing skin sensitisation relies on three in vitro tests, each of which corresponds to a key event in the skin sensitisation adverse outcome pathway (AOP). While the tests for Key Event 1 are completely chemically defined, those for Key Events 2 & 3 contain animal derived components. KeratinoSens is one of the skin sensitisation
tests for Key Event 2. Modified human keratinocyte cells are cultured in the lab and dosed with the item to be tested (e.g. a cosmetic ingredient). Under the correct conditions, the dosed cells generate a luminesence signal which can be detected and quantified. If the detected signal reaches a certain threshold, a positive result (indicating a sensitiser) is obtained. Several animal-derived components are used as standard within the KeratinoSens test. FBS is used in the culture of the keratinocyte
ET50
120 100 80 60 40 20 0
cells, while the enzyme porcine (pig) trypsin is used to detach the cells from the flasks in which they are cultured. For XCellR8’s adaptation to animal-product-
free conditions, the first step is to identify alternatives to the animal components. In this case, human serum - collected from FDA- approved donor sources - was used rather than FBS, and the recombinant protein Trypzean™ in place of porcine trypsin. The test was then validated by testing a
panel of proficiency chemicals from which data exists for the original KeratinoSens method, and by comparison with historic animal test data. All 20 reference chemicals were classified correctly. To have the animal-product-free
adaptation officially recognised and added to the test guideline, the data was assessed by the OECD Expert Working Group on Skin
determination of 3 face mask formulations
Rank order of irritancy using linear extrapolation and logic equation
B ET50 12.86 CS 11 > A 14.42 5 IRRITANCY CLASSIFICATION
B= face mask 2: Very mild A= face mask 1: Very mild C= face mask 3: Non-irritating
1 10 Time (h)
Figure 6: The results of three face mask formulations tested for mildness using the non-animal XtraMild test. Like the surfactants, the ET50 values produced by the test provided the same rank order of irritation as clinical scoring (CS) from human patch testing
www.personalcaremagazine.com June 2022 PERSONAL CARE 100 > C >48 2
Sensitisation and WNT National Co-ordinators committee. Following acceptance by these groups, the adaptation was added as an annex to OECD TG442d in 2018. As illustrated by this example, making
the change to non-animal products is just one step of the process. Correctly performing validation of the method and presenting the result to relevant regulatory/scientific groups is a key consideration, adding up to a lengthy process requiring significant investment. A xeno-free adaptation for a Key Event 3
test (OECD TG 442e h-CLAT) is still underway, and it is unlikely that resources will be invested to adapt every existing non-animal test to animal-product-free conditions. A key step for the future will be encouraging those who develop new test methods to ensure they are animal-product-free from the outset.
100 ET50 10
determination of A 0.3% SLES
140 120 100 80 60 40 20 0
1 Time (h) determination of D 0.3% Novel surfactant 100 ET50
determination of C 0.3% SLS
Test items (0.3%, pH4.7) at 1, 5, 18, 24, 48hrs
Rank order of irritancy using linear extrapolation and logic equation
B
ET50 9.37 CS 14
> A
10.25 9
> C
29.4 4
IRRITANCY CLASSIFICATION
C= SLS: Moderate to mild A= SLES: Moderate to mild B= CAPB: Non-irritating D= Novel surfactant: Non-irritating
> D
38.08 0
33
Percentage of viability relative to untreated control
Percentage of viability relative to untreated control
Percentage of viability relative to untreated control
Percentage of viability relative to untreated control
Percentage of viability relative to untreated control
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