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TESTING 59


volunteers with particular types of skin and make sure that products are not going to cause irritation issues even for user groups with unusually sensitive or easily irritated skin.


Optimising the tests The project’s aim was to develop an extended time point test using the 3D in vitro skin model, looking at the irritancy of a range of ultra-mild items over an extended period of 48 hours. We initially used a range of surfactants to evaluate the model, because we know that surfactants are commonly used in a wide range of cosmetic and personal care formulations. We determined their ET50 values, and then we used that data to develop a prediction model linking the in vitro data to the in vivo human skin patch test. Then we created a database of industry-leading ingredients and formulations which can be used for companies to benchmark their own products against to see how mild their ingredients or their products are compared to the industry average values. Table 1 shows an overview of the in vitro


method. We applied the different surfactants to the surface of the skin model for different time points up to 48 hours. The negative control was not treated. Everything was conducted in triplicate, and we had a positive control which is a non- ionic surfactant called Triton X-100. The measurement, the output of this test, is based on the metabolic activity of the cells, and this is indicated by the conversion of a well-known dye called MTT. This is the same detection method that is used in the standard regulatory skin irritation tests. So we wanted to make sure that we were compatible with those methods as well. The output was measured with the ET50 value and then we compared that with the in vivo information.


In vivo testing We wanted to understand the following factor: How well does patch testing discriminate really mild ingredients and products? This was a good opportunity to systematically look at modern ingredients


Table 1: Optimisation of in vitro methods Test Items


18 17 16 15 14 13 12 11 10 9


1 2 Day Rank order of irritancy E (SLS 70%)


C (SLES/CAPB blend 3) A (SLES/CAPB blend 1) B (SLES/CAPB blend 2) D (Sles/CAPB blend 3) Control (E45 Cream)


Figure 4: Irritation potential of surfactant blends (SLES / CAPB) in vivo.


and modern formulations and see whether we can discriminate even very mild products. We also wanted to understand, for these very mild products, whether the standard method, which is our expert nurses, who have a minimum of ten years of experience of grading erythema and irritation of skin, compared to instrumental methods measuring the redness, the erythema of the skin, and skin temperature. In addition, how well does the human volunteer testing correlate to the in vitro irritation testing? With 48-hour single application patch


tests, which are often performed in the cosmetics industry, it is possible for the test to show that all the products would have passed and to be non-irritant. However, as the patch test evolves and we keep applying the products for several days, up to 14 days, it can show that some of the products would be unacceptable to be released onto the market for consumers. This is why it is important to understand the sensitivity of the method that is been used and whether it generates useful data or just a report which fails to illustrate the real impact of products on the market.


 Surfactants: SLS, SLES, CAPB, a novel biosurfactant  Applied to the skin model surface and incubated for 1, 5, 18, 24 and 48 hours


Controls Measurement Output February 2020


 Negative control: not treated  Positive control: Triton X 100 (non ionic surfactant): 1% solution


 Metabolic activity (conversion of MTT) as an indicator of cell damage  ET50 value (time taken to reduce the viability of the cells to 50% compared with the untreated negative control) PERSONAL CARE EUROPE Cumulative irritation scores


12 4 3 0 0 0


3 4


*


* *


 A  B  C  D  E  Control


chromameter value


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