As part of a government effort to reduce animal testing in cosmetics and other products, researchers have produced a new protocol for screening skin allergens from products. The new method is potentially cheaper and faster than animal testing, while maintaining a similar performance.
Many governing bodies, including the European Union and some states in the U.S., have recently banned the sale of animal-tested cosmetics. However, animal testing is still required in some parts of the world to assess the safety of cosmetics. With regulations varying by region, it has become challenging for certain products to gain approval across the board.
Some animal-free methods of screening for skin allergens already exist, but their widespread adoption has been stymied by several limitations. For example, the direct peptide reactivity assay (DPRA) which requires costly equipment that can only test a handful of samples at a time. The DPRA can be slow as well. To attain statistically meaningful conclusions about a test compound, testers need to repeat the assay’s multi-day process multiple times. The low sample size per test also reduces opportunity for control samples that would allow researchers to identify and minimize factors that may vary between samples and skew measurements.
A new method
To increase confidence in animal-free tests and promote their adoption, several federal agencies are assessing the reliability of new, promising methods. One such method that is much faster than the DPRA, is known as the electrophilic allergen screening assay (EASA).
“The EASA can be used to determine how reactive a chemical would be with human skin proteins, that is, how likely it would be to cause an allergic response. No actual proteins, or the amino acids they are made from, are present in the test. Instead, the researchers use two probe molecules, 4-nitrobenzenethiol (NBT) and pyridoxylamine (PDA), which, when reacting with other chemicals, undergo changes that can be detected optically.” Unfortunately, although the initial version of EASA showed promise, the researchers found it shared weaknesses with the DPRA: The type of instruments needed to run the original EASA method typically only hold up to eight samples at a time and are not readily available to most biological labs.
Scientists “reconfigured EASA so that 96 samples could be analyzed in a single run using plastic well plates and instruments called plate readers — two ubiquitous items in biological labs.
With up to 96 wells to work with, the researchers redesigned the protocol so that many control wells could be tested alongside wells containing test compounds. The controls would allow researchers to find out whether factors such as the solvent used, air bubbles in samples or the optical properties of the test compounds interfered with their measurements.
To validate their redesigned protocol, the researchers used the new EASA to analyze 92 test compounds. Most of the chemicals had been assessed in previously published studies on mice through a standard test called the local lymph node assay (LLNA).
They found that the LLNA and EASA results agreed 77% of the time on which chemicals were allergens and which ones were not. Individual EASA tests were completed within a day, while LLNA tests require at least five days.”
The new EASA also performed similarly to the DPRA, while testing samples at a higher rate and with more accessible equipment. This work provides a robust methodology for evaluating the safety of cosmetics without animals. With further validation, the EASA could become a viable candidate for standardization and eventual adoption by regulatory bodies.
Journal Reference: Elijah J. Petersen et al. Development of a 96-Well Electrophilic Allergen Screening Assay for Skin Sensitization Using a Measurement Science Approach. Toxics, 2022 DOI: 10.3390/toxics10050257