Study gives renewed confidence in animal testing alternatives

As part of a government effort to reduce animal testing, researchers at the National Institute of Standards and Technology (NIST) worked with the Consumer Product Safety Commission (CPSC) and Inotiv Inc. to produce a new protocol for screening for skin allergens. The method is potentially cheaper and faster than animal testing, while maintaining similar performance.

The researchers built on an existing method, improving its efficiency, accessibility and quality controls. Using the new animal-free protocol, they evaluated 92 chemicals and found that their results agreed with those of a common animal testing method for 77% of the compounds. The results, published today in toxic, increase confidence in the new method’s measurements, potentially paving the way for standardization, which could both increase international trade and reduce animal testing.

Many governing bodies, including the European Union and some US states, have recently banned the sale of animal-tested cosmetics. However, animal testing is still needed in some parts of the world to assess the safety of cosmetics. With regulations varying by region, it has become difficult for some products to gain general approval.

Some animal-free skin allergen screening methods exist, but their widespread adoption has been hampered by several limitations. For example, the direct peptide reactivity assay (DPRA) requires expensive equipment that can only test a handful of samples at a time. The DPRA can also be slow. To reach statistically significant conclusions about a test compound, testers should repeat the multi-day process of the test several times.

The small sample size per test also reduces opportunities for control samples that would allow researchers to identify and minimize factors that may vary between samples and measures of bias.

To increase confidence in animal-free testing and promote its adoption, several federal agencies, coordinated by a standing committee of the National Institute of Environmental Health Sciences, are evaluating the reliability of promising new methods. The CPSC, an agency member of the committee, has partnered with NIST to evaluate a method much faster than the DPRA, known as the Electrophilic Allergen Screening Test (EASA).

EASA can be used to determine the reactivity of a chemical with human skin proteins, that is, the likelihood that it will cause an allergic reaction. No actual proteins, or the amino acids they are made of, are present in the test. Instead, the researchers are using two probe molecules, 4-nitrobenzenethiol (NBT) and pyridoxylamine (PDA), which when reacting with other chemicals undergo changes that can be detected optically.

“Both molecules are like substitutes for the amino acids cysteine ​​and lysine,” said Elijah Petersen, NIST researcher and lead author of the paper. “If a test compound binds to NBT and PDA, it is likely to also bind to skin proteins, which is the first step in many adverse effects.”

Although the initial version of EASA showed promise, NIST and CPSC researchers found that it shared weaknesses with the DPRA, Petersen said. 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 laboratories.

Petersen and his colleagues reconfigured the EASA so that 96 samples could be analyzed at once using plastic well plates and instruments called plate readers – two ubiquitous items in biological laboratories.

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 determine if factors such as the solvent used, air bubbles in the samples, or the optical properties of the compounds being tested were interfering with their measurements.

“In this test alone, we have a lot of the information we need to understand the sources of variability,” Petersen said. “I think we really capture more and have more confidence compared to other methods.”

To validate their redesigned protocol, the researchers used the new EASA to analyze 92 test compounds. Most of the chemicals had been evaluated in previously published studies on mice using a standard test called the local lymph node test (LLNA).

They found that the LLNA and AESA results agreed 77% of the time on which chemicals were allergens and which were not. Individual EASA tests were completed in one day, while LLNA tests require at least five days, Petersen said.

The new EASA also operated similarly to the DPRA, while testing samples at a higher rate and with more accessible equipment.

This work provides a robust methodology to assess the safety of animal-free cosmetics. With further validation, EASA could become a viable candidate for standardization and eventual adoption by regulators, according to Petersen.

“This study could support commercial product testing because we’re going to have a really good method that’s cheaper, faster, and of higher quality,” Petersen said. “I think it could also help improve the quality of in vitro test systems in general if people take our approach of adding extensive process controls.”

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