Animal-Test Replacements for Cosmetics

Slowly but surely, the cosmetics industry is moving toward in vitro toxicity testing and away from traditional in vivo tests. The shift is due to regulation as well as a desire to more accurately predict human response and significantly reduce the number of animals required for toxicity testing. The €67 billion ($93 billion = €0.72 = $1) European cosmetics industry, which accounts for 50% of the global market, appears to be leading the effort. Spurred by the cosmetics industry, but likely to encompass other sectors, toxicity testing is shifting to a new paradigm. According to Annamaria A. Bottini et al.’s article “Food for Thought . . . on the Economics of Animal Testing,” published by the EU’s Joint Research Center in 2009, each year, the European cosmetics industry introduces more than 20,000 new products, containing approximately 400 new substances. The article said that in 2005, Europe spent €620 million ($775 million = €0.80 = $1) on toxicity testing. In 2008, more than one million animals were used in toxicity and safety-testing studies in the EU, which accounted for 9% of the total number of animals used, according to a 2010 European Commission (EC) report. A 2011 EC report noted that the EU used 344 animals to assess the safety of cosmetics in 2009.

The Seventh Amendment to the EC’s Cosmetics Directive banned animal testing of final cosmetics products in the EU, as well as the sale of final products and ingredients tested on animals if validated alternatives were available, in 2004. The Amendment banned animal testing of ingredients in the EU and the sale of final products and ingredients on all but five endpoints in 2009. The 2009 law covers endpoints for which in vitro replacement tests have been validated: skin corrosion, skin irritation, dermal absorption and phototoxicity. It also covers endpoints for which partial replacement tests have been validated: eye irritation, acute toxicity and mutagenicity/genotoxicity. None of these tests are intended solely for cosmetics. For endpoints for which in vitro replacement tests are not available—repeated-dose toxicity, skin sensitization, carcinogenicity, reproductive toxicity and toxicokinesis—the Amendment bans animal testing in 2013 for all ingredients used in cosmetics and final cosmetics products sold in the EU. However, in May 2011, the EC issued a report stating that it would not be feasible to conduct in vitro toxicity tests for any of the five endpoints. The EC is currently assessing whether to postpone the 2013 deadline because of the lack of complete replacement methods.

Most of the replacement in vitro toxicity tests are being developed by companies, academia and contract research labs. The EU’s European Framework Programs for Research and Technology Development have provided more than €200 million ($277.8 million) for developing non-animal toxicity tests and models, as well as safety assessments, for various sectors over the past two decades. Cosmetics Europe, which represents EU cosmetics companies, is working with more than 100 partners to develop tests. The EU and Cosmetics Europe are each spending €25 million ($34.7 million) on the first stage of the Safety Evaluation Ultimately Replacing Animal Testing initiative (SEURAT-1). SEURAT-1 is a group of six five-year, complementary repeated-dose toxicity research projects, which began in 2011 and involve more than 70 international research teams. Although SEURAT-1 is not expected to result in available methods for routine use, it is designed to provide information to aid with developing repeated-dose toxicity replacements for animal testing. “As the scientific challenges are complex, we need to pool international expertise in large, high-impact research projects,” explained Valérie Zuang, head of Test Method Validation at the European Union Reference Laboratory for Alternatives to Animal Testing (EURL), part of the European Center for the Validation of Alternative Methods (ECVAM), which was established by the EU in 1991 to research, review and validate alternatives to animal tests.

After replacement-test development, a method, including the assay, is submitted to ECVAM for review and validation. Dr. Zuang told IBO that it takes an average of about two years to validate a well-developed test. Upon validation and an independent peer review of the replacement test, the test is submitted for inclusion into the OECD guidelines.

As of June 2011, 18 partial replacements for animal testing in relation to the 2009 ban have been validated by ECVAM, with another 10 undergoing validation and more in development. It is difficult to predict the timeline for validation for such tests, said Dr. Zuang, as it depends on factors such as the “type and complexity of the test method, advancement of science in the field, knowledge about the modes of action and key steps of the toxicological effect, complexity of the endpoint, and the existence or absence of policy-driven pressure.”

The shift to non animal–based toxicity testing marks a significant change from the approach that has been in place for decades. For example, this system involved post-mortem examination of dosed animals. “The old paradigm is based on high-dose testing in animals to extrapolate the results to low-level exposures in human populations,” explained Robert Fellous, Intertek’s Global business line leader, Fine Chemicals. “This is costly and leads to using a huge number of animals with relatively low throughput. It focuses on explaining high-dose effects, rather than considering the biological basis for dose-response relationships expected in consumers exposed to cosmetics.”

The new model for toxicity testing of cosmetics focuses on understanding how a chemical affects humans and applying that understanding to developing tests. “[It is a] move from the traditional data-driven approach based on observations of effects derived from animal studies toward a knowledge-based framework, where we use our understanding of toxicological modes of action to rationally design integrated assessment and testing systems that are fit for a particular regulatory purpose or endpoint,” said Dr. Zuang. “Understanding the fate of a chemical in the body, i.e., how it is absorbed, distributed, metabolized and excreted, and its so-called mode of action, is essential.”

The new approach to toxicity testing emphasizes two components: analysis of pathways of toxicity (PoT) and implementation of integrating testing strategies (ITS). PoT, as described by David Basketter et al. in the 2012 article, “A Roadmap for the Development of Alternative (Non-Animal) Methods for Systemic Toxicity Testing,” are cellular pathways that lead to adverse health outcomes when altered by a chemical. The goal of the new approach is to develop PoT for each toxicity endpoint. In the case of liver toxicity, for example, understanding what causes the liver to be affected is essential, said Rob Taalman, director of Research at Cosmetics Europe. “If you can identify the critical events that lead to liver toxicity, you can develop your system such that you basically test whether an ingredient has the ability to change that pathway,” he said.

Mapping PoT will allow identification of the modes of action in cells, tissues and organs. “Once we have those [critical events,] we’ll put them in what we call a toolbox of methods, and then companies can use that toolbox and take the tools out that they require to prove the safety of their products,” he said.

ITS is also a necessary component of the new approach. Basketter et al. describe ITS as a battery of tests designed to be utilized together. It is not expected that one individual assay will replace one animal-based test. “To find suitable approaches to model the complex nature of cell-to-cell interactions as they happen in the human body is extremely challenging, especially to predict complex systemic effects,” explained Dr. Zuang. “In this context, there will not be one alternative method to replace one animal-test method.” In regard to in vitro toxicity tests, ECVAM-validated tests for all categories of endpoints utilize ITS. For example, there are two validated tests that measure permeability and opacity to determine severe eye irritation, with two more tests undergoing validation. The goal is to use these four tests together, and perhaps with the others that are in development, to replace the in vivo Draize test.

Cell-based tests are being developed as replacement toxicity tests for animal-based testing for cosmetics. “The aim is to create models that will replicate the activity of a chemical in the body and its interaction with the different cells in the different organs,” said Dr. Zuang. “Recent advances in cell-based research, including the use of stem cells and the development of two-dimensional and three-dimensional cell (co)-cultures, are facilitating the development of much more sophisticated structures [that are] more similar to tissues in the body.”

In addition to the cell-based tests that have been developed as replacement tests, assays are being developed utilizing reconstructed human tissue to mimic organs, generally skin. Also being developed to mimic organs are “human-on-a-chip” tests. These tests are “basically a micro-machine chip with human lung cells that grow on a surface to form a lung-like tissue,” Dr. Taalman explained. Current research is focused on coupling this chip with a heart-like chip that beats and pumps blood. “These systems can help to evaluate how specialized organs like the lung and heart react to a specific chemical.” In vitro cell-based and tissue-based assays are the most likely candidates for replacement tests, said Dr. Fellous, because they will support research on PoT by providing information about the mode of action in target cells, tissues and organs.

Several improvements need to occur before the new wave of tests can fully replace the old tests. Basketter et al. describe the need to better existing in vitro toxicity tests by enhancing their predictability by improving cell conditions like homeostasis, oxygen supply and cell density; moving from tests that use cell lines to ones that utilize primary or stem cells; and using evidence-based approaches for quality assessment. Dr. Fellous echoed the need for better predictability in replacement tests. “The progress achieved in molecular, cellular and computational biology is studying the responses of cells, tissues and organs to chemical stressors,” he said. “These new tools allow the cosmetic industry and regulatory bodies to predict exposure which is expected to be without adverse effects, rather than predicting the incidence of specific adverse responses in human populations.”

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