Meeting the global challenge. A guide to assessing the safety of cosmetics without using animals

Transcription

1 Meeting the global challenge A guide to assessing the safety of cosmetics without using animals Second edition 2017

2 With grateful thanks to The Body Shop for funding the second edition of this guide to testing the safety of cosmetics products and ingredients without the use of animals. Contents 04 About us 06 Introduction to the report 07 Global overview of regulatory progress 08 The benefits of using alternative methods 08 The public does not support the use of live animals 09 Alternatives are usually cheaper and faster than the animal test they replace 09 Alternatives are usually more reliable and accurate than the animal test they replace 10 Product testing requirements 11 Ingredient testing requirements 12 Standard toxicity tests and their alternatives 14 Skin absorption 14 Acute toxicity 15 Skin irritation/corrosion 16 Eye irritation/corrosion 17 Skin sensitisation 18 Mutagenicity/genotoxicity 19 Repeated dose 20 Non-standard toxicity tests and their alternatives 22 Phototoxicity 23 Carcinogenicity 24 Reproductive toxicity 25 Endocrine disruption 26 Toxicokinetics 28 Conclusion 29 References 3

3 About us Photo credit: Philippe Gotteland at EpiSkin Cruelty Free International is the leading organisation working to create a world where nobody wants or believes we need to experiment on animals. Its dedicated team are experts in their fields, combining award-winning campaigning, political lobbying, pioneering undercover investigations, scientific and legal expertise and corporate responsibility. Educating, challenging and inspiring others across the globe to respect and protect animals, it investigates and exposes the reality of life for animals in laboratories, challenges decision-makers to make a positive difference for animals, and champions better science and cruelty free living. Widely respected as an authority on animal testing issues, it is frequently called on by governments, corporations and official bodies for advice or expert opinion. Building relationships with politicians, business leaders and officials, analysing legislation and challenging decision-making panels around the globe, it acts as the voice for animals in laboratories. The Leaping Bunny The Cruelty Free International Leaping Bunny is the most trusted cruelty free certification for non-animal tested products. It is the only international guarantee that companies will not carry out any animal testing for their products in any country in the world. The Leaping Bunny logo is issued for use by companies which produce cosmetics, personal care, household and cleaning products which comply with the rigorous Leaping Bunny certification criteria. More than 700 companies across the globe already hold Leaping Bunny certification, providing real choice for ethical consumers who want to identify and buy products that are free from animal testing. The Body Shop Founded in 1976 in Brighton, England, by Anita Roddick, The Body Shop is a global beauty brand. The Body Shop seeks to make a positive difference in the world by offering high-quality, naturallyinspired skincare, hair care and make-up produced ethically and sustainably. The Body Shop pioneered the philosophy that business can be a force for good and this ethos is still the brand s driving force. The Body Shop has more than 3,000 stores in more than 60 countries. The Body Shop was the first international beauty brand to campaign against the practice of animal testing in cosmetics in 1989, leading the way to a European Union-wide ban on animal testing in This on-going campaign, conducted in partnership with Cruelty Free International, went on to collect 1 million signatures and triggered significant progress across the Asia Pacific region. A UN ban on animal testing for cosmetics In June 2017, The Body Shop and Cruelty Free International launched a new campaign, Forever Against Animal Testing. Together they are working to achieve a resolution for a global ban on cosmetics animal testing by 2020, revolutionising the beauty industry and protecting millions of animals around the world. In the first two months of their campaign they collected 2 million signatures and in 2018 they aim to take the campaign to the highest authority, the United Nations, with 8 million petition signatures, to request an international convention banning cosmetics testing of both ingredients and products on animals

4 Introduction to the report Global overview of regulatory progress Over the past 30 years, public pressure to end the testing of cosmetics on animals has increased around the world and cosmetics animal testing is now banned in a growing number of countries. Most notably, the European Union the world s largest cosmetics market 1 ended the testing of cosmetics products in 2003, the testing of ingredients in 2009 and, in 2013, the sale of any new cosmetics that have been tested outside the EU on animals. Norway, Iceland, India, Israel, Turkey, New Zealand, Guatemala, Serbia and Switzerland also now prohibit the use of animals for cosmetics testing. A phased approach to ending testing is in place in Taiwan and South Korea. Other countries - including the United States, Australia, Canada and Brazil - are currently considering legislation that would see an end to cosmetics testing on animals. Discussions on transitioning to modern alternatives are also underway in countries such as Japan and China. Largely driven by the EU s 2013 deadline and regulatory changes occurring in other parts of the world, research into technologies that replace animal testing by the cosmetics industry and governments has stepped up. As a result, there are now alternatives for the most commonly required safety tests for cosmetics and many of these are now recognised internationally. For tests where alternatives are not recognised internationally, further animal tests can still be avoided depending on the type of ingredient and its intended use. Ethical concern has been the driver for this positive change, and governments can take comfort from the fact that the animal tests traditionally used in the past to ensure the safety of cosmetics now have practical and reliable non-animal alternatives. Since exports of animaltested cosmetics to countries with a marketing ban are impossible, it is important for countries where a ban is not yet in place to provide a way forward for their domestic industry. The second edition of this ground-breaking report explains the current position for each safety test. It will help governments, politicians, regulators and cosmetics manufacturers across the world switch to alternatives to replace animal testing, giving them the confidence that the safest and most modern methods are used and that by moving away from obsolescent technology, access to European and other markets is possible. In the report, Cruelty Free International describes the alternative approaches that are available, and shows how they are not only more ethical but also more reliable, faster and cheaper than the animal tests they replace. The report explains what alternatives are, how cosmetics are tested for product and ingredient safety and which alternatives can be used to replace each traditional animal test. The report then outlines the alternatives for both standard and non-standard animal tests. Standard tests are those most commonly required by national regulators for general purpose cosmetics ingredients. Non-standard tests are not usually a routine requirement for cosmetics, but might be triggered when consumer exposure is expected to be particularly high or the ingredient is expected to have biological activity. The report provides a summary of the validated and, in many cases, internationally accepted, alternatives to these tests on a test-by-test basis, and includes an overview of how new animal tests in non-standard cases can be avoided. Public opinion and consumer demand for cruelty free products have been driving forces behind the global trend towards the phasing out of animal testing for cosmetics and the development of innovative alternatives. The map below depicts the international state of play at time of writing. With momentum building ever-faster for a global ban, please contact Cruelty Free International for an up-to-date overview of the current situation. Bans in place: European Union 28, Norway, Iceland, Serbia, Israel, India, Switzerland, New Zealand, Turkey, Guatemala, San Marino, Liechtenstein Bans in transition: Australia, South Korea, Taiwan Bans under consideration: Brazil, USA, Canada Other progress: Argentina, ASEAN, Viet Nam, Thailand, Russia, China 6 7

5 The benefits of using alternative methods Alternative methods are tests that use simple organisms like bacteria, or tissues and cells from humans (in vitro tests), computer models (in silico approaches) or chemical methods (in chemico tests). Cruelty Free International considers an alternative to be any test method that does not use live vertebrate animals, i.e. live mammals, fish, amphibians, reptiles and birds. It is universally accepted that all vertebrate animals can feel pain and otherwise suffer. Methods that use tissues from vertebrate animals who have already been killed (often for other purposes such as for food) are recognised internationally as alternatives since the animals do not suffer during the test (these are ex vivo tests). Sometimes, the term alternatives is used for methods which use live animals but use fewer animals or cause less suffering. However, this is not how Cruelty Free International or the public understand the term, and it is not how it is used in this report. The public does not support the use of live animals Prior to the implementation of cosmetics testing bans: Czech Republic (2006): 72% of respondents agree with the use of alternatives instead of animal tests for cosmetics. 2 Norway (2002): 81% of respondents have a negative opinion about cosmetics testing. 3 UK (1999): 88% of women want a complete ban on animal testing for cosmetics. 4 New Zealand (2013): 89% of respondents said they do not support testing cosmetics on animals, and 82.9% supported banning the practice. 5 In countries which do not yet have testing bans: USA (2013): 72% of female voters said they oppose testing cosmetics on animals, and three in four said they would feel safer or as safe if non animal-testing methods were used to test the safety of a cosmetic. 6 Canada (2013): 88% of respondents agreed that animal testing can cause pain and suffering to animals and it is not worth causing this kind of suffering just to test the safety of cosmetics, especially when there are safe ingredients available; 80% backed a nationwide ban on testing cosmetic products and ingredients on animals. 7 Australia (2013): 85% of people said they oppose using animals in the development of cosmetics and 81% said they support a national ban on the sale of cosmetics tested on animals. 8 Consumers prefer to buy cosmetics that have not been tested on animals: UK (2004): 79% of people said they would be likely to swap to a brand that was not animal tested if they discovered that their existing brand was tested on animals. 9 USA (2011): 32% of people said they had purchased products labelled as not tested on animals because of their concern for animals. 10 Australia (2014): 43% of women said that Not Tested on Animals was one of the top three features they look for when shopping for cosmetics, ranking higher than quality brand and moisturising. 11 Alternatives are usually cheaper and faster than the animal test they replace The in vitro tests for skin and eye irritation can be conducted in a day, whereas the corresponding rabbit tests take two to three weeks. Similarly, one of the skin sensitisation tests can be conducted in one day, whereas the corresponding test on mice takes at least six times that. These tests can already be conducted at a cost equivalent to the animal test, between 1,000 and 5,000 euros. Methods that avoid the lengthier systemic toxicity tests are much cheaper and faster. For example, computer (QSAR) models can be run at very little cost, assuming some in-house expertise, saving thousands of euros. The cost of an expert to set out a Threshold of Toxicological Concern (TTC) approach or read across argument (see below) could typically be around 3,000 euros, compared to 300,000 euros for a two-generation reproductive toxicity test. The Cell Transformation Assay can cost as little as 500 euros and could avoid the cancer bioassay on rats which takes two years and costs approximately one million euros. Alternatives are usually more reliable and accurate than the animal test they replace Modern alternative methods are required to go through a rigorous validation process to demonstrate they are as or more effective than the animal test they replace. The performance of the alternative is compared to human responses where they are already known, or the existing animal test where they are not. Validated alternative methods are published in the guidelines of international bodies that harmonise the most common methods to assess the safety of chemical substances. These bodies include the Organisation for Economic Cooperation and Development (OECD) which publishes Test Guidelines (TGs) relevant for safety testing of chemicals, including cosmetics. Alternatives will simply not be accepted at international levels by the OECD without sufficient evidence that they reliably detect toxic and non-toxic substances. By contrast, it is important to note that traditional animal tests have never been validated for their use in reliably detecting the safety of cosmetics ingredients. This means that there has not been an independent, controlled assessment of whether the animal test accurately and reliably predicts human reactions using a set of substances for which the human response is known. The validity of existing animal tests is assumed only, based on a history of their use. This is not adequate for today s high safety standards. 8 9

6 Product testing requirements Ingredient testing requirements It is now common practice not to test a specific cosmetics product on animals. Instead, cosmetics companies determine the safety of new formulations made up of existing ingredients by using calculations to determine overall safety factors. 12 Each cosmetics product is considered as a combination of individual cosmetics substances. A qualified safety assessor looks at the data on the ingredients and the extent to which consumers are exposed to the product and comes to a judgement about the safety of the product as a whole. The potential for local effects (irritation, sensitisation), which may occur at the site of contact, needs to be assessed alongside the potential for systemic (internal) effects. The local effects of a product are generally more straightforward to predict based on existing data for individual ingredients, experience of use, the level of individual ingredients and the characteristics and intended use of the product. Companies also gain additional confidence in the local effects of their products by performing compatibility tests using human volunteers. Under strict ethical guidelines and always after the initial safety assessment, volunteers test the products to ensure that the product claims are justified and that there are no skin irritation or sensitivity issues. 13 Companies may also use the alternatives described here for skin and eye irritation to double-check the lack of irritation potential of the whole product before they conduct these volunteer studies. For systemic effects, Margin of Safety (MoS) values are generated for each individual ingredient. These are calculated based on an assessment of the exposure to the human body of the ingredient and the extent to which it is likely to be toxic. The systemic exposure dose (SED) is first calculated based on an assessment of how often and how much of the product is used, the level of ingredient in the product, whether the product is leave on or rinse off and the potential for the product to penetrate the skin. The no observed adverse effect level (NOAEL) is then obtained for the ingredient this is a measure of its toxicity based on new or existing toxicity data. The selected NOAEL is divided by the SED to give the MoS for that ingredient. MoS values of 100 or greater are generally considered to indicate an adequate level of safety; however, higher values may be required for particular ingredients or product types. Avoidance of contamination and impurities can be assured by adherence to Good Manufacturing Practice (GMP) and the relevant ISO and CEN standards for production. Regulators can help improve cosmetics safety by issuing lists of known dangerous substances that should not be used in cosmetics. Regulators can ensure there is traceability of the product and conduct market surveillance. In vitro tests can be carried out to ensure the product does not have a high microbial content and also to determine if preservatives in the product will reduce contamination. Analytical tests can identify if there has been deliberate or inadvertent inclusion of ingredients not listed on the packaging. In the event of any safety issues, companies should declare the quantities of substances in their products, demonstrate GMP and ensure traceability. Animal tests in this scenario will not help identify what the contaminants are, nor will they demonstrate why, if at all, the animals become unwell. They therefore cannot explain any safety issues that may have arisen in the human population as a result. Since the safety of a cosmetics product relies on information about the ingredients, information on the safety of the ingredients is required. For products made up of existing ingredients this is a relatively straightforward task. There are now approximately 30,000 ingredients on the EU s database for which some safety data will already be available. 14 No new animal (or non-animal) safety data is usually required. Exceptions to this may be ingredients which become a concern and for which regulators in a particular region may ask for more data. This has been the case for some specialist cosmetics ingredients such as hair dyes, preservatives and UV filters. The impact of no animal testing for most cosmetics product manufacturers is therefore extremely minimal. Companies can continue to develop their products using existing ingredients or ingredients that become available that have not been animal tested. The proportion of genuinely new ingredients entering the market every year is actually very low. According to Cosmetics Europe, across the industry, new ingredients are introduced at an annual rate of around 4% of the total portfolio. Only a proportion of these are thought to be new to all uses. 15 Those companies that wish to innovate and use genuinely new ingredients have several options: a) use alternative methods such as in vitro or computer based methods like QSARs to determine aspects of the safety of the ingredient; b) determine the safety of new but very similar ingredients based on read across, i.e. extrapolation of the information from data on the original substance; c) use the TTC (threshold of toxicological concern) approach to determine if any further testing is really needed due to low exposure levels. Finally, companies always have the option to continue to innovate but not use, i.e. screen out, ingredients where, exceptionally, safety concerns cannot be alleviated based on these approaches. In this way, human health is best protected

7 Standard toxicity tests and their alternatives This section outlines the standard tests that are most commonly required by national regulators for general purpose cosmetics ingredients. It summarises the validated and, in many cases, internationally accepted alternatives to these tests on a test-by-test basis. Table 1. Standard cosmetics toxicity tests and the available options to avoid animal testing Endpoint Tests for Animal test Options to avoid animal test Skin absorption Acute toxicity Skin irritation/ corrosion Eye irritation/ corrosion Measures the extent to which the substance will penetrate the skin. Assesses the amount of the substance that will cause severe toxic effects if accidentally ingested, inhaled or rubbed on the skin. Measures the extent to which the substance will irritate and damage the skin. Measures the extent to which the substance will irritate the eyes. The substance is rubbed onto the shaved backs of rats who are killed the next day (OECD TG 427). Tends to overpredict. Rats are exposed to a very high dose of the substance such that a number of them are expected to die (OECD TG 402,403, 420,423,425,436). Substance is rubbed into the shaved backs of rabbits who are killed two weeks later (OECD TG 404). Tends to overpredict. Substance is placed in the eyes of live rabbits who are monitored for up to three weeks (OECD TG 405). Notoriously unreliable test. Ex vivo skin based tests for this are well established (Dermal absorption in vitro skin test, OECD TG 428). Not always required because assessing repeated dose toxicity is considered more useful. Cell based tests such as the NRU3T3 can be used to predict lack of toxicity very accurately (ECVAM recommendation 2013) and can be used in a weightof-evidence approach (ECHA guidance 2016). Reconstituted human skin models are now accepted and can be used in most cases (in vitro skin corrosion and irritation tests, OECD TG 431 and 439). Eyes from hens and cows killed for food can now be used to detect non-irritants and severe irritants (BCOP and ICE ex vivo eye models, OECD TG 437 and 438). Detection of non-irritants can be assessed using human corneal epithelial models (OECD TG 492). Endpoint Tests for Animal test Options to avoid animal test Skin sensitisation Mutagenicity/ genotoxicity Repeated dose Measures the likelihood that the substance will cause an allergic reaction if applied to the skin. Assesses the likelihood that the substance will cause genetic damage which could lead to cancer. Measures the effects of repeated exposure to the substance over a long period. The substance is rubbed onto the shaved skin of guinea pigs who are subjectively assessed for allergy (Buehler or GPMT test, OECD TG 406) or painted onto the ears of mice who are killed 6 days later to assess the immune response (LLNA test, OECD TG 429, 442a/b). The more modern test, the LLNA, predicts human reactions only 82% of the time. The substance is force-fed or injected into mice or rats for 14 days who are then killed to look at the effects on their cells (OECD TG 474, 475, 483, 486, 488, 489). Rats (occasionally rabbits, mice or even dogs) are force-fed, forced to inhale or have the substance rubbed onto their shaved skin every day for 28 or 90 days before being killed (OECD TGs ). The ability to correctly predict human reactions (to drugs) using this test is no more than 60%. Several in vitro tests have been accepted; the peptide reactivity (DPRA) test which measures the binding of the substance to proteins, the keratinocyte assay, and the human Cell Line Activation Test (h-clat) based on human skin cells (OECD TG 442c, 442d and 442e respectively). Testing strategies using these methods are already being used by companies and are recognised at the OECD. A battery of two or three cellbased tests is always carried out before conducting an animal test (Bacterial Reverse Mutation Test, OECD 471, in vitro Mammalian Chromosome Aberration Test, OECD 473, in vitro Mammalian Cell Gene Mutation Test, OECD TG 476, in vitro Mammalian Cell Micronucleus Test, OECD 487, in vitro Mammalian Cell Gene Mutation Tests using the thymidine kinase gene, OECD 490). Positives should be assumed to be genotoxic to avoid in vivo follow-up. Can be avoided if the exposure to the substance is likely to be extremely low (TTC concept) or read across can be used if the substance is similar to existing ones that have already been tested

8 Skin absorption Endpoint: The skin absorption test measures the extent to which the substance will penetrate the skin. Animal test: The substance is rubbed on to the shaved backs of rats and they are killed the next day (OECD TG 427). Alternative: Dermal absorption in vitro skin test (OECD TG 428). Determining the extent to which the substance will absorb through the skin and become systemically available is an important step in being able to determine the Margin of Safety (MoS) of an ingredient, and therefore the risk assessment of the product as a whole. In vitro tests for skin absorption are well established and were one of the first alternatives to be approved by the OECD in They measure the extent of absorption of the substance through discs of donated human skin into a fluid reservoir. These tests were shown to accurately reproduce the same absorption through in vivo skin in the 1980s 16 and were accepted for use in the EU in It is known that absorption through rodent skin tends to be higher than it is in humans, and therefore a rodent skin absorption test will overestimate the extent to which the substance will penetrate human skin by a factor of three. 18 This is due to differences in skin thickness, hair follicles and immune responses. In vitro skin absorption methods have the distinct advantage that human skin can be used. The cost of in vivo and in vitro tests is equal because the substance is usually radio labelled in both assays to enable detection of the substance. Acute toxicity Endpoint: The acute toxicity test assesses the amount of the substance that will cause severe toxic effects if accidentally ingested, inhaled or rubbed on to the skin. Animal test: Rats are exposed to a very high dose of the substance such that a number of them are expected to die (OECD TG 402,403, 420,423,425,436). Alternatives: Not always required because assessing repeated dose toxicity is considered more useful. Single dose studies for cosmetics ingredients are not considered useful because these tests were designed years ago as a crude measure of the toxicity of chemicals. Today, it is more common to see repeated dose data instead of LD50 animal test information for cosmetics ingredients. This is because cosmetics are not expected to be very toxic and repeated dose information is usually preferred to directly determine the NOAEL. In the EU, acute toxicity data is not insisted upon if repeated dose information is available. 19 Cell-based tests such as the NRU3T3 (see Phototoxicity) can be used to predict lack of toxicity very accurately. The OECD has issued Guidance Document 129 which outlines the test and how it can be used to estimate the starting dose for an animal test, following a review by US authorities. 20 European validation body ECVAM has recently concluded in a largescale analysis that the test can be safely used to detect non-toxic, non-classified substances. 21 Only one substance (digoxin) that is toxic was not identified by the test (out of 72 substances tested). Since most substances are non-toxic, 22 the use of this test could avoid further testing, however the European Chemicals Agency states it must be used in conjunction with other information to waive the animal test. 23 Skin irritation/corrosion Endpoint: Measures the extent to which the substance will irritate and damage the skin. Animal test: The substance is rubbed into the shaved backs of rabbits who are killed two weeks later (OECD TG 404). Alternatives: Reconstituted human epithelial (RhE) skin models (OECD TG 431 and 439). In vitro models based on reconstituted human epithelial (RhE) skin have been developed since the 1980s. These models comprise small discs of cells grown into an epidermal layer from human skin donated as waste from cosmetic surgery. The models have now been thoroughly validated and internationally approved by the OECD. The tests can be used to classify substances as corrosive (UNGHS category 1; some tests can be used for sub classifications of this category), irritating (UN GHS category 2) and not irritating (not classified). The methods have a wide applicability domain so there are only very limited cases where they could not now be used for this endpoint. EU chemicals legislation now no longer requires the test on rabbits. 24 Skin corrosion can be assessed using RhE skin corrosion model OECD TG 431 or other in vitro models, TG 430 (TER) or TG 435 (Corrositex ). If the test is negative, the RhE skin irritation models (OECD TG 439) should then be used to assess if the substance is irritating or to confirm that it is not irritating. Sometimes companies test their substances using the RhE skin irritation models only since cosmetics ingredients are not usually expected to be corrosive. All OECD TG 439 methods predicted skin irritation to at least 75% accuracy in the validation study, 25 although follow-up studies have shown they are actually more accurate than this. For example, in a study using 184 cosmetics, EpiSkin demonstrated 86% accuracy. 26 Studies show that the methods are more accurate and effective than the Draize rabbit test they replace. For example, a study has confirmed that the test on rabbits tends to overpredict human skin reactions; Epiderm was found to be 76% accurate at predicting human skin patch test results whereas the rabbit test was only correct 56% of the time. 27 The RhE skin models can be purchased as kits from the manufacturers: (EpiDerm ), (EpiSkin, Skin Ethic RHE ) and (epics ). Many contract testing facilities are now familiar with the methods and will use them on behalf of cosmetics companies. Contract testing facilities charge approximately the same as for the test on rabbits, but the kits obtained directly from the manufacturers can be cheaper. Photo credit: Philippe Gotteland at EpiSkin 14 15

9 Eye irritation/corrosion Endpoint: Tests for eye irritation and corrosion measure the extent to which the substance will irritate the eyes if it is accidentally spilt. Animal test: The substance is placed into the eyes of live rabbits who are monitored for up to three weeks (OECD TG 4054). Alternatives: BCOP and ICE ex vivo eye models (OECD TG 437 and TG 438), and HCE eye models (OECD TG 492). Photo credit: BASF Isolated eyes from cows or chickens killed for food purposes can be used to detect severely irritating/corrosive (GHS Cat 1) substances. The OECD TGs were approved in 2009, and updated in 2013 to reflect the fact that they can also be used safely to detect non-irritants (non-classified substances). These ex vivo methods tend to over predict the results of the test on rabbits, which means they always detect severely irritating substances and are therefore protective, but they may also predict that a substance is irritating when it is not. Contract testing facilities charge approximately the same to conduct the ex vivo tests as they do the test on rabbits. The animal test is notoriously cruel and unreliable, with different laboratories often giving very different results 28 and with only low to moderate correlation with human responses as rabbits tend to experience more severe effects than humans 29. More recently, methods based on reconstituted human corneal epithelium (HCE) have been validated and are commercially available, for example, from (EpiOcular ) and (SkinEthic HCE ). OECD TG 492 has been created and includes EpiOcular to detect non-irritant substances (accepted 2015) and will soon be updated to include SkinEthic HCE (updated expected in 2017) and LabCyte (updated expected in 2018). An assessment of 435 cosmetics substances has shown that SkinEthic HCE is 82% accurate 30 and a study by BASF also found EpiOcular to be 82% accurate 31. There are other methods available but they have a more limited applicability: the Fluorescein Leakage Test Method uses an animal-derived epithelial cell line monolayer to identify severe eye irritants (OECD TG 460) and the Short Time Exposure method (STE, OECD TG 491) also uses an animal cell line but can detect both severe irritants and non-irritants. All these methods can be combined in a socalled top down/ bottom up approach using tests in two methods to arrive at a classification. 32 One method (typically an ex vivo method) is used first if it is suspected that the substance is severely irritating (Category 1) whilst another method (typically the HCE methods) is used first if it is suspected that the substance is not irritating (not classified). A final OECD testing strategy outlining this approach is expected in In the meantime, there is already a testing strategy in the current rabbit OECD TG 405 and, for chemicals testing in Europe, a requirement to exhaust testing in vitro/ex vivo before using animals. 33 Currently, however, a combination of alternative methods still does not give confidence in predicting Category 2 (irritation), but this is likely to change in the next couple of years as they are further improved. Skin sensitisation Endpoint: Skin sensitisation is an allergic reaction to a particular substance that results in the development of skin inflammation and itchiness. The skin becomes increasingly reactive to the substance each time it is exposed to it. Animal tests: The substance is rubbed on to the shaved skin of guinea pigs who are subjectively assessed for allergy (Buehler or GPMT test, OECD TG 406), or painted on to the ears of mice who are killed six days later to assess their immune response (LLNA test, OECD TG 429, 442a/b). Alternatives: Several in vitro methods can be used in combination, including the DPRA (OECD TG 442c), KeratinoSens (OECD TG 442d) and h-clat (OECD TG 442e. The mechanism of how skin reacts to sensitising substances to produce an allergic reaction is well understood and is outlined in an OECD document on Adverse Outcome Pathways. 34 This has helped with the development of various alternative methods that address key steps in the pathway. Used in combination these tests can now be used to replace the animal tests for skin sensitisation. The Direct Peptide Reactivity Assay (DPRA) (OECD TG 442c, adopted 2015) is an in chemico method that measures protein reactivity, a key step in the mechanism of skin sensitisation. The ARE-Nrf2 Luciferase Test Method (KeratinoSens ) (OECD TG 442d, adopted 2015) is an in vitro test that uses a human cell line to measure the activation of genes known to be involved in triggering the immune response to contact allergens. The Human Cell Line Activation Test (h-clat) (OECD TG 442e, adopted 2016) is an in vitro test that uses a human cell line that addresses the third key event in skin sensitisation, activation of the dendritic cells. Quantitative Structure-Activity Relationship (QSAR) computer models have particularly strong predictive power for skin sensitisation because reactivity can be predicted from chemical structure. The reactivity of a chemical structure can be predicted based on its structural similarity to other chemicals in the database with known properties. For example, the QSAR model CAESAR is 90% predictive. 35 See for models. Several organisations have successfully assessed the use of these tests in various combinations, usually using two or three of the tests above to predict whether the substance is a sensitiser or not. BASF use an in-house version of KeratinoSens (Lusens, undergoing OECD acceptance), the DPRA and h-clat or Modified myeloid U937 skin sensitisation (mmusst) test (also undergoing OECD acceptance) and a two out of three rule; any two assays must be positive to rate the substance as a skin sensitiser and any two assays must be negative to rate the substance as a non-sensitiser. This strategy has been shown to accurately predict human sensitisers from non-sensitisers 90% of the time 36. The Dutch authorities use a slightly different testing strategy comprising QSAR models and the DPRA followed by the KeratinoSens and the h-clat if the results at that stage are still unclear. Their strategy was found to be accurate 95% of the time using just the DPRA and QSARs, and 100% accurate using all three in vitro tests. 37 The original LLNA test on mice is only up to 82% predictive of human allergic reactions 38 and has been shown to place nearly half of known human strong sensitisers in the wrong sub category. 39 The three in vitro tests can be carried out for approximately the same price as the LLNA animal test (around 4,000 euros). The DPRA takes one day to run, and the KeratinoSens takes four days, whilst the LLNA takes six days not including time for analysis of the results. EU chemicals legislation REACH has recently been updated to permit the use of these alternative tests in combination 40, and the OECD has outlined the various testing strategies in terms of defined approaches to testing and assessment for skin sensitisation. 41 Further work is being done at the OECD to help with the formal recognition of these testing strategies to facilitate their global acceptance

10 Repeated dose The TTC approach Mutagenicity/genotoxicity Endpoint: The mutagenicity/genotoxicity safety test assesses the likelihood that the substance will cause genetic damage to the cells in the body which could lead to cancer. Animal tests: The substance is force-fed or injected into mice or rats for 14 days who are then killed to look at the effects on their cells (OECD TG 474, 475, 483, 486, 488, 489). Alternatives: Bacterial Reverse Mutation Assay (Ames test) (OECD TG 471), In Vitro Mammalian Cell Gene Mutation Test (OECD TG 476), In Vitro Mammalian Chromosome Aberration Test (OECD TG 473), In Vitro Mammalian Cell Micronucleus Test (OECD TG 487) and In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (OECD TG 490). Mutagenicity/genotoxicity is always assessed initially in vitro using bacterial and other cellbased tests. These tests assess the extent of damage to the chromosomes (containing genes) in the cells that could be indicative that the substance causes cancer. In many cases, it is possible to determine whether a substance is likely to be genotoxic by conducting up to three of these cell-based tests, covering effects on gene mutation (TG 471, TG 476 and TG 490) and changes to chromosome structure (TG 473) and number (TG 487). In combination, these tests have been shown to be 85-90% predictive of rodent carcinogenicity test results across a large number of chemicals. 42 These in vitro tests are often accused of being too protective, i.e. safe chemicals can be mistakenly predicted to be genotoxic. However, this is inconclusive as the results are always compared to tests in rats and mice rather than humans. The common approach is to follow up these positive results using an in vivo test on mice or rats. However, follow-up of positive results can be avoided by careful choice of cell type (human cells being preferable), dose levels and method of assessment of the damage. 43 According to the OECD test guidelines for these in vitro tests, cells should also be exposed to the test substance in the presence and absence of an appropriate metabolic activation system. It has been recommended in Europe that only two in vitro tests are required if the In Vitro Mammalian Cell Micronucleus Test (TG 487) is used because it looks at changes to both chromosome structure and number 44. The use of RhE models (see Skin Irritation/Corrosion) is currently being examined by Cosmetics Europe and ECVAM to see if this adds to the assessment, especially since it uses human tissue. 45 Prevalidation studies have already demonstrated that the in vitro human reconstituted skin micronucleus (RSMN) assay can correctly identify positive and negative genotoxicants and further validation studies are currently underway to confirm the sensitivity of the assay. Another test called the reconstructed human skin Comet assay is also undergoing pre-validation studies. 46 Endpoint: Measures the effects of repeated exposure to the substance over a period of time. Animal tests: Rats (occasionally rabbits, mice or even dogs) are force-fed, forced to inhale or have the substance rubbed on to their shaved skin every day for 28 or 90 days before being killed (OECD TGs ). Alternatives: Until a testing strategy using in vitro tests is developed and validated, repeated dose testing can often be avoided using the TTC (threshold of toxicological concern) approach and/or read across approaches. Several reviews of the ability of rodent tests to predict human toxicity, mainly in pharmaceuticals, have found that they are only about 40-60% predictive of human toxicities. 47 Nonetheless, repeated dose information is often required for new cosmetics ingredients to obtain the No Observed Adverse Effect Level (NOAEL) to perform the risk assessment. A variety of cell-based models are available that either use long-lasting liver cells or incorporate a range of cell types into a microchip. These are currently used to screen substances for long term toxicity but do not yet have regulatory acceptance. Because cosmetics substances are often used in very low quantities, in some cases animal tests can be avoided by using the TTC concept. Alternatively, read across or category approaches can be used when there is existing data on a structurally similar substance(s). The TTC approach is based on the concept that for all substances there is a level of exposure below which there is hardly any risk to human health, regardless of how toxic the substance is. If the exposure of a substance in a cosmetics product is known (which it should be as part of the risk assessment; see Product Testing), and if it is very low, then even if the substance is assumed to be toxic, testing will not affect the safety of the product and the TTC approach could apply. Instead of conducting an animal test, the risk assessor will evaluate the likely toxicity class of the substance (based on chemical structural similarity to other substances), then calculate maximum daily exposure. If the substance falls below a certain value, then it can be considered safe. The TTC concept was first used for food additives, but research by the cosmetics industry has shown it to be relevant for cosmetics 48, with examples and databases now available. 49 Cruelty Free International s analysis is that the concept could be used for many common ingredients that are present in small quantities. Read across Substances with physicochemical, toxicological and ecotoxicological properties that are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or category of substances. In this case, existing data on one or more members of the group can be used to provide data (to read across) for the other members, and new testing can be avoided. The OECD has produced guidance on how to use read across as well as a database which can be used to look for similar substances (OECD Toolbox). 50 Read across is well accepted in many regions including the EU. Companies innovating by modifying substances slightly to improve them may well find that they are justified in using read across from the original substance instead of animal testing

11 Non-standard toxicity tests and their alternatives This section provides an overview of the non-standard tests that are not usually a routine requirement for cosmetics. These are tests that might be triggered when consumer exposure is expected to be particularly high or the ingredient is expected to have biological activity. This section summarises the possibilities to avoid new animal tests in these cases on a test-by-test basis. Table 2. Non-standard cosmetics toxicity tests and the available options to avoid animal testing Endpoint Tests for Animal test Options to avoid animal test Endpoint Tests for Animal test Options to avoid animal test Phototoxicity Carcinogenicity Reproductive toxicity Whether the substance will cause a reaction if applied to the skin and the skin is then exposed to sunlight. Assesses the likelihood that a substance will cause cancer if people are exposed to it over a long period. Assesses the likelihood that the substance will reduce fertility or cause developmental problems to the foetus. No suitable animal test exists, the in vitro test is the standard test. Rats or mice are fed the substance for two years to see if they get cancer (OECD TG 451, 452). Costs $2 million and only predicts human cancer 42% of the time. Pregnant female rabbits or rats are force-fed the substance and then killed along with their unborn babies (OECD TG 414). Such tests take a long time and use hundreds of animals. Studies have shown they only detect 60% of known human toxicants. Only required if the substance has UV absorbing properties. Cell based tests have been in place for some time (3T3 NRU cell-based test, OECD TG 432), negative results can be confirmed in human skin tests (in vivo or in vitro). Rarely conducted because it takes so long and is so unreliable. Companies use the genotoxicity tests above and assume if the substance is genotoxic then it may also cause cancer. Cell transformation assays (CTA) have been in use for 50 years, predict 90% of known human carcinogens and can be used for follow up if necessary (OECD guidance documents 214, 231). In some cases, can be avoided if consumer exposure to the substance is likely to be extremely low (TTC concept) or read across from existing substances can be used. Endocrine disruption Toxicokinetics Assesses the likelihood that the substance will interfere with the body s endocrine (hormone) system producing harmful effects. Assesses how the body deals with a substance, i.e. whether it is metabolised and how long it stays in the body. No single established animal test for endocrine disruption exists (nor is likely to). The Hershberger assay looks at the effects on castrated male rats who are injected with or forcefed the substance for 10 days before being killed (OECD TG 441). Rabbits or rats are forced to consume the substance then are placed in cages on their own before being killed and their organs examined (OECD TG 417). Poor estimates based on animal studies are responsible for 30% of drug failures. Not yet seen as a regulatory requirement. Receptor binding assays such as the Stably Transfected Transcriptional Activation assay (STTA) (OECD TG 455) and the BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists (OECD TG 457) and the H295R Steroidogenesis Assay (OECD TG 456) can be used to screen for potential endocrine disrupting properties. Rarely a regulatory requirement in itself. Skin absorption (OECD TG 428) and liver cell metabolism tests (see OECD TG 417) can be put into a PBPK computer model that combines information to predict how the body will react

12 Phototoxicity Endpoint: The phototoxicity test measures the extent to which the substance, if applied to the skin, might react with sunlight and become more dangerous. Animal test: No suitable animal test exists; the in vitro test is the standard test. Alternatives: Not always required; 3T3 NRU cellbased test (OECD TG 432). Information on whether a cosmetics ingredient is likely to cause photo-induced toxicity is only required if the product is intended for use on sunlight-exposed skin, for example face cream, and the substance has UV absorbing properties. The test is used to check that there is no reaction between the substance and sunlight that makes it more toxic, usually more of an irritant. There is no validated animal test for phototoxicity. In vitro tests for phototoxicity have been in place for years; they were validated in the 1990s and approved by the OECD in The NRU3T3 test (OECD TG 432) is based on an animal cell line and measures the number of cells that die when in contact with the substance and radiation. This simple test has been accused of giving false positive results by overpredicting phototoxicity. However, a recent workshop on the use of the test for drug products highlighted that companies need to adhere to the OECD Test Guideline to ensure its correct use and avoid the use of old cells or high doses. 51 In vitro test results can also be followed up by carefully conducted tests in humans (see Product Testing) or reconstituted human skin models (see Skin Irritation). As an analogy, Sun Protection Factor (SPF) claims are tested using human volunteers in a photo patch-testing protocol 52. Carcinogenicity Endpoint: A carcinogen is a substance that causes cancer or increases the likelihood that someone will develop cancer. Animal test: Rats or mice are fed the substance for two years to see if they get cancer (OECD TG 451, 452). Alternatives: Very rarely conducted; carcinogenicity can be assumed from genotoxicity tests or tested using the Cell Transformation Assays (OECD Guidance documents 214 (SHE assay) and 231 (Bhas42 assay)). The carcinogenicity bioassay on rats is a notoriously unreliable test with an estimated prediction of human cancers of only 42% 53. It is expensive (approximately one million euros) and time-consuming (two years minimum) and for these reasons is rarely conducted for cosmetics substances. 54 It is even being phased out for pharmaceuticals. 55 In practice, cosmetics developers use the genotoxicity tests (see Genotoxicity) and assume if the substance is genotoxic then it may cause cancer. Although this may rule out some substances for future use that may be safe, it is normal practice and protects consumers. Follow-up testing can be carried out using the Cell Transformation Assays (CTA) using rodent cells (Syrian Hamster Embryo (SHE), Balb/c3T3 and Bhas42 cells), which detects both genotoxic and non-genotoxic carcinogens. These assays have been in use for over 50 years but have been improved and validated more recently. An OECD review in 2007 concluded that 90-95% of human carcinogens could be detected 56 and guidance documents were published in 2015 and 2016 for the SHE 57 and Bhas42 58 assays respectively. Unfortunately, not all OECD countries accepted the assays as official test guidelines. These tests take 3-6 weeks compared to over two years for the bioassay on rats and costs approximately 500 euros per test compared to 1 million euros for the animal test. In the meantime, these assays have been endorsed by ECVAM for their use in carcinogenicity testing 59 and an assessment of their use in an integrated testing strategy confirmed the findings of the OECD that they can detect 90-95% of genotoxic and non-genotoxic carcinogens. 60 Photo credit: BASF 22 23

13 Reproductive toxicity Endpoint: Reproductive toxicity refers to a wide variety of adverse effects that may occur in different phases within the reproductive cycle, including effects on male and female fertility, sexual behaviour, embryo implantation, embryo development, birth and growth, and development of the young. Animal test: Pregnant female rabbits or rats are force-fed the substance and then killed along with their unborn babies (OECD TG 414). This is the prenatal developmental (or teratology) test. Less commonly conducted for cosmetics but more commonly for chemicals is the twogeneration reproductive toxicity test that monitors the fertility and development of up to two generations of rats (OECD TG 416). Alternatives: Reproductive toxicity tests are not usually a standard requirement. In some cases, they can be avoided using read across or the TTC concept. The embryonic stem cell test (EST) can be used to screen for developmental toxicity. Tests for reproductive toxicity are not considered a core requirement for cosmetics ingredients in Europe and may only be conducted if considerable oral intake or dermal absorption is expected. 61 This is because in many cases, consumers will be exposed to such low levels of the individual substances that reproductive effects, even if the substance has the potential to cause them, are very unlikely to occur. Again, the TTC approach, for which feasibility for reproduction endpoints has been demonstrated for chemicals generally, 62 can also be used (see Repeated Dose). In addition, read across and QSARs can be used for this endpoint (see Repeated Dose). Those companies that voluntarily undertake reproductive toxicity tests usually only carry out the prenatal developmental toxicity test. 63 This test takes at least four weeks, uses hundreds of animals and costs over 60,000 euros. 64 In addition, a number of studies have shown that it only detects about 60% of known human reproductive toxicants 65,66. An in vitro test using animal-based stem cells has been developed, however, to screen for harmful effects on the developing foetus. The embryonic stem cell test (EST) takes advantage of the nature of stem cells to use failure to differentiate into beating heart muscles as an indication of the developmental toxicity potential of a chemical. The EST was fully validated by ECVAM in 2002 and shown to have an overall accuracy of 78% with 20 substances. 67 Although not yet accepted for regulatory purposes, the EST is used by industry for in-house screening purposes. In 2008, Pfizer concluded that the overall performance of the EST was generally good with an accuracy of 75% for 63 chemicals, and that they were confident to use the assay to aid compound-development decisions. 68 Improvements have been made recently to increase applicability 69 and speed of the assay 70 and to account for metabolism 71. The test takes only 10 days to conduct and costs approximately 3,000 euros. Endocrine disruption Endpoint: Tests seek to assess the likelihood that the substance will interfere with the body s endocrine (hormone) system producing harmful effects. Animal tests: No single established animal test for endocrine disruption exists (nor is likely to). The Hershberger assay looks at the effects on castrated male rats who are injected with or force-fed the substance for 10 days before being killed (OECD TG 441). Alternatives: Not a standard endpoint, receptor binding assays (e.g. OECD TG 455,457, 458 and 493) can help screen for endocrine disrupting properties. Although there is much scientific and regulatory interest in the potential for substances to be endocrine disruptors, there are no standard animal or non-animal tests for this endpoint; there is even disagreement about the point at which a substance can be considered an endocrine disruptor. Nonetheless, there are now a range of receptor binding assays that can be used to screen cosmetics ingredients for potential endocrine (hormone) disrupting properties. These assays work by using a labelled compound, which when it binds to a receptor, can be used to detect that receptor. The extent to which the labelled compound can be detected in the presence of the test substance gives a measure of how much the substance has interfered with the receptors related to hormone production. A range of assays are now available, including the Stably Transfected Transcriptional Activation assay (STTA) (OECD TG 455, accepted 2009), Human Recombinant Estrogen Receptor (hrer) In Vitro Assays to Detect Chemicals with ER Binding Affinity (OECD TG 493, accepted 2015) and the BG1Luc estrogen receptor transactivation test method for identifying estrogen receptor agonists and antagonists (OECD TG 457, accepted 2012). The Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (OECD TG 458, accepted 2016) covers male hormones (androgens). More tests for other endocrines are in development and validation

14 Toxicokinetics Endpoint: Toxicokinetics is an assessment of how the body deals with a substance; in other words, whether it is metabolised and how long it stays in the body. This assessment helps to aid decisions on the safety of the substance. Animal test: Rabbits or rats are forced to consume the substance then are placed in cages on their own before being killed and their organs examined (OECD TG 417). Alternatives: Toxicokinetic studies are rarely a legal requirement for the safety assessment of cosmetics. The use of pharmacokinetic computer models, together with in vitro dermal absorption and metabolism data, can adequately replace the key components. prediction rate 74. In fact, before in vitro studies on human cell models were routinely used by the pharmaceutical industry, the failure rate of drugs in clinical trials due to poor prediction of pharmacokinetics was 40% 75 - now it is only 10%. 76 There are companies which offer this as a service. A recent study showed that in vitro liver cell tests with PBPK modelling gave better prediction accuracy for humans compared to in vivo tests on rats and dogs. 77 The option to use computer models and in vitro assays on liver cells to address metabolism has been included in the recently updated OECD TG 417 on toxicokinetics. It is usually not mandatory to have animalbased, toxicokinetic data. In a review of EU cosmetics dossiers, less than 50% of dossiers had toxicokinetic data and the regulator did not request the conduct of an in vivo test. 72 However, toxicokinetic data, or aspects of it, can help in the risk assessment. The skin is the main route for the absorption of cosmetics and can already be modelled using the regulatory approved in vitro skin absorption method (see Skin Absorption). Metabolism can be predicted using high-throughput assays on cultured human hepatocytes (liver cells). Results from these tests can then be run through computer generated, physiologicallybased, toxicokinetic models (PBTK) to predict the distribution and excretion of substances through the human body. These have been used by the pharmaceutical industry with growing sophistication since the 1970s 73 and a number of studies have demonstrated their high 26 27

15 Conclusion References Cruelty Free International estimates that half a million animals suffer in tests for cosmetics around the world every year, and over 80% of the world still allows animal testing for cosmetics. Yet the animal tests that have traditionally been used to assess the safety of cosmetics are cruel, unnecessary, expensive and unreliable. As demonstrated in this report, quicker, cheaper and more reliable modern alternatives have been validated, can be used by companies and should be accepted by regulators worldwide. The public wants to see an end to the suffering of animals used to test cosmetics and personal care products. With better alternatives now available and becoming ever more sophisticated, governments and international decision-making bodies can respond to public opinion and take decisive action to end animal testing for cosmetics whilst also providing better safety of these products. The European Union s trailblazing 2013 ban set a humane example to the world, and has demonstrated that it is possible to have a vibrant, innovative and profitable cosmetics market without the use of animal tests. Other countries are following suit, with progressive legislation in countries as diverse as Guatemala, Iceland, India, Israel, New Zealand, Norway, Serbia, South Korea, Switzerland, Taiwan and Turkey. Whilst rules on animal testing in cosmetics are currently patchwork, with legislation differing around the world, momentum is growing ever-faster for a harmonised global ban which is best for business, consumers and animals. As consumer demand for safe and humane cosmetics increases around the world, assessing the safety of cosmetics without using animals is not only desirable, it is imperative. Governments must now meet the global challenge to do the right thing for their citizens and animals by ending animal testing for cosmetics everywhere and forever. 1. Cosmetics Europe website: cosmetics-industry/ 2. The Public Opinion Research Centre (CVVM) for Svoboda zvírat, Opinion for Dyrevernalliansen, Holdninger til bruk av dyr, landsomfattende omnibus av Opinion Research Business for Cruelty Free International and RSPCA, Horizon poll 2013 see page/338/89-want-cosm 6. PCRM/ORC International endanimaltesting/be_cruelty_free/bcf_canada_poll_ data.pdf 8. Polling conducted in May 2013 by Nexus Research on behalf of Humane Research Australia smallanimaltalk.com/2014/09/should-animals-be-usedfor-cosmetic.html 9. Opinion Research Business for BUAV, The Animal Tracker (Wave 4 - March 2011). Humane Research Council, SCCS The SCCS s Notes of Guidance for the Testing of Cosmetic Substances and their Safety Evaluation, 9th Revision SCCS/1564/ SCCNPC Opinion concerning guidelines on the use of human volunteers in compatibility testing of finished cosmetic products - adopted by the Scientific Committee on Cosmetics and Non-food Products intended for Consumers during the plenary session of 23 June consumer_safety/opinions/sccnfp_opinions_97_04/ sccp_out87_en.htm COLIPA (now Cosmetics Europe) response to the impact assessment of the EU marketing ban ec.europa.eu/consumers/sectors/cosmetics/files/pdf/ animal_testing/at_responses/colipa_ia_2013_1_en.pdf 16. Bronaugh, R. L. et al Methods for in vitro percutaneous absorption studies I: Comparison with in vivo results. Toxicol. Appl. Pharmacol. 62, SCCNFP Basic criteria for the in vitro assessment of percutaneous absorption of cosmetic ingredients. Final guideline adopted by the SCCNFP, 23 June 1999, SCCNFP/0167/ Poet, T.S Assessing dermal absorption. Toxicol. Sci. 58, SCCS The SCCS s Notes of Guidance for the Testing of Cosmetic Substances and their Safety Evaluation, 9th Revision SCCS/1564/ OECD Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests. Series on Testing and Assessment. No EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing. Report EUR EN 22. Bulgheroni A, et al Estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from the 28 day repeated dose toxicity studies in rats. Regul Toxicol Pharmacol. 53, ECHA Guidance on information requirements and chemical safety assessment. 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In Vitro. 13, And Weil, C.S. and Scala, R.A Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxicol. Appl. Pharm. 19,