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Home , Animal testing, Anita Roddick, The Body Shop

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

Second edition 2017 With grateful thanks to for funding the second edition of this guide to testing the safety of cosmetics Contents products and ingredients without the use of animals.

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 tests and their alternatives 14 Skin absorption 14 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

Meeting the global challenge 3 About us

Photo credit: Philippe Gotteland at EpiSkin

Cruelty Free International is the leading organisation The Body Shop working to create a world where nobody wants or believes we need Founded in 1976 in , England, by , The Body to on animals. Its dedicated team are experts in their Shop is a global beauty brand. The Body Shop seeks to make a fields, combining award-winning campaigning, political lobbying, positive difference in the world by offering high-quality, naturally- pioneering undercover investigations, scientific and legal expertise inspired skincare, hair care and make-up produced ethically and and corporate responsibility. Educating, challenging and inspiring sustainably. The Body Shop pioneered the philosophy that business others across the globe to respect and protect animals, it investigates can be a force for good and this ethos is still the brand’s driving and exposes the reality of life for animals in laboratories, challenges force. The Body Shop has more than 3,000 stores in more than 60 decision-makers to make a positive difference for animals, and countries. champions better science and cruelty free living. Widely respected as an authority on issues, it is The Body Shop was the first international beauty brand to campaign frequently called on by governments, corporations and official bodies against the practice of animal testing in cosmetics in 1989, leading for advice or expert opinion. Building relationships with politicians, the way to a -wide ban on animal testing in 2013. business leaders and officials, analysing legislation and challenging This on-going campaign, conducted in partnership with Cruelty Free decision-making panels around the globe, it acts as the voice for International, went on to collect 1 million signatures and triggered animals in laboratories. significant progress across the Asia Pacific region.

The Leaping Bunny A UN ban on animal testing for cosmetics The Cruelty Free International Leaping Bunny is the most trusted In June 2017, The Body Shop and Cruelty Free International launched cruelty free certification for non-animal tested products. It is the only a new campaign, Forever Against Animal Testing. Together they are international guarantee that companies will not carry out any animal working to achieve a resolution for a global ban on cosmetics animal testing for their products in any country in the world. testing by 2020, revolutionising the beauty industry and protecting millions of animals around the world. In the first two months of their The Leaping Bunny logo is issued for use by companies which campaign they collected 2 million signatures and in 2018 they aim to produce cosmetics, personal care, household and cleaning products take the campaign to the highest authority, the , with which comply with the rigorous Leaping Bunny certification criteria. 8 million petition signatures, to request an international convention More than 700 companies across the globe already hold Leaping banning cosmetics testing of both ingredients and products on animals. Bunny certification, providing real choice for ethical consumers who www.thebodyshop.com/ban-animal-testing want to identify and buy products that are free from animal testing.

4 Meeting the global challenge Meeting the global challenge 5 Introduction to the report Global overview of regulatory progress

Over the past 30 years, public pressure to tested cosmetics to countries with a marketing Public opinion and consumer demand for cruelty free products end the testing of cosmetics on animals has ban are impossible, it is important for countries have been driving forces behind the global trend towards increased around the world and cosmetics where a ban is not yet in place to provide a way the phasing out of animal testing for cosmetics and the animal testing is now banned in a growing forward for their domestic industry. development of innovative alternatives. number of countries. Most notably, the European Union – the world’s largest cosmetics market1 The second edition of this ground-breaking The map below depicts the international state of play at time – ended the testing of cosmetics products in report explains the current position for each of writing. With momentum building ever-faster for a global 2003, the testing of ingredients in 2009 and, in safety test. It will help governments, politicians, ban, please contact Cruelty Free International for an up-to-date 2013, the sale of any new cosmetics that have regulators and cosmetics manufacturers across overview of the current situation. been tested outside the EU on animals. , the world switch to alternatives to replace , India, Israel, Turkey, New Zealand, animal testing, giving them the confidence that Guatemala, Serbia and also now the safest and most modern methods are used prohibit the use of animals for cosmetics testing. and that by moving away from obsolescent A phased approach to ending testing is in place technology, access to European and other in Taiwan and South Korea. Other countries - markets is possible. including the United States, , In the report, Cruelty Free International describes and Brazil - are currently considering legislation the alternative approaches that are available, that would see an end to cosmetics testing on and shows how they are not only more ethical animals. Discussions on transitioning to modern but also more reliable, faster and cheaper than alternatives are also underway in countries such the animal tests they replace. as Japan and . The report explains what alternatives are, how Largely driven by the EU’s 2013 deadline and cosmetics are tested for product and ingredient regulatory changes occurring in other parts of safety and which alternatives can be used to the world, into technologies that replace replace each traditional animal test. The report animal testing by the cosmetics industry and then outlines the alternatives for both ‘standard’ governments has stepped up. As a result, there and ‘non-standard’ animal tests. ‘Standard’ tests are now alternatives for the most commonly are those most commonly required by national required safety tests for cosmetics and many regulators for general purpose cosmetics of these are now recognised internationally. ingredients. ‘Non-standard’ tests are not usually For tests where alternatives are not recognised a routine requirement for cosmetics, but might internationally, further animal tests can still be be triggered when consumer exposure is avoided depending on the type of ingredient and expected to be particularly high or the ingredient its intended use. is expected to have biological activity. The Bans in place: report provides a summary of the validated European Union 28, Ethical concern has been the driver for this Norway, Iceland, positive change, and governments can take and, in many cases, internationally accepted, Other progress: Serbia, Israel, Bans in transition: Bans under comfort from the fact that the animal tests alternatives to these tests on a test-by-test basis, Argentina, ASEAN, India, Switzerland, Australia, South consideration: traditionally used in the past to ensure the safety and includes an overview of how new animal Viet Nam, Thailand, New Zealand, Korea, Taiwan Brazil, USA, Canada tests in non-standard cases can be avoided. Russia, China of cosmetics now have practical and reliable Turkey, Guatemala, non-animal alternatives. Since exports of animal- San Marino, Liechtenstein

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

8 Meeting the global challenge Meeting the global challenge 9 Product testing requirements Ingredient testing requirements

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

10 Meeting the global challenge Meeting the global challenge 10 Meeting the global challenge 11 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 Endpoint Tests for Animal test Options to avoid animal test options to avoid animal testing Skin sensitisation Measures the The substance is rubbed Several in vitro tests have been likelihood that the onto the shaved skin of accepted; the peptide reactivity substance will guinea pigs who are (DPRA) test which measures Endpoint Tests for Animal test Options to avoid animal test cause an allergic subjectively assessed for the binding of the substance reaction if applied to allergy (Buehler or GPMT to proteins, the keratinocyte Skin absorption Measures the The substance is rubbed Ex vivo skin based tests for this the skin. test, OECD TG 406) or assay, and the human Cell Line extent to which onto the shaved backs are well established (Dermal painted onto the ears Activation Test (h-CLAT) based on the substance will of rats who are killed the absorption in vitro skin test, OECD of mice who are killed human skin cells (OECD TG 442c, penetrate the skin. next day (OECD TG 427). TG 428). 6 days later to assess 442d and 442e respectively). Tends to overpredict. the immune response Testing strategies using these (LLNA test, OECD TG methods are already being used Acute toxicity Assesses the Rats are exposed to Not always required because 429, 442a/b). The more by companies and are recognised amount of the a very high dose of assessing repeated dose toxicity is modern test, the LLNA, at the OECD. substance that the substance such considered more useful. predicts human reactions will cause severe that a number of them only 82% of the time. toxic effects if are expected to die Cell based tests such as the accidentally (OECD TG 402,403, NRU3T3 can be used to predict Mutagenicity/ Assesses the The substance is force-fed A battery of two or three cell- ingested, inhaled or 420,423,425,436). lack of toxicity very accurately genotoxicity likelihood that or injected into mice or based tests is always carried out rubbed on the skin. (ECVAM recommendation 2013) the substance rats for 14 days who are before conducting an animal test and can be used in a weight- will cause genetic then killed to look at the (Bacterial Reverse Mutation Test, of-evidence approach (ECHA damage which effects on their cells (OECD OECD 471, in vitro Mammalian guidance 2016). could lead to TG 474, 475, 483, 486, Chromosome Aberration Test, Skin irritation/ Measures the Substance is rubbed Reconstituted human skin models cancer. 488, 489). OECD 473, in vitro Mammalian corrosion extent to which into the shaved backs are now accepted and can be Cell Mutation Test, OECD the substance will of who are killed used in most cases (in vitro skin TG 476, in vitro Mammalian irritate and damage two weeks later (OECD TG corrosion and irritation tests, Cell Micronucleus Test, OECD the skin. 404). Tends to overpredict. OECD TG 431 and 439). 487, in vitro Mammalian Cell Gene Mutation Tests using the Eye irritation/ Measures the Substance is placed in Eyes from hens and cows killed thymidine kinase gene, OECD corrosion extent to which the eyes of live rabbits for food can now be used to 490). Positives should be assumed the substance will who are monitored for detect non-irritants and severe to be genotoxic to avoid irritate the eyes. up to three weeks (OECD irritants (BCOP and ICE ex vivo eye follow-up. TG 405). Notoriously models, OECD TG 437 and 438). unreliable test. Detection of non-irritants can be Repeated dose Measures the Rats (occasionally rabbits, Can be avoided if the exposure assessed using human corneal effects of repeated mice or even ) are to the substance is likely to be epithelial models (OECD TG 492). exposure to the force-fed, forced to inhale extremely low (TTC concept) or substance over a or have the substance read across can be used if the long period. rubbed onto their shaved substance is similar to existing skin every day for 28 or 90 ones that have already been days before being killed tested. (OECD TGs 407-413). The ability to correctly predict human reactions (to drugs) using this test is no more than 60%.

12 Meeting the global challenge Meeting the global challenge 13 Skin absorption Acute toxicity Skin irritation/corrosion The RhE skin models can be purchased as kits from the manufacturers: www.mattek.com Endpoint: The skin absorption test measures the Endpoint: The acute toxicity test assesses the Endpoint: Measures the extent to which the (EpiDerm®), www.episkinskinethic.com (EpiSkin®, extent to which the substance will penetrate the amount of the substance that will cause severe substance will irritate and damage the skin. Skin Ethic RHE®) and www.cellsytems.de (epiCS®). skin. toxic effects if accidentally ingested, inhaled or Many contract testing facilities are now familiar rubbed on to the skin. Animal test: The substance is rubbed into the with the methods and will use them on behalf of Animal test: The substance is rubbed on to the shaved backs of rabbits who are killed two cosmetics companies. Contract testing facilities shaved backs of rats and they are killed the next Animal test: Rats are exposed to a very high weeks later (OECD TG 404). charge approximately the same as for the test day (OECD TG 427). dose of the substance such that a number of on rabbits, but the kits obtained directly from the them are expected to die (OECD TG 402,403, Alternatives: Reconstituted human epithelial Alternative: Dermal absorption in vitro skin test manufacturers can be cheaper. 420,423,425,436). (RhE) skin models (OECD TG 431 and 439). (OECD TG 428). Alternatives: Not always required because In vitro models based on reconstituted human Determining the extent to which the substance assessing repeated dose toxicity is considered epithelial (RhE) skin have been developed since will absorb through the skin and become more useful. the 1980s. These models comprise small discs systemically available is an important step in of cells grown into an epidermal layer from being able to determine the Margin of Safety Single dose studies for cosmetics ingredients are human skin donated as waste from cosmetic (MoS) of an ingredient, and therefore the risk not considered useful because these tests were surgery. The models have now been thoroughly assessment of the product as a whole. In vitro designed years ago as a crude measure of the validated and internationally approved by tests for skin absorption are well established and toxicity of chemicals. Today, it is more common to the OECD. The tests can be used to classify were one of the first alternatives to be approved see repeated dose data instead of LD50 animal substances as corrosive (UNGHS category 1; by the OECD in 2004. They measure the extent test information for cosmetics ingredients. This is some tests can be used for sub classifications of of absorption of the substance through discs because cosmetics are not expected to be very this category), irritating (UN GHS category 2) and of donated human skin into a fluid reservoir. toxic and repeated dose information is usually not irritating (not classified). The methods have a These tests were shown to accurately reproduce preferred to directly determine the NOAEL. In wide applicability domain so there are only very the same absorption through in vivo skin in the the EU, acute toxicity data is not insisted upon if limited cases where they could not now be used 1980s16 and were accepted for use in the EU in repeated dose information is available.19 for this endpoint. EU chemicals legislation now 1999.17 no longer requires the test on rabbits.24 Cell-based tests such as the NRU3T3 (see It is known that absorption through rodent skin Phototoxicity) can be used to predict lack of Skin corrosion can be assessed using RhE skin tends to be higher than it is in humans, and toxicity very accurately. The OECD has issued corrosion model OECD TG 431 or other in vitro therefore a rodent skin absorption test will Guidance Document 129 which outlines the models, TG 430 (TER) or TG 435 (Corrositex®). If overestimate the extent to which the substance test and how it can be used to estimate the the test is negative, the RhE skin irritation models will penetrate human skin by a factor of three.18 starting dose for an animal test, following a (OECD TG 439) should then be used to assess This is due to differences in skin thickness, hair review by US authorities.20 European validation if the substance is irritating or to confirm that follicles and immune responses. In vitro skin body ECVAM has recently concluded in a large- it is not irritating. Sometimes companies test absorption methods have the distinct advantage scale analysis that the test can be safely used their substances using the RhE skin irritation that human skin can be used. The cost of in vivo to detect non-toxic, non-classified substances.21 models only since cosmetics ingredients are not and in vitro tests is equal because the substance Only one substance (digoxin) that is toxic was usually expected to be corrosive. All OECD TG is usually radio labelled in both assays to enable not identified by the test (out of 72 substances 439 methods predicted skin irritation to at least detection of the substance. tested). Since most substances are non-toxic,22 75% accuracy in the validation study,25 although the use of this test could avoid further testing, follow-up studies have shown they are actually however the European Chemicals Agency more accurate than this. For example, in a study states it must be used in conjunction with other using 184 cosmetics, EpiSkin® demonstrated information to waive the animal test.23 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

Photo credit: Philippe Gotteland at EpiSkin

14 Meeting the global challenge Meeting the global challenge 15 Eye irritation/corrosion often giving very different results28 and with Skin sensitisation to other chemicals in the database with known only low to moderate correlation with human properties. For example, the QSAR model CAESAR Endpoint: Tests for eye irritation and corrosion responses as rabbits tend to experience more Endpoint: Skin sensitisation is an allergic reaction is 90% predictive.35 See www.antares-life.eu for measure the extent to which the substance will severe effects than humans29. to a particular substance that results in the models. irritate the eyes if it is accidentally spilt. development of skin inflammation and itchiness. More recently, methods based on reconstituted The skin becomes increasingly reactive to the Several organisations have successfully Animal test: The substance is placed into the human corneal epithelium (HCE) have been substance each time it is exposed to it. assessed the use of these tests in various eyes of live rabbits who are monitored for up to validated and are commercially available, for combinations, usually using two or three of the three weeks (OECD TG 4054). example, from www.mattek.com (EpiOcular®) and Animal tests: The substance is rubbed on to the tests above to predict whether the substance is www.skinethic.com (SkinEthic HCE®). OECD TG shaved skin of guinea pigs who are subjectively a sensitiser or not. BASF use an in-house version Alternatives: BCOP and ICE ex vivo eye models 492 has been created and includes EpiOcular® assessed for allergy (Buehler or GPMT test, OECD of KeratinoSens® (Lusens, undergoing OECD (OECD TG 437 and TG 438), and HCE eye models to detect non-irritant substances (accepted 2015) TG 406), or painted on to the ears of mice who acceptance), the DPRA and h-CLAT or Modified (OECD TG 492). and will soon be updated to include SkinEthic are killed six days later to assess their immune myeloid U937 skin sensitisation (mMUSST) test HCE® (updated expected in 2017) and LabCyte response (LLNA test, OECD TG 429, 442a/b). (also undergoing OECD acceptance) and a (updated expected in 2018). An assessment two out of three rule; any two assays must be of 435 cosmetics substances has shown that Alternatives: Several in vitro methods can be positive to rate the substance as a skin sensitiser SkinEthic HCE® is 82% accurate30 and a study by used in combination, including the DPRA (OECD and any two assays must be negative to rate ® BASF also found EpiOcular® to be 82% accurate31. TG 442c), KeratinoSens (OECD TG 442d) and the substance as a non-sensitiser. This strategy h-CLAT (OECD TG 442e. has been shown to accurately predict human There are other methods available but they sensitisers from non-sensitisers 90% of the The mechanism of how skin reacts to sensitising have a more limited applicability: the Fluorescein time36. Leakage Test Method uses an animal-derived substances to produce an allergic reaction is epithelial cell line monolayer to identify severe well understood and is outlined in an OECD The Dutch authorities use a slightly different 34 eye irritants (OECD TG 460) and the Short Time document on Adverse Outcome Pathways. testing strategy comprising QSAR models and Exposure method (STE, OECD TG 491) also uses This has helped with the development of various the DPRA followed by the KeratinoSens® and the an animal cell line but can detect both severe alternative methods that address key steps in the h-CLAT if the results at that stage are still unclear. irritants and non-irritants. pathway. Used in combination these tests can Their strategy was found to be accurate 95% of now be used to replace the animal tests for skin the time using just the DPRA and QSARs, and All these methods can be combined in a so- sensitisation. 100% accurate using all three in vitro tests.37 called ‘top down/ bottom up approach’ using Photo credit: BASF tests in two methods to arrive at a classification.32 The Direct Peptide Reactivity Assay (DPRA) (OECD The original LLNA test on mice is only up to 82% TG 442c, adopted 2015) is an in chemico method 38 Isolated eyes from cows or chickens killed for One method (typically an ex vivo method) is predictive of human allergic reactions and that measures protein reactivity, a key step in the food purposes can be used to detect severely used first if it is suspected that the substance has been shown to place nearly half of known is severely irritating (Category 1) whilst another mechanism of skin sensitisation. The ARE-Nrf2 human strong sensitisers in the wrong sub irritating/corrosive (GHS 1) substances. The ® Luciferase Test Method (KeratinoSens ) (OECD 39 OECD TGs were approved in 2009, and updated method (typically the HCE methods) is used category. The three in vitro tests can be carried TG 442d, adopted 2015) is an in vitro test that in 2013 to reflect the fact that they can also be first if it is suspected that the substance is not out for approximately the same price as the uses a human cell line to measure the activation used safely to detect non-irritants (non-classified irritating (not classified). A final OECD testing LLNA animal test (around 4,000 euros). The DPRA of known to be involved in triggering the ® substances). These ex vivo methods tend to over strategy outlining this approach is expected in takes one day to run, and the KeratinoSens immune response to contact allergens. The predict the results of the test on rabbits, which 2017. In the meantime, there is already a testing takes four days, whilst the LLNA takes six days Human Cell Line Activation Test (h-CLAT) (OECD means they always detect severely irritating strategy in the current rabbit OECD TG 405 and, not including time for analysis of the results. TG 442e, adopted 2016) is an in vitro test that substances and are therefore protective, but they for chemicals testing in Europe, a requirement uses a human cell line that addresses the third EU chemicals legislation REACH has recently may also predict that a substance is irritating to exhaust testing in vitro/ex vivo before using animals.33 Currently, however, a combination of key event in skin sensitisation, activation of the been updated to permit the use of these when it is not. 40 alternative methods still does not give confidence dendritic cells. alternative tests in combination , and the OECD Contract testing facilities charge approximately in predicting Category 2 (irritation), but this is likely has outlined the various testing strategies in Quantitative Structure-Activity Relationship the same to conduct the ex vivo tests as they do to change in the next couple of years as they are terms of ‘defined approaches to testing and (QSAR) computer models have particularly strong 41 the test on rabbits. The animal test is notoriously further improved. assessment for skin sensitisation’. Further work predictive power for skin sensitisation because cruel and unreliable, with different laboratories is being done at the OECD to help with the formal reactivity can be predicted from chemical recognition of these testing strategies to facilitate structure. The reactivity of a chemical structure their global acceptance. can be predicted based on its structural similarity

16 Meeting the global challenge Meeting the global challenge 17 Repeated dose The TTC approach

Endpoint: Measures the effects of repeated The TTC approach is based on the concept that exposure to the substance over a period of time. for all substances there is a level of exposure below which there is hardly any risk to human Animal tests: Rats (occasionally rabbits, mice or health, regardless of how toxic the substance even dogs) are force-fed, forced to inhale or have is. If the exposure of a substance in a cosmetics the substance rubbed on to their shaved skin product is known (which it should be as part of every day for 28 or 90 days before being killed the risk assessment; see Product Testing), and (OECD TGs 407-413). if it is very low, then even if the substance is of these cell-based tests, covering effects on assumed to be toxic, testing will not affect the gene mutation (TG 471, TG 476 and TG 490) and Alternatives: Until a testing strategy using in safety of the product and the TTC approach could changes to chromosome structure (TG 473) and vitro tests is developed and validated, repeated apply. Instead of conducting an animal test, the number (TG 487). In combination, these tests dose testing can often be avoided using the TTC risk assessor will evaluate the likely toxicity class have been shown to be 85-90% predictive of (threshold of toxicological concern) approach of the substance (based on chemical structural rodent carcinogenicity test results across a large and/or read across approaches. similarity to other substances), then calculate number of chemicals.42 maximum daily exposure. If the substance falls Several reviews of the ability of rodent below a certain value, then it can be considered tests to predict human toxicity, mainly in These in vitro tests are often accused of being too ‘safe’. The TTC concept was first used for food pharmaceuticals, have found that they are only protective, i.e. safe chemicals can be mistakenly additives, but research by the cosmetics industry about 40-60% predictive of human .47 predicted to be genotoxic. However, this is has shown it to be relevant for cosmetics48, Nonetheless, repeated dose information is often inconclusive as the results are always compared with examples and databases now available.49 required for new cosmetics ingredients to obtain to tests in rats and mice rather than humans. Cruelty Free International’s analysis is that the Mutagenicity/genotoxicity the No Observed Adverse Effect Level (NOAEL) to The common approach is to ‘follow up’ these concept could be used for many common perform the risk assessment. Endpoint: The mutagenicity/genotoxicity safety positive results using an in vivo test on mice or ingredients that are present in small quantities. test assesses the likelihood that the substance rats. However, follow-up of positive results can A variety of cell-based models are available that will cause genetic damage to the cells in the be avoided by careful choice of cell type (human either use long-lasting liver cells or incorporate body which could lead to cancer. cells being preferable), dose levels and method Read across a range of cell types into a ‘microchip’. These of assessment of the damage.43 According to the are currently used to screen substances for Substances with physicochemical, toxicological Animal tests: The substance is force-fed or OECD test guidelines for these in vitro tests, cells long term toxicity but do not yet have regulatory and ecotoxicological properties that are likely injected into mice or rats for 14 days who are should also be exposed to the test substance acceptance. to be similar or follow a regular pattern as a then killed to look at the effects on their cells in the presence and absence of an appropriate result of structural similarity may be considered (OECD TG 474, 475, 483, 486, 488, 489). metabolic activation system. Because cosmetics substances are often as a group, or ‘category’ of substances. In this used in very low quantities, in some cases Alternatives: Bacterial Reverse Mutation Assay It has been recommended in Europe that only case, existing data on one or more members animal tests can be avoided by using the TTC (Ames test) (OECD TG 471), In Vitro Mammalian two in vitro tests are required if the In Vitro of the group can be used to provide data (to concept. Alternatively, read across or category Cell Gene Mutation Test (OECD TG 476), In Vitro Mammalian Cell Micronucleus Test (TG 487) read across) for the other members, and new approaches can be used when there is existing Mammalian Chromosome Aberration Test (OECD is used because it looks at changes to both testing can be avoided. The OECD has produced data on a structurally similar substance(s). TG 473), In Vitro Mammalian Cell Micronucleus chromosome structure and number44. The use guidance on how to use read across as well as Test (OECD TG 487) and In Vitro Mammalian Cell of RhE models (see Skin Irritation/Corrosion) is a database which can be used to look for similar 50 Gene Mutation Tests Using the Thymidine Kinase currently being examined by Cosmetics Europe substances (OECD Toolbox). Read across is Gene (OECD TG 490). and ECVAM to see if this adds to the assessment, well accepted in many regions including the EU. especially since it uses human tissue.45 Pre- Companies innovating by modifying substances Mutagenicity/genotoxicity is always assessed validation studies have already demonstrated slightly to improve them may well find that initially in vitro using bacterial and other cell- that the in vitro human reconstituted skin they are justified in using read across from the based tests. These tests assess the extent of micronucleus (RSMN) assay can correctly identify original substance instead of animal testing. damage to the chromosomes (containing genes) positive and negative genotoxicants and further in the cells that could be indicative that the validation studies are currently underway to substance causes cancer. In many cases, it is confirm the sensitivity of the assay. Another test possible to determine whether a substance is called the reconstructed human skin Comet likely to be genotoxic by conducting up to three assay is also undergoing pre-validation studies.46

18 Meeting the global challenge Meeting the global challenge 19 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 Whether the No suitable animal test Only required if the substance has Endocrine Assesses the No single established Not yet seen as a regulatory substance will cause exists, the in vitro test is UV absorbing properties. Cell based disruption likelihood that the animal test for endocrine requirement. a reaction if applied the standard test. tests have been in place for some substance will disruption exists to the skin and the time (3T3 NRU cell-based test, OECD interfere with the (nor is likely to). The Receptor binding assays such as the skin is then exposed TG 432), negative results can be body’s endocrine Hershberger assay Stably Transfected Transcriptional to sunlight. confirmed in human skin tests (in vivo (hormone) system looks at the effects on Activation assay (STTA) (OECD or in vitro). producing harmful castrated male rats who TG 455) and the BG1Luc Estrogen effects. are injected with or force- Receptor Transactivation Test Carcinogenicity Assesses the Rats or mice are fed the Rarely conducted because it takes so fed the substance for 10 Method for Identifying Estrogen likelihood that a substance for two years long and is so unreliable. Companies days before being killed Receptor Agonists and Antagonists substance will cause to see if they get cancer use the genotoxicity tests above and (OECD TG 441). (OECD TG 457) and the H295R cancer if people are (OECD TG 451, 452). assume if the substance is genotoxic Steroidogenesis Assay (OECD exposed to it over a Costs $2 million and only then it may also cause cancer. Cell TG 456) can be used to screen long period. predicts human cancer transformation assays (CTA) have for potential endocrine disrupting 42% of the time. been in use for 50 years, predict 90% properties. of known human and can be used for follow up if necessary Toxicokinetics Assesses how Rabbits or rats are Rarely a regulatory requirement in (OECD guidance documents 214, 231). the body deals forced to consume the itself. with a substance, substance then are Reproductive Assesses the Pregnant female rabbits In some cases, can be avoided if i.e. whether it is placed in cages on Skin absorption (OECD TG 428) toxicity likelihood that the or rats are force-fed the consumer exposure to the substance metabolised and their own before being and liver cell metabolism tests (see substance will substance and then killed is likely to be extremely low (TTC how long it stays in killed and their organs OECD TG 417) can be put into a PBPK reduce fertility or along with their unborn concept) or read across from existing the body. examined (OECD TG 417). computer model that combines cause developmental babies (OECD TG 414). substances can be used. Poor estimates based information to predict how the body problems to the Such tests take a long on are will react. foetus. time and use hundreds responsible for 30% of of animals. Studies have drug failures. shown they only detect 60% of known human toxicants.

20 Meeting the global challenge Meeting the global challenge 21 Phototoxicity is no validated animal test for phototoxicity. In Carcinogenicity Follow-up testing can be carried out using the vitro tests for phototoxicity have been in place Cell Transformation Assays (CTA) using rodent Endpoint: The phototoxicity test measures the for years; they were validated in the 1990s and Endpoint: A is a substance that cells (Syrian Hamster Embryo (SHE), Balb/c3T3 extent to which the substance, if applied to the approved by the OECD in 2004. The NRU3T3 test causes cancer or increases the likelihood that and Bhas42 cells), which detects both genotoxic skin, might react with sunlight and become (OECD TG 432) is based on an animal cell line someone will develop cancer. and non-genotoxic carcinogens. These assays more dangerous. and measures the number of cells that die when have been in use for over 50 years but have in contact with the substance and radiation. Animal test: Rats or mice are fed the substance been improved and validated more recently. An Animal test: No suitable animal test exists; the for two years to see if they get cancer (OECD TG OECD review in 2007 concluded that 90-95% in vitro test is the standard test. This simple test has been accused of giving false 451, 452). of human carcinogens could be detected56 and positive results by overpredicting phototoxicity. guidance documents were published in 2015 Alternatives: Not always required; 3T3 NRU cell- Alternatives: Very rarely conducted; However, a recent workshop on the use of and 2016 for the SHE57 and Bhas4258 assays based test (OECD TG 432). carcinogenicity can be assumed from the test for drug products highlighted that respectively. Unfortunately, not all OECD countries companies need to adhere to the OECD Test genotoxicity tests or tested using the Cell Information on whether a cosmetics ingredient accepted the assays as official test guidelines. Guideline to ensure its correct use and avoid Transformation Assays (OECD Guidance is likely to cause photo-induced toxicity is only These tests take 3-6 weeks compared to over the use of old cells or high doses.51 In vitro test documents 214 (SHE assay) and 231 (Bhas42 required if the product is intended for use on two years for the bioassay on rats and costs results can also be followed up by carefully assay)). sunlight-exposed skin, for example face cream, approximately 500 euros per test compared conducted tests in humans (see Product Testing) and the substance has UV absorbing properties. The carcinogenicity bioassay on rats is a to 1 million euros for the animal test. In the or reconstituted human skin models (see Skin The test is used to check that there is no reaction notoriously unreliable test with an estimated meantime, these assays have been endorsed by Irritation). As an analogy, Sun Protection Factor 59 between the substance and sunlight that makes prediction of human of only 42%53. It ECVAM for their use in carcinogenicity testing (SPF) claims are tested using human volunteers it more toxic, usually more of an irritant. There is expensive (approximately one million euros) and an assessment of their use in an integrated in a photo patch-testing protocol52. and time-consuming (two years’ minimum) testing strategy confirmed the findings of the and for these reasons is rarely conducted for OECD that they can detect 90-95% of genotoxic cosmetics substances.54 It is even being phased and non-genotoxic carcinogens.60 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.

Photo credit: BASF

22 Meeting the global challenge Meeting the global challenge 23 Reproductive toxicity for chemicals generally,62 can also be used Endocrine disruption Nonetheless, there are now a range of receptor (see Repeated Dose). In addition, read across binding assays that can be used to screen Endpoint: Reproductive toxicity refers to a and QSARs can be used for this endpoint (see Endpoint: Tests seek to assess the likelihood cosmetics ingredients for potential endocrine wide variety of adverse effects that may occur Repeated Dose). that the substance will interfere with the body’s (hormone) disrupting properties. These assays in different phases within the reproductive endocrine (hormone) system producing harmful work by using a labelled compound, which when cycle, including effects on male and female Those companies that voluntarily undertake effects. it binds to a receptor, can be used to detect fertility, sexual behaviour, embryo implantation, reproductive toxicity tests usually only carry out that receptor. The extent to which the labelled 63 embryo development, birth and growth, and the prenatal developmental toxicity test. This Animal tests: No single established animal test compound can be detected in the presence development of the young. test takes at least four weeks, uses hundreds for endocrine disruption exists (nor is likely to). of the test substance gives a measure of how 64 of animals and costs over 60,000 euros. In The Hershberger assay looks at the effects on much the substance has interfered with the Animal test: Pregnant female rabbits or rats are addition, a number of studies have shown that castrated male rats who are injected with or receptors related to hormone production. A force-fed the substance and then killed along it only detects about 60% of known human force-fed the substance for 10 days before being range of assays are now available, including 65,66 with their unborn babies (OECD TG 414). This reproductive toxicants . An in vitro test using killed (OECD TG 441). the Stably Transfected Transcriptional Activation is the prenatal developmental (or ) animal-based stem cells has been developed, assay (STTA) (OECD TG 455, accepted 2009), Alternatives: Not a standard endpoint, receptor test. Less commonly conducted for cosmetics however, to screen for harmful effects on the Human Recombinant Estrogen Receptor (hrER) In binding assays (e.g. OECD TG 455,457, 458 and but more commonly for chemicals is the two- developing foetus. The embryonic stem cell test Vitro Assays to Detect Chemicals with ER Binding 493) can help screen for endocrine disrupting generation reproductive toxicity test that monitors (EST) takes advantage of the nature of stem cells Affinity (OECD TG 493, accepted 2015) and the properties. the fertility and development of up to two to use failure to differentiate into beating heart BG1Luc estrogen receptor transactivation test generations of rats (OECD TG 416). muscles as an indication of the developmental Although there is much scientific and regulatory method for identifying estrogen receptor agonists toxicity potential of a chemical. and antagonists (OECD TG 457, accepted Alternatives: Reproductive toxicity tests are not interest in the potential for substances to be 2012). The Stably Transfected Human Androgen usually a standard requirement. In some cases, The EST was fully validated by ECVAM in 2002 endocrine disruptors, there are no standard Receptor Transcriptional Activation Assay for they can be avoided using read across or the and shown to have an overall accuracy of animal or non-animal tests for this endpoint; Detection of Androgenic Agonist and Antagonist TTC concept. The embryonic stem cell test (EST) 78% with 20 substances.67 Although not yet there is even disagreement about the point Activity of Chemicals (OECD TG 458, accepted can be used to screen for developmental toxicity. accepted for regulatory purposes, the EST at which a substance can be considered an 2016) covers male hormones (androgens). More is used by industry for in-house screening endocrine disruptor. tests for other endocrines are in development Tests for reproductive toxicity are not considered purposes. In 2008, Pfizer concluded that the and validation. a core requirement for cosmetics ingredients overall performance of the EST was generally in Europe and may only be conducted if good with an accuracy of 75% for 63 chemicals, “considerable oral intake or dermal absorption and that they were confident to use the assay 61 is expected”. This is because in many cases, to aid compound-development decisions.68 consumers will be exposed to such low levels Improvements have been made recently to of the individual substances that reproductive increase applicability69 and speed of the assay70 effects, even if the substance has the potential and to account for metabolism71. The test takes to cause them, are very unlikely to occur. Again, only 10 days to conduct and costs approximately the TTC approach, for which feasibility for 3,000 euros. reproduction endpoints has been demonstrated

24 Meeting the global challenge Meeting the global challenge 25 Toxicokinetics prediction rate74. In fact, before in vitro studies on human cell models were routinely used by Endpoint: Toxicokinetics is an assessment of the , the failure rate of how the body deals with a substance; in other drugs in clinical trials due to poor prediction of words, whether it is metabolised and how long was 40%75 - now it is only it stays in the body. This assessment helps to aid 10%.76 There are companies which offer this as decisions on the safety of the substance. a service. A recent study showed that in vitro liver cell tests with PBPK modelling gave better Animal test: Rabbits or rats are forced to prediction accuracy for humans compared to consume the substance then are placed in cages in vivo tests on rats and dogs.77 The option to on their own before being killed and their organs use computer models and in vitro assays on examined (OECD TG 417). liver cells to address metabolism has been included in the recently updated OECD TG 417 on Toxicokinetic studies are rarely a Alternatives: toxicokinetics. 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.

It is usually not mandatory to have animal- based, 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, physiologically- based, toxicokinetic models (PBTK) to predict the distribution and of substances through the human body. These have been used by the pharmaceutical industry with growing sophistication since the 1970s73 and a number of studies have demonstrated their high

26 Meeting the global challenge Meeting the global challenge 27 Conclusion References

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