Vanity products such as cosmetics are not essential to human health and welfare, but are generally subjected to the same types of animal-based testing protocols as are more "useful" materials. In the United States, such cosmetic tests are not required, but are routine. In Europe, they are mandated, but with some interesting additional provisions. European Union Cosmetics Directives include requirements that animal tests should not be performed if scientifically adequate alternative procedures are "reasonably and practically available." In essence, this represents a built-in restriction on animal (in vivo) tests and promotes alternative (in vitro) tests-at least in principle.
The European Commission provided a further incentive for the development and use of alternative tests when, in 1996, it decided to prohibit cosmetic products containing ingredients or combinations of ingredients tested on animals. Although implementation was postponed from January 1, 1998 to June 30, 2000, this Directive produced a major push for new alternatives. A similar, proactive environment remains lacking in the U.S. regulatory community.
Major problems to the advancement of the alternatives approach remain, but are being actively addressed:
There are more than 7,000 chemicals used in cosmetics. In Europe, more than 400 substances are prohibited, while in the United States, only 14 are restricted. More restrictions correlate with fewer animal tests.
There is a general lack of high-quality in vivo data to serve as a baseline for in vitro validation studies. Considering the multitude of serious deficiencies associated with animal tests, this is not surprising. However, requirements that proposed in vitro replacements be validated against poor quality animal test data, biases such procedures before they begin.
Animal test data may be semi-quantitative, relatively useless, unreliable, and irreproducible, but it is still used to accept or reject many in vitro test proposals.
Regulatory authorities fail to promote alternatives for political rather than scientific reasons.
Corporate product liability lawyers and insurance companies continue to endorse the use of animal tests.
There are no consistent, effective, international efforts to promote alternatives or harmonize testing strategies. The Organization for Economic Cooperation and Development (OECD) provides testing guidelines and procedures, but usually remains significantly out of date with respect to improvements in alternative testing methods.
Despite such difficulties, progress in promoting the alternatives approach to safety testing is being made. Cosmetic products provide several excellent examples.
Skin Corrosivity/ Irritation
Because cosmetics are designed for direct application to the skin, toxic responses of that tissue are of particular interest. Furthermore, if substances are identified as non-corrosive, then further testing for irritancy can be conducted on human volunteers rather than laboratory animals.
There are a wide variety of in vitro or computer-based replacement alternatives for the more traditional and inhumane Draize Rabbit skin tests. These alternative methods use numerous endpoints that provide a relatively complete picture of the potential toxicity of test substances.
The European Center for the Validation of Alternative Methods (ECVAM) recently noted that "it is becoming increasingly apparent that the development and implementation of stepwise (hierarchical) testing strategies" is providing the most effective approaches to predicting the toxicity of new substances and to reducing the number of animals killed in in vivo test procedures.
Quantitative Structure Activity Relationships (QSAR) are particularly useful regarding skin responses to toxic substances. A PC-based system for predicting skin corrosivity is routinely used as an initial screening procedure by companies such as Unilever. Even the OECD recommends that animal tests need not be done if skin corrosion or irritation can be predicted by the basic physiochemical properties of the materials. This is precisely what the QSAR does so well.
The OECD also suggests that "it may not be necessary to test in vivo materials for which corrosive properties are predicted on the basis of results from in vitro tests."
Based on currently available in vitro methods, there are no longer any justifications for the use of animal-based skin tests.
The use of multi-layered, in vitro human skin models is rapidly increasing in both testing and research laboratories. One such in system was recently approved as an in vitro replacement for animal skin corrosivity tests.
According to the ECVAM Scientific Advisory Committee, "the results obtained with the EPISKIN (a test involving the use of a reconstructed human skin model) in the European Center for the Validation of Alternative Methods international validation study on in vitro tests for skin corrosivity, were reproducible, both within and between the three laboratories that performed the test. The EPISKIN test proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols, and soaps/surfactants.
The concordances between the skin corrosivity classifications derived from the in vitro data and from the in vivo data were very good. The test was able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied. The Committee therefore agreed with the conclusion from this formal validation study that the EPISKIN test is scientifically validated for use as a replacement for the animal test, and that it is ready to be considered for regulatory acceptance."
EPISKIN is therefore a valid replacement for the Draize Rabbit Skin test, with the former's basic skin-like gross and microscopic structure, growth characteristics, and biochemical similarities to real human skin.
It is possible to replace the skin of live sentient animals with an in vitro system. Even more astounding possibilities are planned for the future. Work is currently in progress to provide blood vessels and sensory nerves to these artificial human skin equivalents.
If a test substance is found to be non-corrosive, then it becomes possible to determine the potential for skin irritation using human volunteers. A human 4-hour patch test has been developed, is widely used in several companies, and is being considered for endorsement by the OECD. This test was developed by the Unilever Company and validated with more than 65 types of chemicals. It was also optimized for possible ethnic, inter-individual, and seasonal differences in on the skin of volunteers. This represents another ideal replacement for animal-based procedures.
If the properties of the test substance are in doubt, several in vitro methods are also available. Single layer cultures of skin cells are useful for some categories of chemicals. Organ cultures of human skin are suitable and easy to use. Although some laboratories prefer to use animal skin, comparative results suggest that the latter are inappropriate and likely to over-predict toxicity. For example, rabbit skin is far more sensitive than human skin for the same materials.
Perhaps the most useful in vitro method for predicting skin irritation is the three-dimensional, reconstituted human skin equivalents.
As the sophistication of in vitro testing methods continues to increase, justifications and rationalizations used to defend animal testing diminish. As a consequence of this transition to a more rational, alternatives-based testing program, consumers' health and welfare are more adequately protected. As the following brief examples demonstrate, this trend is real and undeniable.
Acute ToxicityFor decades, the routine approach to this concern was mass poisoning large numbers of animals in the classical Lethal Dose 50 test (LD50). The LD50 test is still on the books, but conducted infrequently. It was replaced by several new, less traumatic, but still lethal options. Two of the latter were formally adopted by the OECD and are becoming worldwide standards: the fixed dose and acute toxic class methods.
There has always been a need for a quick, easy in vitro replacement for such lethal tests. Such an alternative-based approach is now available.
The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) program was initiated in 1989. By mid-1996, the 29 contributing laboratories had tested all 50 chemicals in each of the 61 proposed in vitro assays. Evaluation of the data was completed in 1998 with very positive results.
The MEIC found that the toxicity of most chemicals to human cell lines was relevant to acute, lethal effects in humans, with a successful prediction rate of 84%. Of the many methods examined, the MEIC group selected 15 of the best tests as replacement candidates for animal-based acute toxicity methods.
Two simple in vitro methods had an 84¢ prediction success rate for some, and 71% for all of the test substances. Addition of a third technique raised the overall success rate to 77%. If information on certain physiological parameters was added, the rate increased to 83%. What is particularly important about the MEIC effort is the use of human rather than rodent reference data to determine the efficacy of the proposed replacements.
What is also significant is that for the first time, an animal toxicity test was subjected to validation procedures. The mouse LD50 failed in comparison to the in vitro options, achieving only a 66% rate of accurate predictions.
Based on the results of the MEIC study and the creation of batteries of in vitro tests to measure acute toxicity, it is no longer necessary to poison even small groups of animals in order to identify risks resulting from human exposure to new or existing materials. Lethal animal tests are no longer necessary.
A practical, easy-to-use battery of in vitro tests, based on four different toxic endpoints, is now available and was proven to provide better results than traditional rodent-based procedures. The MEIC testing scheme is ready for adoption by companies and regulatory agencies. In addition, the MEIC in vitro methods are ideally suited to study the unknown mechanisms of acute lethal and toxic actions of chemicals, which would contribute to a rationally based approach to product safety.
Eye Irritancy-Draize TestsAbout 20 years ago, the use of rabbits to test the ability of compounds to cause serious eye damage became a major focus of anti-animal testing and the development of replacement alternatives. In the decades that followed, multiple in vitro methods were proposed, tested, semi-validated, but not widely adopted. In large part, this is an artifact of the very serious problems with the unreliability of the original, animal-based Draize tests and a relative absence of suitable human eye exposure information.
Many of the available in vitro alternatives to the Draize clearly provide adequate information on ocular irritation. However, it is difficult to conduct an in vitro replacement validation study when the alternative is expected to favorably compare with in vivo results that are subjective and highly variable. In the long-term, the Draize Eye Irritancy Test and all of the data it has produced should be abandoned and replaced with a new set of well-defined, mechanistically based endpoints to which the proposed in vitro replacements can be compared. Toxicologists and regulatory agencies do not need a substitute for the Draize, but rather an entirely new approach to answering such questions.
As an interim step, a battery of in vitro and computer-based methods could be adopted to provide adequate information to determine the potential eye irritancy of new substances.
Structure Activity Relationship computer models combined with data on basic physiochemical properties can act as a pre-screen. A variety of cell-culture-based assays that have had difficulty passing earlier validation tests should be reconsidered on a case-by-case basis. These can be combined with more recently developed in vitro techniques.
Of particular interest is the HET-CAM assay, which utilizes exposure of the chorio-allantoic membrane of chicken eggs to test substances. This test provides information on inflammatory processes and passed several multi-laboratory validation trials with prediction rates as high as 80%.
The Epi Ocular in vitro system is a multi-layered culture of human cells that very closely mimics the structure of the human cornea. It consists of a metabolically active, stratified, squamous epithelium, with growth and morphological characteristics similar to human tissue. Although man-made, it behaves like the surface of the eye in response to direct exposure to compounds that cause inflammation and irritation.
A number of experimental trials with various chemicals and compounds have shown that the Epi Ocular System can act as a reliable safety test and provide a reproducible and accurate replacement for the Draize Eye Irritation Test. In one instance, the system was exposed to 41 materials, which included final formulations of shampoos, off-the-counter cosmetics, and basic chemicals. Epi Ocular had an 86% correct correlation. Other trials produced similar results.
Although "officially" still "necessary," the Draize test has never been a valid indicator of potential human eye injury. It can and will be replaced with a battery of humane alternatives.
Skin SensitizationBecause cosmetics are designed to make contact with the skin, potential allergic responses are a serious consideration for manufacturers. Traditional testing approaches involve exposure of substances to the skins of guinea pigs coupled with deliberately increased sensitivity. The resulting allergic reactions are observed and ranked.
In the United States, federal regulatory agencies recently adopted a new approach. Substances are applied to a mouse ear. After several days the mice are killed and their lymph nodes are examined for evidence of immune reactions. Although this murine Local Lymph Node Assay represents both a reduction (fewer animals) and refinement (less pain) alternative that is cheaper and quicker than the guinea pig tests, it still involves animal deaths.
In contrast, 1998 Belgian studies using reconstituted, multi-layered in vitro human skin examined potentially sensitizing materials. They concluded, "it may be possible in a single integrated assay to classify and to discriminate between irritant and sensitizing agents." Researchers in California examined the chemical profiles of substances (cytokines) released by humans in response to irritation and allergic reactions. This information may be combined with in vitro tests, making it easier to determine relevant properties of test substances.
Dr. Craig Meyers of Pennsylvania State University uses organotypic raft cultures of simulated human skin to study contact dermatitis. His research, sponsored in part by AAVS' Scientific Affiliate, the Alternatives Research & Development Foundation, is designed to provide a replacement for the tens of thousands of animals currently killed in such dermatitis testing and to provide an in vitro approach to examining treatments for the problem.
Phototoxicity / PhotoirritationThis is a prime example of regulatory necessity producing new in vitro methods. Because some cosmetic preparations are exposed to sunlight, their potential toxic responses need to be identified. The European Union recently announced the formal acceptance of a cell-culture test (3T3 NRU PT) as the officially accepted standard for determining phototoxicity. It was accepted for all types of products, not just cosmetics.
This test uses cells in cultures that are exposed to new substances and UVA light, which simulates sunlight. It was easily reproducible in different laboratories and consistently distinguished between 30 photoirritants and non-irritants. What is even more significant is the effort that produced this alternative also identified five additional in vitro methods that showed significant promise in validation protocols.
Because the 3T3 cell method does not allow for direct application of test materials, as is the case with human skin, researchers in Germany also studied the Epiderm full-skin reconstructed human epidermis as a potential indicator of phototoxicity. Successful tests of 12 chemicals established this as a second reliable in vitro alternative that has the advantage of testing formulations not suitable for use in normal cell culture environments. In combination with the 3T3 cell culture method, these alternatives have eliminated the need for further animal testing of phototoxicity.
Percutaneous AbsorptionaA substance's ability to penetrate the skin is important, since failure to do so would obviate the need for some further types of toxicity testing, either in vivo or in vitro.
The OECD is currently considering guidelines for in vitro tests of percutaneous absorption. This is based on the long-term experience of European chemical, cosmetic, and pesticide manufacturers and has the support of most of the OECD member countries. Unfortunately, as often happens, the United States is on the wrong side of this issue, being opposed to the proposed alternatives.
Non-animal alternatives are available to determine the percutaneous absorption of cosmetics and other compounds. There is no need to continue animal-based procedures for this purpose.
MutagenicityWith all commercial products, especially those intended for deliberate, direct contact with human tissues (i.e., skin), there is a concern about potential carcinogenicity. For this reason, tests to determine a compound's ability to produce mutations are conducted. For several years, in vitro replacement alternatives have been available to measure such mutagenicity. No animal-based methods are needed. In particular, a tri-partite group of in vitro tests are now, or should be, routinely used: 1) reverse mutation assay using bacteria; 2) chromosomal aberration test; and 3) gene mutation assay.
ConclusionTraditional animal-based toxicity tests were never necessary for cosmetic and personal care products, as tacitly acknowledged in U.S. regulations. Their use has been optional, but widespread. As stated, for seven of the eight types of tests considered, in vitro replacement alternatives are available and should be adopted by manufacturers and regulatory agencies. The final test, the Draize Eye Irritancy test, may already have adequate in vitro substitutions, but problems inherent in the validation process have thus far excluded them. That situation should change in the very near future.
There are no compelling scientific reasons why the new millennium cannot begin with widespread use of cruelty-free, humane, in vitro approaches to toxicity testing of cosmetics, in particular, and other substances, in general.
(article by John McArdle, Ph.D., AAVS' Science Advisor)
Please visit the following links related to animal testing for cosmetics - you will learn about the many alternatives that a compassionate consumer have, and with choosing cruelty-free products you are going to actively help many innocente animals - Thank You!
- Leaping Bunny
- Choose Cruelty Free
- Compassionate Consumer
- P&G Kills-
Uncaged - Boycott Procter & Gamble
+ Animal Tests / Vivisection links database
