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  Issue 1 (2003)

Authors' Article
Horst Spielmann
New Methods for Animal-Experiment-Free Tolerability Tests of Cosmetics

Manuscript following the lecture at 7th Annual Meeting of the GD Gesellschaft für Dermopharmazie e.V. in Bonn on 2 April 2003

Summary: The tolerability test of cosmetics in animal experiments is subject to severe criticism in the public in Germany and all EU member states. This criticism, which emanated from animal protection associations, to begin with has been taken up in the meantime by consumers, authorities and governments of the EU member states. After several years' discussion, the 7th amendment of the EU Cosmetics Directive 76/768/EEC (EU Commission 1976), proposed by the EU Parliament, was accepted (EU directive 2003/15/EG) on 27 February 2003 by the Council of Ministers of the EU member states. This amendment designates a gradual restriction of animal experiments for cosmetics and a ban in 10 years. The reason for this gradual way of proceedings is the fact that at the time being not all animal experiments provided for testing of cosmetics can be replaced by animal-experiment-free methods. At the same time considerable progress has been achieved in the development, validation and international regulatory approval of novel, animal-experiment-free test methods in the last 10 years so that the replacement of animal experiments in the tolerability testing of cosmetics will be feasible in the near future. Therefore, the progress attained is introduced which is based on mutual efforts of scientists in laboratories of the cosmetics industry and other research institutes in the development of alternative methods for animal experiments.

Legal Stipulations for
Safety-toxicological Tests of
Cosmetics in the EU
It has to be considered for the implementation of the EU Cosmetics Directive 97/18/EG that on the one hand there are substances which are to be exclusively safety-toxicologically tested according to the stipulations of the EU Cosmetics Directive and contrariwise, there are substances which are applied as ingredients in cosmetic products and toxicologically tested as industrial chemicals as they are primarily not employed in cosmetics. The latter substances undergo very thorough testing according to the chemical law pursuant to sanitary and environmental aspects. It is moreover important that cosmetical ready-to-use products may no longer be tested in the animal experiment since the amendment of the Animal Welfare Act in Germany in 1987. This legal stipulation is in the meantime adhered to by all EU member states owing to a self-obligation of the cosmetics industry since the year 2000, because considerable progress has been achieved in the development of animal-experiment-free test methods and at the same time no drawbacks have become known for the customer so far. This promising development confirm both the European association of the manufacturers of cosmetics COLIPA as well as the body of experts of the EU commission for the sanitary assessment of cosmetics SSCNF, the Scientific Committee on Cosmetic Products, and Non-Food Products. Remarkable in this context is that from a scientific, economical and ethical view, encouraging progress has been achieved by a voluntary commitment by the manufacturer of cosmetics, without the EU cosmetics directive stipulating a marketing ban for cosmetical ready-to-use products to date.

General Legal Stipulations for
the Realization of Animal
Experiments in the EU

NAccording to EU Directive 86/609/EWG (EU 1986) relating to the dealing with test animals which also represents the legal basis for the German Act for the Prevention of Cruelty to Animals, according to § 7.2 "an animal experiment may not be performed if a scientifically established animal-experiment-free method is available which has been experimentally proven in practice". Further, article 23 of the same EU directive reads as follows: "EU Commission and member states are to advance research for the development and validation of alternative methods which supply the same information as the animal experiment but require a lower number of animals or are less stressing, and they are to strengthen the general conditions for this research". These legal stipulations have been implemented by several EU member states in a different manner. Germany, for example, founded already in 1989 the Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch ZEBET (Central Clearing Office for the Registration and Assessment of Replacement and Supplementary Methods for Animal Experiments) in the former Bundesgesundheitsamt (BGA) (Federal Public Health Authority), whereas most EU member states - with only few exceptions - have not initiated any own supporting programmes for alternative methods of animal experiments and rely on the activities of the EU commission.

Figure 1 shows the European centers for the furtherance of alternative methods. The illustration explains that accordingly in Great Britain, Nottingham, the foundation FRAME (Fund for the Replacement of Animals in Medical Experiments) and in the Netherlands the NCA (National Center for Alternatives) in the Reichsgesungheitsamt (RIVM) have been founded as well as the EU Validierungszentrum ECVAM (European Center for the Validation of Alternative Methods) in the Gemeinsame Forschungsstelle (GFS) (Common Research Center) in ISPRA/Italy by the EU Commission in 1992. Because of the success of the European Centers, the Validation Center ICCVAM (Interagency Coordinating Committee for the Validation of Alternative Methods) was founded in the frame of the National Toxicology Program NTP outside of Europe by the US government in the year 1995 and Poland, as future joining member of the EU established the Zentrum für Alternativmethoden "Vitryna" (Center for Alternative Methods) in Lodsz in 2001.

Figure 1
Centers for the development and validation of alternative methods for animal experiments in Europe. Since 1995 there is a validation center of the Federal Authorities ICCVAM in the USA and since 2001 the Center for Alternative Methods Vitryna in Poland.

Enlarged Version

7th Amendment of the EU
Cosmetics Directive Adopted in 2003

The 7th amendment directive 2003/15/EG (EU 2003) concerning the EU Cosmetics Directive of 1976 (EU 1976) is to lead to a ban of marketing of cosmetics in single steps, the ingredients of which have been tested in animal experiments. This gradual procedure is to entail a replacement of the majority of safety-toxicological animal tests usual so far by animal-experiment-free "Alternative Methods" within 10 years, at which the cells and tissue cultures as well as the molecular biological and molecular genetical methods are employed. In 7th amendment directive, the EU Commission has stipulated a very precise way of proceedings.

1. Already in 2003 the following animal-experiment-free methods have to be applied:

For the test of all ready-to-use products and test of ingredients on penetration through the skin as to caustic and phototoxic properties.

2.  Until end of 2006, the following animal tests will be given up:

Tests on eye-irritating, skin-irritating and skin-sensitizing properties.

3. Until end of 2012, i. e. in 10 years, the following animal tests will be renounced.

Tests on embryo-toxic properties.

4.  In the year 2013 the following complex animal experiments will still be required according to the assessment of the EU commission for the testing of cosmetics:

Tests on fertility-inhibiting and carcinogenic properties as well as the determination of the distribution and metabolism of foreign materials in the organism (toxico-kinetics).

In general, irrespective of this scheduling in 7th amendment directive of the EU Cosmetics Decree, all safety-toxicological alternative methods have to be applied in lieu of animal tests if they have been included as official test method for chemical substances in annex V of the EU Dangerous Substance Act (EU Directive 67/548/EEC, EU 1967) after experimental validation by the EU.

Replacement of Safety Toxicological
Animal Experiments by
Animal-test-free Methods

Safety-toxicological tests are mandatory for all chemical substances, in fact due to reasons of industrial safety in order to protect workers who get in contact with them in the course of the manufacturing process. Further tests for the consumer protection are obligatory by considering of the application, e. g. for cosmetics, food additives and drugs. Moreover, since a few years, tests for the environmental compatibility of novel substances have to be carried out. Internationally, it has proven successful in toxicology to determine the dangerous properties of a substance in a first step in internationally standardized animal tests, e. g. in Europe according to the stipulations of the EU (EU Dangerous Substance Act, EU Directive 67/548/EEC) and worldwide according to the test methods of the OECD (Organization for Economic Cooperation and Development) (OECD 1982). The toxicological test methods provided by the EU are published in annex V of the Dangerous Substance Act. They are summarized in Table 1, whereas the methods are particularly stressed by which the local tolerability of the ingredients of cosmetics is tested.

Table 1:
Safety-toxicological Test Methods of EU and OECD
  Acute tests, with nonrecurring application
Acute toxicity, dermal & oral
Eye irritation/ -causticity
Skin irritation/-causticity
Skin sensitization
Skin penetration
II. Long-term tests with multiple applications
  Sub-acute toxicity
  Sub-chronical toxicity
  > chronical toxicity
III. Special and organ-specific toxicology
  Metabolism & toxico-kinetics (ADME)
  Neurotoxicity & immuno-toxicity
  Teratogenicity / embryotoxicity
  Reproduction toxicology
Genotoxicity / mutagenicity
  • = these tests methods are indispensable for the tolerability test of ingredients for cosmetics.

If the safety-toxicological animal experiments listed in Table 1 have to be substituted by animal-experiment-free methods according to 7th amendment of the EU Cosmetics directive 76/768/EEC, the new methods have to be experimentally validated pursuant to the recommendations of the EU and the OECD (OECD 1996) in order to prove that the results which are obtained with the new methods allow an assessment of the dangerous properties of new substances in the same way as the established animal experiments. The general way of proceedings in the experimental validation of toxicological test methods in several laboratories under blind conditions with a sufficiently large number of test substances is schematically illustrated in Figure 2 (OECD 1996). It is obvious that it is relatively time-consuming and very costly till a new method has successfully undergone the long way of validation and is also internationally recognized by all OECD member states, i. e. by all major industrial nations. So far only 4 animal experiment free alternative methods have been included after an approximately ten-year experimental validation into the official test directives, namely tests for the examination of caustic and phototoxic properties as well as penetration through the skin and on sensitizing or allergenic effect on skin.

Figure 2
Experimental validation of toxicological test methods: the essential elements of the procedure for the development and experimental validation of toxicological methods, harmonized by the OECD in 1996, which apply in the same way to animal experiments and animal experiment-free alternative methods (OECD 1996) are described.

Enlarged version

The progress achieved so far concerning the development, validation and official approval of animal-experiment free safety-toxicological test methods are explained in the following:

1. Eye Irritation

The simple claim to substitute the severely stressing Draize-test at the rabbit eye for the examination of eye-irritating properties by means of one or several in-vitro methods has not been fulfilled despite large-scale validation studies. The reason is that

1.   Draize tests are applied for various purposes, namely for tests of substances as to

- harmlessness for the application at the eye as well as
- minor and
- severely eye-irritating properties.

2.  Moreover, by means of the Draize test reactions at various parts of the organ eye (mucous membrane, cornea and ocular lens) are assessed and

3. urthermore, at international consideration, the reactions achieved in the Draize-test are rated according to very differing standards by national authorities.

A fundamentally well-known problem of the Draize-test at the rabbit eye is moreover the limited reproducibility which was already proven in a comprehensive validation study by Weil and Scala in 1971 and the result of which is shown in Figure 3.

Figure 3
Validation of Draize-test at the rabbit eye in toxicological laboratories in the USA (Weil and Scala, 1971). The three substances which are depicted in different colors are tested in 24 laboratories (A-X) in the Draize-test and in doing so, considerably varying classifications reaching from non-irritating to severely irritating, for the same substance, have been achieve.

Draize-Test in-vivo in Comparison: 3 Substances in 24 Laboratories (A-X). According to Weil & Scala: Toxicol. Appl. Pharmacol. 19: 276-360 (1971)

Enlarged version

In Europe, a large number of alternative methods for the replacement of the Draize-Test has been developed and validated. To date the application of the new methods is only admitted for the classification and characterization of substances with severely eye-irritating properties. In Figure 4 the four alternative methods are listed which have been developed and validated in different EU member states. Test results, which have been obtained with the four in-vitro tests, are approved by all EU member states. Less positive is the situation outside of Europe, although the OECD calls with emphasis for the development of alternative methods for the Draize-test at the rabbit eye by its member states. Animal-experiment free in-vitro tests for a differentiation of non- and minor eye-irritating properties which are of importance for the cosmetics industry, have not yet been accepted for official purposes despite intensive experimental validation. The 7th amendment of the EU Cosmetics Directive takes that into consideration.

Figure 4
Alternative methods for tests of substances with severely eye-irritating properties, which have been approved for official tests in the EU. It concerns the HET-CAM test at the incubated hen's egg, the BCOP (bovine cornea opacity and permeability) test, the IRE (isolated rabbit eye) test at the rabbit eye and the ICE (isolated chicken eye) test at the chicken eye. The eyes, respectively, are from slaughterhouse material.

Enlarged version

2. Irritating Effect on the Skin

The results of the Draize-test at the rabbit skin for testing of skin-irritating properties are - according to expert opinions of the DG Gesundheit und Verbraucherschutz (Health and Consumer Protection) - only partly transferable to humans. ECVAM and the OECD are in favor of a new test strategy in which in-vitro methods, which have been proven in toxicological laboratories of the industry, are combined with a subsequent tolerability test at human voluntary testees. Several EU and OECD member states, such as for instance Germany and Austria decline in principle the test at humans instead of animals test due to ethical reasons. An ECVAM validation study with artificial human skin (Model EpiDerm ), performed from 1999 to 2000, has unfortunately not yet yielded the successful result, which had been expected, due to the insufficient reproducibility of the biological damaging parameters. In the year 2003 the ECVAM has started jointly with the US validation center ICCVAM an international validation study with commercial human skin models. It will depend on the outcome of the study, in which all major producers of cosmetics participate whether or not tests of the examination of skin irritating properties can be renounced in the end of 2006, without endangering the consumer protection, as designated in 7th amendment of the EU Cosmetics Directive.

3. Causticity on Skin and Mucous Membranes

In 1998 ECVAM has successfully terminated a validation study with three in-vitro methods for the testing of causticity on the skin, namely with rat skin and artificial human skin (Episkin & EpiDerm ). This concerns biotechnologically produced, commercially marketed human skin models. In the year 2000, these validated methods were entered as official test methods in annex V of the Dangerous Substance Act (EU Directive 67/548/EEC) (EU Commission, 2000a) by the EU commission. Therefore, they have to be applied under EU directive 86/609/EWG for the protection of test animals and in the EU member states animal experiments for this purpose are prohibited. However, particularly encouraging is the fact that in 2002 the expert commission of the OECD accepted a special method to test the causticity with human skin models for the worldwide application (OECD 2002a). The successful development and validation of this in-vitro method with the skin model EpiDerm has been coordinated by the German validation center ZEBET.

4. Skin Sensitization

Sensitizing or allergenic effects are the most frequent unwanted property of chemical substances (10 percent). In the case of substances, which are to be applied in cosmetics, sensitizing properties are not acceptable. Therefore, a test on sensitizing properties in the safety-toxicological test of cosmetics is indispensable. The sensitization test in the skin of guinea pigs according to Magnusson and Kligman, usual so far, is severely stressing because adjuvants for the reaction enhancing are injected in the skin and the immunological reaction of the damaged guinea pig skin is tested. Since 2001 the EU member states, the USA and all OECD member states accept the considerably less stressing LLNA (local lymph node assay), which is performed at the ear-lymph nodes of mice, the growth inhibition of which is tested in in-vitro culture (OECD 2002b). The LLNA is in fact no pure in-vitro test but rather an "ex-vivo test", at which the stress for the test animal is considerably reduced.

5. Penetration Through the Skin

For substances, which are to be applied in cosmetics or pesticides, the absorption through the skin is decisive for the effect in the entire body as to the safety-toxicological assessment. The EU expert commission for cosmetics, the SCCNFP, has fundamentally decided that ingredients of cosmetics do not have to be toxicologically tested on systemic effects if they are not absorbed by the skin. Therefore, the test of the skin penetration is of major importance for the tolerability test of cosmetics. In June 2001, the OECD recommended the test of skin penetration with human skin, which originates from operation material as one of the first worldwide valid in-vitro toxicity test for approval (OECD 2002c).

5. Photo-toxicity

Since 1992, ZEBET has coordinated a EU/COLIPA pre-validation and validation study of in-vitro photo-toxicity tests that was successfully terminated in 1998 with the experimental validation of the 3T3 NRU in-vitro photo-toxicity tests (3T3 NRU PT test) at which a fibroblast cell line of mice is applied. Figure 5 illustrates a typical example for a phototoxic substance in 3T3 NRU in the in vitro photo-toxicity test. The results of the validation study with 30 test substances under blind conditions in 10 laboratories in Europe and the USA as well as an additional validation study with 10 UV-filter substances which are applied in sun protection products, confirmed that the results in the 3T3 test allow a better prediction of the photo-toxic properties for humans than with every other in-vivo or in-vitro test method (Spielmann et al., 1998 a,b). Therefore, in 2000 the EU commission included the 3T3 NRU PT test as single photo-toxicity test in annex V of the Dangerous Substance Act (EU Commission, 2001c) so that only this in-vitro test can be applied for official purposes in Europe and moreover that animal experiments for this purpose are prohibited. Further, in the year 2002, an expert commission of the OECD has accepted the 3T3 NRU PT as official worldwide test method for the photo-toxicity test (OECD, 2002d). Thus, the 3T3 NRU PT test is the first in-vitro-toxicity test, which is accepted worldwide after successful experimental validation for official purposes.

Figure 5
Example for a phototoxic substance (Ketoprofene), which entails a positive result in the 3T3 NRU PT in-vitro photo-toxicity test. The test is performed in a micro-titer plate with 96 reaction receptacles. UV radiation leads to a phototoxic reaction with the phototoxic substance in the lower plate that entails relation-dependent to decoloring.

Enlarged version

Animal Protection and
Development of Novel Cosmetics are Compatible

The scientific advisory board of the EU validation center ECVAM assessed in 2001, in connection with the proposal of the EU Commission for a new chemicals policy that in approximately five years there will be animal experiment-free-alternative methods for toxicological test methods available. The assessment summarized in Figure 6 clarifies that for the most important fields in toxicology there will be animal-experiment-free methods available (EU Commission 2001) very soon.

Figure 6
Special fields of toxicology in which, according to the opinion of the scientific advisory board of the EU validation center ECVAM, validated animal-experiment-free toxicological test methods will be available in the coming 5 years.

Enlarged version

The results presented explain that the EU legislation in the sector cosmetics for which a particularly high priority is given to the EU Directive 86/609/EWG for the protection of test animals does not lead to a standstill of research and new development, as is often maintained, but the cosmetics industry in Europe also takes the leading position scientifically in the field of toxicological safety tests in comparison with Japan and the USA. The EU Animal Protection Act has therefore lead to the market leadership in the world market in the cosmetics sector. The 7th amendment of the Cosmetics Directive of the EU is an important milestone for the strengthening of the position of the European cosmetics industry in the world market. It is, furthermore, conceived following the reality and takes primarily the consumer requirements into consideration without neglecting the scientific progress and animal protection. The example of the cosmetics industry explains that innovative economical developments connected with a leading position in the world market and topical animal protection is not mutually exclusive but complements one another.

Figure 7
Test animals do not have to suffer any longer today from safety toxicological tests of cosmetics.

Enlarged version


EU Kommission (1976) EU Richtlinie 76/768/EEC zur Angleichung der gesetzlichen Grundlagen für kosmetische Produkte in den Mitgliedsstaaten. Brüssel, Belgien: EU DG Umwelt.

EU Kommission (1986) EU Richtlinie 86/609/EEC zu Schutz von Versuchstieren. Brüssel, Belgien: EU DG Umwelt.

EU Kommission (1983) EU Richtlinie 83/467EEC zur fünften technischen Anpassung der EU Richtlinie 67/548/EEC an die Gesetzgebung für die Klassifizierung, Verpackung und Kennzeichnung von Gefahrstoffen. Brüssel, Belgien: EU DG Umwelt.

EU Kommission (2000a) EU Richtlinie 2000/33/EU für die 21st Änderung von Anhang V der EU Richtinie 67/548/EEC zur Klassifizierung, Verpackung und Kennzeichnung von Gefahrstoffen: Test Methode B-40 "Hautätzung - in vitro Methode”. O. J. der EU Kommission vom 8. Juni 2000, L136, 85-97.

EU Kommission (2000b) EU Richtlinie 2000/33/EU für die 21st Änderung von Anhang V der EU Richtinie 67/548/EEC zur Klassifizierung, Verpackung und Kennzeichnung von Gefahrstoffen: Test Methode B-41 "Phototoxizität – in vitro 3T3 NRU Phototoxizitätstest ”. O. J. der EU Kommission vom 8. Juni 2000, L136, 85-97.

EU Kommission (2001). Weissbuch für eine neue Chemikalienpolitik. Brüssel, Belgien im Internet:

EU Kommission (2003) EU Richtlinie 2003/15/EG zur Änderung der Richtlinie 76/768/EWG des rates zur Angleichung des rechtsvorschriften der Mitgliedsstaaten über kosmetische Mittel. O. J. der EU Kommission vom 11. März 2003, L66, 26-35

OECD (Organisation for Economic Co-operation and Development) (1982) OECD Guidelines for Testing of Chemicals. Paris, Frankreich: OECD Publikation Office.

OECD (1996) Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods. Paris, Frankreich: OECD Publication Office.

OECD (2002a) OECD guidelines for the testing of chemicals: Test Guideline 431 "In vitro skin corrosion: human skin model ". Paris, Frankreich, OECD Publication Office.

OECD (2002b) OECD guidelines for the testing of chemicals: Test Guideline 429 "Skin sensitisation: Local Lymph Node Assay. Paris, Frankreich, OECD Publication Office

OECD (2002c) OECD guidelines for the testing of chemicals: Test Guideline 428 "Skin absorption: in vitro method ". Paris, Frankreich, OECD Publication Office.

OECD (2002d) OECD guidelines for the testing of chemicals: Test Guideline 432 "In vitro 3T3 NRU phototoxicxity test". Paris, Frankreich, OECD Publication Office.

Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch G., de Silva, O., Holzhütter, H.G., Clothier, R., Desolle, P., Gerberick, G.F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J.M., Csato, M., Sladowski, D., Steiling, W. und Brantom, P.:  The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU Phototoxicity test. Toxic. in Vitro 12, 305-327, 1998a

Spielmann, H., Balls, M. Dupuis, J., Pape, W.J.W., de Silva, O., Holzhütter, H.G., Gerberick, F., Liebsch, M., Lovell, W.W., Pfannenbecker, U. und Brantom, P.: A special study on the phototoxic potential of UV filter chemicals from Annex II of the REU Directive 76/86 in the 3T3 NRU in vitro phototoxicity test. ATLA 26, 979-708, 1998b

Weil, C.S. und Scala, R.A.: Study of intra- and interlaboratory variability in the results of rabbit eye and skin irrtiation tests. Toxicol. Appl. Pharmacol. 19, 276-360; 1971


Professor Dr. med. Horst Spielmann

Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET) (Central Association for the Registration and Assessment of Substitution- and Supplementary Methods for Animal Experiments) at the Bundesinstitut für Risikobewertung (BfR) (Federal Institute for Risk Assessment) in Berlin


Dr. med. Horst Spielmann
Direktor und Professor
Leiter ZEBET im BfR
Diedersdorfer Weg 1
12277 BERLIN

Tel: 01888-412-2270
Fax: 01888-412-2958


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