Alternatives to the Use of Animals in Safety Testing as Required by the EU-Cosmetics Directive 2009

every new toxicological test method must pass through a defined procedure of validation and regulatory acceptance. This worldwide agreed procedure is modular, which allows a flexible application of only those modules that are necessary for a final implementation of a certain new method. As noted in Table 1, a formal validation process aims predominantly at defining the relevance and reliability of a new testing method. This process is usually completed by a scientific review and a statement of the ECVAM Scientific Advisory Committee (ESAC). Without a positive eSAC statement, no test may be designated as “validated”. However, a test method labelled as validated is not automatically applicable for regulatory purposes. Acceptance for regulatory use can be achieved only by standardisation and implementation into a test guideline, e.g. at the OECD level. Although the process of development, validation and implementation of alternative methods is extremely time consuming, the work of several institutions worldwide has been very successful, and the German ZeBet has played an important role in this process over the last 20 years. A list of alternatives to the use of animals in safety testing, which covers the requirements of the 7th amendment of the EU Cosmetics Directive, which entered into force on March 11, 2009, is presented.


Richard Vogel
Bundesinstitut für Risikobewertung (BfR), ZeBet, Berlin, Germany 1 Establishing alternative methods every new toxicological test method must pass through a defined procedure of validation and regulatory acceptance.This worldwide agreed procedure is modular, which allows a flexible application of only those modules that are necessary for a final implementation of a certain new method.As noted in Table 1, a formal validation process aims predominantly at defining the relevance and reliability of a new testing method.This process is usually completed by a scientific review and a statement of the ECVAM Scientific Advisory Committee (ESAC).Without a positive eSAC statement, no test may be designated as "validated".However, a test method labelled as validated is not automatically applicable for regulatory purposes.Acceptance for regulatory use can be achieved only by standardisation and implementation into a test guideline, e.g. at the OECD level.Although the process of development, validation and implementation of alternative methods is extremely time consuming, the work of several institutions worldwide has been very successful, and the German ZeBet has played an important role in this process over the last 20 years.A list of alternatives to the use of animals in safety testing, which covers the requirements of the 7 th amendment of the EU Cosmetics Directive, which entered into force on March 11, 2009, is presented.

+ In Vitro test for Percutaneous Absorption
OECD Test Guideline 428, accepted on April 13, 2004 No animals are necessary to test the uptake of chemical substances via skin.

Acute oral toxicity
• Fixed Dose Procedure (FDP)   OECD Test Guideline 420, accepted on December 17, 2001 • Acute toxic Class Method (AtC)   Invited paper, reviewed for publication 16th April 2009.The author is a senior scientist at the Bundesinstitut für Risikobewertung (BfR) in Berlin, Germany.The conclusions in the paper are not necessarily in line with the official BfR position.

Tab. 1: Steps towards a new test method according to a harmonised OECD validation concept (1996)
Period (approx.)

Regulatory implementation
• Establishment of a directive (e.g.OECD) 10 years 4. Validation ESAC statement

Eye irritation
All existing alternative approaches are far from being validated or even standardised.Thus a test guideline is not expected soon.However, at least strong eye irritancy effects can likely be detected by ICE or BCOP.This will be a matter of discussion in the eye corrosion expert group.• In Vitro Micronucleus test OECD Test Guideline 487, acceptance expected 2009 Several in vitro methods to detect different kinds of mutagenic effects, like gene mutations, chromosome mutations or genome mutations, have been accepted as guideline tests during the last decades.Unfortunately, only clear negative results are acceptable, excluding a mutagenic potential with sufficient certainty.However, the high rate of false positive results necessarily leads to follow up in vivo tests.Therefore, the alternatives in the field of mutagenicity testing are as yet only able to reduce the animal numbers but not replace in vivo tests completely.

Phototoxicity
OECD Test Guideline 423, accepted on December 17, 2001 • Up-and-Down Procedure (UDP) OECD Test Guideline 425, accepted on October 03, 2008 -Deletion of the Acute Oral Toxicity Test, Lethal Dose (LD 50 ) OECD Test Guideline 401, deleted in 2001 Three alternatives to the classical LD 50 test reduce the animal numbers markedly, but still require animals.On the other hand the LD 50 test was replaced completely and could be deleted from the OECD guidelines for testing of chemicals.be possible by introducing the so-called Halle-Register.2. Since a battery of in vitro methods has been available for many years, the creation of a new testing strategy combining these could make mutagenicity testing possible without animals.This is also the task of a working group of the German section of the european environment Mutagen Society (EEMS).

Skin corrosion
It is expected that both endpoints can be ascertained without using animals in the near future!

Conclusion
Alternative methods to animal experiments are described in Annex IX of the EU Cosmetics Directive.This Annex should be updated as follows: Annex IX This Annex lists the alternative methods accepted worldwide as OECD guidelines, which are, therefore, available to meet the requirements of this Directive.As animal testing may not be replaced completely by an alternative method, it should be mentioned in Annex IX whether the alternative method fully or partially replaces animal testing (Tab.2).
Such an Annex would keep the producers of cosmetic ingredients informed on the current status of alternative methods, especially which already meet the requirements of the Directive and which do not.Consequently, short term activities of institutions like ZeBet should focus on the following endpoints: 1.Without prejudice to the general obligations deriving from Article 2, Member States shall prohibit: (a) the marketing of cosmetic products where the final formulation, in order to meet the requirements of this Directive, has been the subject of animal testing using a method other than an alternative method after such alternative method has been validated and adopted at Community level with due regard to the development of validation within the OECD; (b) the marketing of cosmetic products containing ingredients or combinations of ingredients which, in order to meet the requirements of this Directive, have been the subject of animal testing using a method other than an alternative method after such alternative method has been validated and adopted at Community level with due regard to the development of validation within the OECD; (c) the performance on their territory of animal testing of finished cosmetic products in order to meet the requirements of this Directive; (d) the performance on their territory of animal testing of ingredients or combinations of ingredients in order to meet the requirements of this Directive, no later than the date on which such tests are required to be replaced by one or more validat-A derogation shall only be granted if: (a) the ingredient is in wide use and cannot be replaced by another ingredient able to perform a similar function; (b) the specific human health problem is substantiated and the need to conduct animal tests is justified and is supported by a detailed research Protocol proposed as the basis for the evaluation.
the decision on the authorisation, the conditions associated with it and the final result achieved shall be part of the annual report to be presented by the Commission in accordance with Article 9.

Article 9
every year the Commission shall present a report to the european Parliament and the Council on: (a) progress made in the development, validation and legal acceptance of alternative methods.The report shall contain precise data on the number and type of experiments relating to cosmetic products carried out on animals.The Member States shall be obliged to collect that information in addition to collecting statistics as laid down by Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes.The Commission shall in particular ensure the development, validation and legal acceptance of alternative test methods which do not use live animals; (b) progress made by the Commission in its efforts to obtain acceptance by the OECD of alternative methods validated at Community level and recognition by non-member countries of the results of the safety tests carried out in the Community using alternative methods, in particular within the framework of cooperation agreements between the Community and these countries; (c) the manner in which the specific needs of small and mediumsized enterprises have been taken into account.and toxicokinetics, for which there are no alternatives yet under consideration.Information about the provisional and final results of these studies should form part of the yearly reports presented pursuant to Article 9.
On the basis of these annual reports, the timetables established in accordance with paragraph 2 may be adapted within a maximum time limit of six years as referred to in paragraph 2 or 10 years as referred to in paragraph 2.1 and after consultation of the entities referred to in paragraph 2.
2.3.The Commission shall study progress and compliance with the deadlines as well as possible technical difficulties in complying with the ban.Information about the provisional and final results of the Commission studies should form part of the yearly reports presented pursuant to Article 9.If these studies conclude, at the latest two years prior to the end of the maximum period referred to in paragraph 2.1, that for technical reasons one or more tests referred to in paragraph 2.1 will not be developed and validated before the expiry of the period referred to in paragraph 2.1 it shall inform the European Parliament and the Council and shall put forward a legislative proposal in accordance with Article 251 of the Treaty.
2.4.In exceptional circumstances where serious concerns arise as regards the safety of an existing cosmetic ingredient a Member Statemay request the Commission to grant a derogation from paragraph 1.The request shall contain an evaluation of the situation and indicate the measures necessary.On this basis, the Commission may, after consultation of the SCCNFP and by means of a reasoned decision, authorise the derogation in accordance with the procedure referred to in Article 10(2).This authorisation shall lay down the conditions associated with this derogation in terms of specific objectives, duration and reporting of the results.

ANNEX IX List of validated alternative methods to animal testing
this Annex lists the alternative methods validated by the european Centre on Validation of Alternative Methods (eCVAM) of the Joint Research Centre available to meet the requirements of this Directive and which are not listed in Annex V to Council Directive 67/548/EEC on the approximation of laws, regula-tions and administrative provisions relating to the classification, packaging and labelling of dangerous substances.As animal testing may not be replaced completely by an alternative method, it should be mentioned in Annex Ix whether the alternative method fully or partially replaces animal testing.
1. Testing of acute toxicity: a further reduction of the animal numbers or even a replacement of the animal tests appears to ed alternative methods listed in Annex V to Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances or in Annex IX to this Directive.No later than 11 September 2004 the Commission shall, in accordance with the procedure referred to in Article 10(2) and after consultation of the Scientific Committee on Cosmetic Products and Non-Food Products intended for consumers (SCCNFP) establish the contents of Annex IX. 2. The Commission, after consultation of the SCCNFP and of the european Centre for the Validation of Alternative Methods (eCVAM) and with due regard to the development of validation within the OECD, shall establish timetables for the implementation of the provisions under paragraph 1(a), (b) and (d), including deadlines for the phasing-out of the various tests.The timetables shall be made available to the public not later than 11 September 2004 and be sent to the European Parliament and the Council.The period for implementation shall be limited to a maximum of six years after the entry into force of Directive 2003/15/EC in relation to paragraph 1(a), (b) and (d).2.1.In relation to the tests concerning repeated-dose toxicity, reproductive toxicity and toxicokinetics, for which there are no alternatives yet under consideration, the period for implementation of paragraph 1(a) and (b) shall be limited to a maximum of 10 years after the entry into force of Directive 2003/15/EC.2.2.The Commission shall study possible technical difficulties in complying with the ban in relation to tests, in particular those concerning repeated-dose toxicity, reproductive toxicity Annex For additional information, parts of the articles 4a and 9 as well as the Annex IX of the EU Cosmetics Directive are cited in their present form: 1976L0768-EN-24.04.2008 -021.002-

•
Improvement of the test protocol epiSkin tM , Human Skin Model OECD Test Guideline 431, accepted on April 13, 2004 + EpiDerm tM , Human Skin Model OECD Test Guideline 431, accepted on April 13, 2004

•
Rat transcutaneous electrical Resistance (teR)OECD Test Guideline 430, accepted on April 13, 2004 While the TER-test only reduces the number of animals, artificial models of human skin, EpiDerm and EpiSkin, replace animal tests on skin corrosion.EpiDerm tM , Human Skin Model OECD Test Guideline, acceptance expected in 2009 EpiDerm and EpiSkin are most promising to replace animal testing on skin irritation as well.Both tests are already part of the eU collection of test guidelines and will be standardised by the OECD this year. Tab.

2: Alternatives to animal testing of different toxicolocal endpoints Toxicological endpoint Alternatives to replace animal tests Alternatives to reduce animal numbers Dermal absorption OECD 428
bold = OECD-replacement methods available or expected soon standard = OECD-reduction methods available, replacement methods or strategies in preparation italic = no validated or standardised alternative methods available