I *** COMMISSION OF THE EUROPEAN COMMUNITIES 
Brussels, 24.07.1996 
COM(96) 365 final 
REPORT FROM THE COMMISSION 
DEVELOPMENT, VALIDATION AND LEGAL ACCEPTANCE OF 
ALTERNATIVE METHODS TO ANIMAL EXPERIMENTS IN THE FIELD OF 
COSMETIC PRODUCTS 
1995 

DEVELOPMENT, VALIDATION AND LEGAL ACCEPTANCE OF 
ALTERNATIVE METHODS TO ANIMAL EXPERIMENTS IN THE FIELD OF 
COSMETIC PRODUCTS 
1995 
SUMMARY 
I INTRODUCTION 
The Commission's obligations 
Article 4(ij of Directive 76/768/EEC, as amended by Directive 93/35/EEC, specifies that 
"Member States shall prohibit the marketing of cosmetic products containing ... 
ingredients or combinations of ingredients tested on animals after 1 January 1998...". 
The ambiguity of the expression "combinations of ingredients" has been resolved as far 
as the Commission is concerned, which considers that the ban also covers finished 
cosmetic products. 
The implementation of this ban is linked to the development of "satisfactory methods to 
replace animal testing ... scientifically validated as offering an equivalent level of 
protection for the consumer". 
With a view to providing regular information on how the situation is developing it is 
specified that "ftjhe Commission shall present an annual report to the European 
Parliament and the Council on progress in the development, validation and legal 
acceptance of alternative methods to those involving experiments on animals. That report 
shall contain precise data on the number and type of experiments related to cosmetic 
products carried out on animals". 
1994 Report 
A first report was presented in 1994. It described interesting but limited results, 
concluding that "animal models cannot be replaced, though they can contribute to 
reducing the number of animals used...". However, the outlook was reasonably optimistic 
as regards the validation of alternative methods for eye irritation, percutaneous absorption, 
phototoxicity/photoirritancy and basic mutagenicity tests. It was also underlined that given 
the current stage of the art it was unlikely that animal tests could be totally replaced in 
toxicity studies for evaluating the systemic risk, i.e. action via the circulatory system on 
the organism in its entirety after percutaneous absorption. 
H 1995 REPORT 
The objectives of the report and the notion of the potential risk for human health are the 
same as in the 1994 report. 
The players involved in implementing this instrument are: 
the European Commission: DG XI, DG-JRC, DG XXIV, DG XII 
the European industry, represented by COLIPA (the European Cosmetic, Toiletry 
and Perfume Association) 
- the American administration and the American Cosmetology Federation, CFTA 
- the Japanese administration and the Japanese Cosmetology Federation, JCIA 
the OECD. 
1 Clarification of the validation stages 
Validation is the procedure via which the reliability and relevance of a procedure are 
established to a specific end. This process turned out to be more complex than envisaged 
and will take more time than foreseen. It has been necessary to define more precisely the 
validation stages of the new tests (development of a test and production of a protocol; 
prevalidation; validation proper; objective and independent evaluation of the study, 
progress towards legal acceptance). 
2 The initiatives 
The various players have taken numerous initiatives with a view to achieving the 
prescribed objectives. 
PGXI 
DG XI manages Directive 86/609/EEC on the protection of animals used for 
experimental and other scientific purposes and, to this end, is responsible for 
collecting statistics on the animals used. It also manages Directive 67/548/EEC 
on the classification and labelling of dangerous chemical substances, whose 
annexes define the test methods recognised by the EU. 
DG-JRC: 
ECVAM (European Centre for the Validation of Alternative Methods) has 
already organised 14 seminars on the development and validation of 
alternative methods. Seven, of these seminars concerned the safety of 
cosmetic products and the reports have been published. Two other reports 
on the prevalidation and validation procedures are also available. 
• ECB (European Chemicals Bureau) provides the technical and scientific 
support needed by DG XI. 
DG XXTV/CSC (Scientific Committee on Cosmetology): 
• DG XXIV plays a motor role in implementation, being responsible for the 
Cosmetic Products Directive, and must prepare for the Commission the 
draft measures postponing the deadline for the ban on animal tests, should 
this be necessary. 
• The CSC and its subcommittee more specifically responsible for 
alternative methods have evaluated various documents and dossiers 
presented by COLIPA. They have also adopted a document titled "The 
information required for the scientific evaluation of validation studies of 
11 
alternative tests with a view to their utilisation in evaluating the safety of 
cosmetic products" 
D3XII 
DG XII has funded various research programmes on the development and 
validation of alternative methods. 
COLIPA/SCAAT (Steering Committee on Alternatives to Animal Testing) 
Besides preparing dossiers and participating in numerous studies COLIPA 
organised an international scientific symposium on alternatives to animal tests in 
Brussels on 29 and 30 November 1995. 
r 
USA 
• The American government is actively seeking international cooperation 
with a view to issuing recommendations. 
• The FDA (Food and Drug Administration), which is the agency 
responsible for the safety of cosmetic products, has expressed its 
misgivings as regards the ban on animal experiments in the 
implementation of Directive 93/35/EEC. 
OECD 
The OECD publishes guidelines on toxicity tests, approved by the Member States. 
A guideline on the in vitro control of percutaneous absorption is currently under 
discussion. 
3 Statistics on animal experiments 
The compilation of data specifically relating to cosmetic ingredients and products is 
difficult because the collection of data on the number of animals used for experimental 
and other scientific purposes in application of Directive 86/609/EEC is not foreseen on 
an annual basis. 
Eight Member States have declared that animal tests for finished cosmetic 
products have not been conducted on their territory (Italy, Greece, Belgium, 
Ireland, Sweden, Finland, Luxembourg, Germany). 
- Six Member States have declared that animal tests for cosmetic ingredients have 
not been conducted on their territory (Greece, Netherlands, Ireland, Sweden, 
Finland, Luxembourg). 
- Three Member States (Austria, France, United Kingdom) have communicated 
figures while specifying in some cases that these figures are unreliable and not 
interprétable. 
It should also be noted that certain Member States that do not produce ingredients may 
use ingredients tested in other Member States or in third countries and that certain 
ingredients may have been tested for other purposes. 
cc/ag 
Conclusions and perspectives relating to the development of alternative methods 
for the various toxicity tests used in the evaluation of the safety of cosmetic 
ingredients and cosmetic products. 
4-1 Ingredients 
Phototoxicitv/photoirritancv (skin reaction after epicutaneous application of a 
chemical substance in the presence of UV radiation). This concerns in particular 
UV filters. 
The results of Phase II of the EU/COLIPA international validation study on in 
vitro photoirritancy tests have already allowed considerable progress to be made. 
A Phase HI is envisaged. A supplementary study on UV filters will be necessary. 
Validation should be possible in the near future. 
Percutaneous absorption 
A new guideline on in vitro tests for percutaneous absorption could be approved 
by the Member States and the OECD in the near future. 
Skin irritation 
It is proposed to evaluate skin irritation of cosmetic ingredients on-human 
volunteers. This could be done provided prior studies concerning the toxic risk 
furnish adequate guarantees. 
Skin sensitisation 
In vitro evaluation of skin sensitisation requires more in-depth research into the 
mechanistic basis of sensitisation before a validation study can be considered. 
Eve irritation 
The results of the validation studies of alternative methods to the Draize eye 
irritation tests have been disappointing (EU/Home Office study and COLIPA) and 
none of the tests used met the studies' objective. A more flexible approach to 
experimentation could be encouraged by accepting a combination of scientifically-
validated methods and evaluating the toxicity of the substances under 
consideration by comparison with appropriate benchmark substances. 
Photomutagenicitv (all modifications of the information content of the genetic 
material arising in the presence of UV radiation) 
Recommendations have been proposed on the criteria to be adopted with a view 
to defining the in vitro test protocols. 
Skin corrosivity 
Corrosive ingredients are not used in cosmetic products. However, the ECVAM 
validation study of in vitro tests on skin corrosivity could be used at the screening 
phase and provide essential information, before considering studies on human 
volunteers. 
IV 
Mutagenicity 
In vitro tests to evaluate the mutagenic potential of cosmetic ingredients are in 
current use. In vivo tests are carried out only when the in vitro tests results are 
unsatisfactory. 
It will probably be impossible to totally replace in vivo tests to evaluate the 
systemic risk, such as tests of acute toxicity, subchronic toxicity, carcinogenicity, 
toxicity of reproduction and development, in the foreseeable future. However by 
refining the methods used it has already been possible to reduce substantially the 
number of animals used. 
4-2 Finished cosmetic products 
Eye tolerance and skin tolerance of finished cosmetic products can normally be 
evaluated in vitro provided the data relating to the ingredients' toxicity and their 
physico-chemical properties are known and provide the necessary guarantees. 
Skin compatibility (absence of skin irritation) of finished cosmetic products can 
be evaluated in man provided this is done in the context of strictly controlled 
clinical studies. 
It is important to emphasise that the use of human volunteers as a way to replace 
animal tests must be considered with the greatest prudence. Such studies, whose 
ethical dimension is obvious, should only be authorised after in vitro and in vivo 
tests have demonstrated mat there is no risk of serious consequences. 
In summary: - The validation of alternative methods for evaluating 
percutaneous absorption and photoirritancy in the case of 
ingredients, and for evaluating eye tolerance and skin 
tolerance in the case of finished products, can be envisaged 
in the near future. 
Progress in alternative methods for evaluating eye irritation, 
skin irritation and skin sensitisation in relation to 
ingredients requires that additional studies first be carried 
out; 
The development of in vitro methods in the domains 
involving a systemic risk is not likely in the foreseeable 
future, even if it may be possible to reduce the number of 
animals used. 
GLOSSARY 
CAPT: Committee of Adaptation to Technical Progress 
CAAT: Centre for Alternatives to Animal Testing (John Hopkins University. USA) 
CFTA: Cosmetic, Toiletry and Fragrance Association (USA) 
COLIPA: European Cosmetic, Toiletry and Perfumery Association 
DEREK: Deductive Estimation of Risk from Existing Knowledge 
ECB: European Chemicals Bureau 
ECVAM: European Centre for the Validation of Alternative Methods 
ERGATT: European Research Group for Alternatives in Toxicity Testing 
ES AC: ECVAM Scientific Advisory Committee 
EU: European Union 
EU/HO international validation study: 
European Union/Home Office international validation study 
FDA: Food and Drug Administration (USA) 
FDCA: Food, Drug and Cosmetics Act (USA) 
FLT: Fluorescein Leakage Test * 
FRAME: Fund for the Replacement of Animals in Medical Experiments 
GLP: Good Laboratory Practice 
ICC V AM: US Inter-Agency Coordinating Committee for the Validation of Alternative 
Methods 
IF AW: International Fund for Animal Welfare 
IRAG: Interagency Regulatory Alternatives Group 
IVTIP: In Vitro Testing Industrial Platform 
JCIA: Japanese Cosmetics Industry Association 
JRC: Joint Research Centre 
MMAS: Modified Maximum Average Score 
MHW: Ministry for Health and Welfare (Japan) 
NIEHS: National Institute of Environmental Health Sciences (USA) 
NRU: Neutral Red Uptake Assay 
NTP: National Toxicology Program (USA) 
PM: Prediction Model 
OECD: Organisation for Economic Cooperation and Development 
QSAR: Quantitative Structure-Activity Relationships 
RBC: Red Blood Cell Haemolysis Test 
SCAAT: Steering Committee on Alternatives to Animal Testing 
SCC: Scientific Committee on Cosmetology 
SLAT: Scweizerisches Institut fur Alternativen zu Tierversuchen 
TEA: Tissue Equivalent Assay 
UV: Ultra Violet 
ZEBET: Zentralstelle zur Erfassung und Bewertung von Ersatz und 
Erganzungsmethoden zum Tierversuch im Bundesgesundheitsamt 
vi 
CONTENTS 
A. 
B. 
D. 
E 
F. 
G. Introduction 
Clarification of the stages in validation 
- Validation of alternative methods used for different purposes 
- Stages in the evaluation of new tests 
Initiatives in 1995 
The Players 
The initiatives 
1. European Union 
DGXI 
DG-JRC (ECVAM-ECB) 
DG XXIV/SCC 
DG XII 
COLIPA/SCAAT 
2. 
3. 
4 USA 
Japan 
OECD 
The state of play 
Statistics on animal experiments 
Conclusions 
The outlook PAGES 
2 
2-5 
3 
3 
5-13 
5 
6 
6 
6 
8 
10 
11 
12 
12 
13 
13-17 
17-18 
18-19 
19-20 
A. INTRODUCTION 
This is the second of the reports which the Commission must present annually to the 
European Parliament and Council on progress in the development, validation and legal 
acceptance of methods which could replace animal experiments for cosmetics testing. 
'The Commission shall present an annual report to the European Parliament and 
the Council on progress in the development, validation and legal acceptance of 
alternative methods to those involving experiments on animals. That 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 Directive 
86/609/EEC on the protection of animals used for experimental and other scientific 
purposes. The Commission shall in particular ensure the development, validation 
and legal acceptance of experimental methods which do not use live animals. ' 
(Article 4(i) of Directive 76/768/EEC, as modiûeé by Directive 93/35/EEC). 
With the exception of the objectives, players and notion of potential risk for human health, 
which remain unchanged, the points developed in the first annual report (COM(94) 606) 
are reviewed: 
Clarification of the stages in validation (B) 
Initiatives in 1995 (C) 
The state of play (D) 
Statistics on animal experiments (E) 
Conclusions (F) 
The outlook (G) 
B. CLARIFICATION OF THE STAGES IN 
VALIDATION 
The Commission's main objective remains that of encouraging the development, 
validation and legal adoption of alternative methods which can offer the consumer a 
level of protection at least equivalent to that achieved by using animal studies. 
Validation is the process by which the reliability and relevance of a procedure are 
established for a specific purpose. 
The stages involved in achieving this objective are more numerous, and the process a 
more complex one, than initially foreseen. 
The first Amden workshop (held in Switzerland in 1990) laid down the theoretical basis 
which is essential for the validation process, viz. intra-laboratory validation, inter-
laboratory evaluation, the development of databases, and evaluation of the results. 
Experience with validation studies conducted since 1990 has shown that it is necessary to 
better define the various objectives of the validation studies and to integrate new stages 
into the process of evaluating new tests. 
ECVAM (European Centre for the Validation of Alternative Methods), a unit of the 
Environment Institute of the Joint Research Centre (ISPRA), is at the hub of the 
discussions on validation, and major contributions have been made in the following fields: 
* practical aspects of validation 
* prevalidation scheme 
* international discussions with ICCVAM (US Inter-Agency Coordinating Committee 
for the Validation of Alternative Methods) and the OECD (Organisation for 
Economic Cooperation and Development) on the harmonisation of validation and 
acceptance criteria. 
Recommendations concerning the practical and logistical aspects of validating alternative 
tests were made at the second Amden Workshop (24-28 January 1994), organised jointly 
by ECVAM and ERGATT (the European Research Group for Alternatives in Toxicity 
Testing), and are set out in ECVAM workshop report No 5. 
Validation of alternative methods used for diffèrent purposes 
Validation of alternative tests can be conducted with four main types of objectives in 
mind. A distinction is made between: 
1. Validation of alternative procedures for the use in non-regulatory studies. 
2. Validation of alternative tests for inclusion as part of regulatory guidelines. 
3. Validation of alternative tests to replace existing guidelines. 
4. Validation of alternative tests which are designed to provide part of the information 
required by a regulatory guideline. 
Stages in the evaluation of new tests 
1. Development of a test and production of a protocol 
The criteria taken into consideration in developing a test mainly comprise a 
description of the basis of the method, the definition of its scientific objective, the 
specification of its biological endpoint, and the expression and interpretation of the 
results, via a prediction model (PM). 
The prediction model must enable the result obtained with an alternative method 
to be converted into a correct prediction of in vivo toxicity. 
The prediction model is critical for the success of a validation programme. It 
enables a clearly defined hypothesis to be tested and an objective evaluation of test 
performance, and it serves as a guide for planning the validation study. 
2. Prevalidation 
Experience has shown that the outcome of large and expensive validation studies 
can be compromised if their managers do not insist that optimised test protocols 
and proof of their performance are submitted before the start of the formal 
validation study. The objectives of prevalidation, and a scheme of the prevalidation 
process, are described in the first report of the ECVAM Prevalidation Task Force. 
The aims of prevalidation are: 
to optimise and standardise the protocol 
to evaluate the method's transferability. 
The prevalidation exercise comprises three phases: 
refinement of the protocol, involving collaboration between the laboratory 
which has developed the method and the laboratory designated to optimise 
the method 
transfer of the protocol, involving collaboration between the first two 
laboratories and the laboratory designated for the transfer 
a blind study, involving, as a minimum, participation of the three 
laboratories responsible for studying the protocol's performance. 
3. Validation 
The validation stage comprises a formal inter-laboratory study. 
The main stages are: 
study design 
selection of the tests 
selection of the laboratories 
selection and distribution of the test materials 
collection and analysis of the results 
evaluation of the study. 
4. Independent assessment of the conduct and outcome of the study. 
This should include an objective evaluation of the value of the scientifically 
validated tests by comparison with other tests, taking into account the validation 
objectives. 
5. Progression towards legal acceptance 
It is essential that any new method that is considered to be adequately validated as 
a replacement for an existing method receives as widespread international 
recognition as possible. For example, the OECD test guidelines are particularly 
important in this respect, since they are used for tests conducted in member 
countries in Europe and North America, and in Japan, Australia and New Zealand. 
Furthermore, under the OECD Mutual Acceptance of Data Agreement, member 
countries have agreed to accept data from tests performed according to OECD test 
guidelines, provided that the principles of Good Laboratory Practice (GLP) are 
observed. The OECD has established a procedure for updating test guidelines and 
for the introduction of new test methods. 
C. INITIATIVES IN 1995 
THE PLAYERS 
For the record, the players involved in encouraging the research, development and 
validation of alternative methods are: 
1. EUROPEAN UNION 
The European Commission 
Several Commission services are involved in issues relating to animal 
experimentation and the development and evaluation of alternative methods. 
DG XI, Environment, Nuclear Safety and Civil Protection manages and is 
responsible for Directive 86/609/EEC, on the protection of animals used for 
experimental or other scientific purposes, and for Directive 67/548/EEC, on the 
classification, packaging and labelling of dangerous substances. 
DG- JRC, Joint Research Centre 
ECVAM, European Centre for the Validation of Alternative Methods 
ESAQ ECVAM Scientific Advisory Committee 
ECB, European Chemicals Bureau 
DG XXIV: Consumer Policy 
Unit A3 - Products, manages Directive 76/768/EEC (the Cosmetics 
Directive) 
SCO. the Scientific Committee on Cosmetology, a Commission advisory 
committee 
DG XII, Science, Research and Development funds research programmes: 
European industry 
COLIPA - Comité de Liaison Européen des Industrie Cosmétiques, des Produits de 
Toilette et de la Parfumerie, has set up SCAAT. 
SCAAT - Steering Committee on Alternatives to Animal Testing; supervises 
COLIPA's validation studies and coordinates the validation efforts of the 
cosmetics industry on a worldwide basis. 
2. USA 
CTFA - Cosmetic, Toiletry and Fragrance Association 
3. JAPAN 
J CIA - Japanese Cosmetics Industry Association 
4. ORGANISATION FOR ECONOMIC COOPERATION AND 
DEVELOPMENT (OECD) 
The OECD publishes test guidelines, as approved by consensus among its member 
countries. These guidelines are almost identical to the Annex V test methods of the 
EU, and, in association with the principle of Mutual Acceptance of Data, provide 
an opportunity for international harmonisation and for a rational approach to the 
application of the Three Rs. 
THE INITIATIVES 
1. EUROPEAN UNION 
DGXI 
DG XI, Environment, Nuclear Safety and Civil Protection has reached agreement with 
the Member States on the best approach to be adopted in gathering, on a bi-annual basis, 
the statistical data required under Articles 13 and 26 of Directive 86/609/EEC on the 
protection of animals used for experimental and other scientific purposes. The table which 
the Member States must complete includes a figure for the total number of animals used 
for cosmetic testing. DG XI is also responsible for Directive 67/548/EEC and for the EU 
test methods, as detailed in Commission Directive 87/302/EEC and in the Annex to 
Commission Directive 92/69/EEC (Part B - Methods for the Determination of Toxicity). 
DG XI supported the EU/HO international validation study on alternatives to the Draize 
eye irritation test, and also the EU/COLIPA international validation study on in vitro 
phototoxicity. 
DGJRC 
ECVAM (the European Centre for the Validation of Alternative Methods) is at the 
service of all the DGs concerned with the issues associated with alternative methods. 
ECVAM was established by the European Commission in accordance with a 
Communication from the Commission to the Council and the European Parliament in 
October 1991, and in response to Article 23 of Directive 86/609/EEC, which stated that: 
"The Commission and Member States should encourage research into the development and 
validation of alternative techniques which could provide the same level of information as 
that obtained in experiments using animals, but which involve fewer animals or which 
entail less painful procedures, and shall take such other steps as they consider appropriate 
to encourage research in this field". 
ECVAM has been set up to: 
1. Coordinate the validation of alternative test methods at the European Union level. 
2. Act as a focal point for the exchange of information on the development of 
alternative test methods. 
3. Establish, maintain and manage a database on alternative procedures. 
4. Promote dialogue among legislators, industries, biomedical scientists, consumer 
organisations and animal welfare groups, with a view to the development, 
validation and international recognition of alternative test methods. 
The ECVAM opening symposium organised at ISPRA on 18 October 1994 was devoted to 
discussion of the validation of replacement alternative methods. At this symposium, 
ECVAM's role in the development, validation and acceptance of alternative tests and test 
strategies, including those for cosmetics testing, was considered, and may be summarised 
as follows: 
contribute to the development of guidelines via the validation process itself, since 
this is linked to the development and acceptance of pertinent and reliable tests for 
specific objectives; 
identify priorities, taking into account the state of the art in specific fields; 
ensure that the alternative tests relate to simple, distinct and well-defined biological 
outcomes whose mechanistic basis is sufficiently well known; 
promote a multi-disciplinary approach with a view to developing integrated test 
strategies, incorporating quantitative structure-activity relationships (QSAR), 
biokinetics and cell and tissue culture methods; 
address the difficulties resulting from the lack of test materials with associated 
high-quality in vivo data; 
encourage the réévaluation of the existing guidelines and regulatory procedures; 
achieve consensus by working with the appropriate government, industrial, 
academic and other bodies; 
draw up criteria for validation, for independent evaluation of the results of 
validation studies, and for the legal acceptance of alternative tests and tests 
strategies, through active cooperation with such bodies as the OECD and ICCVAM 
(Inter-Agency Coordinating Committee for the Validation of Alternative Methods, 
USA). 
To date, ECVAM has organised several workshops on the development and validation of 
alternative tests and has published the reports of 14 of these workshops. ECVAM has 
given priority to alternatives for cosmetics testing. The reports of direct relevance to 
evaluating the safety of cosmetics are: 
report 2 In vitro phototoxicity testing (1994) 
report 5 Practical aspects of the validation of toxicity test procedures (1995) 
report 6 A prevalidation study on in vitro skin corrosivity testing (1995) 
report 7 Development and validation of non-animal tests and testing strategies: 
the identification of a coordinated response to the challenge and the 
opportunity presented by the Sixth Amendment to the Cosmetic 
Directive (1995) 
report 8 The integrated use of alternative approaches for predicting toxic hazard 
(1995) 
report 11 The Three Rs: the way forward (1995) 
report 13 Methods for assessing percutaneous absorption (published in 1996) 
Two other reports were published in 1995 on: 
the validation of alternative test methods (a joint statement by ECVAM and the 
ECB) 
the first ECVAM Prevalidation Task Force Report. 
ECVAM's priorities in the field of cosmetics testing for 1995-1998 include: 
developmental toxicity 
metabolism 
corrosivity 
phototoxicity 
dermal penetration 
ocular and dermal irritation 
sensitisation 
ECB (the European Chemicals Bureau) provides technical and scientific support for 
DGXI on the classification and labelling of dangerous substances, on notification of new 
substances, on existing chemicals, on export/import control of dangerous substances, and 
on testing methods (according to Annex V of Directive 67/548/EEC). An effective liaison 
is being developed between the ECB and ECVAM with respect to replacement alternative 
test methods, and modifications to animal procedures which reduce the numbers of 
animals required or lessen the suffering of any animals necessarily used in complying with 
regulations and guidelines. 
DG XXIV / SCC 
DG XXIV Consumer Policy, which manages the Cosmetic Directive (Directive 
76/768/EEC), remains the driving force and has expanded its activities with a view to the 
fullest possible compliance with the provisions of Directive 93/35/EEC within the agreed 
time limits. 
If alternative methods have not been scientifically validated and legally accepted, 
DG XXIV has been instructed to prepare for the Commission draft measures 
designed to extend the time limit for the ban on animal tests in accordance with the 
Committee for Adaptation to Technical Progress Procedure (Article 10, 
Directive 76/768/EEC). 
8 
This Committee (CATP) is made up of representatives of the Member States and is 
chaired by a Commission representative. The Chairman submits to the Committee the 
draft measures to be adopted. 
The opinion is delivered by qualified majority. 
In compliance with the new co-decision procedure, the European Parliament will be 
informed at the same time and under the same conditions of all draft measures submitted 
to the CATP. 
If there has been insufficient progress in developing satisfactory methods to replace 
animal testing, and in particular in those cases where alternative methods of 
testing, despite all reasonable endeavours, have not been scientiûcally validated as 
offering an equivalent level of protection for the consumer, taking into account 
OECD toxicity test guidelines, the Commission shall, by 1 January 1997, submit 
draft measures to postpone the date of implementation of this provision, for a 
sufficient period, and in any case for no less than two years, in accordance with 
the procedure laid down in Article 10. Before submitting such measures, the 
Commission will consult the Scientific Committee on Cosmetology. (Article 4(i) of 
Directive 76/768/EEC, as modified by Directive 93/35/EEC). 
On several occasions DG XXIV has urged the Member States to meet their obligations 
regarding the collection of precise data on animal experiments concerning ingredients and 
finished cosmetic products. The statistical data to be collected under Directive 93/35/EEC 
with regard to cosmetic products go beyond the data which Member States must supply to 
comply with the requirements of Directive 86/609/EEC. The Member States have been 
requested to submit these more detailed data on an annual basis. 
DG XXIV has liaised regularly with DG XI and with ECVAM, and has facilitated 
communication between all players. 
SCC: the Scientific Committee on Cosmetology, a Commission advisory committee, has 
been requested to deliver a scientific opinion to DG XXIV on the applicability of validated 
alternative methods for ^evaluating the safety of cosmetic products for human use. 
The SCC1 s subcommittee on alternative methods and guidelines organised two 
working meetings (2 February, 11 May) and two joint meetings with SCAAT/COLIPA 
(19 September, 1 December) during which the following actions were developed. 
1. Evaluation of documents and dossiers submitted by COLIPA 
In vitro photoirritation (EU/COUPA) 
An analysis of the data communicated by COLIPA - viz. the partial results 
published in the literature, in regard to the prevalidation phase, and partial 
information on phase 2 has not allowed the SCC to deliver a scientific opinion on 
the draft as a whole. 
However, the SCC emphasises that the animal models are inadequate for predicting 
the phototoxic effects in man and considers that a study of Min vitro" phototoxic 
potential is an important step in the process of evaluating the safety of 
cosmetic products containing UV filters. 
In vitro percutaneous absorption (COLIPA) 
After analysis of the documents presented by COLIPA, viz. guidelines for in vitro 
percutaneous absorption tests, standard protocols and a general overview of in 
vitro/in vivo correlations, the SCC recommends presenting the "in vitro" 
percutaneous absorption methodology in a standard manner and supplementing the 
methodology with intra- and inter-laboratory results obtained on suitably chosen 
test materials and vehicles. 
Photomutagenicity (COLIPA) 
The COLIPA recommendations in the final report transmitted to the SCC indicate 
the criteria to be used in defining the test protocols. 
The SCC regrets that it cannot yet base the evaluation of the photomutagenic 
potential of UV filters on validated tests, despite the fact that it developed general 
criteria for realising photomutagenicity tests in 1990 (SPC/803/90). 
2. Information required for the scientific evaluation of the validation studies of 
the alternative tests with a view to using them in evaluating the safety of 
cosmetic products 
Taking into consideration the problems arising in connection with validation of the 
alternative methodologies, the SCC considered there was a need to prepare a 
document that would be of direct use for future validation studies. 
The qualitative and quantitative aspects of the validation process are taken into 
account in this report. The criteria selected: 
concern the scientific validation of the tests 
make it possible to measure a degree of toxicity 
are applicable to cosmetic ingredients 
involve definition of the statistical analysis procedures 
The need to provide evidence of protocol optimisation and to formalise the link 
between in vitro and in vivo data in the form of a mathematical relationship are 
highlighted. 
DGXII 
DG XII, Science, Research and Development funds research programmes, notably 
including the development and validation of alternative methods according to Decision N° 
1110/94/EC of the European Parliament and the Council of 26 April 1994 concerning the 
Fourth Framework Programme for Research and Technology Development, which clearly 
set out instructions as follows: "Whenever possible, experimentation and testing on 
10 
animals should be replaced by in vitro or other methods... 
Several programmes on the development and validation of in vitro tests have been 
supported by DG XII or are in progress. Some of the projects could be of use for the 
cosmetic sector: 
BRIDGE - Final report (EUR 15777, published in 1995) 
Development of a predictive in vitro test for the detection of sensitising 
compounds. 
BIOTECH (1992-1994) 
Some projects concern in vitro developmental toxicology. 
BIOTECH (1994-1998) 
The work programme foresees prenormative research on in vitro alternatives to 
animal experiments in pharmacotoxicology. 
rVTIP (In Vitro Testing Industrial Platform): forum organised to improve contacts 
between technology producers and users. Two meetings were organised in 1995, on 
progress with the Fourth Framework Programme and on an overview of progress made 
with validation. 
COLEPA/SCAAT 
For each field of investigation SCAAT has established specific working parties made up of 
various experts to implement the validation work. 
An international scientific symposium on alternatives to animal experiments was organised 
by COLIPA in Brussels on 29 and 30 November 1995, with representatives of industry, 
the scientific community, the Commission and the European Parliament, and animal 
protection societies. 
The first session, devoted to VALIDATION, emphasised the critical role of prediction 
models and surveyed ongoing validation studies on photoirritation and eye irritation. The 
session ended with a round table on the perspectives of a regulatory approach. 
The second session, devoted to DEVELOPMENT, included presentations on percutaneous 
absorption, skin compatibility and skin sensitisation. 
The third session, devoted to SAFETY EVALUATION, addressed the initiatives taken by 
the SCC as well as the approaches of the British and German regulatory authorities. 
Emphasis was placed on the importance of providing scientific data in support of the 
cosmetics industry's "in-house" studies and test strategies for evaluating the safety of 
finished products. 
The presentations were supplemented by a poster exhibition. In the context of this 
exhibition, a brochure setting out the approach of IFAW (the International Fund for 
Animal Welfare) was provided to the participants. As there is clearly no question of 
11 
replacing animal tests by a simple in vitro test, IFAW presents a phase-based strategy 
based essentially on alternatives which focus on the use of cells and tissues of human 
origin, excluding all in vivo animal experiments and recommending in vivo tests on 
volunteers. 
The symposium highlighted the important role of COLIPA (European cosmetics industry) 
in undertaking research into the development and validation of alternative methods. The 
work being done by the major European cosmetic companies is very substantial when one 
considers the number of animals used in cosmetic experiments and the knowledge acquired 
greatly benefits other industries. 
Moreover, the symposium provided the different players involved with precise scientific 
information and gave rise to an interesting exchange of views. A joint approach of 
industry and the Commission was called for, so that animal tests in the fields of skin 
compatibility, photoirritation and percutaneous absorption can rapidly be replaced. 
2. USA 
The US government is keen on international cooperation to recommend an approach and 
to define the criteria for the regulatory adoption of new alternative methods, clearly 
establishing the conditions under which these methods are used (biological outcomes, 
relevance for classes of substances, etc.). ICCVAM organised a workshop on the 
validation and regulatory acceptance of alternative toxicological testing methods, sponsored 
by the National Institute of Environmental Health Sciences (NIEHS) and the National 
Toxicology Program (NTP), in December 1995 in Arlington, Virginia. The purpose of the 
workshop was to discuss the draft ICCVAM report containing recommendations about 
criteria and processes for the validation and regulatory acceptance of new and revised 
toxicological testing methods. 
The Food and Drug Administration (FDA) is the US agency responsible for cosmetics 
safety. The FDA has interactive relations with the public and private agencies involved in 
the development and validation of alternative tests, and focuses on methods which generate 
reliable scientific data. 
The FDA has misgivings about the prohibition of animal experiments in the 
implementation of Directive 93/35/EEC. Certain cosmetics manufactured in the EU 
might not satisfy the safety requirements (for health) laid down by the US FDCA (Food, 
Drug and Cosmetics Act). 
3. JAPAN 
The Japanese Ministry for Health and Welfare (MHW) is responsible for testing and test 
guidelines in Japan. Japanese research groups are particularly involved in the validation of 
alternatives to the Draize eye test. The acceptance of the alternatives depends on their 
specific role in the evaluation of eye irritation (screening or replacement; individual test or 
batteries of tests). JCIA considers that it would be difficult to recommend for regulatory 
testing purposes for evaluating the safety of cosmetic products, a test or test battery 
whose predictivity was less than that of in vivo methods. 
12 
4. OECD 
In October 1994, the 5th meeting of the National Coordinators of the OECD Test 
Guidelines Programme agreed that an attempt should be made to internationally harmonise 
the various published and advocated concepts for the validation of alternative test methods. 
In view of the international debate on the issue, it was considered timely for the OECD to 
step in and provide a platform for all parties involved, through which it might be possible 
to reach international consensus on validation and acceptance criteria. The National 
Coordinators emphasised that existing proposals should be used as the basis for an 
internationally acceptable approach, rather than the development of yet another concept. In 
this respect, the work of centres such as CAAT (Johns Hopkins University Center for 
Alternatives to Animal Testing) in the US, ECVAM in the EU, ERGATT, and various 
national centres and committees (e.g. FRAME in the UK, IRAG and ICCVAM in the US, 
NCA in the Netherlands, SIAT in Switzerland, and ZEBET in Germany) was well-
recognised. 
The National Coordinators agreed that an OECD Workshop would be the best approach, 
since such a meeting would offer ample opportunity for all parties having an interest in the 
subject to discuss the issue and to seek consensus. Furthermore, it was considered of 
crucial importance that member countries would include in their nominations individuals 
having responsibilities in the regulatory area. Sweden offered to host the workshop. 
A Steering Committee was established in January 1995 to advise the OECD Secretariat on 
the scope and structure of the Workshop, and to assist in the development of the 
programme for the Workshop, which was scheduled for 22-24 January 1996, in Solna, 
Sweden. ECVAM and ZEBET were represented on this Steering Committee. 
D. THE STATE OF PLAY 
METHODS UNDER DEVELOPMENT AND VALIDATION 
Skin sensitisation 
Sensitisation is an area of considerable research and is extremely relevant to cosmetics 
safety. An interesting approach to the in vitro evaluation of skin sensitisation was 
presented at the symposium organised by COLIPA (29-30 November 1995). An ECVAM 
workshop on skin sensitisation was helcl during 1995, the report of which will be 
published in 1996. 
The chemical structures and sensitising potentials of various compounds have been 
extensively examined and expert computer systems, such as DEREK (Deductive 
Estimation of Risk from Existing Knowledge), are being developed to identify structural 
alerts associated with skin sensitisation potential. A possible predictive approach could be 
based on measurement of the molecular signals induced in cell cultures by sensitising 
substances. 
13 
Notably in this domain it should be emphasised that prediction of sensitisation potential in 
humans remains a major problem because of the inter-individual variability in response. 
Photomutagenicity Validation (COLIPA) 
Since 1990, COLIPA has submitted dossiers on UV filters containing photomutagenicity 
data obtained from different types of tests. 
COLIPA organised a ring test to define a suitable protocol by using two substances whose 
photomutagenic potentials were known, and which were activated by UV-A radiation and 
different bacterial strains. The results of this ring test have made it possible to draft 
recommendations on the criteria for selecting bacterial strains or cell cultures, the solar 
simulator, the technique of sample irradiation, UV radiation doses, doses of substances to 
be tested, and the use of positive controls. It is difficult to plan a validation study in the 
absence of in vivo reference data. 
Percutaneous absorption 
ECVAM, COLIPA and the OECD have collaborated to facilitate the international adoption 
of a testing guideline on in vitro percutaneous absorption. 
Pbotoinitation/phototoxicity: Validation - phase U (EU/COUPA) 
ECVAM and DG XI are represented on the Management Team for the study, which is 
being coordinated by ZEBET, and is examining the possibility of a supplementary 
validation study designed to predict the photoirritant potential of UV filters. 
In the first (prevalidation) phase of the study, analysis of the results obtained in a cell 
viability test made it possible to determine a photoirritancy factor for distinguishing 
photoirritant substances from non-photoirritant substances. The objective of the second 
(validation) phase, planned as a blind trial, was to determine whether a cell toxicity test 
and a test measuring cell damage (the NRU1 and the combined test RBC2 [photohaemolysis 
+ haemoglobin oxidation]) could correctly predict the photoirritant potential of 32 
chemical substances, which have been administered systemically or topically in man. 
The pretiminary results of phase II, which ended in 1995, are confined to the NRU test 
used in all the laboratories. The RBC protocol was used only in three laboratories. 
Supplementary statistical evaluations were carried out by an independent biostatistician and 
will be available during the first half of 1996. 
'NRU: neutral red uptake assay|§| 
2RBC: red blood cell haemolysis test; used to evaluate damage to the cell membranes 
14 
Eye irritation: final report on the EC/HO study on alternatives to the Draize eye 
irritation test 
The objective of this study was to determine whether nine alternative tests, alone or in 
combination, could replace the Draize test for severely irritant test materials or evaluate 
the irritant potential of chemical substances, with or without regard to the chemical class 
and over the entire range of measurable potentials. 
A total of 60 chemical substances were analysed independently in 37 laboratories. The 
results were compared with the MMAS (modified maximum average score) obtained in the 
Draize test and submitted for statistical evaluation. 
With the exception of predicting the irritant potential of surfactants, none of the nine tests 
achieved any of the envisaged objectives. 
Many valuable lessons were learned during this study, which were taken into account in 
the planning of the COLIPA study. They also led to the ECVAM workshop on practical 
aspects of validation, and to the development of the ECVAM prevalidation scheme. The 
variablility of the in vivo data was identified as a major obstacle for the establishment of 
the relevance and reliability of the in vivo tests. 
Eye irritation: Validation (COIJPA) 
The validation exercise was conducted on 23 cosmetic ingredients (surfactants, alcohols, 
preservatives), of which 20 were the same as those tested in the EC/HO study, and on 32 
cosmetic formulations (with a large range of physico-chemical forms). The test materials 
were tested in vivo in compliance with OECD guideline 405. 
The specific objectives of this study were to determine whether the data obtained on the 
basis of 10 in vitro protocols currently used in the cosmetic industry (of which five had 
also been evaluated in the EC/HO study): 
correlate acceptably with the MMAS (modified maximum average score); 
correlate acceptably with the individual scores and recovery times, such as those 
described in OECD guideline 405; 
correctly predict the eye irritation potential in the rabbit on the basis of algorithms 
for the alternative method. 
For the first time in a validation study, prediction models (PMs) were laid down 
before the start of the study. They define precisely the biological outcomes obtained for 
each in vitro model and how to convert each in vitro biological outcome into a prediction 
of eye irritation potential. The PMs used in this study are based on historical data 
available for the ingredients and formulations of interest to the cosmetics industry, notably 
for substances with a low to moderate irritation potential. 
The programme did not permit complete validation because of the small number of 
laboratories that participated in assays specific to the programme. None of the 
methods tested can yet validly replace the Draize eye irritation test. 
15 
The reliability of the PMs was evaluated by determining whether the inter-laboratory 
results were reproducible and whether the data were distributed within the prediction 
intervals of the predefined PMs. 
The RBC test showed good inter-laboratory reproducibility and also satisfied the reliability 
criteria. 
Three of the protocols tested conformed reasonably with their prediction models (FLT3, 
RBC, TEA4), but supplementary studies are necessary to resolve several technical 
problems associated with the PMs before final conclusions about their performances can 
be drawn. 
This validation study highlights the importance of the availability of good-quality in 
vivo data. 
The in vivo data available are adequate for evaluating the risk. However, in order to 
compare the in vivo and in vitro data, the two sets of data should theoretically have as 
small a variability as possible, to permit precise statistical evaluation. Experience has 
shown that this is not always the case. 
Examination of the results indicate that certain PMs could be refined and tested in a future 
study. 
Skin corrosivity: Prevalidation (ECVAM) 
Four skin corrosivity tests are being evaluated and optimised (TER - rat skin 
transcutaneous electrical resistance assay, CORROSITEX, Skin2 and EPISKIN) with a 
view to planning a formal validation study for 1996. 
Skin compatibility (COUPA) 
Skin compatibility is defined as the absence of skin irritation in normal conditions of 
use and in reasonably foreseeable conditions of improper use. 
Seven COLIPA member companies evaluate the skin compatibility of their finished 
products in man under controlled conditions, because the irritation potentials of most 
cosmetic products are very low. 
COLIPA has developed guidelines for evaluating the compatibility of finished cosmetic 
products on human skin: 
3FLT: fluorescein leakage test: measures the damage caused to a cell barrier. 
*TEA: tissue equivalent assay; measures the time required to cause a 50% reduction in the viability of 
cells in reconstituted human skin 
16 
taking ethical requirements into account, studies on man must be conducted in 
accordance with the Helsinki Declaration (1964, 1989); 
a prudent approach to tests, stage by stage, is essential; 
prior knowledge of the composition and stability of the products tested, as well as 
prior evaluation of toxicity data on the product ingredients, is necessary. 
Guidelines on the skin compatibility of cosmetic ingredients are currently being developed. 
Human volunteer studies (ECVAM) 
Human volunteer studies are often listed along with other replacement alternatives for 
animal tests. The ethical, legal, safety, logistic and scientific problems associated with 
such studies are being investigated by ECVAM, in collaboration with the University of 
Pavia, the University of Nottingham, and a number of cosmetic companies. 
E. STATISTICS ON ANIMAL EXPERIMENTS 
The collection of data on the number of animals used for experimental or other scientific 
purposes pursuant to Articles 13 and 26 of Directive 86/609/EEC is not planned on an 
annual basis. 
This partly explains the difficulties encountered in collecting annual data specifically 
concerning cosmetic ingredients and products. 
However, the Commission has on numerous occasions urged the Member States to obtain 
the statistics required, as is recalled in relation to the activities of DG XXIV and DG XI. 
(1) Eight Member States have declared that animal tests for finished cosmetic products 
have not been carried out on their territory (Italy, Greece, Belgium, Ireland, 
Sweden, Finland, Luxembourg, Germany). 
(2) Six Member States have declared that animal tests for cosmetic ingredients have 
not been carried out on their territory (Greece, Netherlands, Ireland, Sweden, 
Finland, Luxembourg). 
(3) Only three Member States (Austria, France, United Kingdom) have communicated 
figures relating to the number of animals used, while pointing out however that 
these figures cannot be interpreted and that uncertainties remain as to their 
correspondence to reality. Under these circumstances this report cannot present 
comparative tables consisting of numerical data because such a table would give a 
false view of reality. 
(4) As regards the declarations that tests have not been carried out, it should also be 
noted that certain Member States that do not produce ingredients may of course use 
ingredients tested in other Member States or in third countries and that certain 
ingredients used for cosmetic products may have been tested for other purposes. 
17 
The Commission strongly regrets that it does not have more precise data at this moment, 
but it depends on the Member States for the collection of these data. 
F. CONCLUSIONS 
Progress during 1995 with regard to alternative test methods and their validation can be 
summarised as follows: 
1. The report on Phase II of the EU/COLIPA international validation study on in vitro 
tests for phototoxicity (photoirritancy) will be made available in 1996, but it is 
already clear that the validation of the NRU cell culture test has been very 
successful. Those involved in the study are confident that an acceptable OECD 
guideline can be drafted within the next two years, i.e. after the final report on the 
study has been published and minor refinements to protocols have been made. 
2. Experience in European industry on the critical issue of percutaneous absorption 
has led to a concrete proposal for an OECD guideline for an in vitro test. ECVAM 
and COLIPA are working with the OECD and others, to facilitate the adoption of 
an in vitro test guideline by the OECD Member Countries. 
3. The results of the EC/HO international validation study on alternatives for the 
Draize eye irritancy test were disappointing, in that none of the tests met the 
goals of the study. However, much of value was learned during this study, as a 
result of which the quality of future validation studies will be markedly improved. 
4. The first phase of the COLIPA evaluation of alternatives to the Draize eye test, 
conducted on a more-limited range of materials than the EC/HO study, produced 
more-promising results, and showed the value of incorporating prediction models 
into test protocols. 
5. As a result of a COLIPA study, recommendations have been drafted on criteria to 
be adopted in defining in vitro test protocols for photomutagenicity. 
6. As a result of another COLIPA initiative, guidelines on the skin compatibility 
testing of cosmetic products and their ingredients in man have been developed. 
7. The use of human volunteers as a replacement alternative deserves further scrutiny, 
but it must be emphasised that such studies should only be conducted after results 
from previous in vivo and/or in vitro studies permit avoidance of the risk of any 
serious consequences. 
8. Validation has turned out to be even more difficult, more time-consuming and 
more costly than had been expected. Indeed, discussions are still going on about 
18 
what validation actually means and how the relevance and reliability of alternative 
methods can best be established. ECVAM is at the heart of these discussions, and 
particularly important contributions have been made in a number of ways. 
9. The ECVAM prevalidation scheme, which is currently under international 
evaluation, is likely to improve the success rate of validation studies, and to reduce 
the time needed to conduct them, as well as their cost. 
10. International discussions have taken place within Europe, and also with the USA 
and Japan, on the harmonisation of criteria for the validation and acceptance of 
replacement alternative test methods. 
G. THE OUTLOOK 
i 
1. The emergence of more-realistic expectations of the validation process, and the 
success of international discussions on criteria for the validation and acceptance of 
replacement alternative methods can be expected to result in substantial progress in 
the future. 
2. After the conclusion of a further small study on chemicals of particular concern to 
the SCC, it can be expected that a draft regulatory guideline for in vitro 
phototoxicity testing will be produced and proposed for acceptance by the 
regulatory authorities. 
3. It is hoped that a new guideline on in vitro tests for percutaneous absorption will 
shortly be accepted by the EU Member States and the OECD Member Countries. 
4. An acceleration of progress toward the development of standardised and 
appropriate in vitro methods for detecting sensitisation potential can be expected. 
5. A reconsideration of what is expected of eye irritancy tests (in vivo and in vitro) 
will be essential. Meanwhile, a more flexible approach to testing could be 
encouraged, for example, by accepting the evaluation of the toxicities of new 
products and ingredients by comparison with knowledge of appropriate benchmark 
substances. 
6. Greater use could be made of the fact that the eye tolerance and skin tolerance of 
finished cosmetic products can be evaluated in vitro, provided one crucial 
condition is met, viz. prior knowledge of the toxicity data pertaining to the 
ingredients and their physico-chemical properties. In addition, the skin 
compatibility of finished cospietic products can be evaluated in the context of 
strictly controlled clinical studies. 
19 
7. A successful outcome can be expected for the ECVAM validation study on tests for 
skin corrosivity, which could then be used, for example, to provide part of the 
information considered essential before human volunteer studies can be considered 
permissible. 
8. There will be greater emphasis on the integrated use of various different 
approaches (e.g. computer-based predictions, in vitro tests), and on hierarchical 
testing strategies, to reduce, refine and replace the use of animals for testing 
purposes. 
The Sixth Amendment to Directive 76/768/EEC provides a unique opportunity for sensible 
progress toward the replacement of animal testing in the field of cosmetics. Such progress 
is being made, but its continuation will depend upon the maintenance of good relationships 
and effective collaborations between all the parties involved, i.e. the Commission and its 
advisory committees, the Member States, the cosmetics industry, and the regulatory 
authorities. 
20 

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