REPORT FROM THE COMMISSION - DEVELOPMENT, VALIDATION AND LEGAL ACCEPTANCE OF ALTERNATIVE METHODS TO ANIMAL EXPERIMENTS IN THE FIELD OF COSMETIC PRODUCTS - 1995
Date: 1996-07-24
Institutions: European Commission
Subjects: animal experimentation, bioprocess, cosmetic product, protection of animals, research method

COMMISSION  OF THE  EUROPEAN  COMMUNITIES 

I 

* ** 

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. 

P G XI 
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" 

D 3 X II 
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 
the 
regards 
implementation  of Directive 93/35/EEC. 

the  ban  on  animal  experiments 

in 

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  substances  under 
consideration  by  comparison  with  appropriate  benchmark  substances. 

the  toxicity  of 

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 
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. 

irritation  and  skin  sensitisation 

GLOSSARY 

CAPT: 
CAAT: 
CFTA: 
COLIPA: 
DEREK: 
ECB: 
ECVAM: 
ERGATT: 
ES AC: 
EU: 
EU/HO international  validation study: 

Committee of Adaptation to Technical  Progress 
Centre for Alternatives to Animal Testing (John Hopkins University. USA) 
Cosmetic, Toiletry  and Fragrance Association  (USA) 
European Cosmetic, Toiletry  and Perfumery  Association 
Deductive Estimation of Risk from Existing Knowledge 
European Chemicals Bureau 
European Centre  for the Validation of  Alternative Methods 
European Research Group for Alternatives in Toxicity  Testing 
ECVAM Scientific  Advisory  Committee 
European Union 

FDA: 
FDCA: 
FLT: 
FRAME: 
GLP: 
ICC V AM: 

IF AW: 
IRAG: 
IVTIP: 
JCIA: 
JRC: 
MMAS: 
MHW: 
NIEHS: 
NRU: 
NTP: 
PM: 
OECD: 
QSAR: 
RBC: 
SCAAT: 
SCC: 
SLAT: 
TEA: 
UV: 
ZEBET: 

European Union/Home  Office  international  validation study 
Food and Drug Administration  (USA) 
Food, Drug and Cosmetics Act (USA) 
Fluorescein Leakage Test  * 
Fund for the Replacement of Animals in Medical  Experiments 
Good Laboratory Practice 
US Inter-Agency Coordinating Committee for the Validation of Alternative 
Methods 
International  Fund for Animal  Welfare 
Interagency  Regulatory  Alternatives Group 
In Vitro Testing Industrial  Platform 
Japanese Cosmetics Industry  Association 
Joint Research Centre 
Modified  Maximum Average Score 
Ministry  for Health  and Welfare (Japan) 
National  Institute of Environmental  Health  Sciences  (USA) 
Neutral Red Uptake Assay 
National Toxicology  Program (USA) 
Prediction Model 
Organisation for Economic  Cooperation and Development 
Quantitative  Structure-Activity  Relationships 
Red Blood Cell Haemolysis Test 
Steering  Committee on Alternatives to Animal Testing 
Scientific  Committee on Cosmetology 
Scweizerisches Institut fur Alternativen zu Tierversuchen 
Tissue Equivalent Assay 
Ultra Violet 
Zentralstelle 
Erganzungsmethoden zum Tierversuch im Bundesgesundheitsamt 

zur  Erfassung  und  Bewertung  von  Ersatz  und 

vi 

CONTENTS 

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 

A. 

B. 

D. 

E 

F. 

G. 

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. 

2. 

3. 

4. 

Validation  of alternative  procedures  for  the use in  non-regulatory  studies. 

Validation  of alternative  tests for  inclusion  as part of regulatory  guidelines. 

Validation  of alternative  tests to replace existing  guidelines. 

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, 
laboratories  responsible  for  studying  the protocol's  performance. 

involving,  as  a  minimum,  participation  of  the  three 

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 
experimentation  and  the development  and evaluation  of alternative  methods. 

services  are 

involved 

relating 

issues 

in 

to  animal 

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 
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  European  Commission 

"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. 
2. 

3. 
4. 

Coordinate  the  validation  of alternative  test  methods at  the  European  Union  level. 
Act  as  a  focal  point  for  the  exchange  of  information  on  the  development  of 
alternative  test  methods. 
Establish,  maintain  and  manage a database on  alternative  procedures. 
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: 

incorporating  quantitative  structure-activity 

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, 
(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 
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). 

the  appropriate  government, 

relationships 

industrial, 

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 
report  5 
report  6 

In vitro phototoxicity  testing  (1994) 
Practical  aspects of the validation  of toxicity  test procedures  (1995) 
A prevalidation  study  on  in vitro  skin corrosivity  testing  (1995) 

report  7 

report  8 

report  11 
report  13 

Development  and  validation  of  non-animal  tests  and  testing  strategies: 
the  identification  of  a  coordinated  response  to  the  challenge  and  the 
to  the  Cosmetic 
opportunity  presented  by 
Directive  (1995) 
The  integrated  use  of  alternative  approaches  for  predicting  toxic  hazard 
(1995) 
The Three  Rs: the  way  forward  (1995) 
Methods for  assessing percutaneous  absorption  (published  in  1996) 

the  Sixth  Amendment 

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 
(Article  10, 
Committee 
Directive 76/768/EEC). 

to  Technical  Progress  Procedure 

for  Adaptation 

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 
the  "in  vitro" 
vitro/in  vivo  correlations, 
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. 

the  SCC  recommends  presenting 

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 
compounds. 

in  vitro  test  for 

the  detection  of  sensitising 

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) 

(2) 

(3) 

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). 

ingredients  have 
Six  Member  States  have  declared  that  animal  tests  for  cosmetic 
not  been  carried  out  on  their  territory  (Greece,  Netherlands,  Ireland,  Sweden, 
Finland,  Luxembourg). 

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. 

2. 

3. 

4. 

5. 

6. 

7. 

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. 

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. 

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. 

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. 

As  a  result  of  a  COLIPA  study,  recommendations  have  been  drafted  on  criteria  to 
be adopted  in defining  in vitro test protocols for  photomutagenicity. 

As  a  result  of  another  COLIPA  initiative,  guidelines  on  the  skin  compatibility 
testing of cosmetic products and their ingredients in  man have been  developed. 

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. 

The  ECVAM  prevalidation  scheme,  which 
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. 

is  currently  under 

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. 

9. 

10. 

G. 

THE  OUTLOOK 

1. 

2. 

3. 

4. 

5. 

6. 

i 

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. 

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. 

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. 

An  acceleration  of  progress 
the  development  of  standardised  and 
appropriate  in vitro  methods for  detecting  sensitisation  potential  can be  expected. 

toward 

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. 

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 
skin 
ingredients  and 
compatibility  of  finished  cospietic  products  can  be  evaluated  in  the  context  of 
strictly  controlled  clinical  studies. 

their  physico-chemical  properties. 

In  addition, 

the 

19 

7. 

8. 

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. 

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 

ISSN 0254-1475 

COM(96) 365  final 

DOCUMENTS 

EN 

10  15  05  03 

Catalogue number  : CB-CO-96-366-EN-C 

ISBN 92-78-07193-5 

Office for Officiai Publications of the European Communities 

L-2985 Luxembourg 

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