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Directorate-General for Health and Food Safety
Terms of reference
DG III requests the opinion of the Scientific Committee on Cosmetic and Non-Food Products (SCCNFP) as to the status of alternative methods for the safety assessment of cosmetic ingredients according to the current state of the art. Specifically DG III requests that the SCCNFP assesses the possibility of replacing data obtained on the basis of animal tests by data obtained making use of alternative methods in the safety evaluation of cosmetic ingredients (XXIV/1890/98)
1- Background
UV-absorbing chemicals are employed as ingredients of various cosmetic products. Guidelines for the safety testing of cosmetics require a test for photo-irritation potential of this type of compounds. Testing usually is done on animals, although an accepted protocol to test in vivo for photo-irritation potential does not exist.
2- Different phases in the study
2-1. In a first phase in 1992-1993, a joint EU/COLIPA prevalidation study was designed to identify in vitro test procedures for a validation trial under blind conditions. Twenty chemicals with known phototoxicity properties were selected according to scientific criteria by an independent COLIPA task force of experts. The chemicals underwent different tests e.g. photohaemolysis test, histidine oxidation test, Candida albicans test, SOLATEX PI®, Skin2®, Testskin® and the 3T3 mouse fibroblast test. It came out that the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) with mouse fibroblasts using a sun simulator UV source (UVA 5J / cm2) was giving better overall correlation to in vivo data than results from any of the other tests.
2-2. At ZEBET a prediction model for the 3T3 NRU PT test was developed, which took the IC-50 values from cytotoxicity dose-concentration curves in the presence and absence of exposure to UV-light into account. A photo-irritation factor (PIF) was calculated which is the ratio of IC50 (-UV) / IC50 (+UV).
Discriminant analysis showed that a PIF of 5.0 provided the best prediction to discriminate between phototoxic and non-phototoxic chemicals.
(Spielmann et al.1994b, 1995).
s published independently from the validation study (Holzhütter 1997).
2-4. The results of the 3T3 NRU PT test were reproducible and correlated well with the in vivo data. Therefore, in 1997, the ECVAM Scientific Advisory Committee (ESAC) and in 1998 DG III and DG XI of the European Commission concluded from the formal validation study under blind conditions "that the 3T3 NRU PT is a well validated test and ready to be considered for regulatory acceptance" (Anon. 1998).
s can be correctly assessed by the 3T3 NRU PT test.
2-6. In 1998, the SCCNFP reviewed carefully the publications from the validation studies, the ESAC statement and the application study of the UV filters. Critical questions were posed to the management team. These were all answered using appropriate scientific criteria.
3- Opinion of the SCCNFP
Taking the results obtained in the prevalidation and formal validation study of the 3T3 NRU PT test and the results of the application study of this test to the UV filters of Annex VII of the Directive 76/768/EEC into account, the SCCNFP proposes the use of the 3T3 NRU PT test as the standard method for testing the UV light absorbing cosmetic ingredients or mixtures of ingredients for phototoxic potential.
4- References
1. Spielmann H., Lovell W.W., Hölzle E., Johnson B.E., Maurer T., Miranda M.A., Pape W.J.W., Sapora O.H. & Sladowski D.
In vitro phototoxicity testing. The report and recommendations of ECVAM Workshop 2.
ATLA (1994 a) 22, 314-348.
2. Spielmann H., Balls M., Brand M., Döring B., Holzhütter H.G., Kalweit S., Klecak G., L'Eplattenier H., Liebsch M., Lovell W.W., Maurer T., Moldenhauer F., Moore L., Pape W.J.W., Pfannenbecker U., Potthast J., de Silva O., Steiling W. & Willshaw A.
EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a Balb/c 3T3 cell phototoxicity assay.
Toxicology in vitro (1994 b) 8, 793-796.
3. Spielmann H., Liebsh M., Pape W.J.W., Balls M., Dupuis J., Klecak G., Lovell W.W., Maurer T., de Silva O. & Steiling W.
EEC/COLIPA in vitro Photoirritancy Program : Results of the first stage of validation.
In : Elsner P., Maibach HI (eds) : Irritant Dermatitis. New Clinical and Experimental Aspects.
Curr. Probl. Dermatol. Basel, Karger (1995), vol. 23, pp 256-264.
4. EC - DG XXIV
Notes of guidance for testing of cosmetic ingredients for their safety evaluation. Annex 8.
(1997) XXIV/1878/97
5. Holzhütter H.G.
A general measure of in vitro phototoxicity derived from pairs of dose response curved and its use for predicting in vivo phototoxicity of chemicals.
ATLA (1997) 25, 445-462.
6. Spielmann H., Balls M., Dupuis J., Pape W.J.W., Pechovitch G.¸ de Silva O., Holzhütter H.G., Clothier R., Desolle P., Gerberick G.F., Liebsch M., Lovell W.W., Maurer T., Pfannenbecker U., Potthast J.M., Csato M., Sladowski D., Steiling W. & Brantom P.
The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU Phototoxicity test.
Toxicology in vitro (1998a) 12, 305-327.
7. Spielmann H., Balls M., Dupuis J., Pape W.J.W., de Silva O; Holzhütter H.G., Gerberick F., Liebsch M., Lovell W.W. & Pfannenbecker U.
A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test.
ATLA (1998 b) 26, 679-708.
8. Anon.
Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxic potential).
ATLA (1998) 26, 7-8.
9. Guillot J.P., Gonnet J.F., Loquerie J.F., Martin M.C., Convert P. & Cotte J. (1985).
A new method for the assessment of phototoxic and photoallergic potentials by topical applications in the albino guinea pig.
J Toxicol Cut & ocular toxicol. (1985) 4, 117-134.
10. Kaidbey K.H., Kligman A.M.
Photomaximisation tests for identifying photoallergic contact sensitisers.
Contact Dermatitis (1980) 6, 161-169.
11. Mandate for SCCNFP Specific Working Group on alternative methods, for the safety evaluation of cosmetic products (XXIV/1890/98) adopted on 20 May 1998.
DG III requests the opinion of the Scientific Committee on Cosmetic and Non-Food Products (SCCNFP) as to the status of alternative methods for the safety assessment of cosmetic ingredients according to the current state of the art. Specifically DG III requests that the SCCNFP assesses the possibility of replacing data obtained on the basis of animal tests by data obtained making use of alternative methods in the safety evaluation of cosmetic ingredients (XXIV/1890/98)
1- Background
UV-absorbing chemicals are employed as ingredients of various cosmetic products. Guidelines for the safety testing of cosmetics require a test for photo-irritation potential of this type of compounds. Testing usually is done on animals, although an accepted protocol to test in vivo for photo-irritation potential does not exist.
2- Different phases in the study
2-1. In a first phase in 1992-1993, a joint EU/COLIPA prevalidation study was designed to identify in vitro test procedures for a validation trial under blind conditions. Twenty chemicals with known phototoxicity properties were selected according to scientific criteria by an independent COLIPA task force of experts. The chemicals underwent different tests e.g. photohaemolysis test, histidine oxidation test, Candida albicans test, SOLATEX PI®, Skin2®, Testskin® and the 3T3 mouse fibroblast test. It came out that the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) with mouse fibroblasts using a sun simulator UV source (UVA 5J / cm2) was giving better overall correlation to in vivo data than results from any of the other tests.
2-2. At ZEBET a prediction model for the 3T3 NRU PT test was developed, which took the IC-50 values from cytotoxicity dose-concentration curves in the presence and absence of exposure to UV-light into account. A photo-irritation factor (PIF) was calculated which is the ratio of IC50 (-UV) / IC50 (+UV).
Discriminant analysis showed that a PIF of 5.0 provided the best prediction to discriminate between phototoxic and non-phototoxic chemicals.
(Spielmann et al.1994b, 1995).
s published independently from the validation study (Holzhütter 1997).
2-4. The results of the 3T3 NRU PT test were reproducible and correlated well with the in vivo data. Therefore, in 1997, the ECVAM Scientific Advisory Committee (ESAC) and in 1998 DG III and DG XI of the European Commission concluded from the formal validation study under blind conditions "that the 3T3 NRU PT is a well validated test and ready to be considered for regulatory acceptance" (Anon. 1998).
s can be correctly assessed by the 3T3 NRU PT test.
2-6. In 1998, the SCCNFP reviewed carefully the publications from the validation studies, the ESAC statement and the application study of the UV filters. Critical questions were posed to the management team. These were all answered using appropriate scientific criteria.
3- Opinion of the SCCNFP
Taking the results obtained in the prevalidation and formal validation study of the 3T3 NRU PT test and the results of the application study of this test to the UV filters of Annex VII of the Directive 76/768/EEC into account, the SCCNFP proposes the use of the 3T3 NRU PT test as the standard method for testing the UV light absorbing cosmetic ingredients or mixtures of ingredients for phototoxic potential.
4- References
1. Spielmann H., Lovell W.W., Hölzle E., Johnson B.E., Maurer T., Miranda M.A., Pape W.J.W., Sapora O.H. & Sladowski D.
In vitro phototoxicity testing. The report and recommendations of ECVAM Workshop 2.
ATLA (1994 a) 22, 314-348.
2. Spielmann H., Balls M., Brand M., Döring B., Holzhütter H.G., Kalweit S., Klecak G., L'Eplattenier H., Liebsch M., Lovell W.W., Maurer T., Moldenhauer F., Moore L., Pape W.J.W., Pfannenbecker U., Potthast J., de Silva O., Steiling W. & Willshaw A.
EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a Balb/c 3T3 cell phototoxicity assay.
Toxicology in vitro (1994 b) 8, 793-796.
3. Spielmann H., Liebsh M., Pape W.J.W., Balls M., Dupuis J., Klecak G., Lovell W.W., Maurer T., de Silva O. & Steiling W.
EEC/COLIPA in vitro Photoirritancy Program : Results of the first stage of validation.
In : Elsner P., Maibach HI (eds) : Irritant Dermatitis. New Clinical and Experimental Aspects.
Curr. Probl. Dermatol. Basel, Karger (1995), vol. 23, pp 256-264.
4. EC - DG XXIV
Notes of guidance for testing of cosmetic ingredients for their safety evaluation. Annex 8.
(1997) XXIV/1878/97
5. Holzhütter H.G.
A general measure of in vitro phototoxicity derived from pairs of dose response curved and its use for predicting in vivo phototoxicity of chemicals.
ATLA (1997) 25, 445-462.
6. Spielmann H., Balls M., Dupuis J., Pape W.J.W., Pechovitch G.¸ de Silva O., Holzhütter H.G., Clothier R., Desolle P., Gerberick G.F., Liebsch M., Lovell W.W., Maurer T., Pfannenbecker U., Potthast J.M., Csato M., Sladowski D., Steiling W. & Brantom P.
The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU Phototoxicity test.
Toxicology in vitro (1998a) 12, 305-327.
7. Spielmann H., Balls M., Dupuis J., Pape W.J.W., de Silva O; Holzhütter H.G., Gerberick F., Liebsch M., Lovell W.W. & Pfannenbecker U.
A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test.
ATLA (1998 b) 26, 679-708.
8. Anon.
Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxic potential).
ATLA (1998) 26, 7-8.
9. Guillot J.P., Gonnet J.F., Loquerie J.F., Martin M.C., Convert P. & Cotte J. (1985).
A new method for the assessment of phototoxic and photoallergic potentials by topical applications in the albino guinea pig.
J Toxicol Cut & ocular toxicol. (1985) 4, 117-134.
10. Kaidbey K.H., Kligman A.M.
Photomaximisation tests for identifying photoallergic contact sensitisers.
Contact Dermatitis (1980) 6, 161-169.
11. Mandate for SCCNFP Specific Working Group on alternative methods, for the safety evaluation of cosmetic products (XXIV/1890/98) adopted on 20 May 1998.
