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Protocol no. 78
3T3 NRU PHOTOTOXICITY TEST

  • The cytotoxocity of the test compound to 3T3 cells is assessed by Neutral Red uptake following exposure in the presence or absence of UVA light.
     Contact Person
    Dr. med. Horst Spielmann
    Direktor und Professor
    Head of ZEBET

    ZEBET at the BgVV
    Diedersdorfer Weg 1
    D-12277 Berlin, Germany
    phone +49.1888.412.2270
    fax   +49.1888.412.2958
    e-mail: zebet@bgvv.de
    & spielmann.zebet@bgvv.de

    • June 8 of the year 2000 
    The EU Commission has finaly published Commission Directive 2000/33/EC in the Official Journal of the EU Commission on pages L36/90-L136/107

    Commission Directive 2000733/EC of April 25 2000 is entitled "adapting to technicalprogress for the 27th time Councoil Directive
    67/548/EEC oin the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of
    dangerous substances".

    The 27th amendment of EU Dir. 67/548/EEC is regulating for the first time the regulatory acceptance of 2 in vitro toxicity tests into Annex V
    to the Directive, which contains the guidelines for  toxicity testing that are approved for regulatory purposes in EU member states.
    The 2 in vitro toxicitxy tests that have been accepted into Annex V are Method B 40 SKIN CORROSION (Annex I to EU Dir. 2000/33/EC), pg L136/91-97 and Method B 41 PHOTOTOXICITY - IN VITRO 3T3 NRU PHOTOTOXICITY TEST
    (Annex I to EU Dir. 2000/33/EC), pg. L136/98-107.

    l_13620000608en00010089.pdf = English version
    l_13620000608de00010089.pdf = German version

    The Ofifcial Journal, where you can download the information from the internet:
    http://europa.eu.int/eur-lex/en/dat/2000/l_136/l_13620000608en00900107.pdf


    REFERENCES

    (1) Spielmann, H., Balls, 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., Pfannbecker, U., Potthast, J., De
    Silva, O., Steiling, W. and Willshaw, A. ( 1994), EEC/COLIPA project on in vitro phototoxicity testing:
    First results obtained with a Balb/c 3T3 cell phototoxicity assay, Toxicology in Vitro 8, pp. 793-796.
    (2) Anon (1998), Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for
    phototoxicity), European Commission, Joint Research Centre: ECVAM and DGXI/E/2, 3 November
    1997, ATLA 26, pp. 7-8.
    (3) Spielmann, H., Balls, M., Dupuis, J., Pape, W. J. W., Pechovitch, G., De Silva, O., Holzhütter, H. G.,
    Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W. W., Maurer, T., Pfannenbecker, U.,
    Potthast, J. M., Csato, M., Sladowski, D., Steiling, W. and Brantom, P. (1998), EU/COLIPA iUIn vitro
    phototoxicityl, validation study, results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test,
    Toxicology in Vitro 12, pp. 305-327.
    (4) OECD Test Guidelines Programme, ENV/MC/CHEM/TG(96)9: Final Report of the OECD Workshop on
    Harmonisation of Validation and Acceptance Criteria of Alternative Toxicological Test Methods, OECD
    Publications Office, Paris, 1996.
    (5) Lovell, W.W. (1993), A scheme for in vitro screening of substances for photoallergenic potential,
    Toxicology in Vitro 7, pp. 95-102.
    (6) Santamaria, L. and Prino, G. (1972), List of the photodynamic substances, Research progress in organic,
    biological and medicinal chemistry Vol. 3 Part 1, North Holland Publishing Co, Amsterdam, pp. XI-XXXV.
    (7) Spielmann, H., Lovell, W.W., Hölzle, E., Johnson, B.E., Maurer, T., Miranda, M.A., Pape, W.J.W., Sapora,
    O. and Sladowski, D. (1994), In vitro phototoxicity testing: The report and recommendations of ECVAM
    workshop 2, ATLA 22, pp. 314-348.
    (8) Spikes, J.D. (1989), Photosensitization, The science of photobiology, edited by KC Smith, Plenum Press,
    New York, 2nd edition, pp. 79-110.
    (9) Borenfreund, E. and Puerner, J.A. (1985), Toxicity determination in vitro by morphological alterations
    and neutral red absorption, Toxicology Letters 24, pp. 119-124.
    (10) Lambert L. A, Warner W.G. and Kornhauser A. (1996), Animal models for phototoxicity testing,
    Dermatotoxicology, edited by FN Marzulli and HI Maibach, published by Taylor & Francis, Washington
    DC, 5th Edition, pp. 515-530.
    (11) Tyrrell R.M. and Pidoux M (1987), Action spectra for human skin cells: estimates of the relative
    cytotoxicity of the middle ultraviolet, near ultraviolet and violet regions of sunlight on epidermal
    keratinocytes, Cancer Research 47, pp. 1825-1829.
    (12) ZEBET/ECVAM/COLIPA, Standard Operating Procedure: Balb/c 3T3 NRU Phototoxicity Test, drafted 23
    December 1997 by M. Liebsch and approved 6 March 1998 by the Management Team of the
    EU/COLIPA project irIn Vitro Photoirritationli.
    (13) Spielmann, H., Balls, M., Dupuis, J., Pape, W. J. W., De Silva, O., Holzhütter, H. G., Gerberick, F.,
    Liebsch, M., Lovell, W. W. and Pfannenbecker (1998), A Study on the Phototoxic Potential of UV Filter
    Chemicals from Annex VII of the EU Directive 76/768/EEC in the 3T3 NRU In Vitro Phototoxicity Test,
    ATLA 26, pp. 679-708.
    (14) Holzhütter, H.G. and Quedenau, J. (1995), Mathematical modelling of cellular responses to external
    signals, Journal of Biological Systems 3, pp. 127-138.
    (15) Holzhütter, H.G. (1997), A general measure of in vitro phototoxicity derived from pairs of dose-response
    curves and its use for predicting the in vivo phototoxicity of chemicals, ATLA 25, pp. 445-462.

    IP-78 June 2000