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ACUTE DERMAL TOXICITY USING THE OECD TG

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					                                                        ACUTE DERMAL TOXICITY USING THE OECD TG 404
                                         INTEGRATED TESTING STRATEGY COMBINING THE EpiSkin VALIDATED TEST METHODS.

                                                                                                         N Alépée • MH Grandidier • N Seyler • F Soler • J Cotovio • JR Meunier
                                                                                                                                                       L’Oréal Research & Innovation, France.
                                                                                                                                                              nalepee@rd.loreal.com



INTRODUCTION                                                                                                                                                    RESULTS
1       Skin corrosion
        1998: ECVAM/ESAC statement on the EpiSkin skin corrosion test method
                                                                                                                                                                    Impact of the interference signal due to MTT-reducing chemicals                                   Application of the testing strategy of TG 404 to chemicals listed by
        2000: Acceptance as EU Test Methods B.40 bis
        2004: OECD adoption of Test Guideline 431
                                                                                                                                                                                                                                                                                              NICEATM / ICCVAM
                                                                                                                                                                  MTT reducers test substances are able to cause a non specific change of color of
                                                                                                                                                                  MTT. The MTT reducers, which can interfere, show a potential for under-classification,               In 2009, the USA has a concern with that false negative corrosives are not detected
2       Skin Irritation
                                                                                                                                                                  leading to false negative outcome.                                                                  by the irritation in vitro validated test methods. Based on the review of data from
        2007: ECVAM/ESAC statement of EpiSkin under EU DSD
                                                                                                                                                                  Adapted killed tissues controls were established to defined the non specific MTT va-                  ECVAM validation studies, ICCVAM identified 15 test substances with high tendency
        2008: ECVAM/ESAC statement on the performance under UN GHS / EU CLP of EpiSkin
                                                                                                                                                                  lue and determine the accurate skin corrosion potential of MTT reducer agents.                      for misclassification as false negative corrosives. The substances represent various
        2009: Acceptance of Reconstructed human Epidermis as EU Test Method B.46
                                                                                                                                                                  The non-specific reduction of MTT by the test substances was assessed by measuring                   categories of chemicals such as amines, organic saturated acids, metal salts ….
        2010: OECD adoption of Test Guideline 439
                                                                                                                                                                  the relative conversion of MTT using killed tissues (KT). The treated tissue true MTT
                                                                                                                                                                  metabolic conversion was calculated to measure the viability of living cells.                       • Among those 15 in vivo corrosives, 12 test substances were correctly predicted as
3       Considering that now in vitro Test Guidelines for skin corrosion and irritation (TGs 431 and 439) have been adopted, the OECD TG 404
                                                                                                                                                                                                                                                                        skin corrosives. One of the 3 non-corrosive substances was predicted as irritating.
        Integrated Testing Strategy (ITS) should be revised. The purpose of the study was to evaluate the possible combinations of the in vitro skin
                                                                                                                                                                                                                                                                      • Applying the testing strategy of the OECD TG 404 (Figure 1), that combines
        corrosion and skin irritation EpiSkin validated test methods.
                                                                                                                                                                                                                                                                        measurement of pH, QSAR…, skin corrosion and skin irritation tests with EpiSkin,
                                                                                                                                                                                                                                                                        it was demonstrated that a probability for misclassification of these 15 corrosive
                                                                                                                                                                                                                                                                        substances is minimal.




                                                                                                 * Without any appropriate controls for MTT reducers

                                                                                                                            Table1:
                                                                                                                      Predictive Capacity
                                                                                                                                                                    • Among 9 in vivo corrosive MTT reducers, 8 test substances were correctly predicted as skin
                                                                                                            of the EpiSkin Validated Test methods                     corrosives.
                                                                                                                                                                    • No over-prediction using adapted controls, leading to false positive result was observed with
                            Figure 1:                                                                                                                                 the in vivo non corrosive substances.
        OECD Test Guideline 404 – Testing and evaluation
                                                                                                                                                                      ➡ APPLICATION OF THE SKIN CORROSION PROTOCOLS TO MTT
            strategy for dermal Irritation/Corrosion
                                                                                                                                                                        REDUCERS MIGHT BE REASONABLY RECOMMENDED.
                                                                                                                                                                      ➡ THE USE OF NON COLORIMETRIC APPROACH MIGHT BE CONSIDERED
                                                                                                                                                                        FOR TOO STRONG MTT INTERFERING REDUCERS.
METHODS
1       In vitro model
        Human reconstructed epidermis grown at air liquid interface.                                                                                                                                                             OECD TG404 application: a weight of evidence strategy
        EpiSkin tissues were supplied by SkinEthic Laboratories (www.skinethic.com).
                                                                                                                                                                  • Further testing with 41 additional substances,
2       Test substances                                                                                                                                             in both skin corrosion and irritation tests was
        More than 60 chemicals covering main in vivo skin corrosion/irritation classes, were evaluated.                                                             performed in EpiSkin to confirm whether these
                                                                                                                                                                    chemicals would not be missed when using
3       In vitro Skin Corrosion/Irritation Tests - Prediction Models                                                                                                top-down or bottom-up testing strategies.
        Each chemical was applied topically onto tissues. At the end of the treatment period, cytotoxicity was determined by the MTT conversion                   • The substances represent various categories
                                                                                                                                                                                                                                                                                                         Figure 2: Top-Down and Bottom-up approaches to all
        test. Adapted controls were added for MTT reducers and coloring/tissue staining test substances.                                                            of chemicals covering the in vivo skin corrosion/                                                                                                          chemicals
                                                                                                                                                                    irritation classes (31 non label; 15 irritants and                                                                                  for skin corrosion & skin irritation: Weight of Evidence
                                                                                                                                                                    13 corrosives).                                                                                                                                   with 59 chemicals in EpiSkin

                                                                                                                                                                  • None of the skin corrosive chemicals was
                                                                                                                                                                    predicted as non-irritating in the EpiSKin.
                                                                                                                                                                  • 9 out of 10 irritants were well classified.
                                                                                                                                                                    Over prediction was observed for non irritants
                                                                                                                                                                    such as cinnamaldehyde, known to be a
                                                                                                                                                                    sensitizer.

4       Performance
        Sensitivity (% of corrosives/irritants correctly predicted in vitro), specificity (% of No Label/No Category chemicals) and the concordance
        (% of chemicals correctly classified) were calculated.



CONCLUSIONS
    1    OECD TG 431 and SOP of EpiSkin test method for skin corrosion should be updated to include instructions for colored chemicals and
         direct MTT-reducers, as already included in skin irritation OECD = OECD TG 439.
    2    In vitro test methods that have been validated for skin corrosion or skin irritation under EU DSD should be considered valid also for UN
         GHS and EU CLP.
    3    Based on the skin corrosion validation study, EpiSkin is able to distinguish Category 1A from Cat 1B/C corrosives, essential for
         transportation of the substances (UN GHS and Transport of Dangerous Goods-Regulations).
    4    With the adoption of OECD TG 439 on in vitro skin irritation, the acute dermal irritation/corrosion of chemicals (OECD TG 404) can be
         fully addressed in vitro.
    5    Integrated Testing Strategy is not a strict sequence. The step-wise procedure, weight of evidence and finally testing should be
         considered as an acceptable approach to structure the relevant information that can be used for hazard assessment.

				
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