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