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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 29, 2018 to June 14, 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
Type of study:
other: peptide depletion
Justification for non-LLNA method:
OECD 442C cites the DPRA model as a validated method for skin sensitization testing in the context of an integrated approach to testing and assessment.

Test material

Constituent 1
Chemical structure
Reference substance name:
2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)
EC Number:
Cas Number:
Molecular formula:
2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)
Test material form:
solid: particulate/powder

In vitro test system

Details on the study design:
Characterisation of the test method:
The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-led validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Method workflow summary
Test items were incubated for 24 hours (±2hours) at 25±2.5°C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an HPLC system (20-minute-runtime) using gradient elution and UV detection at 220nm to measure peptide concentration. Test items were compared to reference controls containing the test item solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion Relative percent peptide depletion values were used in a prediction model that assigns test items to one of four reactivity classes.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: 1
other: Cys % depletion
1:10 Cysteine
Vehicle controls validity:
other: mean % depletion
Cysteine 1:10/lysine 1:50
Vehicle controls validity:
Other effects / acceptance of results:
All acceptance criteria were met in the Cysteine Run 1. However, multiple criteria for Lysine were not met in Run 2 (orange cells) and in addition there was significant co-elution of the test item, in the lysine peptide buffer. Hence the lysine data was not subsequently used and the Cysteine data and cysteine 1:10 prediction model alone were used.

Any other information on results incl. tables

Table 1. Acceptance criteria of Cysteine and Lysine

Criterion Run 1
Run 2
Std Curve r^2>0.99 0.993 0.988 PASS/FAIL
PC 60.8% to 100% depletion Cys 69.689% N/A PASS
PC 40.2% to 69.4% depletion Lys N/A 5.555% FAIL
SD Cys Depletion PC<14.9% 0.437% N/A PASS
SD Lys Depletion PC<11.6% N/A No Data FAIL
RefA Mean Conc 0.50±0.05mM 0.523 M 0.515 mM PASS/PASS
Peak Area CV RefB<15.0% 1.661% 77.573% PASS/FAIL
Peak Area CV RefC<15.0% 0.552% 173.205% PASS/FAIL
SD Cys Depletion Test Item<14.9% 0.135% N/A PASS
SD Lys Depletion Test Item<11.6% N/A 1.795% PASS
RefC Mean Conc 0.50±0.05mM 0.524 mM 0.184 mM PASS/FAIL

Cys = Cysteine, Lys = Lysine, SD = Standard Deviationm CV = Coefficient of Variation, PC = Positive Control.

Note that standard 2 and standard 7 for Cysteine was excluded from the standard curve as they were outlier values. The standard curve was produced successfully using the remaining 5 points, therefore there was no impact upon the final data.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
The final mean %peptide depletion observed in the DPRA was 92.957%. Therefore, CR SR94 was classified as Positive with Moderate as per the Cysteine 1:10 prediction model.
Executive summary:

In this study, the skin sensitization potential of CR SR94 was assessed using the In Chemico Direct Peptide Reactive Assay (DPRA) method according to OECD442C. After a 24h incubation with both Cysteine and Lysine containing peptides, the percent peptide depletion was measured by High Performance Liquid Chromatography (HPLC).

For assessment of CR SR94 the Cysteine peptide 1:10 prediction model was used. The final mean % peptide depletion observed using this model was 92.957%. Therefore, CR SR94 was classified as Positive with Moderate reactivity as per the prediction model.