2,7-Naphthalenedisulfonic 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)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 29, 2018 to June 14, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes

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

Test material information

Constituent 1

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:

695-779-2

Cas Number:

80019-35-8

Molecular formula:

C29H21ClN8O17S5.4Na

IUPAC 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)

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

Results

Results 1

Run / experiment:

other: 1

Parameter:

other: Cys % depletion

Remarks:

1:10 Cysteine

Value:

0.135

Vehicle controls validity:

valid

Results 2

Parameter:

other: mean % depletion

Remarks:

Cysteine 1:10/lysine 1:50

Value:

92.957

Vehicle controls validity:

valid

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 (Cysteine)	Run 2 (Lysine)	Outcome
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

Conclusions:

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.