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)

Endpoint summary

Description of key information

For OECD 442C, the final mean % 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.

For OECD 442D, the test item induced statistically significant luciferase induction >1.5 in all 3 repetitions. The respective EC1.5 values were calculated as 1.47 µM, 7.03 µM and 31.25 µM for repetitions 1 to 3 respectively. The statistical significance viability, dose response and dose acceptance criteria were all met. Therefore, the test item was classified as positive using the KeratinoSens prediction model.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

Run / experiment:

other: 1

Parameter:

other: Cys % depletion

Remarks:

1:10 Cysteine

Value:

0.135

Vehicle controls validity:

valid

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 16, 2018 to April 30, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes

Type of study:

other: immotalized adherent cell line derived from HaCaT human keratinocytes

Details on the study design:

Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, passage number 17.
Day 2: 24 hours after seeding, the test and control items were applied and plates were incubated at 37°C, 5% CO2, ≥95% relative humidity for 48±2 hours.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates).

Run / experiment:

other: 1

Parameter:

other: EC1.5 value

Remarks:

µM

Value:

1.47

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: EC1.5 value

Remarks:

µM

Value:

7.03

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: EC1.5 value

Remarks:

µM

Value:

31.25

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Solubility Assessment

The test concentrations of CR SR94 used in the KeratinoSens TM method were selected on the basis of solubility test carried out during the study:

Solubility of the test item in was confirmed up to 200mM in DMSO. Subsequent dilution in cell culture medium gave a top concentration of 2000µM.

Determination of the skin sensitisation potential of CR SR94

Table 1. Sensitisation Potential of the Test Item: Repetition 1

Repetition 1	Test item concentration (µM)
	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125.00	250.00	500.00	1000.00	2000.00
Mean fold induction	1.007	1.987	1.793	1.767	1.785	1.951	2.067	2.053	2.366	5.255	64.448	0.060
Viability%	242.951	103.104	101.248	96.697	77.872	95.418	99.617	93.515	88.35	85.518	78.605	7.929
T-test	9.29E-01	9.14E-08	1.10E-08	7.22E-11	8.80E-13	1.68E-14	2.61E-18	5.12E-18	7.70E-22	8.70E-41	8.13E-32	1.41E-17
SD	0.099	1.021	0.654	0.418	0.280	0.378	0.238	0.244	0.319	0.614	18.226	0.034
Imax	64.448 at 1000µM
EC1.5	1.47µM
IC30	1121.75µM
IC50	1404.73µM

Table 2. Sensitisation Potential of the Test Item: Repetition 2

Repetition 2	Test item concentration (µM)
	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125.00	250.00	500.00	1000.00	2000.00
Mean fold induction	0.881	1.131	0.989	1.628	2.076	1.35	1.249	1.191	0.844	1.773	5.917	0.002
Viability%	116.410	129.377	138.189	140.778	136.881	122.87	120.811	118.448	95.299	73.029	29.512	7.094
T-test	1.37E-01	9.12E-02	9.00E-01	1.54E-09	5.36E-13	7.39E-05	3.66E-03	1.87E-02	4.85E-02	8.27E-13	1.64E-29	1.08E-18
SD	0.167	0.066	0.288	0.307	0.623	0.222	0.232	0.159	0.112	0.255	1.473	0.003
Imax	5.917 at 1000µM
EC1.5	7.03µM
IC30	534.80µM
IC50	764.60µM

Table 3. Sensitisation Potential of the Test Item: Repetition 3

Repetition 3	Test item concentration (µM)
	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125.00	250.00	500.00	1000.00	2000.00
Mean fold induction	0.937	1.060	1.129	1.376	1.394	1.500	1.741	1.783	1.753	2.021	12.944	4.095
Viability%	122.173	124.664	135.718	130.892	121.706	141.866	126.699	132.592	140.955	126.376	114.69	38.931
T-test	4.16E-01	4.32E-01	1.03E-01	1.22E-05	4.43E-06	6.83E-08	1.62E-12	3.95E-14	1.99E-12	6.03E-16	1.83E-43	3.93E-14
SD	0.108	0.052	0.134	0.142	0.117	0.193	0.223	0.123	0.255	0.359	1.980	0.221
Imax	12.944 at 1000µM
EC1.5	31.25µM
IC30	1589.90µM
IC50	1853.89µM

Dtermination criteria for the skin sensitisation potential of the test item

	REP 1	REP 2	REP 3
Does at least one concentration of Test Item induce luciferase activity≥1.5-fold?	Yes	Yes	Yes
Does the first concentration inducing luciferase activity equal to or above 1.5, have a viability above 70%?	Yes	Yes	Yes
Is p value<0.05 for at least one concentration that yielded≥1.5-fold induction with viability above 70%?	Yes	Yes	Yes
Does EC1.5 value occur at a concentration <1000µM (<200µg/mL)	Yes	Yes	Yes
Does the test item induce the luciferase in a dose-dependent manner?	Yes	Yes	Yes
Classification	Positive	Positive	Positive

Assay Acceptance Criteria (Mean of 3 repetitions)

Criteria		Result	PASS or FAIL
1	Positive Control (PC) (Cinnamic aldehyde) induction≥1.5-fold in at least one concentration	Yes (4/5)	PASS
2	Average induction of PC at 32µM is [1.6~3.0]	Yes (2.161)	PASS
3	EC1.5 value is [6-39µM]	Yes (14.024µM)	PASS
4	CV% of blank values <20%	Yes (10.1038%)	PASS

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item induced statistically significant luciferase induction >1.5 in all 3 repetitions. The respective EC1.5 values were calculated as 1.47 µM, 7.03 µM and 31.25 µM for repetitions 1 to 3 respectively. The statistical significance viability, dose response and dose acceptance criteria were all met. Therefore, the test item was classified as positive using the KeratinoSens prediction model.

Executive summary:

The human skin sensitisation potential of CR SR94 was assessed using the validated in vitro method, the KeratinoSens TM assay, adapted to animal product-free conditions by XCellR8, and validated in-house to determine keratinocyte activation.

 

After 48h exposure of cells with 12 concentrations of CR SR94, Luciferase measurements and MTT viability testing were performed.

 

CR SR94 was classified as Positive according to the KeratinoSens prediction model.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation.