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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

Referenceopen allclose all

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.

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.