Registration Dossier

Administrative data

Description of key information

Weight of Evidence: non-skin sensitising, 2020

1. Molecular initiating Key Event 1: negative (or minimal peptide reactivity class), DPRA, OECD TG 442C, 2019

2. Molecular initiating Key Event 2: negative (no LC induction > 1.5 in 2 of 2 experiments at up to 2000 µM) (n=3 replicates), KeratinoSens, OECD TG 442D, 2019

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08-08-2019 to 27-08-2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v) and isopropanol (IPA). At a concentration of 100 mM, ST 10 C 19 was soluble in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), IPA, ethanol and methanol, but not in MQ and ACN:MQ (1:1, v/v). Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- HPLC-PDA (UV) methodology are reported in the full study report.
- Preparation of synthetic peptide solutions [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.7 mg of SPCC in 21.36 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.506 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.505 ± 0.002 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
2. Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.3 mg of SPCL in 19.88 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes. SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. The SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.505 ± 0.004 mM while the mean peptide concentration of Reference Controls C was 0.515 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Sample incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 23 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 1.000 and SPLC r2 = 0.9997)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 71.0% ± 0.6% and SPCL 63.1% ± 0.6%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.6% and SPCL PC : SD = 0.6%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.506 ± 0.002 mM, and 0.505 ± 0.005 mM ; Lysine A, C reference controls: 0.505 ± 0.004 mM and 0.515 ± 0.004 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.3% ; Lysine: Reference Controls B and C was 1.1%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 36.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.82. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile (ACN)
Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)
Positive control results:
- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 71.0% ± 0.6% (high reactivity)
- PC LYS-peptide depletion (mean): 63.1% ± 0.6% (high reactivity)
Key result
Run / experiment:
other: Mean (%)
Parameter:
other: Cys-peptide depletion
Value:
5.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Negative : No or minimal reactivity
Remarks:
n = 3 ; See 'any other information on results incl. tables' for further information
Key result
Run / experiment:
other: Mean (%)
Parameter:
other: Lys-peptide depletion
Value:
1.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
n = 3 ; Negative: No or minimal reactivity ; See 'any other information on results incl. tables' for further information
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.
- Other: In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples. In the Lysine reactivity assay: upon preparation as well as after incubation a phase separation was observed in the CC and the test item samples. In this case, it could not be precise the amount of test item remained in the solution.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 1.000 and SPLC r2 = 0.9997)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 71.0% ± 0.6% and SPCL 63.1% ± 0.6%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.6% and SPCL PC : SD = 0.6%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.506 ± 0.002 mM, and 0.505 ± 0.005 mM ; Lysine A, C reference controls: 0.505 ± 0.004 mM and 0.515 ± 0.004 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.3% ; Lysine: Reference Controls B and C was 1.1%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 36.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.82. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.

Table 1.0 – Acceptability of the DPRA

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

1.000

>0.99

0.9997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.506 ± 0.002

0.50 ± 0.05

0.505 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.505 ± 0.002

0.50 ± 0.05

0.515 ± 0.004

 

 

 

 

 

CV (%) for RC samples

B and C

<15.0

0.3

<15.0

1.1

Mean peptide depletion PC (cinnamic aldehyde) (%)

60.8-100

71.0

40.2-69.0

63.1

SD of peptide depletion PC (cinnamic aldehyde)

(%)

<14.9

0.6

<11.6

0.6

SD of peptide depletion for the test item

(%)

<14.9

0.6

<11.6

1.2

Where: RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

 

Table 2.0 – Results of the DPRA with the test item

SPCC depletion (CYSTEINE)

SPCL depletion (LYSINE)

Mean of SPCC and SPCL depletion

Mean

± SD

Mean

± SD

Test item

5.4%

±0.6%

1.4%

±1.2%

3.4%

 

Interpretation of results:
other: The test item gave a negative in DPRA and was classified in the “Negative: No or minimal reactivity class” using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for C&L purposes
Conclusions:
The test item gave a negative in DPRA and was classified in the “Negative: No or minimal reactivity class” using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes
Executive summary:

The study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. Upon preparation as well as after incubation of the SPCC test item samples, no precipitate or phase separation was observed in any of the samples. Upon preparation as well as after incubation of the SPCL test item samples, a phase separation was observed. Since phase separation was observed after the incubation period for SPCL, it cannot be precise the amount of test item which remained in the solution to react with the peptide. In the cysteine reactivity assay the test item showed 5.4% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 3.4% and as a result the test item was considered to be negative in the DPRA and classified in the “negative or no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met with acceptable restrictions.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01-08-2019 to 16-08-2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).
Type of study:
activation of keratinocytes
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422D – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method and EURL ECVAM DB-ALM Protocol no. 155: KeratinoSens™, (Adopted March, 2018).
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- A solubility test was performed. The test item was suspended or dissolved in DMSO to a final concentration of 200 mM (clear colourless). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation). This concentration was selected as highest concentration for the main assay.
- Test Item preparation and concentrations: In the main experiments the test item was dissolved in DMSO at 200 mM (colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Positive Control preparation and concentrations: The positive control was Ethylene dimethacrylate glycol (EDMG), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted to the final concentration ranges of 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.
- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 63 µM; Experiment 2: 36 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 63 µM and 36 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (2.65-fold and 2.96-fold in experiment 1 and 2, respectively).
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 5.6% ; Experiment 2: 3.9%.

Cell line used:
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene. The cell line was developed by supplier (full details in the full study report). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell culture and exposure:
Cells are grown for 24 h in 96-well plates. The maintainece medium was Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL). One day prior to testing cells were harvested and distributed into 96-well plates (10,000 cells/well) in basic medium (Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum). Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25). For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1 and P+8 in experiment 2. The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. The exposure medium was Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum. Three wells per plate were left empty (no cells and no treatment) to assess background values.

The following parameters are calculated in the KeratinoSens test method:
(i) The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
(ii) The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
(iii) The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

- Cell viability assay MTT:
Test item IC50: IC50 value as the concentration in μM reducing the viability by 50%
Experiment 1: mean (n=3) 667 μM ; and Experiment 2: mean (n=3) 462 μM
Test item IC30: IC30 value as the concentration in μM reducing the viability by 30%
Experiment 1: mean (n=3) 537 μM ; and Experiment 2: mean (n=3) 367 μM

- Luciferase assay
Imax indicating maximum fold-induction up to concentration 2000 μM
Experiment 1: mean (n=3) 1.25 and Experiment 2: mean (n=3) 1.39

- Determinations:
EC1.5
Experiment 1: could not be determined and Experiment 2: could not be determined ; due to no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations.

Evaluation criteria
Test item considered ‘negative’ where
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 µg/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

Results:
0 out of 2 positive experiments (each in triplicate). The test item showed toxicity with IC30 values of 537 μM and 367 μM and IC50 values of 667 μM and 462 μM in experiment 1 and 2, respectively. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.25-fold and 1.39-fold in experiment 1 and 2, respectively. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test item is classified as negative in the KeratinoSens assay since positive results (>1.5-fold induction) were not observed at test concentrations up to 2000 µM (and/or with a cell viability of > 70% compared to the vehicle control).
Positive control results:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 63 µM; Experiment 2: 36 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 63 µM and 36 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (2.65-fold and 2.96-fold in experiment 1 and 2, respectively).
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 5.6% ; Experiment 2: 3.9%.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 341 valid runs are presented in the full study report.
Key result
Run / experiment:
other: mean Experiment 1 (n = 3)
Parameter:
other: EC1.5
Remarks:
/ µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No EC1.5 > 1.5 could be determined ; no luciferase activity induction
Key result
Run / experiment:
other: mean Experiment 2 (n=3)
Parameter:
other: EC1.5
Remarks:
/ µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No EC1.5 > 1.5 could be determined ; no luciferase activity induction
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: - The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 63 µM; Experiment 2: 36 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 63 µM and 36 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (2.65-fold and 2.96-fold in experiment 1 and 2, respectively).
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 5.6% ; Experiment 2: 3.9%.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 341 valid runs are presented in the full study report.
Interpretation of results:
other: The test item gave 0 out of 2 positive experiments (each in triplicate). The result will be considered within a weight of evidence assessment for C&L purposes
Conclusions:
Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item gave 0 out of 2 positive experiments (each in triplicate) with >1.5-fold induction observed at test concentrations up to 2000 µM (and/or with a cell viability of >70% compared to the vehicle control).
Executive summary:

The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between two standard deviations of the historical mean (63 µM and 36 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 2 (2.65-fold and 2.96-fold in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.6% and 3.9% in experiment 1 and 2, respectively). The test item showed toxicity with IC30 values of 537 μM and 367 μM and IC50 values of 667 μM and 462 μM in experiment 1 and 2, respectively. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.The maximum luciferase activity induction (Imax) was 1.25-fold and 1.39-fold in experiment 1 and 2, respectively. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test item is classified as negative in the KeratinoSens assay since no positive results (>1.5-fold induction) were observed at test concentrations up to 2000 µM (and/or with a cell viability of > 70% compared to the vehicle control). All relevant test acceptability criteria were met.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2019 : The study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. Upon preparation as well as after incubation of the SPCC test item samples, no precipitate or phase separation was observed in any of the samples. Upon preparation as well as after incubation of the SPCL test item samples, a phase separation was observed. Since phase separation was observed after the incubation period for SPCL, it cannot be precise the amount of test item which remained in the solution to react with the peptide. In the cysteine reactivity assay the test item showed 5.4% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 3.4% and as a result the test item was considered to be negative in the DPRA and classified in the “negative or no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met with acceptable restrictions.

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2019: The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between two standard deviations of the historical mean (63 µM and 36 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 2 (2.65-fold and 2.96-fold in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.6% and 3.9% in experiment 1 and 2, respectively). The test item showed toxicity with IC30 values of 537 μM and 367 μM and IC50 values of 667 μM and 462 μM in experiment 1 and 2, respectively. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.The maximum luciferase activity induction (Imax) was 1.25-fold and 1.39-fold in experiment 1 and 2, respectively. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test item is classified as negative in the KeratinoSens assay since no positive results (>1.5-fold induction) were observed at test concentrations up to 2000 µM (and/or with a cell viability of > 70% compared to the vehicle control). All relevant test acceptability criteria were met.

 

Weight of Evidence Conclusion:

The applicant assesses by expert judgement the available information and indicates based on the weight of evidence that there is no evidence of skin sensitisation in two validated in chemico (DPRA) and in vitro (KeratinoSens) assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitisation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation.