p-benzoquinone dioxime

Endpoint summary

Description of key information

Weight of Evidence: skin sensitising, 2018

1. Molecular initiating Key Event 1: positive (high peptide reactivity class), DPRA, OECD TG 442C, 2018

2. Molecular initiating Key Event 2: positive (LC induction > 1.5 in 2 of 2 experiments (n=3 replicates), KeratinoSens, OECD TG 442D, 2018

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-09-2018 to 26-09-2018

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-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:

uideline study performed under GLP. All relevant validity criteria were met. 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 of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

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, the test item was not soluble in ACN, MQ or ACN:MQ (1:1, v/v), but was soluble in IPA. Therefore, IPA solvent was used to dissolve the test item. 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 10.2 mg of SPCC in 20.36 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. In addition, a RCcysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL IPA. 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).
2. Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.6 mg of SPCL in 20.46 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. In addition, a RClysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL IPA. 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).
- 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 24.5 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 = 0.998 and SPLC r2 = 0.9942)
(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 70.4% ± 1.1% and 50.5% ± 3.8%)
(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 = 1.1% and SPCL PC : SD = 3.8%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C and CIPA reference controls: 0.514 ± 0.022 mM, 0.504 ± 0.002 mM and 0.489 ± 0.022 mM ; Lysine A, C and CIPA reference controls: 0.515 ± 0.015 mM, 0.529 ± 0.009 mM and 0.523 ± 0.012 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 = 2.3% ; Lysine: Reference Controls B and C was 3.2%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCC, however, at 258 nm a peak was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCC depletion, there was no interference of the test item with SPCC. It was not possible to calculate the mean SPCC A220/A258 area ratio since the test item displayed high reactivity towards SPCC. Overall, it was concluded that the test item did not interfere with SPCC at a wavelength of 220 nm for quantitative analysis purposes. Within the Lysine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCL whereas a small response at 258 nm was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCL depletion, there was no interference of the test item with SPCL. For the A-lys samples, the mean SPCL A220/A258 area ratio was 6.13. This was outside the 15.16-18.52 range, however, since the test item did not interfere with SPCL at 220 nm, it can be concluded that the responses at a wavelength of 220 nm were suitable for quantitative analysis purposes.
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 = Isopropanol

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)

Vehicle / solvent:

isopropanol

Positive control:

cinnamic aldehyde

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 70.4% ± 1.1% (high reactivity)
- PC LYS-peptide depletion (mean): 50.5% ± 3.8% (moderate reactivity)

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

100 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: HIGH REACTIVITY CLASS

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.6 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

n = 3 ; Test item appears to have no or minimal reactivity to Lys peptide ; See 'any other information on results incl. tables' for further information

Outcome of the prediction model:

high reactivity [in chemico]

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 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 = 0.998 and SPLC r2 = 0.9942)
(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 70.4% ± 1.1% and 50.5% ± 3.8%)
(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 = 1.1% and SPCL PC : SD = 3.8%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C and CIPA reference controls: 0.514 ± 0.022 mM, 0.504 ± 0.002 mM and 0.489 ± 0.022 mM ; Lysine A, C and CIPA reference controls: 0.515 ± 0.015 mM, 0.529 ± 0.009 mM and 0.523 ± 0.012 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 = 2.3% ; Lysine: Reference Controls B and C was 3.2%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCC, however, at 258 nm a peak was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCC depletion, there was no interference of the test item with SPCC. It was not possible to calculate the mean SPCC A220/A258 area ratio since the test item displayed high reactivity towards SPCC. Overall, it was concluded that the test item did not interfere with SPCC at a wavelength of 220 nm for quantitative analysis purposes. Within the Lysine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCL whereas a small response at 258 nm was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCL depletion, there was no interference of the test item with SPCL. For the A-lys samples, the mean SPCL A220/A258 area ratio was 6.13. This was outside the 15.16-18.52 range, however, since the test item did not interfere with SPCL at 220 nm, it can be concluded that the responses at a wavelength of 220 nm were suitable for quantitative analysis purposes. 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	0.998	>0.99	0.994
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.514 ± 0.022	0.50 ± 0.05	0.515 ± 0.015
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.504 ± 0.002	0.50 ± 0.05	0.529 ± 0.009
Mean peptide concentration RC-CIPA samples (mM)	0.50 ± 0.05	0.489 ± 0.022	0.50 ± 0.05	0.523 ± 0.012
CV (%) for RC samples B and C	<15.0	2.3	<15.0	3.2
Mean peptide depletion PC (cinnamic aldehyde) (%)	60.8-100	70.4	40.2-69.0	50.5
SD of peptide depletion PC (cinnamic aldehyde) (%)	<14.9	1.1	<11.6	3.8
SD of peptide depletion for the test item (%)	<14.9	0.0	<11.6	0.5

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	100.0%	±0.0%	0.6%	±0.5%	50.3%

Interpretation of results:

other: The test item gave a positive in DPRA and was classified in the “high 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

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to the skin. The test item indicated a positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

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. Isopropanol (IPA) was found to be an appropriate solvent to dissolve the test item. Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. After incubation of the SPCC and SPCL test item samples, a precipitate was observed. In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.3% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17-07-2018 to 17-08-2018

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 211 mM (black suspension or solution; due to the dark colour it was not possible to judge whether the stock a solution or suspension). The stock was sonicated (Time: 11 min; Temp.: 21 - 28 °C). The 100-fold dilution of the 211 mM DMSO stock in DMEM glutamax formed a clear solution. The 2000 µM (200 mM stock) concentration in DMSO was selected as highest concentration for the main assay.
- Test Item preparation and concentrations: In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 200 mM (brown suspension). The stock was sonicated (Time: 10 min; Temp.: 21 - 27 °C). 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 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: 81 µM; Experiment 2: 31 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 81 µM and 31 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (1.87-fold and 3.07-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: 19.0% ; Experiment 2: 7.2%.

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+9 in experiment 1 and P+11 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) 794 μM ; and Experiment 2: mean (n=3) 387 μM
Test item IC30: IC30 value as the concentration in μM reducing the viability by 30%
Experiment 1: mean (n=3) 713 μM ; and Experiment 2: mean (n=3) 342 μM

- Luciferase assay
Imax indicating maximum fold-induction up to concentration 2000 μM
Experiment 1: mean (n=3) 3.36 and Experiment 2: mean (n=3) 8.11

- Determinations:
EC1.5
Experiment 1: mean (n=3) 81 μM and Experiment 2: mean (n=3) 31 μM

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:
2 out of 2 positive experiments (each in triplicate). 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 positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM 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: 81 µM; Experiment 2: 31 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 81 µM and 31 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (1.87-fold and 3.07-fold in experiment 1 and 2, respectively).

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 279 valid runs are presented in the full study report.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Remarks:

/ µM

Value:

81 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

mean Experiment 1 (n = 3)

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Remarks:

/ µM

Value:

31 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

mean Experiment 2 (n=3)

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: 81 µM; Experiment 2: 31 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 81 µM and 31 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold in experiment 2 (1.87-fold and 3.07-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: 19.0% ; Experiment 2: 7.2%.

Interpretation of results:

other: The test item gave 2 out of 2 positive experiments (each in triplicate). The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to the skin. The test item gave 2 out of 2 positive experiments (each in triplicate) >1.5-fold induction observed at test concentrations < 1000 µM 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 suspended in dimethyl sulfoxide at 200 mM. From the stock solution, 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 (81 µM and 31 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 (1.87-fold and 3.07-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% (19% and 7.2% in experiment 1 and 2, respectively). The test item showed toxicity (IC30 values of 713 µM and 342 µM and IC50 values of 794 µM and 387 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 22 µM and 17 µM in experiment 1 and 2) was measured in both experiments. The maximum luciferase activity induction (Imax) was 3.36-fold and 8.11-fold in experiment 1 and 2. The test item is classified as positive in the KeratinoSens assay since positive results (> 1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of > 70% compared to the vehicle control. All relevant test acceptability criteria were met.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2018 : 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. Isopropanol (IPA) was found to be an appropriate solvent to dissolve the test item. Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. After incubation of the SPCC and SPCL test item samples, a precipitate was observed. In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.3% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met.

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2017: 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 suspended in dimethyl sulfoxide at 200 mM. From the stock solution, 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 (81 µM and 31 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 (1.87-fold and 3.07-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% (19% and 7.2% in experiment 1 and 2, respectively). The test item showed toxicity (IC30 values of 713 µM and 342 µM and IC50 values of 794 µM and 387 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 22 µM and 17 µM in experiment 1 and 2) was measured in both experiments. The maximum luciferase activity induction (Imax) was 3.36-fold and 8.11-fold in experiment 1 and 2. The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM 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 this study by expert judgement and indicates that the weight of evidence that there is evidence of skin sensitisation in two validated in chemico (DPRA) and in vitro (KeratinoSens) skin sensitization assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitization. There is also by expert judgement evidence of reliable skin sensitization alerts in relevant in silico models. The weight of evidence is indicative of skin sensitisation potential of the substance in accordance with the Regulation (EC) 1272/2008 criteria.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation: category 1A: H317: May cause an allergic skin reaction