2-octyldodecyl 5-oxo-L-prolinate

Endpoint summary

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:

key study

Study period:

From July 03, 2017 to July 14, 2018

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))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Details on the study design:

See below 'Any other information for materials and methods incl. tables' for details on study design.

Reference Substance
Reference Substance Name: Cinnamic aldehyde, kosher Test Facility Test Item Number: RS473/A
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKBP1014V
Purity: 98.4%
Test item storage: In the refrigerator (2-8°C)
Stable under storage conditions until: 31 May 2018
Purity/composition correction factor: Yes

Run / experiment:

other: 3

Parameter:

other: % Mean Cysteine Peptide Depletion

Value:

7.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: % Mean Lysine Peptide Depletion

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3 each Cys and Lys

Parameter:

other: Mean % Peptide Depletion (Cys + Lys)

Value:

5.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Since precipitation was observed in both peptide solutions (Cys and Lys), amount of test substance that remained in the solution to react with the peptides is unknown. Consequently, this negative result is uncertain.

Other effects / acceptance of results:

All acceptance/validation criteria were met.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and C(isopropanol), the Coefficient of Variation for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test substance, were all within the acceptability criteria for the DPRA.

The means of Reference and Positive Control samples in either of Cysteine or Lysine Reactivity Assay were within the acceptance criteria, this confirms the suitability of the HPLC system and indicates that the solvent used to dissolve the test substance did not impact the % Peptide Depletion.

The area ratio (A220/A258) of the Reference Control samples in either of Cysteine or Lysine Reactivity Assay were within range (12.77-19.59) giving indication that no co-elution occurred.

Results

 

Solvent Selection

Isopropanol was found to be an appropriate solvent to dissolve the test substance and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

Acceptance criteria

Acceptance criteria for all controls and the test substance were met in both Cysteine or Lysine Reactivity Assay 

Acceptability of the Direct Peptide Reactivity Assay (DPRA)
	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability	Results for	Acceptability	Results for
	criteria	SPCC	criteria	SPCL
Correlation coefficient (r2)	>0.99	0.991	>0.99	0.994
standard calibration curve
Mean peptide concentration	0.50 ± 0.05	0.517 ± 0.008	0.50 ± 0.05	0.521 ± 0.013
RC-A samples (mM)
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.511 ± 0.008	0.50 ± 0.05	0.528 ± 0.009
Mean peptide concentration	0.50 ± 0.05	0.468 ± 0.006	0.50 ± 0.05	0.522 ± 0.017
RC-Cwatersamples (mM)
CV (%) for RC samples	<15.0	1.4	<15.0	2.5
B and C
Mean peptide depletion	60.8-100	71.2	40.2-69.0	50.5
cinnamic aldehyde (%)
SD of peptide depletion	<14.9	0.2	<11.6	1.8
cinnamic aldehyde (%)
SD of peptide depletion for the test substance (%)	<14.9	1.2	<11.6	2.5
RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Results Summary

The test substance produced 5.20% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. However, since precipitation was observed upon addition of the test substance to the peptide solutions, Amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test substance
SPCC		SPCL		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
depletion		depletion
Mean	± SD	Mean	± SD	5.20%	Cysteine 1:10 / Lysine 1:50 prediction model
7.10%	±1.2%	3.30%	±2.5%		Negative: No or minimal reactivity
SD = Standard Deviation

 

Conclusions:

the test substance was determined to be non-sensitising. However, since precipitation was observed upon addition of the test substance to the peptide solutions, the amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care.

Executive summary:

A study was conducted to determine the skin sensitisating potential of the test substance according to OECD Guideline 442C, in chemico Direct Peptide Reactivity Assay (DPRA), in compliance with GLP. Test samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) at 25 ± 2.5˚C for 24 - 30 h. After incubation, the relative peptide concentration was determined by HPLC using gradient elution and photodiode array (PDA) detector to measure peptide concentration. Test substances were compared to reference controls in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes for skin sensitisation. Acceptance criteria for all controls and the test substance were met in either of Cysteine or Lysine Reactivity Assay. The test substance produced 5.2% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitising with no or minimal reactivity. Under study conditions, the test substance was determined to be non-sensitising. However, since precipitation was observed upon addition of the test substance to the peptide solutions, the amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care (Reinen, 2017).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 01, 2017 to October 06, 2017

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)

Version / remarks:

February, 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Details on the study design:

Principle of the Test:
The test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an Antioxidant response element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 (transcription factor involved in the antioxidant response pathway) dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Test chemicals are considered positive if they induce a statistically significant induction of the luciferase activity above a given threshold (i.e. > 1.5 fold or 50% increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 µM and at a concentration at which the cellular viability is above 70%).

Experimental Materials
- Test System Source and Maintainence: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). 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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell Culture medium:
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Test substance, controls and working solutions:
- A correction factor was used to correct for the purity of of the test substance
- Vehicle: Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
- Negative Control: The negative control is the vehicle of the test substance.
- Positive Control (RS582): Ethylene dimethacrylate glycol (Batch# SHBG0572V)
- The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
- On each plate three blank wells were tested (no cells and no treatment).

Test substance solubility test: The test substance was suspended in DMSO to a final concentration of 200 mM. The 100-fold dilution of the 200 mM DMSO stock in DMEM formed a homogeneous solution (slight precipitation). At concentrations of 100 mM and lower the test substance was fully soluble. In the main experiments the test substance was suspended in dimethyl sulfoxide (DMSO), and followed dilution series to get following test concentrations:
Three experiments were conducted: Initially, experiment 1 and 2 obtained different results for luciferase activity and therefore did not pass all the acceptability criteria and therefore was repeated to complete 2 valid experiments.
- experiment 1: 875, 438, 219, 109, 55, 27, 14, 6.8, 3.4, 1.7, 0.85 and 0.43 µM
- experiment 2: 851, 426, 213, 106, 53, 27, 13, 6.6, 3.3, 1.7, 0.83 and 0.42 µM
- experiment 3: 1000, 100, 67, 44, 30, 20, 13, 8.8, 5.9, 3.9, 2.6 and 1.7 µM
- All formulations formed a clear solution.

Number of Run: All concentrations of the test substance and positive controls were tested in triplicate.


Experimental Design
- Treatment of Cells: One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. 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 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. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 h at 37±1.0ºC in the presence of 5% CO2. In total 3 valid experiments were performed.

- Luciferase Activity Measurement: After the 48 h exposure the medium was removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

- For cell viability assay, medium was replaced after the 48 h exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 h at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

- Environmental condition: Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations were not considered to affect the study integrity.

Positive control results:

Results of Positive control passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (81 µM, 34 µM and 84 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.17-fold, 3.13-fold and 2.35-fold in experiment 1, 2 and 3, respectively).

Key result

Run / experiment:

other: 1,3

Parameter:

other: EC1.5 value

Remarks:

Test substance concentration (μM) needed for a statistically significant (p<0.001) induction of the luciferase activity

Value:

4.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments.

Other effects / acceptance of results:

- The average coefficient of variation of the luminescence reading for the negative (solvent) control was below 20% (6.9%, 6.3% and 5.9% in experiment 1, 2 and 3, respectively). Overall it was concluded that the test conditions were adequate and that the test system functioned properly.

- The test substance showed toxicity (IC₃₀values of 11 µM, 5.4 µM and 12 µM and IC₅₀values of 12 µM, 5.9 µM and 13 µM in experiment 1, 2 and 3, respectively).

- In the first experiment, a statistically significant induction of the luciferase activity (EC_1.5 value 4.2 µM; p<0.001 Student’s t test) was measured) The maximum luciferase activity induction (I_max) was 1.68 -fold.

- In the second experiment, no biologically relevant luciferase induction was observed at all experimental concentrations, therefore no EC_1.5 could be calculated.

- Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC_1.5 value 5.0 µM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (I_max) was 1.92 -fold.

- Overall, the test substance was classified as positive in the KeratinoSensTM assay since positive results (>1.5 -fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments. In conclusion, test substance was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Table 1: Overview Luminescence Induction and Cell Viability in Experiment 1, 2 and 3
Exp.1	Concentration (µM)	0.43	0.85	1.7	3.4	6.8	14	27	55	109	219	438	875
	luminescence	1.21	1.16	1.27	1.44	1.68***	0.08	0	0	0	0	0	0
	viability (%)	106	100.3	100	123	154	11.8	0.1	-0.1	14	-0.2	-0.2	-0.1
Exp.2	Concentration (µM)	0.42	0.83	1.7	3.3	6.6	13	27	53	106	213	426	851
	luminescence	0.72	0.91	0.96	1.37	0.32	0.09	0	0	0	0	0	0
	viability (%)	114	109.3	118	140.7	24.8	2.7	0.1	-0.1	-0.1	-0.1	-0.1	0.1
Exp.3	Concentration (µM)	1.7	2.6	3.9	5.9	8.8	13	20	30	44	67	100	1000
	luminescence	1.1	1.22	1.39	1.60***	1.92***	0.55	0.4	0.1	0	0.01	0	0
	viability (%)	113	122.1	132	152.5	148.6	43.3	22	5.2	0.8	-0.1	0	-0.1
*** p<0.001 Student’s t test

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2
Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.13	1.1	1.23	1.38	1.80***	2.17***
Exp 1 viability (%)	111	116	108	117.3	121.4	121.8
Exp 2 luminescence	1.26	1.34	1.48	1.73***	2.49***	3.13***
Exp 2 viability (%)	104	116	117	120.3	120	123.5
Exp 3 luminescence	1.1	1.2	1.29	1.44	1.61***	2.35***
Exp 3 viability (%)	99.3	101	108	113.5	114.2	115.1
*** p<0.001 Student’s t test

Table 3: Overview EC1.5, Imax, IC30 and IC50 Values
	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test substance Experiment 1	4.2	1.68	11	12
Test substance Experiment 2	NA	1.37	5.4	5.9
Test substance Experiment 3	5	1.92	12	13
Pos Control Experiment 1	81	2.17	NA	NA
Pos Control Experiment 2	34	3.13	NA	NA
Pos Control Experiment 3	84	2.35	NA	NA
NA = Not applicable

Conclusions:

Under study conditions, the test substance was concluded to be skin sensitiser

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase (KeratinoSens) test method according to OECD Guideline 442D, in compliance with GLP. Three independent experiments were performed. Cells were incubated with test substance at concentration ranges of 0.43 – 875 µM, 0.42 – 851 µM and 1.7 - 1000 µM for 48 h in the first, second and third experiment, respectively. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. The EC1.5 value observed at test concentrations with a cell viability of >70% compared to the vehicle control was considered positive for skin sensitisation. The test substance showed toxicity [IC30 values (concentration effecting reduction of cellular viability by 30%) of 11 µM, 5.4 µM and 12 µM in experiment 1, 2 and 3, respectively]. In the first experiment, a statistically significant induction of the luciferase activity (EC1.5 value 4.2 µM) was measured. In the second experiment, no biologically relevant luciferase induction was observed at all experimental concentrations, therefore no EC1.5 could be calculated. Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 5.0 µM) was measured. Overall, the test substance was classified as positive in the KeratinoSens assay since positive results (>1.5 -fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments. Under the study conditions, the test substance was concluded to be a skin sensitiser (Gijsbrechts, 2017).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 18, 2017 to November 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: U937 cell line activation Test)

Version / remarks:

OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U- SENS™)’ (9 October 2017)

Deviations:

no

GLP compliance:

yes

Type of study:

other: Drift in CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells

Justification for non-LLNA method:

U937 cell line activation test (U-SENS™) method was recommended by EURL ECVAM to be used as part of an IATA to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling. However, it may also potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA. Nevertheless, further work, preferably based on human data, is required to determine how U-SENS™ results may possibly inform potency assessment (OECD 442 E, 9 October 2017)

Details on the study design:

Principle of Test:
The U937 cell line activation Test (U-SENS™) method is an in vitro assay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The CD86 surface marker is one typical marker of U937 activation. CD86 is known to be a co-stimulatory molecule that may mimic monocytic activation, which plays a critical role in T-cell priming. The changes of CD86 cell surface marker expression are measured by flow cytometry following cell staining typically with fluorescein isothiocyanate (FITC)-labelled antibodies. Cytotoxicity measurement is also conducted [e.g. by using propidium iodide (PI)] concurrently to assess whether upregulation of CD86 cell surface marker expression occurs at sub-cytotoxic concentrations. The stimulation index (S.I.) of CD86 cell surface marker compared to solvent/vehicle control is calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

Test System:
U937 human monocytes (Inducible CD86-expressing cells)
Source: ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM.
- Stock cultures of these cells were stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line were checked for infection with a mycoplasma detection test. Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Reference substances:
- Negative Control: Lactic Acid (LA, RS471; Batch# BCBK8387V) was used as negative control. On the treatment day, a solution at 10 mg/mL was prepared in culture medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).
- Positive Control: 2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599; Batch# BCBR6376V) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).
- Vehicle: 0.4 % dimethyl sulfoxide (DMSO, Sigma, Zwijndrecht, The Netherlands) in Cell Culture medium (RPMI-1640, Life Technologies, Bleiswijk, The Netherlands).

Dose Levels:
Two independent experiments were performed. Test substance was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to a below final test concentrations:
Experiment 1: 200, 100, 50, 20, 10 and 1.0 µg/mL
Experiment 2: 30, 25, 20, 15, 10, 5.0, 4.0 and 1.0 µg/mL
- A correction factor of 1.13 was used to correct for the purity of the test substance.

Replicates:
At least two experiments were conducted per test substance concentration to demonstrate reproducibility of the results and conclusion. Three replicates of complete medium untreated control, solvent/vehicle control, negative and positive controls were tested.

Treatment of Cells:
Cells are treated for 45 ± 3 h with the selected doses. A negative untreated control (culture medium), a vehicle control and the positive and negative control items were included. The final volume in the wells was 200 µL.
Precipitate evaluation: Before and after 45 ± 3 h of exposure, wells were checked for precipitate. If any, precipitate is documented in the raw data and reported in the table of the results.

Cell antibodies staining for IgG1 and CD86:
Treated Cells are washed, re-suspended with buffer and stained with below Fluorescein Isothiocyanate (FITC)-conjugated antibodies (used for both IgG1 and CD86 staining), by incubating refrigerated in the dark for 30 minutes- Mouse IgG1 of unknown specificity, for isotypic control
- Human CD86 specific mouse IgG1

Flow cytometry analysis:
- Expression level of CD86 and cell viability are analysed using flow cytometry.
- For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) denoted as 'CV70'; and The stimulation index (S.I.) of CD86 cell surface marker was calculated.
- Refer to the section 'Any other information on materials and methods incl. tables' for calculation details

Positive control results:

Experiment 1: The positive control (TNBS) showed a S.I. ≥ 351% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 557% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
- Both tests passed the acceptance criteria

Key result

Run / experiment:

other: 1

Parameter:

other: EC150 (Test substance concentration (µg/mL) that induces CD86 stimulation index (S.I.) of 150%)

Value:

5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test substance cytotoxicity concentration for 70% viability (CV70) was 26 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: EC150 (Test substance concentration (µg/mL) that induces CD86 stimulation index (S.I.) of 150%)

Value:

6.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test substance cytotoxicity concentration for 70% viability (CV70) was 13 µg/mL

Other effects / acceptance of results:

Experiment 1: The negative control (LA) showed a S.I. < 91% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The negative control (LA) showed a S.I. < 74% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Both tests passed the acceptance criteria:

- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (97% in experiment 1 and 97% in experiment 2).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.
- The CD86 basal expression of untreated U937 cells is within the range of ≥2% and ≤25%.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥0.6% and <1.5% in both experiments.

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Experiment 1

No precipitation was observed at the start and end of the incubation period in the 96 well plates.

Test Substance showed toxicity, the calculated CV70 was 26 µg/mL.

A biologically relevant increase in the expression of CD86 was observed after treatment with the test substance, the EC150: 5.0 µg/mL.

Experiment 2

No precipitation was observed at the start and end of the incubation period in the 96-well plates.

Test Substance showed toxicity, the calculated CV70 was 13 µg/mL.

A biologically relevant increase in the expression of CD86 was observed after treatment with the test substance, the EC150: 6.3 µg/mL.

No drift in CD86 expression was observed in the untreated controls and negative controls.In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test substance was classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, test substance was classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions.

Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2
Dose (µg/mL)	% Viability (Mean)		CD86-IgG1 S.I.		Colour Interference S.I.
	Experiment		Experiment		Experiment
	1	2	1	2	1	2
1	98	99	109	84	96	95
4	-	99	-	126	-	88
5	-	99	-	132	-	89
10	98	97	201	201	115	99
15	-	43	-	282	-	188
20	81	62	293	241	97	139
25	-	81	-	192	-	148
30	-	14	-	391	-	94
50	25	-	699	-	118	-
100	20	-	896	-	157	-
200	26	-	270	-	152	-
- Not Applicable

Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control
Control substances	% Viability (Mean)		CD86-IgG1 S.I.
	Experiment		Experiment
	1	2	1	2
Negative Control; LA1	97	99	91	70
Negative Control; LA2	97	99	80	72
Negative Control; LA3	97	98	78	74
Positive Contyrol; TNBS1	96	96	356	557
Positive Contyrol; TNBS2	96	95	351	575
Positive Contyrol; TNBS3	96	94	386	674
DMSO	98	99	143	136
	IgG1 value (%)		CD86 basal expression (%)
	Experiment		Experiment
	1	2	1	2
Vehicle Control; RPMI1	0.9	0.8	4.4	5.8
Vehicle Control; RPMI2	0.7	0.9	4.2	5.6
Vehicle Control; RPMI3	0.5	0.5	4.7	5.3
RPMI Mean Viability			97	97
RPMI Drift			20%	-1%
LA Drift			-9%	4%

Overview EC150 and CV70 Values
	EC150 (µg/mL)	CV70 (µg/mL)
Test substance Experiment 1	5	26
Test substance Experiment 2	6.3	13

Conclusions:

Under the study conditions, the test substance was determined to be a skin sensitiser.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the U937 cell line activation test method according to OECD Guideline 442E, in compliance with GLP. The U937 cell line activation test (U-SENS™) method is an in vitro assay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The changes of CD86 cell surface marker expression was measured by flow cytometry following cell staining with fluorescein labelled antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of CD86 cell surface marker expression occurs at sub-cytotoxic concentrations. Results were derived based on the CV70 value (i.e. a test substance concentration showing 70% of U937 cell survival) and the EC150 value (i.e. the concentration at which the test substance induces a CD86 stimulation index (S.I.) of 150%). Test substance was tested at eleven different concentrations in two independent run. In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. The test substance showed cytotoxicity (CV70 values of 26 µg/mL and 13 µg/mL in experiment 1 and 2, respectively) and biologically relevant induction of the CD86 activity (EC150 values of 5.0 µg/mL and 6.3 µg/mL in experiment 1 and 2, respectively). Based on the prediction model, the test substance was classified as positive in the assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. Under the study conditions, the test substance was predicted to be a skin sensitiser (Eurlings, 2018).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 22, 2017 to January 29, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay

Version / remarks:

July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Remarks:

Inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: Approx. 10 weeks old
- Weight at study initiation: 16.7 to 25.0 g
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum): Pelleted rodent diet, (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) (ad libitum)
- Water (e.g. ad libitum): Municipal tap water (ad libitum)
- Acclimation period: 5 d
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

(AcOO) (Merck, Darmstadt, Germany and Fagron, Cappele a/d IJssel, the Netherlands). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at test facility and on information provided by the Sponsor.

Concentration:

- Dose groups: 0 (vehicle control), 5, 10, 25 % w/w

- Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 h after adding the vehicle to the test substance. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test substance, since the test method requires a logical concentration range rather than specific dose levels.

No. of animals per dose:

5 females/dose group

Details on study design:

PRE-SCREEN TESTS: A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study, except that the animals were approximately 10-13 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-13 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Initially, two test substance concentrations were tested; 50% and 100%. Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.

- Compound solubility: The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Irritation:
- Systemic toxicity:
- Ear thickness measurements:
- Erythema scores:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT : Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean, except for one animal which weighed 24% less than the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Name of test method: LLNA


Experiment Design:
- Days 1, 2 and 3: Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals.
- Day 6: Excision, Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After 5 h, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Day 6: Tissue Processing for Radioactivity: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.
- Day 7: Radioactivity Measurements: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

- Criteria used to consider a positive response: A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test substance is regarded as a skin sensitizer. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation

Other Observation:
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily
- Clinical Observations: Post dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 h after dosing). All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded
- Body Weights: Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy)
- Irritation: Observations for erythema and eschar formation were performed once daily on Days 1-6 (on Days 1-3 within 1 h after dosing). A description of all other (local) effects was recorded.
- Erythema and eschar formation Scoring system: No erythema = 0, Very slight erythema (barely perceptible = 1, Well-defined erythema = 2, Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) = 3, Severe erythema (beet redness) to eschar formation preventing grading of erythema = 4
- Pathology: No necropsy was performed, since all animals survived until the end of the observation

Positive control substance(s):

other: For both scientific and animal welfare reasons, no concurrent positive control group was included in the study

Positive control results:

The results of reliability test with 3 concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials and essential procedures served as Positive Control

Key result

Parameter:

SI

Value:

ca. 6.6

Test group / Remarks:

Test substance concentration 25% (highest dose tested)

Remarks on result:

other: Skin sensitizer

Key result

Parameter:

SI

Value:

ca. 1.9

Test group / Remarks:

Test substance concentration 10%

Key result

Parameter:

SI

Value:

ca. 1.7

Test group / Remarks:

Test substance concentration 5%

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

Control group

Key result

Parameter:

EC3

Remarks:

Estimated test substance concentration (% w/w) that will give a SI =3

Value:

ca. 13.5

Test group / Remarks:

Calculated based on dose- response data

Remarks on result:

other: Skin sensitizer (Category 1B) according to CLP

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Radioactivity Measurements and SI Values: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 909, 987 and 3483 DPM, respectively. The mean DPM/animal value for the vehicle control group was 530 DPM. The SI values calculated for the test substance concentrations 5, 10 and 25% were 1.7, 1.9 and 6.6, respectively. The data showed a dose- response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 13.5% was calculated.



SKIN REACTIONS / IRRITATION: very slight irritation of the ears as shown by two animals treated at 25% on Day 4 was considered not to have a toxicologically significant effect on the activity of the nodes.
SYSTEMIC TOXICITY: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
MACROSCOPIC EXAMINATION OF THE LYMPH NODES AND SURROUNDING AREA: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Pre-Screen Test: At 100% and 50% test substance concentrations, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At 25% and 10% test item concentrations, no signs of systemic toxicity were noted and no to very slight irritation were observed and therefore 25% was selected as highest concentration for the main study.

Table 1: Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Dose Groups	Animal	Size nodes*		DPM/animal	Mean DPM	±SEM	Mean SI	±SEM
		Left	right
1 (control)	1	n	n	270	530	101	1	0.2
	2	n	n	387
	3	n	n	651
	4	n	n	845
	5	n	n	498
2 (5% w/w)	1	n	n	789	909	55	1.7	0.2
	2	n	n	1012
	3	n	n	890
	4	n	n	1058
	5	n	n	797
3 (10% w/w)	1	n	n	496	987	203	1.9	0.4
	2	+	n	1245
	3	n	n	1386
	4	n	n	820
	5	+	+	14047**
4 (25% w/w)	1	n	n	1911	3483	1149	6.6	2.2
	2	n	n	4207
	3	n	n	1196
	4	n	n	7626
	5	n	n	2475
* Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal). **Value rejected and not used for interpretation (outlier based on the Dixon’s Q-test). SEM = Standard Error of the Mean.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the study condition, the test substance was determined to be a skin sensitiser, with a classification of Skin Sens. 1B – H317 (May cause an allergic skin reaction) according to CLP (EC 1272/2008) criteria.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the Local Lymph Node Assay according to OECD Guideline 429, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a pre-screen. In the main study, three experimental groups of five female CBA/J mice were treated at concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (acetone/olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after 5 h the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 909, 987 and 3483 DPM, respectively. The mean DPM/animal value for the vehicle control group was 530 DPM. The SI values calculated for the test substance concentrations 5, 10 and 25% were 1.7, 1.9 and 6.6, respectively. These results indicate that the test substance could elicit a SI ≥ 3. The data showed a dose response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 13.5% was calculated. Under the study condition, the test substance was determined to be a skin sensitiser, with a classification of Skin Sens. 1B – H317 (May cause an allergic skin reaction) according to CLP (EC 1272/2008) criteria (van Sas, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Study 1 (in vitro):

A study was conducted to determine the skin sensitisating potential of the test substance according to OECD Guideline 442C, in chemico Direct Peptide Reactivity Assay (DPRA), in compliance with GLP. Test samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) at 25 ± 2.5˚C for 24 - 30 h. After incubation, the relative peptide concentration was determined by HPLC using gradient elution and photodiode array (PDA) detector to measure peptide concentration. Test substances were compared to reference controls in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes for skin sensitisation. Acceptance criteria for all controls and the test substance were met in either of Cysteine or Lysine Reactivity Assay. The test substance produced 5.2% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitising with no or minimal reactivity. Under study conditions, the test substance was determined to be non-sensitising. However, since precipitation was observed upon addition of the test substance to the peptide solutions, the amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care (Reinen, 2017).

Study 2 (in vitro):

A study was conducted to determine the skin sensitisation potential of the test substance using the U937 cell line activation test method according to OECD Guideline 442E, in compliance with GLP. The U937 cell line activation test (U-SENS™) method is an in vitro assay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The changes of CD86 cell surface marker expression was measured by flow cytometry following cell staining with fluorescein labelled antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of CD86 cell surface marker expression occurs at sub-cytotoxic concentrations. Results were derived based on the CV70 value (i.e. a test substance concentration showing 70% of U937 cell survival) and the EC150 value (i.e. the concentration at which the test substance induces a CD86 stimulation index (S.I.) of 150%). Test substance was tested at eleven different concentrations in two independent run. In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. The test substance showed cytotoxicity (CV70 values of 26 µg/mL and 13 µg/mL in experiment 1 and 2, respectively) and biologically relevant induction of the CD86 activity (EC150 values of 5.0 µg/mL and 6.3 µg/mL in experiment 1 and 2, respectively). Based on the prediction model, the test substance was classified as positive in the assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. Under the study condition, the test substance was predicted to be a skin sensitiser (Eurlings, 2018).

Study 3 (in vitro):

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase (KeratinoSens) test method according to OECD Guideline 442D, in compliance with GLP. Three independent experiments were performed. Cells were incubated with test substance at concentration ranges of 0.43 – 875 µM, 0.42 – 851 µM and 1.7 - 1000 µM for 48 h in the first, second and third experiment, respectively. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. The EC1.5 value observed at test concentrations with a cell viability of >70% compared to the vehicle control was considered positive for skin sensitisation. The test substance showed toxicity [IC30 values (concentration effecting reduction of cellular viability by 30%) of 11 µM, 5.4 µM and 12 µM in experiment 1, 2 and 3, respectively]. In the first experiment, a statistically significant induction of the luciferase activity (EC1.5 value 4.2 µM) was measured. In the second experiment, no biologically relevant luciferase induction was observed at all experimental concentrations, therefore no EC1.5 could be calculated. Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 5.0 µM) was measured. Overall, the test substance was classified as positive in the KeratinoSens assay since positive results (>1.5 -fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments. Under the study conditions, the test substance was concluded to be a skin sensitiser (Gijsbrechts, 2017).

Evaluation of the in vitro studies:

A DPRA assay, a KeratinoSens assay and a U-SENS assay were performed in accordance with the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

OECD Toolbox v3.4 revealed structural alerts for protein binding of the test substance via acylation in some protein binding profilers, but gave no alert in others.

In a DPRA assay, the mean of the SPCC and SPCL depletion was 5.2% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. It is of note that upon preparation as well as after incubation a precipitate was observed, which can indicate that the outcome is an underestimation of the protein binding capacity of the test item.

In the KeratinoSens assay, the test itemis classified as positive,since positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments.

Furthermore, the test substance was classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) based on the results of the U-SENS assay.

In conclusion, two out of three in vitro tests indicated that the substance has skin sensitizing properties, whereas the outcome on protein binding capacity was inconclusive. Since these results cannot be used to conclude on the potency of the substance an in vivo study to determine the skin sensitizing potency was conducted.

Study 4 (in vivo):

A study was conducted to determine the skin sensitisation potential of the test substance using the Local Lymph Node Assay according to OECD Guideline 429, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a pre-screen. In the main study, three experimental groups of five female CBA/J mice were treated at concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (acetone/olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after 5 h the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 909, 987 and 3483 DPM, respectively. The mean DPM/animal value for the vehicle control group was 530 DPM. The SI values calculated for the test substance concentrations 5, 10 and 25% were 1.7, 1.9 and 6.6, respectively. These results indicate that the test substance could elicit a SI ≥ 3. The data showed a dose response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 13.5% was calculated. Under the study condition, the test substance was determined to be a skin sensitiser, with a classification of Skin Sens. 1B – H317 (May cause an allergic skin reaction) according to CLP (EC 1272/2008) criteria (van Sas, 2018).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of an LLNA study, the substance was determined to be a skin sensitiser, with a classification of Skin Sens. 1B – H317 (May cause an allergic skin reaction) according to CLP (EC 1272/2008) criteria.