4-bromobenzyl alcohol

Endpoint summary

Description of key information

In an in chemico direct protein reactivity assay (DPRA) according to OECD Guideline 442C, the test item showed minimal reactivity towards both peptides (reference 7.4.1 -1). An in silico assessment of the test item did not indicate a skin sensitisation potential (reference 7.4.1 -2). In an in vitro skin sensitisation study according to OECD 442D the test item did not induce luciferase activity in the transgenic KeratinoSens™ cell line in at two independent experiment runs. Therefore the test item is considered not to be a skin sensitiser.

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:

19 October 2017 - 27 October 2017

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system)

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 260515HS_DWW1115
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 120514HSDW_W0517

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls (RCs): cysteine or lysine peptide in acetonitrile with and without test item; Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 22.31 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (41.27 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 21.3 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.76 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis, if a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 -400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides via HPLC.

HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A (0.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
PPD = (1 - (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%.

Key result

Run / experiment:

other: mean depletion of both peptide depletions

Parameter:

other: combined peptide depletion [%]

Value:

1.31

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

1.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

1.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Precipitation and phase seperation was observed for the positive control samples. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	809.6354	0.1341	74.93	75.35	0.40	0.53
	794.7175	0.1317	75.39
	783.8397	0.1299	75.72
Test Item	3200.8433	0.5195	0.87	1.34	0.41	30.95
	3175.2896	0.5154	1.66
	3181.4165	0.5164	1.47

  

Table 2: Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1768.9691	0.2117	57.97	56.98	1.07	1.87
	1804.7944	0.2160	57.12
	1858.1759	0.2224	55.85
Test Item	4166.7764	0.4987	0.99	1.29	0.28	22.00
	4152.8242	0.4971	1.32
	4142.9829	0.4959	1.56

 

Table 8: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	1.31	Minimal Reactivity	no sensitiser	1.34	Minimal Reactivity	no sensitizer
Positive Control	66.16	High Reactivity	sensitizer	75.35	Moderate Reactivity	sensitizer

Interpretation of results:

other: The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in chemico direct protein reactivity assay (DPRA) according to OECD Guideline 442C, the test item showed minimal reactivity towards both peptides. The test item can be considered as “non-sensitiser”.

Executive summary:

In the present study the test item was assessed for skin sensitising properties by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C according to OECD Guideline 442C (DPRA). Subsequently samples were analysed by HPLC. For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation (small droplets on the bottom of the vial) was observed for the samples of the positive control (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%. No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (1.31%). Based on the prediction model 1 (Cystein 1:10 / Lysine 1:50 Prediction Model) the test item can be considered as non-sensitiser.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

no guideline available

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Specific details on test material used for the study:

SMILES: C1(=CC=C(C=C1)CO)Br

Key result

Parameter:

other: alerts

Value:

0

Remarks on result:

no indication of skin sensitisation

Remarks:

QSAR predicted value. The substance is within the applicability domain of the model.

Interpretation of results:

other: Derek result: No alters matched.

Conclusions:

Using Derek Nexus v5.0, no skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2017).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-18 to 2018-03-22

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 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

July 1, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Justification for test system:
The induction of the Keap1-Nrf2-ARE signaling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Controls:
- Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
- Negative control: DMSO at a final concentration of 1 % (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
- Positive control: Cinnamic aldehyde was used as positive control. It was dissolved in DMSO and the final concentration range was 4 - 64 µM. The final concentration of the solvent DMSO was 1 % (v/v) for all wells.

Test item preparation:
The test item was dissolved in DMSO. The final concentration of the solvent was 1 % (v/v) in all test item concentrations.

Concentrations:
- Negative control: 1 % (v/v) DMSO in test item exposure medium
- Positive control: 4, 8, 16, 32, 64 µM
- Test item: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cell line:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 12 in experiment 1; P 4 in experiment 2) were used. Cells were cultured in 75 cm^2 culture flasks (Greiner) in maintenance medium at 37 ± 1 °C and 5 % CO2. For test item exposure, cells were cultured in medium for test item exposure.

Number of replicates:
- Blank: 1 replicate/plate in every independent run
- Negative control: 6 replicates/plate in every independent run
- Positive control: 3 replicates in every independent run
- Test item: 3 replicates in every independent run

Experimental Procedure:
A cell suspension of 8 x 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 x 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ±1 h in assay medium at 37 °C ±1 °C and 5 % CO2. Thereafter, the assay medium was discarded and replaced by 150 L test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5 % CO2.

Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5 % CO2. Afterwards the medium was removed and replaced by 200 µL 10 % SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5 % CO2 overnight (experiment 1) and over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at a wavelength of 600 nm.

Prediction Model:
The test item was considered positive in accordance with UN GHS "Category 1" if the following conditions were met in at least two independently prepared test repetitions:
- Imax was >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability was >70 % at the lowest concentration with an induction of luciferase activity >1.5
- EC15 value was <1000 µM an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 uM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Acceptance Criteria:
The test met acceptance criteria if:
- the luciferase activity induction of the positive control was statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM was between 2 and 8
- the EC1.5 value of the positive control was within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO was <20 % in each repetition.

Positive control results:

No cytoxicity was detected at all concentrations of the positive control. Overall statistically significant Luciferase induction was detected for the positive control at 32 and 64 µM.

Key result

Run / experiment:

other: overall (2000 µM as highest dose for test item)

Parameter:

other: Luciferase activity fold induction

Value:

0.66

Vehicle controls validity:

valid

Remarks:

1.0

Negative controls validity:

valid

Remarks:

same as vehicle control

Positive controls validity:

valid

Remarks:

3.71 (64 µM as highest dose)

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Induction of Luciferase Activity - Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.13	1.24	1.19	0.08
	8.00	1.13	1.17	1.15	0.03
	16.00	1.39	1.59	1.49	0.14
	32.00	1.86	2.06	1.96	0.14	*
	64.00	4.26	3.17	3.71	0.77	*
Test Item	0.98	1.13	1.16	1.15	0.03
	1.95	1.15	1.04	1.10	0.08
	3.91	1.05	1.07	1.06	0.02
	7.81	0.99	1.10	1.05	0.08
	15.63	1.02	1.08	1.05	0.04
	31.25	1.05	0.97	1.01	0.06
	62.50	1.10	1.03	1.06	0.05
	125.00	0.95	0.94	0.94	0.00
	250.00	0.86	0.90	0.88	0.03
	500.00	0.73	0.80	0.77	0.05
	1000.00	0.69	0.79	0.74	0.07
	2000.00	0.62	0.70	0.66	0.06

*=significant induction according to Student’s t-test, p<0.05

 

Table 2: Additional parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5 [µM]	n/a	n/a	-	-
Imax	1.15	1.16	1.16	0.01
IC30 [µM]	965.66	895.43	930.54	49.66
IC50 [µM]	1978.60	n/a	1978.60	-

Interpretation of results:

other: The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in vitro skin sensitidation study according to OECD 442D the test item did not induce luciferase activity in the transgenic KeratinoSens™ cell line in at two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

Executive summary:

In the present study the sensitising potential of a test item to skin was assessed through the activation of keratinocytes by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.The test item was dissolved in DMSO for the study according to OECD 442D. Based on the molecular weight of the test item a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100 % solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. Both, in the first and second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA) and a ARE-Nrf2 Luciferase Test. Furthermore, an in silico assessment using Derek Nexus v5.0 was carried out.

 

DPRA (reference 7.4.1 -1)

In the study according to OECD Guideline 442C (DPRA) the test item was assessed for skin sensitising properties by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation (small droplets on the bottom of the vial) was observed for the samples of the positive control (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%. No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (1.31%). Based on the prediction model 1 (Cystein 1:10 / Lysine 1:50 Prediction Model)the test item can be considered as non-sensitiser.

 

ARE-Nrf2 Luciferase Test (reference 7.4.1-3)

The sensitising potential of a test item to skin was assessed through the activation of keratinocytes by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.The test item was dissolved in DMSO for the study according to OECD 442D. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. Both, in the first and second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.

 

In silico assessment (reference 7.4.1 -2)

For in silico assessment, Derek Nexus v5.0 was used. No skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

 

Conclusion

The test item showed minimal reactivity towards the peptides (DPRA, OECD 442C, reference 7.4.1 -1) and no dose response for luciferase activity induction (OECD 442D, reference 7.4.1-3). In addition, an in silico assessment was performed (reference 7.4.1 -2) also showing no indication of skin sensitisation. Therefore, the test item is considered not to have a skin sensitising potential (UN GHS: no category).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.