Sodium 2-bromoethanesulphonate

Endpoint summary

Description of key information

Skin:

In an in chemico skin sensitisation assay according to OECD Guideline 442C (DPRA), the test item is not considered as skin sensitising (reference 7.4.1-1).

In an in vitro skin sensitisation assay according to OECD Guideline 442D (ARE-Nrf2 Luciferase Test; KeratinoSens™), the test item did not show skin sensitising properties (reference 7.4.1-2).

An in silico assessment of the test item did not indicate a skin sensitisation potential (reference 7.4.1 -3).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 May 2017 - 06 September 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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS "Category 1". Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as an IATA, combining them with other complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. The induction of the Keap1-Nrf2-ARE signalling 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.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.26 (experiment 1); 3.39 (experiment 2); 5.70 (experiment 3)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.22

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: luciferase activity

Value:

1.06

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 3

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

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

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	100.0	100.0	100.0	100.0	0.0
Positive Control	4.00	92.5	96.3	102.4	97.1	5.0
	8.00	99.0	105.7	105.5	103.4	3.8
	16.00	93.9	103.9	112.2	103.3	9.2
	32.00	91.4	99.8	106.2	99.1	7.4
	64.00	85.0	101.6	121.1	102.6	18.1
Test Item	0.98	110.9	89.0	95.0	98.3	11.3
	1.95	121.2	83.4	108.1	104.2	19.2
	3.91	116.8	70.8	111.6	99.7	25.2
	7.81	114.8	62.5	106.9	94.8	28.2
	15.63	102.6	64.0	111.1	92.6	25.1
	31.25	84.9	63.7	104.8	84.5	20.5
	62.50	64.1	58.2	106.9	76.4	26.6
	125.00	55.4	57.9	107.3	73.5	29.3
	250.00	50.4	62.4	106.8	73.2	29.7
	500.00	41.3	62.8	102.7	68.9	31.2
	1000.00	34.7	79.0	107.7	73.8	36.7
	2000.00	32.5	93.3	103.3	76.4	38.3

 

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.07	1.21	1.03	1.10	0.10
	8.00	1.20	1.31	1.50	1.34	0.15
	16.00	1.31	1.34	1.39	1.35	0.04
	32.00	1.43	1.98	2.32	1.91	0.45	*
	64.00	2.71	4.17	2.90	3.26	0.79	*
Test Item	0.98	1.25	1.35	0.97	1.19	0.19
	1.95	1.15	1.01	0.90	1.02	0.12
	3.91	1.14	0.94	0.95	1.01	0.11
	7.81	0.96	0.93	0.94	0.94	0.02
	15.63	1.11	1.04	0.79	0.98	0.17
	31.25	1.28	0.90	0.80	0.99	0.26
	62.50	1.60	0.93	1.08	1.20	0.35
	125.00	1.22	1.07	0.82	1.04	0.20
	250.00	1.41	0.86	0.79	1.02	0.34
	500.00	1.22	1.38	0.84	1.15	0.28
	1000.00	0.94	0.93	0.92	0.93	0.01
	2000.00	1.26	1.13	1.43	1.27	0.15

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

 

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.12	1.11	1.05	1.09	0.04
	8.00	1.18	1.16	1.02	1.12	0.09
	16.00	1.32	1.28	1.23	1.28	0.04
	32.00	1.63	1.65	1.86	1.71	0.12	*
	64.00	3.25	3.46	3.47	3.39	0.12	*
Test Item	0.98	1.00	0.83	1.06	0.96	0.12
	1.95	1.10	1.38	1.04	1.17	0.18
	3.91	1.08	1.18	0.91	1.06	0.14
	7.81	1.01	1.28	1.27	1.19	0.15
	15.63	0.96	1.09	0.92	0.99	0.09
	31.25	0.97	1.20	0.95	1.04	0.14
	62.50	1.04	1.50	0.87	1.13	0.32
	125.00	0.96	1.27	0.94	1.06	0.18
	250.00	0.92	1.16	0.95	1.01	0.13
	500.00	1.03	1.28	0.90	1.07	0.19
	1000.00	1.09	1.63	0.94	1.22	0.36
	2000.00	1.18	1.39	1.02	1.20	0.18

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

Table 4: Induction of Luciferase Activity Experiment 3

Experiment 3	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.33	1.21	1.24	0.08
	8.00	1.35	1.34	1.08	1.25	0.15
	16.00	1.46	1.55	1.27	1.43	0.14
	32.00	2.11	1.98	1.64	1.91	0.24	*
	64.00	6.96	5.89	4.26	5.70	1.36	*
Test Item	0.98	1.05	0.91	1.02	0.99	0.07
	1.95	0.82	0.81	0.82	0.82	0.01
	3.91	0.87	0.84	0.83	0.85	0.02
	7.81	0.88	0.89	0.83	0.87	0.03
	15.63	0.88	0.92	0.85	0.88	0.04
	31.25	0.92	0.99	0.87	0.93	0.06
	62.50	1.11	0.98	0.83	0.97	0.14
	125.00	0.91	0.92	0.84	0.89	0.04
	250.00	0.90	1.01	0.78	0.90	0.12
	500.00	1.05	0.98	0.88	0.97	0.09
	1000.00	1.05	1.16	0.98	1.06	0.09
	2000.00	1.00	1.17	0.90	1.02	0.14

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

Table 5: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.10	1.09	1.24	1.14	0.08
	8.00	1.34	1.12	1.25	1.24	0.11
	16.00	1.35	1.28	1.43	1.35	0.07
	32.00	1.91	1.71	1.91	1.84	0.11	*
	64.00	3.26	3.39	5.70	4.12	1.37	*
Test Item	0.98	1.19	0.96	0.99	1.05	0.12
	1.95	1.02	1.17	0.82	1.00	0.18
	3.91	1.01	1.06	0.85	0.97	0.11
	7.81	0.94	1.19	0.87	1.00	0.17
	15.63	0.98	0.99	0.88	0.95	0.06
	31.25	0.99	1.04	0.93	0.99	0.06
	62.50	1.20	1.13	0.97	1.10	0.12
	125.00	1.04	1.06	0.89	0.99	0.09
	250.00	1.02	1.01	0.90	0.98	0.07
	500.00	1.15	1.07	0.97	1.06	0.09
	1000.00	0.93	1.22	1.06	1.07	0.15
	2000.00	1.27	1.20	1.02	1.16	0.13

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

Table 6: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5[µM]	n.a.	n.a.	n.a.	-	-
Imax	1.27	1.22	1.06	1.19	0.11
IC30[µM]	53.62	n.a.	n.a.	53.62	-
IC50[µM]	261.95	n.a.	n.a.	261.95	-

n.a. = not applicable

 

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin sensitisation assay according to OECD Guideline 442D (ARE-Nrf2 Luciferase Test; KeratinoSens™), the test item did not show an induction of Luciferase activity and therefore no skin sensitising properties. The data generated with this method may be not 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.

Executive summary:

In an in vitro skin sensitisation assay according to OECD Guideline 442D (ARE-Nrf2 Luciferase Test; KeratinoSens™), the skin sensitising potential of the test substance was determined. The test item was dissolved in DMSO. Based on a molecular weight of 211.01 g/mol 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. 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.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Within this first experiment a cytotoxic effect could be observed starting from 62.50 µM onwards. In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, the data showed a cytotoxic effect for the concentration range from 7.81 µM up to 500 µM. However, this effect showed no dose-effect relationship and could not be observed microscopically. In the third experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, no cytotoxic effect was observed within the third experiment. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Solvent and positive controls were valid. Under the condition of this study the test item is therefore considered as non sensitiser.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 April 2017 - 12 July 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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical. This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS "Category 1". Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Peptides:
19.33 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.535 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM (JPT Peptide Technologies GmbH; > 95%; Lot. No.:
111016HS-MHeW0117).
19.85 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 (37.083 mL) to reach a concentration of 0.667 mM (JPT Peptide Technologies GmbH; > 95%; Lot. No.:
220114HSDWW0117).
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups:
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 (Fisher Chemical; Cat. No. 10398233; > 99.9%; CAS No.: 75-05-8; Lot. No.: 1673078).
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 (Fisher Chemical; Cat. No. 10398233; > 99.9%;
CAS No.: 75-05-8; Lot. No.: 1673078).
Reference Control C (solvent control): undiluted
Test Item: 100 mM stock solution in water
Positive Control: 100 mM stock solution (Sigma Aldrich; Cat. No.: W228613; > 95%; CAS No.: 104-55-2; Lot. No.: MKBS2662V)
Co-elution Controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Incubation of the Test Item with the Cysteine and Lysine Peptide:
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 using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions.

HPLC Preparation and Analysis:
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 and 50% phase B 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.

Key result

Run / experiment:

other: Lysine and Cysteine

Parameter:

other: depletion of peptides (%)

Value:

3.44

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes

Depletion of the 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	1325.06	0.1445	71.28	71.95	0.69	0.95
	1261.79	0.1375	72.65
	1295.50	0.1412	71.92
Test Substance	4573.02	0.5021	0.31	1.74	1.34	76.96
	4498.50	0.4939	1.94
	4450.95	0.4886	2.98

Depletion of the 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	1766.7614	0.2083	56.65	56.79	1.06	1.87
	1715.1394	0.2021	57.91
	1801.3400	0.2123	55.80
Test Substance	4011.6292	0.4740	0.31	5.13	4.97	96.77
	3829.0969	0.4524	4.85
	3612.4436	0.4268	10.23

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Predicition 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)
	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Substance	3.44	Minimal Reactivity	no sensitizer	1.74	Minimal Reactivity	no sensitizer
Positive Control	64.37	High Reactivity	sensitizer	71.95	High Reactivity	sensitizer

 

Interpretation of results:

GHS criteria not met

Conclusions:

In an in chemico skin sensitisation assay according to OECD Guideline 442C (DPRA), the test substance induced a mean peptide depletion of 3.44% and is therefore not considered as skin sensitising. The data generated with this method may be not 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.

Executive summary:

In an in chemico skin sensitisation assay according to OECD Guideline 442C (DPRA), the direct peptide binding potetial of the test item was determined. The test item was completely soluble in water and the resulting solution was used for further testing. Based on a molecular weight of 211.01 g/mol a 100 mM stock solution was prepared. The test item solutions were tested 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 stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine or cysteine peptide solution, respectively. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. A slight precipitation was observed for the samples of the positive 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.

No co-elution of test item with the peptide peaks was observed. Sensitizing 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 C (RC C). Results of the positive control were valid. The refenrce controls confirmed the validity of the study for both, the cysteine and the lysine peptide.

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% (3.44%). In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item might be considered as "non-sensitiser".

Reference 3

Endpoint:

skin sensitisation: in vitro

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: [Na+].[O-]S(=O)(=O)CCBr

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 alerts matched.

Conclusions:

Using Derek Nexus v5.0.1, 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 isapplicable to thequery 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 isrelevant for theregulatory 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use. The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.

In an in chemico skin sensitisation assay according to OECD Guideline 442C (DPRA) (reference 7.4.1 -1), the direct peptide binding potetial of the test item was determined. The test item was completely soluble in water and the resulting solution was used for further testing. Based on a molecular weight of 211.01 g/mol a 100 mM stock solution was prepared. The test item solutions were tested 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 stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine or cysteine peptide solution, respectively. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. A slight precipitation was observed for the samples of the positive 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. No co-elution of test item with the peptide peaks was observed. Sensitizing 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 C (RC C). 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% (3.44%). In this study under the given conditions the test substance showed minimal reactivity towards both peptides. The test item might be considered as "non-sensitiser".

In an in vitro skin sensitisation assay according to OECD Guideline 442D (ARE-Nrf2 Luciferase Test; KeratinoSens™) (reference 7.4.1 -2), the skin sensitising potential of the test item was determined. The test item was dissolved in DMSO. Based on a molecular weight of 211.01 g/mol 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. 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. In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Within this first experiment a cytotoxic effect could be observed starting from 62.50 µM onwards. In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, the data showed a cytotoxic effect for the concentration range from 7.81 µM up to 500 µM. However, this effect showed no dose-effect relationship and could not be observed microscopically. In the third experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, no cytotoxic effect was observed within the third experiment. 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.

For in silico assessment, Derek Nexus v5.0.1 was used (reference 7.4.1-3). 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.

According to the IATA, three key events of the AOP should be analysed within a testing battery for a conclusive determination of the skin sensitising properties. However, if two out of three key events of the test battery the test substance can be considered as non-sensitiser. Since the DPRA and the KeratinoSens™ assay were negative, no h-CLAT (OECD 442E) had to be performed. In conclusion, regarding the results of the in chemico/in vitro studies (no direct peptide binding, no keratinocate activation) within the scope of the IATA, the test substance should not be considered as skin sensitising. Additionally, the results of an in silico prediction did not indicate a skin sensitising potential of the test item.

Therefore, the test item is considered not to have a skin sensitising potential (UN GHS: No Category).

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 test item is not considered to be classified for skin sensitisation (UN GHS: No Category) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.