Sodium 2-bromoethanesulphonate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

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.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-bromoethanesulphonate

EC Number:

224-244-4

EC Name:

Sodium 2-bromoethanesulphonate

Cas Number:

4263-52-9

Molecular formula:

C2H5BrO3S.Na

IUPAC Name:

sodium 2-bromoethanesulphonate

Test material form:

solid

In vitro test system

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.

Results and discussion

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)).

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.