[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

adopted Feb 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”

Version / remarks:

adopted Feb 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

In vitro test system

Details on the study design:

TEST CELL LINE
- Type: LuSens cell line
- Passage number: 8 (cytotoxicity range finder assay), 10 (experiment I and II)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 9% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

TEST CONCENTRATIONS
0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 and 400 μg/mL (cytotoxicity range finder assay)
53.8, 64.6, 77.5, 93.0, 111.6, 134.0, 160.8, 192.9, 231.5, 277.8, 333.3 and 400.0 μg/mL (experiment I and II)

CONTROLS
Solvent control:
- Substance: DMEM
Positive control:
- Substance: p-phenylenediamine
- Final concentration: 80 µM
Negative control:
- Substance: DL-lactic acid
- Final concentration: 5000 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 h (cytotoxicity range finder assay); 24 h (experiment I) and 25 and 45 min (experiment II)
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 2 h
- Device: plate reader
- Wavelength: 600 nm
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

ACCEPTANCE CRITERIA
- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70%.
- The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20%.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70%.

EVALUATION CRITERIA
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70%) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid experiments.
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed. A negative result obtained with test chemicals that do not form a stable dispersion and/or were not tested up to 2000 μM (or 2000 μg/mL for test chemicals with no defined molecular weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.
In order to come to a conclusion on the skin sensitization hazard of a substance, a minimum of two valid and independent experiments needs to indicate a positive or negative result according to the above-described criteria. If the first two experiments come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two experiments give discordant results (i.e. one is negative and the other is positive), a third independent experiment needs to be conducted to complete the study. The skin sensitizing potential (corresponding to the potential to activate the Nrf2 transcription factor) of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three experiments are negative/positive, the substance is considered as negative/positive.

Results and discussion

Positive control results:

The positive control p-phenylenediamine induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control for both experiments. The relative viability was 85.4 and 84.9% in experiment I and II, respectively.

In vitro / in chemico

Results

Key result

Run / experiment:

other: Experiment I and II

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: induction is reported as "x fold induction" therefore no unit is provided

Other effects / acceptance of results:

No significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative and growth control induced a 1.0- and 1.1-fold induction in experiment I and a 1.0- and 1.0-fold induction in experiment II, respectively. The cell viabilities were 103.6, 99.9, 105.1 and 101.2%. Since the average induction of the negative and growth control were < 1.5 fold and the viability was at least 70%, the egative and growth control results were considered to be valid.
- Acceptance criteria met for positive control: The positive control p-phenylenediamine induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control for both experiments. The relative viability was 85.4 and 84.9% in experiment I and II, respectively. Since the average induction of the positive control was ≥ 2.5 fold and the viability was at least 70%, the positive control results were considered to be valid.
- Acceptance criteria met for variability between replicate measurements: The average percentage standard deviation was 6.02 and 6.88% in experiment I and II, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.

Any other information on results incl. tables

Table 1: Results of Experiment I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	6.02	100.0	2.22	2.22
Growth Control	-	1.0	0.07	7.14	99.9	3.91	3.91
Negative Control	5000 µM	1.1	0.05	5.01	103.6	2.24	2.16
Positive Control	80 µM	5.9	0.28	4.74	85.4	2.26	2.65
Test item	53.8	1.0	0.14	13.57	104.5	1.94	1.86
Test item	64.6	1.1	0.09	8.65	99.2	0.45	0.46
Test item	77.5	1.0	0.04	3.69	98.3	1.20	1.22
Test item	93.0	1.1	0.05	4.58	99.0	2.25	2.28
Test item	111.6	1.0	0.02	1.99	97.8	2.85	2.92
Test item	134.0	1.1	0.12	10.90	96.9	2.94	3.03
Test item	160.8	1.1	0.06	6.09	97.8	2.96	3.03
Test item	192.9	1.0	0.07	6.44	96.5	3.75	3.88
Test item	231.5	1.1	0.06	5.38	96.0	4.04	4.20
Test item	277.8	1.1	0.07	6.20	97.6	1.54	1.58
Test item	333.3	1.0	0.03	2.87	98.1	1.70	1.73
Test item	400.0	1.1	0.01	0.86	99.0	1.14	1.15

Table 2: Results of Experiment II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.07	6.88	100.0	2.39	2.39
Growth Control	-	1.0	0.06	5.54	101.2	3.04	3.01
Negative Control	5000 µM	1.0	0.06	5.95	105.1	2.80	2.66
Positive Control	80 µM	5.9	0.59	9.94	84.9	3.01	3.55
Test item	53.8	1.1	0.08	7.66	105.4	0.56	0.53
Test item	64.6	1.1	0.04	4.08	99.4	1.47	1.47
Test item	77.5	1.1	0.02	2.16	99.1	0.90	0.91
Test item	93.0	1.0	0.05	4.54	97.9	1.32	1.34
Test item	111.6	1.0	0.04	3.68	95.9	4.17	4.35
Test item	134.0	1.1	0.06	5.70	96.4	6.49	6.74
Test item	160.8	1.1	0.03	2.64	97.3	2.44	2.51
Test item	192.9	1.0	0.04	4.39	98.7	1.91	1.94
Test item	231.5	1.1	0.04	4.00	99.3	2.77	2.79
Test item	277.8	0.9	0.04	4.58	97.8	1.19	1.21
Test item	333.3	1.1	0.01	0.84	97.7	2.25	2.30
Test item	400.0	1.1	0.04	4.07	99.4	2.37	2.38

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event "activation of keratinocytes"

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is no sensitiser in KeratinoSens Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No. 1272/2008.