1,2-difluoro-3-methoxy-6-[4-(trans-4-propylcyclohexyl)-1-cyclohexen-1-yl]-benzene

Endpoint summary

Description of key information

OECD 442 C: no prediction due to precipitation of the test material

OECD 442 D: negative

OECD 442 E: positive

As a worst case approach, the test item is considered to be a skin sensitizer.

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:

2017-11-23 to 2018-01-20

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))

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

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 65.38%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0.37

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.8700	0.5340	4254.6284	0.5340
STD2	9.0120	0.2670	2161.7087	0.2670
STD3	4.5180	0.1335	1051.8925	0.1335
STD4	2.2520	0.0667	525.2503	0.0667
STD5	1.1010	0.0334	260.5259	0.0334
STD6	0.5300	0.0167	130.8325	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

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	4.4470	0.1325	74.33	74.15	0.18	0.25
	4.4770	0.1334	74.15
	4.5100	0.1344	73.96
Test Item	16.9410	0.5052	0.26	0.55	0.25	46.16
	16.8690	0.5031	0.68
	16.8640	0.5029	0.71

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	1675.9452	0.2100	57.93	56.62	1.13	2.00
	1753.5808	0.2197	55.98
	1754.5223	0.2198	55.95
Test Item	4019.5232	0.5032	0.26	0.37	0.13	35.68
	4016.2871	0.5028	0.34
	4009.0984	0.5019	0.52

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

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	--	--	--	0.55	Minimal Reactivity	negative
Positive Control	65.38	High Reactivity	positive	74.15	Moderate Reactivity	positive

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation the prediction
model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary
information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study the test item was dissolved in DMF, based on the results of the pre-experiments. A maximum solubility of 37.5 mM was determined. However, in the main experiments stock solutions of 100 mM were prepared accidentally. It is assumed that not the whole amount of test item was dissolved. Since with this concentration the highest possible concentration was guaranteed, the experiments were not repeated. For reasons of simplicity the concentrations of the stock solutions are reported as 100 mM. Based on a molecular weight of 348.47 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 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the co-elution control of the positive control sample.

A minor co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion and were not used for evaluation. The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide (0.55%). According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

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 65.38%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-14 to 2018-06-18

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the 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.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.40 (experiment 1); 3.90 (experiment 2) and 3.79 (experiment 3)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

145.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 15.63 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

108.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

12.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Key result

Run / experiment:

other: 3

Parameter:

other: luciferase activity

Value:

1.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 42.22 µM

Key result

Run / experiment:

other: 3

Parameter:

other: cell viability [%]

Value:

149.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: EC 1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is 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 is between 2 and 8
- the EC1.5 value of the positive control is 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 is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Cytotoxicity

Results of the Cytotoxicity Measurement

	Conc. [µM]	Cell Viability [%]				Exp 3
		Exp 1	Exp 2	Mean	SD	Conc. [µM]	Cell Viability [%]
Positive Control	4.00	99.3	106.9	103.1	5.4	4.00	105.8
	8.00	102.3	114.7	108.5	8.8	8.00	99.1
	16.00	97.6	104.6	101.1	4.9	16.00	112.3
	32.00	97.9	113.5	105.7	11.0	32.00	118.6
	64.00	90.6	108.0	99.3	12.3	64.00	122.5
Test Item	0.98	125.0	105.9	115.5	13.6	4.24	109.1
	1.95	122.3	106.7	114.5	11.0	5.65	106.3
	3.91	138.1	103.7	120.9	24.3	7.53	101.1
	7.81	152.8	106.9	129.8	32.5	10.03	101.4
	15.63	158.4	108.9	133.7	34.9	13.37	104.4
	31.25	145.3	81.8	113.5	44.9	17.82	99.2
	62.50	134.4	0.1	67.2	95.0	23.76	115.9
	125.00	130.2	0.2	65.2	92.0	31.67	132.4
	250.00	122.2	0.6	61.4	86.0	42.22	149.7
	500.00	128.0	0.6	64.3	90.1	56.28	145.7
	1000.00	123.3	0.4	61.9	86.9	75.02	130.2
	2000.00	129.3	0.4	64.8	91.2	100.00	110.5

Conc.: Concentration; Exp: Experiment

Induction of Luciferase Activity Experiment 1

Experiment1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Positive Control	4.00	1.02	1.08	1.05	1.05	0.03
	8.00	1.64	1.51	1.45	1.53	0.10	*
	16.00	1.61	1.80	1.84	1.75	0.12	*
	32.00	3.20	3.64	4.58	3.81	0.70	*
	64.00	7.99	6.39	7.83	7.40	0.88	*
Test Item	0.98	1.20	1.17	1.16	1.18	0.02
	1.95	1.17	1.12	1.18	1.15	0.03
	3.91	1.25	1.29	1.33	1.29	0.04
	7.81	1.40	1.34	1.33	1.35	0.04
	15.63	1.44	1.35	1.34	1.38	0.05
	31.25	1.68	1.38	1.39	1.48	0.17
	62.50	1.30	1.28	1.52	1.37	0.13
	125.00	1.28	1.11	1.27	1.22	0.09
	250.00	1.14	1.20	1.08	1.14	0.06
	500.00	1.24	1.23	1.25	1.24	0.01
	1000.00	1.22	1.18	1.07	1.16	0.08
	2000.00	1.42	1.17	1.03	1.21	0.20

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

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Positive Control	4.00	1.22	1.16	1.04	1.14	0.09
	8.00	1.42	1.26	1.19	1.29	0.12
	16.00	1.64	1.74	1.56	1.65	0.09	*
	32.00	1.89	2.27	2.23	2.13	0.21	*
	64.00	3.27	4.16	4.28	3.90	0.55	*
Test Item	0.98	1.30	1.27	1.35	1.30	0.04
	1.95	1.05	1.11	1.22	1.13	0.09
	3.91	1.36	1.36	1.19	1.30	0.10
	7.81	1.38	1.21	1.36	1.32	0.09
	15.63	1.67	1.86	1.39	1.64	0.23	*
	31.25	1.65	1.16	1.65	1.48	0.28
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

Induction of Luciferase Activity Experiment 3

Experiment 3	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Positive Control	4.00	1.21	1.28	1.49	1.32	0.15
	8.00	1.11	1.37	1.29	1.26	0.13
	16.00	1.15	1.44	1.35	1.32	0.15
	32.00	1.83	1.70	2.13	1.88	0.22	*
	64.00	3.77	3.63	3.97	3.79	0.17	*
Test Item	4.24	1.18	1.24	1.25	1.23	0.04
	5.65	0.98	0.91	1.10	1.00	0.09
	7.53	1.09	1.05	1.14	1.09	0.05
	10.03	0.86	0.91	1.09	0.95	0.12
	13.37	0.90	1.02	1.17	1.03	0.14
	17.82	0.84	0.91	1.10	0.95	0.14
	23.76	0.98	1.12	1.19	1.10	0.11
	31.67	0.98	1.19	1.31	1.16	0.17
	42.22	1.59	1.27	1.48	1.45	0.16
	56.28	1.33	1.44	1.54	1.44	0.11
	75.02	1.30	1.35	1.39	1.35	0.05
	100.00	1.21	1.59	1.32	1.37	0.19

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

Induction of Luciferase Activity – Overall Induction

	Conc. [µM]	Fold Induction				Exp 3
		Exp 1	Exp 2	Mean	SD	Conc. [µM]]	Fold Induction
Positive Control	4.00	1.05	1.14	1.09	0.06	4.00	1.32
	8.00	1.53	1.29	1.41	0.17	8.00	1.26
	16.00	1.75	1.65	1.70	0.07	16.00	1.32
	32.00	3.81	2.13	2.97	1.18	32.00	1.88
	64.00	7.40	3.90	5.65	2.47	64.00	3.79
Test Item	0.98	1.18	1.30	1.24	0.09	4.24	1.23
	1.95	1.15	1.13	1.14	0.02	5.65	1.00
	3.91	1.29	1.30	1.30	0.01	7.53	1.09
	7.81	1.35	1.32	1.34	0.03	10.03	0.95
	15.63	1.38	1.64	1.51	0.19	13.37	1.03
	31.25	1.48	1.48	1.48	0.00	17.82	0.95
	62.50	1.37	0.00	0.68	0.97	23.76	1.10
	125.00	1.22	0.00	0.61	0.86	31.67	1.16
	250.00	1.14	0.00	0.57	0.81	42.22	1.45
	500.00	1.24	0.00	0.62	0.88	56.28	1.44
	1000.00	1.16	0.00	0.58	0.82	75.02	1.35
	2000.00	1.21	0.00	0.60	0.86	100.00	1.37

Conc.: Concentration; Exp: Experiment

Additional Parameters

Parameter	Exp. 1	Exp. 2	Exp. 3	Mean	SD
EC1.5[µM]	n.a.	12.25	n.a.	12.25	-
Imax	1.48	1.64	1.45	1.52	0.10
IC30[µM]	n.a.	35.75	n.a.	35.75	-
IC50[µM]	n.a.	43.40	n.a.	43.40	-

Exp: Experiment; n.a.: not applicable

Acceptance Criteria

Criterion	Range	Exp 1	pass/fail	Exp 2	pass/fail		Exp 3	pass/fail
CV DMSO Solvent Control	< 20%	10.3	pass	11.5		pass	9.3	pass
CV THF Solvent Control		10.3	pass	15.0		pass	9.6	pass
No. of PC concentration steps with significant luciferase activity induction >1.5	≥ 1	4.0	pass	3.0		pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	7.72	pass	12.71		pass	21.19	pass
Induction PC at 64 µM	2.00 < x < 8.00	7.40	pass	3.90		pass	3.79	pass

Exp: Experiment

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

Interpretation of results:

other: 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.

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
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 the present study the test material was dissolved in THF. Based on a molecular weight of 348.47 g/mol a stock solution of 200 mM was prepared for experiment 1 and 2. In order to verify the first and second experiment, a third experiment with adapted concentrations was performed. For experiment 3 a stock solution of 10 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 for experiment 1 and 2 and a constant dilution factor of 1:1.33 for experiment 3. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

Experiment 1 and 2:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Experiment 3:

100, 75.02, 56.28, 42.22, 31.67, 23.76, 17.82, 13.37, 10.03, 7.53, 5.65, 4.24 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, nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

In the second experiment, a max luciferase activity (I_max) induction of 1.64 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 108.9%. No furthersignificant luciferase induction > 1.5 was found in the tested concentration range.The calculated EC_1.5 was < 1000 µM (12.25 µM).

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.

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-28 to 2018-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (242% experiment 1; 351% experiment 2, 370% experiment 3) and 200% for CD54 (251% experiment 1; 252% experiment 2, 292% experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

132

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 250 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

117

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 69.77 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

189

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 208.33 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

122

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 83.72 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

152

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 173.61 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

129

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 83.72 µg/mL

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Results of the Cell Batch Activation Test (Batch 1)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	87.5	373	>150	88.1	358	>200	yes	pass
NiSO4	100 µg/mL	83.5	295	>150	82.1	603	>200	yes	pass
LA	1000 µg/mL	95.7	81	≤150	95.8	100	≤200	no	pass

Results of the Cell Batch Activation Test (Batch 2)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	83.4	364	>150	82.3	419	>200	yes	pass
NiSO4	100 µg/mL	79.1	351	>150	77.9	688	>200	yes	pass
LA	1000 µg/mL	96.2	77	≤150	95.9	105	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	93.40
Solvent Control	THF	--	95.10
Test item	C8	3.91	95.30
	C7	7.81	95.60
	C6	15.63	96.10
	C5	31.25	96.30
	C4	62.50	94.90
	C3	125.00	95.50
	C2	250.00	94.90
	C1	500.00	94.70
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	93.2	93.6	92.5	3139	1213	684	2455	529	92	92	459	177
Solvent Control 2 (THF)	0.20%	92.6	93.2	92.7	3075.0	1289.0	714.0	2361	575	100	100	431	181
Solvent Control 1 (DMSO)	0.20%	92.5	91.9	91.9	3337	1245	668	2669	577	100	100	500	186
DNCB	4.00	78.9	79.2	78.5	7147	2123	675	6472	1448	242	251	1059	315
Test item	250	90.3	90.2	90.0	3884	1414	776	3108	638	132	111	501	182
	208.33	91.5	91.3	91.1	3527	1378	771	2756	607	117	106	457	179
	173.61	90.3	90.4	90.3	3652	1377	790	2862	587	121	102	462	174
	144.68	91.1	92.1	91.7	3541	1454	785	2756	669	117	116	451	185
	120.56	91.7	91.4	91.5	3194	1359	767	2427	592	103	103	416	177
	100.47	92.1	91.6	90.8	3326	1424	775	2551	649	108	113	429	184
	83.72	91.7	91.6	91.3	3276	1398	991	2285	407	97	71	331	141
	69.77	91.5	92.0	91.3	3403	1448	778	2625	670	111	117	437	186

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	95.2	95.2	94.3	2540	1237	551	1989	686	90	113	461	225
Solvent Control 2 (THF)	0.20%	95.3	95.0	94.8	2264	1220	595	1669	625	100	100	381	205
Solvent Control 1 (DMSO)	0.20%	95.4	94.9	94.3	2777	1168	562	2215	606	100	100	494	208
DNCB	4.0	77.0	77.2	75.8	8350	2099	569	7781	1530	351	252	1467	369
Test item	250.00	93.50	92.80	93.80	3571	1335	643	2928	692	175	111	555	208
	208.33	92.40	92.10	93.00	3818	1267	666	3152	601	189	96	573	190
	173.61	93.00	93.50	92.90	3513	1356	648	2865	708	172	113	542	209
	144.68	92.90	92.70	92.80	2847	1275	655	2192	620	131	99	435	195
	120.56	94.10	94.40	93.10	2766	1306	662	2104	644	126	103	418	197
	100.47	94.10	93.40	92.90	2712	1103	658	2054	445	123	71	412	168
	83.72	94.40	94.60	94.70	2453	1396	633	1820	763	109	122	388	221
	69.77	93.40	93.20	93.00	3221	1353	656	2565	697	154	112	491	206

CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	95.2	94.9	94.6	1666	937	628	1038	309	85	65	265	149
Solvent Control 2 (THF)	0.20%	96.5	95.8	95.9	1689	911	504	1185	407	100	100	335	181
Solvent Control 1 (DMSO)	0.20%	95.2	95.2	95.1	1713	971	496	1217	475	100	100	345	196
DNCB	4.0	83.3	84.2	84.4	5085	1972	586	4499	1386	370	292	868	337
Test item	250.0	90.7	94.2	93.6	2174	1034	568	1606	466	136	115	383	182
	208.33	93.7	93.2	92.3	2184	989	573	1611	416	136	102	381	173
	173.61	92.8	92.0	92.5	2415	1069	618	1797	451	152	111	391	173
	144.68	93.8	93.5	93.2	2261	960	576	1685	384	142	94	393	167
	120.56	94.2	94.2	93.7	2080	1004	553	1527	451	129	111	376	182
	100.47	93.5	93.5	93.5	2108	1016	567	1541	449	130	110	372	179
	83.72	93.4	94.1	94.1	2169	1098	575	1594	523	135	129	377	191
	69.77	94.4	94.6	94.0	2087	1075	593	1494	482	126	118	352	181

Acceptance Criteria

Acceptance Criterion

Range

Exp. 1

pass/fail

Exp. 2

pass/fail

Exp. 3

pass/fail

cell viability solvent controls [%]

>90

91.9

-

93.6

pass

94.3

-

95.4

pass

94.6

-

96.5

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

8

pass

RFI of positive control of CD86

≥150

242

pass

351

pass

370

pass

RFI of positive control of CD54

≥200

251

pass

252

pass

292

pass

RFI of DMSO solvent control of CD86

<150

109

pass

111

pass

117

pass

RFI of DMSO solvent control of CD54

<200

109

pass

88

pass

154

pass

RFI of THF solvent control of CD86

<150

96

pass

84

pass

114

pass

RFI of THF solvent control of CD54

<200

109

pass

91

pass

132

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

459

pass

461

pass

265

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

500

pass

494

pass

345

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

431

pass

381

pass

335

pass

MFI ratio CD54/IgG1for medium control [%]

>105

177

pass

225

pass

149

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

186

pass

208

pass

196

pass

MFI ratio CD54/IgG1 for THF control [%]

>105

181

pass

205

pass

181

pass

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	96.2	1.6	216
number of test doses with viability >50%	-	-	555
RFI of positive control of CD86	358.9	90.1	36
RFI of positive control of CD54	435.7	285.8	36
RFI of solvent control (THF) of CD86	102.9	17.4	36
RFI of solvent control (THF) of CD54	102.0	15.4	36
MFI ratio IgG1/CD86 for medium control [%]	285.6	124.3	36
MFI ratio IgG1/CD86 for THF control [%]	270.8	105.0	36
MFI ratio IgG1/CD54 for medium control [%]	308.6	137.1	36
MFI ratio IgG1/CD54 for THF control [%]	158.2	28.4	36

Interpretation of results:

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

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test material was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 250 mg/mL to 1.95 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

500, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL

In all experiments precipitation of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 90.3% (CD86), 90.2% (CD54) and 90.0% (isotype IgG1 control) in the first experiment, to 93.5% (CD86), 92.8% (CD54) and 93.8% (isotype IgG1 control) in the second experiment and to 90.7 % (CD86), 94.2 % (CD54) and 93.6 % (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 175%, 189%, 172% (250 µg/mL to 173.61 µg/ml) and 154% (69.77 µg/mL) in the second experiment and to 152% (173.61 µg/mL) in the third experiment. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since one of the cell surface markers exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The provided information indicates need for classification for skin sensitisation class 1 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/ 776.