3-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

Endpoint summary

Description of key information

The potential of the test material to cause skin sensitisation was assessed in vitro by means of AOP related assays:

The OECD 442 C study provided inconclusive results, the OECD 442 D and OECD 442 E experiments were both positive. Two out of three key events for skin sensitisation were thus positive indicating that the test material causes a potential for skin sensitisation.

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:

2017-11-16 to 2017-12-23

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:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.32 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.475 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
22.01 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 (41.50 mL) to reach a concentration of 0.667 mM.
- Preparation of the test chemical solutions: The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Reference control A & B: Peptide stock solution was dissolved in acetonitrile.
Reference control C: RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilize the test item.
Co-elution controls: were set up in parallel to sample preparation but without the respective peptide solution.
INCUBATION
- Incubation conditions: 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.
- Precipitation noted: Precipitation was observed for the samples of the test item (including the co-elution control of the test item) after the 24 h +/- 2 h incubation period but prior to the HPLC analysis.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: 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.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

other: N,N-dimethylformamide

Positive control:

cinnamic aldehyde

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 63.50%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.3 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

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	5221.6792	0.5340	4311.3857	0.5340
STD2	2648.5273	0.2670	2143.2170	0.2670
STD3	1327.5009	0.1335	1043.3549	0.1335
STD4	670.7400	0.0667	519.3511	0.0667
STD5	334.8562	0.0334	259.5146	0.0334
STD6	166.3505	0.0167	130.4601	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	1609.4324	0.1633	68.11	68.17	0.05	0.08
	1604.4860	0.1628	68.21
	1605.4248	0.1629	68.19
Test Item	4967.2417	0.5065	0.03	0.30	0.37	122.03
	4961.0894	0.5059	0.15
	4932.7520	0.5030	0.72

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	1589.2585	0.1982	59.95	58.82	0.99	1.68
	1662.4030	0.2073	58.11
	1650.5492	0.2058	58.41
Test Item*	4055.4836	0.5032	0.00	0.00	0.00	n/a
	4028.6870	0.4999	0.00
	4043.9385	0.5018	0.00

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	-	-	-	0.30	Minimal Reactivity	no sensitiser
Positive Control	63.50	High Reactivity	sensitiser	68.17	Moderate Reactivity	sensitiser

Interpretation of results:

other: Prediction cannot be made. The result must be considered in the context of integrated approach.

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. However, 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 material, was dissolved in DMF, based on the results of the pre-experiments.

Based on a molecular weight of 296 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 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. Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples were 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 was observed for the samples of the positive control (including the co-elution control of the positive control). Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples of the test item were centrifuged at 400g for 5 minutes prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_DMF). However, precipitation was observed in the cysteine experiment as well.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of cysteine peptide was ≤ 6.38% (0.30%).

According to the evaluation criteria in the guideline, if a precipitation 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 63.50%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-14 to 2018-02-05

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 of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in Tetrahydrofuran. A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium.
- Preparation of the test chemical serial dilutions: Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. Since the test item was dissolved in THF, DMSO was added at a final concentration of 1% (v/v).
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
- Preparation of the positive controls: Cinnamic aldehyde was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells. Furthermore, 1% (v/v) of THF was added to the positive control.
- Preparation of the solvent, vehicle and negative controls: 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. Furthermore, 1% (v/v) of THF was added to the negative control.
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 µM
- Cytotoxicity assessment performed: Cell viability was measured.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 2
- Number of repetitions: 3
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125.0, 250.0, 500.0, 1000.0, 2000.0 µM
- Exposure time: 48 h +/- 1 h
- Description on study acceptance criteria:
- 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.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): 10E+4 cells per well; passage 7 in experiment 1 and passage 3 in experiment 2
- Incubation conditions: cells were grown for 24 h +/- 1 h at 37°C and 5% CO2
- Washing conditions: After exposure, the supernatant was aspirated from the white assay plates. Cells were washed once with DPBS

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: plate reader for luminescence measurement
- Plate used: white 96-well plates (flat bottom)
- Lysate preparation: After incubation with the test item, cells were washed once with DPBS. Subsequently, 20 µL of passive lysis buffer were added into each well and the plate were incubated for 20 min at room temperature in the absence of light.

DATA EVALUATION
- Cytotoxicity assessment: Cell viability was measured by using the MTT Assay.
- Prediction model used:
The test item is considered positive (UN GHS Category 1) if:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <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 μM 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.

Vehicle / solvent control:

other: DMSO and THF in test item exposure medium

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.60 (experiment 1) and 3.43 (experiment 2)).

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

2.59 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

max luciferase acitivity (Imax) induction of 2.64 was determined at a test item concentration of 125 µM. The corresponding cell viability was 213.6%.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

1.4 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Imax induction of 2.79 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 146.6%.

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.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	105.2	83.5	94.4	15.3
	8.00	108.8	87.0	97.9	15.4
	16.00	114.3	96.3	105.3	12.7
	32.00	123.0	100.6	111.8	15.9
	64.00	167.6	111.7	139.6	39.5
Test Item	0.98	84.6	110.8	97.7	18.6
	1.95	84.9	113.3	99.1	20.1
	3.91	99.1	136.3	117.7	26.3
	7.81	138.9	150.9	144.9	8.5
	15.63	195.8	146.6	171.2	34.8
	31.25	221.9	124.6	173.3	68.8
	62.50	225.5	121.6	173.5	73.5
	125.00	213.6	93.2	153.4	85.1
	250.00	164.6	123.9	144.2	28.8
	500.00	101.4	128.4	114.9	19.1
	1000.00	195.0	147.1	171.0	33.9
	2000.00	192.3	149.9	171.1	30.0

 

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	1.44	1.05	1.43	1.31	0.22
	8.00	1.26	1.24	1.48	1.33	0.13
	16.00	1.70	1.25	1.80	1.58	0.29	*
	32.00	2.46	1.88	2.36	2.23	0.31	*
	64.00	5.70	3.23	4.86	4.60	1.26	*
Test Item	0.98	1.13	1.09	1.19	1.14	0.05
	1.95	1.44	1.34	1.30	1.36	0.07
	3.91	2.04	1.83	1.50	1.79	0.27	*
	7.81	2.26	2.06	1.68	2.00	0.29	*
	15.63	2.68	2.29	2.27	2.41	0.23	*
	31.25	2.90	2.50	2.42	2.61	0.26	*
	62.50	2.95	2.57	2.37	2.63	0.29	*
	125.00	2.99	2.51	2.42	2.64	0.31	*
	250.00	2.90	2.12	2.14	2.39	0.45	*
	500.00	2.89	2.53	2.18	2.53	0.36	*
	1000.00	2.69	2.26	2.06	2.34	0.32	*
	2000.00	2.37	2.00	1.78	2.05	0.30	*

* = 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
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.05	1.07	1.12	1.08	0.04
	8.00	1.17	1.29	1.25	1.24	0.07
	16.00	1.31	1.35	1.68	1.45	0.20
	32.00	1.93	1.85	1.69	1.83	0.12	*
	64.00	3.67	2.88	3.74	3.43	0.48	*
Test Item	0.98	1.16	1.27	1.36	1.26	0.10
	1.95	1.86	1.80	1.75	1.81	0.05	*
	3.91	2.07	2.23	2.26	2.19	0.10	*
	7.81	2.71	2.57	2.67	2.65	0.07	*
	15.63	2.79	2.89	2.68	2.79	0.10	*
	31.25	2.42	2.75	2.75	2.64	0.19	*
	62.50	2.81	2.55	2.49	2.61	0.17	*
	125.00	2.54	2.66	2.35	2.52	0.16	*
	250.00	2.62	2.79	2.68	2.69	0.09	*
	500.00	2.48	2.49	2.70	2.55	0.13	*
	1000.00	2.47	2.27	2.70	2.48	0.22	*
	2000.00	2.44	2.14	2.27	2.28	0.15	*

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

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	1.31	1.08	1.19	0.16
	8.00	1.33	1.24	1.28	0.06
	16.00	1.58	1.45	1.51	0.10	*
	32.00	2.23	1.83	2.03	0.29	*
	64.00	4.60	3.43	4.01	0.83	*
Test Item	0.98	1.14	1.26	1.20	0.09
	1.95	1.36	1.81	1.58	0.32
	3.91	1.79	2.19	1.99	0.28	*
	7.81	2.00	2.65	2.32	0.46
	15.63	2.41	2.79	2.60	0.26	*
	31.25	2.61	2.64	2.62	0.02	*
	62.50	2.63	2.61	2.62	0.01	*
	125.00	2.64	2.52	2.58	0.09	*
	250.00	2.39	2.69	2.54	0.22	*
	500.00	2.53	2.55	2.54	0.01	*
	1000.00	2.34	2.48	2.41	0.10	*
	2000.00	2.05	2.28	2.17	0.17	*

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	2.59	1.40	2.00	0.84
Imax	2.64	2.79	2.71	0.10
IC30[µM]	n/a	n/a	-	-
IC50[µM]	n/a	n/a	-	-

n/a: not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	10.1	pass	9.1	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	13.39	pass	18.27	pass
Induction PC at 64 µM	2.00 < x < 8.00	4.60	pass	3.43	pass

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 generated should be considered in the context of integrated approached such as IATA.

Conclusions:

In this study under the given conditions the test item did 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 sensitiser.
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 test item was dissolved in tetrahydrofuran.

Based on a molecular weight of 296 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 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.

In both experiment no cytotoxic effect was observable.

In the first experiment, a max luciferase activity (I_max) induction of 2.64 was determined at a test item concentration of 125 µM. The corresponding cell viability was 213.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.79) was found to be 3.91 µM. The corresponding cell viability was >70% (99.1%). The calculated EC_1.5 was <1000 µM (2.59 µM).

In the second experiment, a max luciferase activity (I_max) induction of 2.79 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 146.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.81) was found to be 1.95 µM. The corresponding cell viability was >70% (113.3%). The calculated EC_1.5 was < 1000 µM (1.40 µM). A 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 sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-05-07 to 2018-06-12

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: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

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 of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution:
The test item was soluble in Tetrahydrofuran (THF) at a concentration of 500 mg/mL. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.

- Preparation of the test chemical serial dilutions:
The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

- Preparation of the positive controls:
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted, resulting in a final DMSO concentration of 0.2% (v/v).

- Preparation of the solvent, vehicle and negative controls:
A medium control was included in the test.
Since the test item was solubilized in THF, a THF (0.20%) control served as solvent control for the test item. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control.
- Log Kow of the test chemical: > 6.5

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 1000 µg/mL
- Solubility in solvents: The test item was soluble in THF at a concentration of 500 mg/mL.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: No cytotoxicity was observed.
- Final concentration range selected on basis of: Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 1
- Number of repetitions: 2
- Test chemical concentrations: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL
- Application procedure: The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
- Exposure time: 24 h
- Description on study 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%.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 E+6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2E+6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1E+6 cells/well).

- Incubation conditions: Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
- Washing conditions: Cells were washed twice with FACS buffer.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT
- Flow cytometry used: Yes
- Plate used: 96-well V-bottom plate.
- Propidium iodide staining/cytotoxicity measurements: PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
- Preparation for CD54 and/or CD86 expression measurements/cell staining: Cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner.

DATA EVALUATION
- Cytotoxicity assessment: Cell viability was analysed by flow cytometry (wavelength > 650 nm) using PI staining.
- Prediction model used: Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

Vehicle / solvent control:

other: THF (0.2%) served as solvent control for the test item

Negative control:

other: medium control

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (330% experiment 1; 305% experiment 2) and 200% for CD54 (304% experiment 1; 303% experiment 2) were clearly exceeded.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI/CD86 >= 150%

Value:

210 %

Cell viability:

91.6%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI/CD54 >= 200%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no relevant increase

Remarks:

The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI/CD86 >= 150%

Value:

215 %

Cell viability:

92.30%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI/CD54 >= 200%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no relevant increase

Remarks:

The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

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 20 Activation Test

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	89.2	266	>150	88.4	206	>200	yes	pass
NiSO4	100 µg/mL	82.3	220	>150	80.6	283	>200	yes	pass
LA	1000 µg/mL	96.2	79	≤150	96.9	101	≤200	no	pass

 Results of the Cell Batch 21 Activation Test

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	85.5	251	>150	84.7	287	>200	yes	pass
NiSO4	100 µg/mL	74.9	249	>150	75.5	518	>200	yes	pass
LA	1000 µg/mL	94.8	67	≤150	95.0	108	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	92.20
Solvent Control	THF	--	93.00
Test item	C8	7.81	92.50
	C7	15.63	93.50
	C6	31.25	94.80
	C5	62.50	93.90
	C4	125.00	94.40
	C3	250.00	93.50
	C2	500.00	93.40
	C1	1000.00	93.90
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	-	96.4	96.0	95.8	2089	1122	641	1448	481	94	96	326	175
Solvent Control 2 (THF)	0.20%	96.3	96.0	95.8	1975.0	1166.0	609.0	1366	557	100	100	324	191
Solvent Control 1 (DMSO)	0.20%	96.3	95.4	95.9	2160	1124	621	1539	503	100	100	348	181
DNCB	4.00	84.3	83.6	84.1	5750	2207	677	5073	1530	330	304	849	326
Test item	1000	93.2	92.6	93.0	2937	1146	607	2330	539	171	97	484	189
	833.33	91.6	91.2	90.8	3507	1205	641	2866	564	210	101	547	188
	694.44	92.3	91.7	92.4	3327	1223	632	2695	591	197	106	526	194
	578.70	91.9	92.2	90.4	3369	1177	640	2729	537	200	96	526	184
	482.25	91.8	92.0	92.5	3300	1230	679	2621	551	192	99	486	181
	401.88	93.1	92.1	92.4	2980	1161	621	2359	540	173	97	480	187
	334.90	92.0	92.1	92.5	3158	1122	606	2552	516	187	93	521	185
	279.08	93.5	94.6	94.4	2746	1128	626	2120	502	155	90	439	180

 

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	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	95.7	95.7	95.3	2063	980	607	1456	373	93	83	340	161
Solvent Control 2 (THF)	0.20%	95.8	96.3	95.8	1801	1005	624	1177	381	100	100	289	161
Solvent Control 1 (DMSO)	0.20%	95.5	95.0	95.5	2153	1035	588	1565	447	100	100	366	176
DNCB	4.0	84.3	83.6	83.7	5409	1994	641	4768	1353	305	303	844	311
Test item	1000.00	92.00	91.30	91.30	3085	1045	675	2410	370	205	97	457	155
	833.33	92.30	92.50	91.90	3192	1051	662	2530	389	215	102	482	159
	694.44	92.90	93.00	93.70	2893	1065	611	2282	454	194	119	473	174
	578.70	93.00	92.60	93.30	2952	1047	637	2315	410	197	108	463	164
	482.25	93.10	93.40	93.50	2671	1024	712	1959	312	166	82	375	144
	401.88	93.90	93.00	93.80	2730	1007	636	2094	371	178	97	429	158
	334.90	92.90	92.40	92.80	2897	1004	594	2303	410	196	108	488	169
	279.08	94.40	94.30	94.50	2363	1022	599	1764	423	150	111	394	171

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.4	-	96.4	pass	95.0	-	96.3	pass
number of test dosed with viability >50% CD86	≥4	8			pass	8			pass
number of test dosed with viability >50% CD54	≥4	8			pass	8			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	8			pass
RFI of positive control of CD86	≥150	330			pass	305			pass
RFI of positive control of CD54	≥200	304			pass	303			pass
RFI of DMSO solvent control of CD86	<150	106			pass	107			pass
RFI of DMSO solvent control of CD54	<200	105			pass	120			pass
RFI of THF solvent control of CD86	<150	94			pass	81			pass
RFI of THF solvent control of CD54	<200	116			pass	102			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	326			pass	340			pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	348			pass	366			pass
MFI ratio IgG1/CD86 for THF control [%]	>105	324			pass	289			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	175			pass	161			pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	181			pass	176			pass
MFI ratio IgG1/CD54 for THF control [%]	>105	191			pass	161			pass

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	97.0	1.3	672
number of test doses with viability >50%	-	-	1786
RFI of positive control of CD86	401.0	146.8	112
RFI of positive control of CD54	576.6	312.0	112
RFI of solvent control of CD86	115.0	15.1	112
RFI of solvent control of CD54	118.8	25.5	112
MFI ratio IgG1/CD86 for medium control [%]	202.4	50.0	112
MFI ratio IgG1/CD86 for DMSO control [%]	221.6	58.5	112
MFI ratio IgG1/CD54 for medium control [%]	141.0	24.7	112
MFI ratio IgG1/CD54 for DMSO control [%]	147.7	25.6	112

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.
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 500 mg/mL to 3.91 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:

1000; 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

The test item precipitates in all test item concentrations in the dose finding assay and in the main experiments 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 93.2% (CD86), 92.6% (CD54) and 93.0% (isotype IgG1 control) in the first experiment and to 92.0% (CD86), 91.3% (CD54) and 91.3% (isotype IgG1 control)in the second experiment.

The expression of the cell surface marker CD86 was upregulated to a maximum of 210% (experiment 1) and 215% (experiment 2). The upregulation above the threshold of 150% was observed in all concentrations (experiment 1) and at concentrations of 334.90 - 1000.00 µg/mL (experiment 2). In contrast, the expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Direct Peptide Reactivity Assay (OECD 442 C)

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of cysteine peptide was≤ 6.38% (0.30%).

According to the evaluation criteria in the guideline, if a precipitation 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 63.50%.

ARE-Nrf2 Luciferase Test Method (OECD 442 D)

In both experiment no cytotoxic effect was observable.

In the first experiment, a max luciferase activity (Imax) induction of 2.64 was determined at a test item concentration of 125 µM. The corresponding cell viability was 213.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.79) was found to be 3.91 µM. The corresponding cell viability was >70% (99.1%). The calculated EC1.5 was <1000 µM (2.59 µM).

In the second experiment, a max luciferase activity (Imax) induction of 2.79 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 146.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.81) was found to be 1.95 µM. The corresponding cell viability was >70% (113.3%). The calculated EC1.5 was < 1000 µM (1.40 µM). A 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 sensitiser.

Human Cell Line Activation Test (OECD 442 E)

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 93.2% (CD86), 92.6% (CD54) and 93.0% (isotype IgG1 control) in the first experiment and to 92.0% (CD86), 91.3% (CD54) and 91.3% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to a maximum of 210% (experiment 1) and 215% (experiment 2). The upregulation above the threshold of 150% was observed in all concentrations (experiment 1) and at concentrations of 334.90 - 1000.00 µg/mL (experiment 2). In contrast, the expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments

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

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is need for classification according to the EU Regulation (EC) No 1272/2008 on Classification,Lab elling and Packaging of Substances and Mixtures. The test material should be classified for skin sensitisation category 1 and labelled with H317.