1,4-Cyclohexanedicarboxylic acid, 1,4-bis[4-[6-[(1-oxo-2-propen-1-yl)oxy]hexyl]phenyl] ester, trans-

Endpoint summary

Description of key information

OECD 442C: skin sensitizer

OECD 442D: non-sensitizer to skin

OECD 442 E: 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:

weight of evidence

Study period:

2018-09-05 to 2018-09-12

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 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 66.92%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

3.28

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:

100

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	15.3680	0.5340	16.0900	0.5340
STD2	7.7670	0.2670	8.0180	0.2670
STD3	3.8360	0.1335	4.0080	0.1335
STD4	1.8710	0.0667	1.9940	0.0667
STD5	0.9040	0.0334	0.9980	0.0334
STD6	0.4260	0.0167	0.5000	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	4.2840	0.1493	72.34	72.48	0.20	0.27
	4.2280	0.1473	72.70
	4.2760	0.1490	72.39
Test Item	14.7380	0.5110	4.85	3.28	1.92	58.61
	15.3130	0.5308	1.13
	14.8890	0.5162	3.87

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	5.8160	0.1934	61.85	61.36	0.58	0.95
	5.8670	0.1950	61.51
	5.9890	0.1991	60.71
Test Item	0.0000	0.0003	100.00	100.00	0.00	0.00
	0.0000	0.0003	100.00
	0.0000	0.0003	100.00

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	51.64	High Reactivity	positive	3.28	Minimal Reactivity	negative
Positive Control	66.92	High Reactivity	positive	72.48	Moderate Reactivity	positive

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 showed high reactivity towards both peptides. The test item is 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 the test material was dissolved in acetonitrile, based on the results of the pre-experiments.

Based on a molecular weight of 632.78 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.

All test item solutions were freshly prepared immediately prior to use.

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 samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

Precipitation of the test item with both peptide peaks was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was> 6.38% (51.64%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as sensitiser.

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 66.92%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-09-14 to 2018-11-05

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 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 (6.02 (experiment 1) and 2.35 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 2000 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

58.6

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:

1.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 2000 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

171.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [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.

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	93.7	100.0	96.8	4.5
	8.00	97.8	93.9	95.8	2.8
	16.00	102.6	103.1	102.8	0.4
	32.00	103.8	105.8	104.8	1.4
	64.00	103.8	83.1	93.5	14.6
Test Item	0.98	84.9	76.8	80.9	5.7
	1.95	79.9	83.0	81.5	2.3
	3.91	83.4	83.4	83.4	0.0
	7.81	77.2	78.1	77.7	0.6
	15.63	74.6	81.7	78.2	5.0
	31.25	68.5	75.7	72.1	5.1
	62.50	72.3	69.6	71.0	1.9
	125.00	73.9	72.1	73.0	1.2
	250.00	61.9	80.6	71.2	13.2
	500.00	69.7	83.6	76.7	9.8
	1000.00	75.3	80.4	77.9	3.6
	2000.00	58.6	171.8	115.2	80.1

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	1.03	1.07	1.09	1.07	0.03
	8.00	1.29	1.31	1.39	1.33	0.06
	16.00	1.54	1.76	1.65	1.65	0.11	*
	32.00	2.51	2.09	2.48	2.36	0.23	*
	64.00	6.93	4.60	6.53	6.02	1.25	*
Test Item	0.98	0.80	0.93	0.81	0.85	0.07
	1.95	0.88	1.17	0.90	0.98	0.16
	3.91	0.76	0.86	0.70	0.77	0.08
	7.81	0.82	1.16	0.76	0.91	0.21
	15.63	0.78	0.77	0.77	0.77	0.01
	31.25	0.79	0.87	0.80	0.82	0.04
	62.50	0.82	0.79	0.73	0.78	0.05
	125.00	0.80	0.84	0.79	0.81	0.03
	250.00	0.79	0.86	0.73	0.79	0.07
	500.00	0.91	1.00	0.89	0.94	0.06
	1000.00	0.89	0.83	0.83	0.85	0.04
	2000.00	1.31	0.93	0.89	1.04	0.23

* = 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	1.13	0.99	1.07	1.07	0.07
	8.00	1.02	1.34	1.38	1.25	0.20
	16.00	1.37	1.26	1.69	1.44	0.23
	32.00	1.53	1.88	2.01	1.81	0.25	*
	64.00	2.28	2.25	2.52	2.35	0.15	*
Test Item	0.98	0.73	1.16	0.78	0.89	0.24
	1.95	0.86	1.07	1.14	1.02	0.15
	3.91	0.70	0.98	1.00	0.89	0.17
	7.81	0.79	0.87	0.80	0.82	0.04
	15.63	0.72	0.78	0.83	0.78	0.06
	31.25	0.82	0.88	1.00	0.90	0.09
	62.50	0.82	0.78	0.83	0.81	0.02
	125.00	0.87	0.85	0.89	0.87	0.02
	250.00	0.99	1.02	0.98	1.00	0.02
	500.00	0.95	0.97	1.04	0.99	0.05
	1000.00	0.96	0.98	1.03	0.99	0.03
	2000.00	1.09	1.14	1.92	1.38	0.46

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

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.07	1.07	1.07	0.00
	8.00	1.33	1.25	1.29	0.06
	16.00	1.65	1.44	1.55	0.15
	32.00	2.36	1.81	2.08	0.39
	64.00	6.02	2.35	4.18	2.59
Test Item	0.98	0.85	0.89	0.87	0.03
	1.95	0.98	1.02	1.00	0.03
	3.91	0.77	0.89	0.83	0.09
	7.81	0.91	0.82	0.87	0.06
	15.63	0.77	0.78	0.77	0.00
	31.25	0.82	0.90	0.86	0.06
	62.50	0.78	0.81	0.79	0.02
	125.00	0.81	0.87	0.84	0.04
	250.00	0.79	1.00	0.90	0.14
	500.00	0.94	0.99	0.96	0.04
	1000.00	0.85	0.99	0.92	0.10
	2000.00	1.04	1.38	1.21	0.24

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	n.a.	n.a.
Imax	1.04	1.38	1.21	0.24
IC30[µM]	591.52	n.a.	n.a.	n.a.
IC50[µM]	n.a.	n.a.	n.a.	n.a.

n.a.: not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	4.0	pass	13.7	pass
CV Solvent Control TI (1% THF)	<20%	9.0	pass	14.2	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	2.0	pass
EC1.5 PC	± 2 x SD of historical mean	12.28	pass	18.54	pass
Induction PC at 64 µM	2 .00 < x < 8.00	6.02	pass	2.35	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.5	96
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.4	0.6	96
EC1.5 PC	7 < x < 34 µM	18.5	6.0	96
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.5	96

Interpretation of results:

other: The result 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 632.78 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.

After incubation, precipitations were observed microscopically in cell culture medium at a concentration range from 15.6 µM – 2000 µM in the first main experiment and at a concentration of 2000 µM in the second main experiment.

In the first experiment, a slight cytotoxicity in the concentrations 31.25 µM, 250 µM, 500 µM and 2000 µM was observed, the concentrations were excluded from evaluation. No significant luciferase induction > 1.5 was found in the remaining test concentrations. Therefore, no EC_1.5 value could be calculated.

In the second experiment, only at 62.5 µM a slight cytotoxicity was observed, the concentration was excluded from evaluation. 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:

weight of evidence

Study period:

2018-12-10 to 2019-02-07

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 (296% experiment 1; 350% experiment 2; 408% experiment 3) and 200% for CD54 (299% experiment 1; 358% experiment 2; 409% experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

214

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1000 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

137

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 578.70 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

107

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 482.25 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 279.08 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

306

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 482.25 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

162

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 279.08 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 3

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	85.4	306	>150	84.7	220	>200	yes	pass
NiSO4	100 µg/mL	81.8	251	>150	80.9	363	>200	yes	pass
LA	1000 µg/mL	94.0	93	≤150	93.9	93	≤200	no	pass

Results of the Cell Batch Activation Test 4

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	84.5	342	>150	85.8	334	>200	yes	pass
NiSO4	100 µg/mL	81.7	280	>150	81.7	578	>200	yes	pass
LA	1000 µg/mL	95.3	90	≤150	95.6	123	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	94.50
Solvent Control	THF	--	95.50
Test material	C8	7.81	95.20
	C7	15.63	95.80
	C6	31.25	94.60
	C5	62.50	94.80
	C4	125.00	95.20
	C3	250.00	95.40
	C2	500.00	94.80
	C1	1000.00	95.00
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		NoSD

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	-	97.5	97.2	96.4	1230	761	518	712	243	90	93	237	147
Solvent Control 2 (THF)	0.20%	97.0	97.0	96.7	1115.0	743.0	514.0	601	229	100	100	217	145
Solvent Control 1 (DMSO)	0.20%	96.8	96.9	96.9	1289	760	500	789	260	100	100	258	152
DNCB	4.00	85.8	84.9	84.0	2958	1396	619	2339	777	296	299	478	226
Test material	1000	95.3	94.5	94.4	1853	854	566	1287	288	214	126	327	151
	833.33	96.6	97.0	96.8	1452	843	542	910	301	151	131	268	156
	694.44	96.6	96.6	95.9	1602	837	536	1066	301	177	131	299	156
	578.70	97.4	97.5	97.1	1350	856	542	808	314	134	137	249	158
	482.25	96.3	97.0	96.9	1446	868	558	888	310	148	135	259	156
	401.88	97.2	97.0	97.0	1375	826	547	828	279	138	122	251	151
	334.90	97.2	97.4	97.2	1344	801	537	807	264	134	115	250	149
	279.08	96.4	96.8	96.6	1370	786	545	825	241	137	105	251	144

 

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	-	96.1	96.5	96.9	1548	914	513	1035	401	133	172	302	178
Solvent Control 2 (THF)	0.20%	96.9	95.7	96.8	1570	1043	519	1051	524	100	100	303	201
Solvent Control 1 (DMSO)	0.20%	96.7	96.9	96.7	1297	751	518	779	233	100	100	250	145
DNCB	4.0	76.9	84.0	83.0	3392	1502	667	2725	835	350	358	509	225
Test material	1000.00	96.20	95.50	96.10	1592	825	584	1008	241	96	46	273	141
	833.33	95.90	95.80	95.60	1604	831	577	1027	254	98	48	278	144
	694.44	96.00	96.40	96.10	1557	806	561	996	245	95	47	278	144
	578.70	96.30	96.50	95.80	1492	818	572	920	246	88	47	261	143
	482.25	96.70	96.30	95.90	1668	777	542	1126	235	107	45	308	143
	401.88	96.90	94.90	93.50	1560	762	558	1002	204	95	39	280	137
	334.90	96.40	96.20	96.40	1472	863	592	880	271	84	52	249	146
	279.08	96.90	96.90	96.60	1704	882	593	1111	289	106	55	287	149

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	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	97.3	97.1	96.7	1019	832	586	433	246	101	101	174	142
Solvent Control 2 (THF)	0.20%	96.9	95.9	95.3	951	868	570	381	298	100	100	167	152
Solvent Control 1 (DMSO)	0.20%	97.5	97.3	97.4	963	779	535	428	244	100	100	180	146
DNCB	4.0	86.4	88.2	87.4	2328	1577	580	1748	997	408	409	401	272
Test material	1000.0	95.8	95.6	95.7	969	1190	712	257	478	67	160	136	167
	833.33	96.3	96.3	95.9	1534	1006	589	945	417	248	140	260	171
	694.44	96.2	95.8	96.0	1731	1014	706	1025	308	269	103	245	144
	578.70	95.1	94.7	95.5	1829	1149	695	1134	454	298	152	263	165
	482.25	96.2	96.1	96.3	1787	1070	623	1164	447	306	150	287	172
	401.88	96.4	96.2	96.3	1693	985	611	1082	374	284	126	277	161
	334.90	96.3	96.3	96.2	1750	1244	810	940	434	247	146	216	154
	279.08	96.9	96.7	96.3	1667	1100	616	1051	484	276	162	271	179

Acceptance Criteria

Acceptance Criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

Experiment 3

pass/fail

cell viability medium and solvent controls [%]

>90

96.4

-

97.5

pass

95.7

-

96.9

pass

95.3

-

97.5

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

296

pass

350

pass

408

pass

RFI of positive control of CD54

≥200

299

pass

358

pass

409

pass

RFI of DMSO solvent control of CD86

<150

111

pass

75

pass

99

pass

RFI of DMSO solvent control of CD54

<200

107

pass

58

pass

99

pass

RFI of THF solvent control of CD86

<150

84

pass

102

pass

88

pass

RFI of THF solvent control of CD54

<200

94

pass

131

pass

121

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

237

pass

302

pass

174

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

258

pass

250

pass

180

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

217

pass

303

pass

167

pass

MFI ratio CD54/IgG1 for medium control [%]

>105

147

pass

178

pass

142

pass

MFI ratio CD54/IgG1 for DMSO control [%]

>105

152

pass

145

pass

146

pass

MFI ratio CD54/IgG1 for THF control [%]

>105

145

pass

201

pass

152

pass

Historical Data

Criterion	mean	SD	N
Viability Solvent Control [%]	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 (THF) of CD86	115.0	15.1	112
RFI of solvent control (THF) of CD54	118.8	25.5	112
MFICD86 / MFIIgG1 for medium control [%]	202.4	50.0	112
MFICD86 / MFIIgG1 for THF control [%]	221.6	58.5	112
MFICD54 / MFIIgG1 for medium control [%]	141.0	24.7	112
MFICD54 / MFIIgG1 for THF control [%]	147.7	25.6	112

Interpretation of results:

other: The result 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 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 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

In all experiments, dose finding experiment and main experiment, precipitation of the test item was observed for all the 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 95.3% (CD86), 94.5% (CD54) and 94.4% (isotype IgG1 control) in the first experiment, 96.20% (CD86), 95.50% (CD54) and 96.10% (isotype IgG1 control) in the second experiment and to 95.8% (CD86), 95.6% (CD54) and 95.7% (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 214% in the first experiment and to 306% in the third experiment. The upregulation above the threshold of 150% was observed at concentrations of 1000, 833.33 and 694.44 µg/mL in the first experiment and at concentrations of 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, and 279.08 µg/mL in the third experiment. 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 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

Based on the provided information there is sufficient information for classification for skin sensitisation class 1 (H317) according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.