4-(6-acryloyloxyhexyloxy)benzoic acid (4-(trans-4-propylcyclohexyl)phenyl ester)

Endpoint summary

Description of key information

Skin sensitisation is considered as an allergic response following skin contact. The sensitisation potential can be assessed by numerous assays including in vivo studies and in vitro

experiments.

The in vitro assays performed show that due to the physicochemical properties of the compound, namely logP and limited solubility, the in vitro approach is technically challenging. Two of the three assays performed are inconclusive thus a conclusion based on in vitro data is not possible.

However, QSAR analysis of the chemical structure provides additional information. This analysis resulted in two structural alerts for skin sensitisation. The subsequent EC3 prediction based on information of close structural analogues reveals a moderate potential for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance on IR/CSA R.6 QSARs and grouping of chemicals

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Specific details on test material used for the study:

see QPRF

Parameter:

other: Structural Alert for skin sensitisaton

Value:

2

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Two alerts for skin sensitisation have been identified. See QPRF for details.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Using Derek and Meteor Nexus , the skin sensitising potential assessment resulted in two alerts for skin sensitisation. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate. Due to the prediction result, the compound should be regarded as moderate skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-06-25 to 2018-06-28

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 65.38%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.52

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

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	15.0360	0.5340	14.1920	0.5340
STD2	7.5920	0.2670	7.1540	0.2670
STD3	3.7630	0.1335	3.5770	0.1335
STD4	1.8500	0.0667	1.7790	0.0667
STD5	0.8890	0.0334	0.8770	0.0334
STD6	0.4300	0.0167	0.4310	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	4.1310	0.1469	70.73	71.32	0.53	0.74
	4.0270	0.1433	71.47
	3.9870	0.1418	71.75
Test Item	14.4290	0.5114	0.00	0.52	0.89	172.02
	14.1140	0.5002	0.01
	13.8970	0.4925	1.54

 

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	5.3010	0.1990	60.30	59.44	0.84	1.41
	5.4220	0.2036	59.39
	5.5240	0.2074	58.63
Test Item	13.4640	0.5057	0.00	0.00	0.00	-
	13.3870	0.5028	0.00
	13.4230	0.5042	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	0.26	Minimal Reactivity	-	0.52	Minimal Reactivity	-
Positive Control	65.38	High Reactivity	sensitiser	71.32	Moderate Reactivity	sensitiser

Interpretation of results:

other: The result should be considered in the context of IATA.

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. 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 acetonitrile, based on the results of the pre-experiments.

Based on a molecular weight of 492 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.

Forthe 100 mM stock solution of the test item turbidity 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 turbidity 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). Precipitation was observed for the samples of the test item (including the co-elution control). Test item samples were 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.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.26%).Since precipitation wasobserved, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline,no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.Therefore, 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 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-07-23 to 2018-09-04

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 (6.98 (experiment 1); 3.11 (experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.56

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:

125.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

1 867.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: clearly above threshold of 1000 µM

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.44

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:

117.6

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	80.8	102.0	91.4	15.0
	8.00	83.6	101.1	92.3	12.3
	16.00	84.6	107.0	95.8	15.9
	32.00	84.8	109.9	97.3	17.7
	64.00	83.4	111.1	97.3	19.6
Test Item	0.98	122.0	127.7	124.9	4.0
	1.95	132.1	118.6	125.4	9.6
	3.91	116.0	103.8	109.9	8.6
	7.81	75.1	100.4	87.7	17.9
	15.63	114.4	106.8	110.6	5.4
	31.25	84.5	95.3	89.9	7.6
	62.50	30.2	86.2	58.2	39.6
	125.00	78.2	85.1	81.6	4.9
	250.00	29.0	87.7	58.3	41.5
	500.00	71.9	91.7	81.8	14.0
	1000.00	91.0	107.1	99.1	11.4
	2000.00	125.4	117.6	121.5	5.5

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	1.22	1.07	1.13	1.14	0.08
	8.00	1.26	1.49	1.28	1.34	0.12
	16.00	1.72	1.96	1.73	1.80	0.14	*
	32.00	2.50	2.81	2.72	2.68	0.16	*
	64.00	6.59	7.67	6.67	6.98	0.60	*
Test Item	0.98	1.42	1.40	1.31	1.38	0.06
	1.95	1.24	1.09	1.16	1.16	0.07
	3.91	1.53	1.12	1.19	1.28	0.22
	7.81	1.18	1.42	1.14	1.24	0.15
	15.63	1.10	1.11	1.08	1.10	0.01
	31.25	1.11	1.01	0.88	1.00	0.12
	62.50	1.07	0.86	0.88	0.94	0.12
	125.00	0.78	0.89	0.86	0.84	0.06
	250.00	0.72	1.11	0.75	0.86	0.22
	500.00	0.78	1.13	0.89	0.93	0.18
	1000.00	1.03	1.33	1.00	1.12	0.18
	2000.00	1.40	1.93	1.34	1.56	0.32	*

* = 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	1.32	1.19	1.14	1.22	0.09
	8.00	1.35	1.27	1.30	1.31	0.04
	16.00	1.50	1.29	1.47	1.42	0.11
	32.00	2.00	1.78	2.00	1.93	0.13	*
	64.00	3.66	2.55	3.11	3.11	0.55	*
Test Item	0.98	1.23	1.47	1.58	1.43	0.18
	1.95	1.07	1.17	1.17	1.14	0.06
	3.91	0.96	1.07	1.08	1.03	0.07
	7.81	1.09	1.27	1.03	1.13	0.12
	15.63	0.94	1.12	0.95	1.01	0.10
	31.25	0.94	1.09	0.89	0.98	0.10
	62.50	0.79	0.85	0.76	0.80	0.05
	125.00	0.91	0.91	0.83	0.88	0.05
	250.00	0.92	0.94	0.84	0.90	0.06
	500.00	0.95	1.10	0.90	0.98	0.11
	1000.00	1.03	1.26	0.94	1.08	0.16
	2000.00	1.28	1.70	1.34	1.44	0.23

* = 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	1.14	1.22	1.18	0.06
	8.00	1.34	1.31	1.32	0.03
	16.00	1.80	1.42	1.61	0.27
	32.00	2.68	1.93	2.30	0.53
	64.00	6.98	3.11	5.04	2.74
Test Item	0.98	1.38	1.43	1.40	0.03
	1.95	1.16	1.14	1.15	0.02
	3.91	1.28	1.03	1.16	0.18
	7.81	1.24	1.13	1.19	0.08
	15.63	1.10	1.01	1.05	0.07
	31.25	1.00	0.98	0.99	0.02
	62.50	0.94	0.80	0.87	0.10
	125.00	0.84	0.88	0.86	0.03
	250.00	0.86	0.90	0.88	0.03
	500.00	0.93	0.98	0.96	0.04
	1000.00	1.12	1.08	1.10	0.03
	2000.00	1.56	1.44	1.50	0.08

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

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	12.7	pass	9.4	pass
CV Solvent Control TI (1% THF)	<20%	14.9	pass	18.6	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	10.74	pass	18.50	pass
Induction PC at 64 µM	2.00 < x < 8.00	6.98	pass	3.11	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 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 492 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 the first experiment, a max luciferase activity (I_max) induction of 1.56 was determined at a test item concentration of 2000 µM.

The corresponding cell viability was 125.4%. No further significant luciferase induction > 1.5 was found in the tested concentration range.The calculated EC_1.5 was > 1000 µM (1867.82 µM).

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

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 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-10-02 to 2018-11-16

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 both
experiments. The threshold of 150% for CD86 (323% experiment 1; 365% experiment 2) and
200% for CD54 (446% experiment 1; 387% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 625 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 900 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

119

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 900 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 251.17 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	--	--	96.80
Solvent Control	THF	--	96.20
test material	C8	7.03	96.40
	C7	14.06	96.20
	C6	28.13	96.90
	C5	56.25	96.80
	C4	112.50	96.80
	C3	225.00	96.10
	C2	450.00	96.50
	C1	900.00	94.90
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	95.8	95.7	1402	882	533	869	349	79	78	263	165
Solvent Control 2 (THF)	0.20%	95.2	95.2	95.2	1555.0	942.0	552.0	1003	390	100	100	282	171
Solvent Control 1 (DMSO)	0.20%	95.5	95.9	95.5	1635	987	538	1097	449	100	100	304	183
DNCB	4.00	80.1	80.6	78.8	4099	2559	556	3543	2003	323	446	737	460
test material	900	94.6	95.5	95.6	1632	975	577	1055	398	105	102	283	169
	750	94.6	94.5	94.3	1722	975	596	1126	379	112	97	289	164
	625	94.0	94.1	94.9	1740	951	595	1145	356	114	91	292	160
	520.83	95.3	94.8	94.8	1660	921	579	1081	342	108	88	287	159
	434.03	94.6	95.0	94.7	1719	963	600	1119	363	112	93	287	161
	361.69	94.3	95.5	94.5	1680	957	598	1082	359	108	92	281	160
	301.41	94.9	95.2	94.9	1728	977	584	1144	393	114	101	296	167
	251.17	95.2	95.0	94.9	1632	955	600	1032	355	103	91	272	159

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	-	94.6	94.1	94.1	2259	1199	592	1667	607	107	88	382	203
Solvent Control 2 (THF)	0.20%	93.8	94.0	93.8	2053	1259	589	1464	670	100	100	349	214
Solvent Control 1 (DMSO)	0.20%	94.5	94.6	93.8	2124	1258	569	1555	689	100	100	373	221
DNCB	4.0	80.9	80.6	79.7	6271	3252	589	5682	2663	365	387	1065	552
test material	900	93.70	93.70	93.50	2380	1272	632	1748	640	119	96	377	201
	750	93.40	94.00	94.20	2322	1218	634	1688	584	115	87	366	192
	625	93.50	94.00	93.70	2324	1249	631	1693	618	116	92	368	198
	520.83	94.70	94.80	94.40	2083	1238	623	1460	615	100	92	334	199
	434.03	93.90	93.10	94.00	2335	1240	641	1694	599	116	89	364	193
	361.69	92.80	93.10	93.00	2340	1253	643	1697	610	116	91	364	195
	301.41	93.60	93.90	93.70	2367	1304	634	1733	670	118	100	373	206
	251.17	94.00	94.20	93.60	2349	1329	632	1717	697	117	104	372	210

 

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.2	-	96.4	pass	93.8	-	94.6	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	323			pass	365			pass
RFI of positive control of CD54	≥200	446			pass	387			pass
RFI of DMSO solvent control of CD86	<150	126			pass	93			pass
RFI of DMSO solvent control of CD54	<200	129			pass	114			pass
RFI of THF solvent control of CD86	<150	115			pass	88			pass
RFI of THF solvent control of CD54	<200	112			pass	110			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	263			pass	382			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	304			pass	373			pass
MFI ratio CD86/IgG1 for THF control [%]	>105	282			pass	349			pass
MFI ratio CD54/IgG1for medium control [%]	>105	165			pass	203			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	183			pass	221			pass
MFI ratio CD54/IgG1for THF control [%]	>105	171			pass	214			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 result should be considered in the context of IATA.

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item has to be considered as inconclusive.
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 at the highest soluble concentration of 450 mg/mL. For the dose finding assay stock solutions with a concentration range of 450 mg/mL to 3.52 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:

900; 750, 625, 520.83, 434.03, 361.69, 301.41, 251.17 µg/mL

In all experiments (dose finding assay and main 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 94.6% (CD86), 95.5% (CD54) and 95.6% (isotype IgG1 control) in the first experiment and to 93.7% (CD86), 93.7% (CD54) and 93.5% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Because of a Log K_OW value above 3.5 a negative result cannot be considered. Therefore, the test item has to be considered as inconclusive.

Reference 5

Endpoint:

skin sensitisation, other

Type of information:

other: Weight of Evidence Justification

Adequacy of study:

other information

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Key result

Parameter:

other:

Value:

1

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evi-dence for a classification in class 1B for skin sensitisation for the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evidence for a classification in class 1B for skin sensitisation.