4-Ethoxy-2,3-difluorphenylboronic acid

Endpoint summary

Description of key information

OECD 442D (KeratinoSens): skin sensitiser

OECD 442C (DPRA): no prediction can be made

OECD 442E (h-CLAT): skin sensitiser

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:

2018-03-27 to 2018-04-07

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))

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 68.26%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

9.04

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	5138.8999	0.5340	4282.5977	0.5340
STD2	2651.5103	0.2670	2162.7876	0.2670
STD3	1325.9341	0.1335	1090.6255	0.1335
STD4	655.7342	0.0667	532.2374	0.0667
STD5	319.7461	0.0334	267.5374	0.0334
STD6	151.5604	0.0167	134.4307	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	1463.3074	0.1502	70.30	70.42	0.10	0.15
	1455.7604	0.1494	70.46
	1453.6044	0.1492	70.50
Test Item	4825.3594	0.4981	2.08	9.04	11.27	124.66
	4779.7603	0.4933	3.00
	3841.2102	0.3962	22.05

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	1317.4542	0.1636	67.49	66.09	1.31	1.98
	1382.0761	0.1716	65.90
	1422.7880	0.1767	64.89
Test Item	4074.0474	0.5068	0.00	0.00	0.00	-
	4059.1592	0.5050	0.00
	4066.8315	0.5059	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	4.52	Minimal Reactivity	no sensitiser	9.04	Minimal Reactivity	no sensitiser
Positive Control	68.26	High Reactivity	sensitiser	70.42	Moderate Reactivity	sensitiser

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 showed minimal reactivity towards both peptides. The test item might be considered as “non-sensitiser”, however, due to the borderline depletion no prediction can be made.
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 ACN, based on the results of the pre-experiments. Based on a molecular weight of 201.96 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 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. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. 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.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_ACN).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (4.52%). Based on the prediction model 1 the test item can be considered as non-sensitiser. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered. 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 68.26%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-04 to 2018-04-30

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 (3.47 (experiment 1); 4.34 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

4.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1000 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

82.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

84.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

5.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1000 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

64.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

102.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

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	103.3	101.3	102.3	1.4
	8.00	98.6	100.5	99.5	1.4
	16.00	93.0	98.8	95.9	4.1
	32.00	99.2	101.8	100.5	1.9
	64.00	95.0	96.8	95.9	1.2
Test Item	0.98	103.6	107.8	105.7	3.0
	1.95	100.7	107.2	103.9	4.6
	3.91	91.4	105.1	98.3	9.6
	7.81	94.3	107.3	100.8	9.1
	15.63	95.8	106.7	101.3	7.7
	31.25	97.4	105.8	101.6	5.9
	62.50	98.6	107.2	102.9	6.1
	125.00	96.6	101.1	98.8	3.2
	250.00	91.3	99.1	95.2	5.6
	500.00	91.8	90.2	91.0	1.1
	1000.00	82.4	64.3	73.3	12.9
	2000.00	14.0	84.6	49.3	49.9

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	1.24	0.88	0.99	1.03	0.18
	8.00	1.13	1.00	1.16	1.10	0.09
	16.00	1.53	1.42	1.44	1.46	0.06
	32.00	1.72	1.41	1.58	1.57	0.16	*
	64.00	3.75	2.81	3.86	3.47	0.58	*
Test Item	0.98	1.26	1.11	0.81	1.06	0.23
	1.95	0.95	0.79	0.79	0.84	0.10
	3.91	1.54	0.88	0.84	1.09	0.39
	7.81	1.04	0.95	0.93	0.97	0.06
	15.63	1.13	0.94	0.96	1.01	0.11
	31.25	1.11	1.04	1.04	1.06	0.04
	62.50	1.68	1.33	1.26	1.42	0.23
	125.00	1.77	1.58	1.57	1.64	0.12	*
	250.00	2.15	2.62	2.35	2.37	0.24	*
	500.00	2.86	2.81	2.95	2.87	0.07	*
	1000.00	4.70	3.71	3.78	4.06	0.55	*
	2000.00	1.26	1.09	4.77	2.37	2.08

* = 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	1.14	1.06	0.99	1.07	0.07
	8.00	1.20	1.39	1.54	1.37	0.17
	16.00	1.28	1.59	1.57	1.48	0.18
	32.00	2.36	2.74	2.58	2.56	0.19	*
	64.00	4.04	5.02	3.97	4.34	0.58	*
Test Item	0.98	1.04	1.03	1.18	1.09	0.08
	1.95	0.90	0.92	0.95	0.92	0.03
	3.91	0.94	0.96	0.87	0.92	0.04
	7.81	0.81	0.87	0.98	0.88	0.09
	15.63	1.07	0.89	0.95	0.97	0.09
	31.25	0.83	0.90	1.09	0.94	0.13
	62.50	1.19	1.38	1.22	1.26	0.10
	125.00	1.58	1.69	1.62	1.63	0.06	*
	250.00	1.68	1.93	1.71	1.77	0.14	*
	500.00	2.97	2.99	2.34	2.76	0.37	*
	1000.00	6.24	5.68	4.28	5.40	1.01	*
	2000.00	3.44	3.80	2.79	3.35	0.51	*

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

Induction of Luciferase Activity – 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	1.03	1.07	1.05	0.02
	8.00	1.10	1.37	1.24	0.20
	16.00	1.46	1.48	1.47	0.01
	32.00	1.57	2.56	2.06	0.70
	64.00	3.47	4.34	3.91	0.62	*
Test Item	0.98	1.06	1.09	1.07	0.02
	1.95	0.84	0.92	0.88	0.06
	3.91	1.09	0.92	1.00	0.12
	7.81	0.97	0.88	0.93	0.06
	15.63	1.01	0.97	0.99	0.03
	31.25	1.06	0.94	1.00	0.09
	62.50	1.42	1.26	1.34	0.11
	125.00	1.64	1.63	1.63	0.00	*
	250.00	2.37	1.77	2.07	0.42
	500.00	2.87	2.76	2.82	0.08	*
	1000.00	4.06	5.40	4.73	0.94	*
	2000.00	2.37	3.35	2.86	0.69

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	84.95	102.82	93.88	12.64
Imax	4.06	5.40	4.73	0.94
IC30[µM]	1181.78	889.34	1035.56	206.78
IC50[µM]	1474.24	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	< 20%	10.6	pass	7.0	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	21.51	pass	16.27	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.47	pass	4.34	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 results 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 the test item was dissolved in DMSO.

Based on a molecular weight of 201.96 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.

In the first experiment, a max luciferase activity (I_max) induction of 4.06 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 82.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 125 µM. The corresponding cell viability was >70% (96.6%).The calculated EC_1.5 was < 1000 µM (84.95 µM).

In the second experiment, a max luciferase activity (I_max) induction of 5.40 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 64.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.63) was found to be 125 µM. The corresponding cell viability was >70% (101.1%).The calculated EC_1.5 was < 1000 µM (102.82 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:

02 July 2018 - 16 October 2018

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Skin sensitisation (In vitro test system) - Details on 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
experiments.

The following relative fluorescense intensities and viabilities (values in %) have been detected:

Run Concentr. / [µg/mL] CD54 CD86 viability (CD54) viability (CD86)
1 4 205 232 81.6 81.8
2 4 240 247 83.3 83.3


The threshold of 150% for CD86 and 200% for CD54 were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: max relative fluorescence intensity CD86 (%)

Value:

226

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: c = 307.35 µg/mL; cell viability = 85.1 %

Key result

Run / experiment:

other: 1

Parameter:

other: max relative fluorescence intensity CD54 (%)

Value:

40

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: c = 307.35 µg/mL; cell viability = 86.2 %

Key result

Run / experiment:

other: 2

Parameter:

other: max relative fluorescence intensity CD86 (%)

Value:

296

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: c = 386.82 µg/mL; cell viability = 78.8%

Key result

Run / experiment:

other: 2

Parameter:

other: max relative fluorescence intensity CD54 (%)

Value:

62

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: c = 177.86 µg/mL; cell viability = 84%

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: cell viability > 50%; RFI values of the solvent control is not >= 150% for CD86 and not >= 200% for CD54
- Acceptance criteria met for positive control: RFI values of the positive control (DNCB) is >= 150% for CD86 and >= 200% for CD54 at a cell viability of 50%
- Further acceptance criteria: The cell viability of at least four tested doses of the test item in each run is > 50%. The MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control is > 150%.
- Range of historical values if different from the ones specified in the test guideline:
mean SD N
cell viability solvent controls (%) 97 1.3 672
number of test doses with viability > 50% - - 1786
RFI of positive control of CD86 401 146.8 112
RFI of positive control of CD54 576.6 312 112
RFI of solvent control of CD86 115 15.1 112
RFI of solvent control of CD54 118.8 25.5 112
MFI ratio IgG1/CD86 for medium control (%) 202.4 50 112
MFI ratio IgG1/CD86 for DMSO control (%) 221.6 58.5 112
MFI ratio IgG1/CD54 for medium control (%) 141 24.7 112
MFI ratio IgG1/CD54 for DMSO 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:

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

Executive summary:

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.

In the present study the test was dissolved in DMSO. 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.

A CV75 of 531.10 ± 19.56 μg/mL was derived in the dose finding assay.Based on the CV75, the main experiment was performed covering the following concentration steps:

637.32, 531.10, 442.58, 368.82, 307.35, 256.12, 213.44, 177.86 μg/mL

In the dose-finding assays and the main experiments precipitates were observed for all concentrations when diluted 1:250 in cell culture medium. Sonication was used to aid solubilisation. 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.

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 75.8% (CD86), 75.5% (CD54) and 73.8% (isotype IgG1 control) in the first experiment and to 75.4% (CD86), 75.4% (CD54) and 74.6% (isotype IgG1 control) in the second experiment.

In experiment 1, the expression of the cell surface marker CD86 was upregulated to a maximum of 226% above the threshold of 150% at a concentration of 307.35μg/mL. An upregulation above the threshold of 150% was observed in the whole concentration range except for the two highest concentrations. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%.

In experiment 2, the expression of the cell surface marker CD86 was upregulated to a maximum of 296% above the threshold of 150% at a concentration of 386.82μg/mL. An upregulation above the threshold of 150% was observed in the whole concentration range. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%.

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.

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the test item was investigated by performing the in vitro Skin Sensitisation battery (OECD 442D, OECD 442C, and OECD 442E).

KeratinoSens Assay (OECD 442D)

In the first experiment, a max luciferase activity (Imax) induction of 4.06 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 82.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 125 µM. The corresponding cell viability was >70% (96.6%).The calculated EC1.5was< 1000 µM (84.95 µM).

In the second experiment, a max luciferase activity (Imax) induction of 5.40 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 64.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.63) was found to be 125 µM. The corresponding cell viability was >70% (101.1%).The calculated EC1.5was< 1000 µM (102.82 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.

DPRA (OECD 442C)

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptideswas≤6.38% (4.52%).Based on the prediction model 1 the test item can be considered as non-sensitiser.According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered. Therefore, no prediction can be made.

h-CLAT (OECD 442E)

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 75.8% (CD86), 75.5% (CD54) and 73.8% (isotype IgG1 control) in the first experiment and to 75.4% (CD86), 75.4% (CD54) and 74.6% (isotype IgG1 control) in the second experiment.

In experiment 1, the expression of the cell surface marker CD86 was upregulated to a maximum of 226% above the threshold of 150% at a concentration of 307.35μg/mL. An upregulation above the threshold of 150% was observed in the whole concentration range except for the two highest concentrations. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%.

In experiment 2, the expression of the cell surface marker CD86 was upregulated to a maximum of 296% above the threshold of 150% at a concentration of 386.82μg/mL. An upregulation above the threshold of 150% was observed in the whole concentration range. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%.

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.

Overall conclusion

Since two out of three test were positive, the test item is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on data provided, the test item is classified for skin sensitisation category 1 and labelled with H317 according to Regulation (EC) No 1272/2008.