4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Endpoint summary

Description of key information

The results in the in vitro assays performed along with the absence of any structural alert for skin sensitisation in the test compound and in the metabolites generated provide sufficient arguments to conclude that there is no potential for skin sensitisation associated with the test material. (for details see WoE justification report)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-13 to 2018-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

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

Details on the study design:

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

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.17% (experiment 1) and 67.80% (experiment 2).

Key result

Run / experiment:

other: cysteine run 1

Parameter:

other: mean peptide depletion [%]

Value:

19.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run 1

Parameter:

other: mean peptide depletion [%]

Value:

0.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: cysteine run 2

Parameter:

other: mean peptide depletion [%]

Value:

4.73

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: lysine run 2

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

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 (experiment 1)

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4943.5503	0.5340	4663.5073	0.5340
STD2	2498.8708	0.2670	2361.9275	0.2670
STD3	1258.2625	0.1335	1192.6749	0.1335
STD4	633.7274	0.0667	602.5394	0.0667
STD5	325.8550	0.0334	302.3090	0.0334
STD6	170.8465	0.0167	151.3044	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Cysteine and Lysine Values of the Calibration Curve (experiment 2)

Sample	Cystein Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4904.8657	0.5340	14.4730	0,5340
STD2	2485.3242	0.2670	7.2720	0,2670
STD3	1239.3314	0.1335	3.6620	0,1335
STD4	613.7947	0.0667	1.8200	0,0667
STD5	299.9632	0.0334	0.9210	0,0334
STD6	145.2999	0.0167	0.4500	0,0167
STD7	0.0000	0.0000	0.0000	0,0000

Since the measurement was performed on a different HPLC system the values of the peak areas are not comparable between the two experiments.

Depletion of the Cysteine Peptide (experiment 1)

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	1489.4670	0.1594	69.37	69.23	0.18	0.25
	1505.8701	0.1612	69.03
	1493.4833	0.1598	69.29
Test Item	3986.6003	0.4295	18.02	19.67	1.45	7.37
	3879.8984	0.4179	20.22
	3853.3762	0.4151	20.76

Depletion of the Cysteine Peptide (experiment 2)

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1471.0265	0.1596	69.33	69.51	0.61	0.88
	1486.4678	0.1613	69.01
	1429.7356	0.1551	70.19
Test Item	4517.5327	0.4904	5.82	4.73	0.94	19.96
	4596.3301	0.4990	4.17
	4595.4790	0.4989	4.19

Depletion of the Lysine Peptide (experiment 1)

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	1539.7554	0.1748	64.31	63.12	1.08	1.71
	1602.9802	0.1820	62.84
	1630.8385	0.1852	62.20
Test Item	4332.6734	0.4947	0.00	0.43	0.43	99.25
	4295.2358	0.4904	0.44
	4277.1602	0.4883	0.86

Depletion of the Lysine Peptide (experiment 2)

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.5140	0.1660	66.56	66.10	0.50	0.75
	4.5670	0.1680	66.16
	4.6470	0.1709	65.57
Test Item	13.5890	0.5008	0.00	0.00	0.00	-
	13.6350	0.5025	0.00
	13.5890	0.5008	0.00

Since the measurement was performed on a different HPLC system the values of the peak areas are not comparable between the two experiments.

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 (Experiment 1)

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	10.05	Low Reactivity	sensitiser	19.67	Low Reactivity	sensitiser
Positive Control	66.17	High Reactivity	sensitiser	69.23	Moderate Reactivity	sensitiser

Categorization of the Test Item (Experiment 2)

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item 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	2.36	Minimal Reactivity	no sensitiser	4.73	Minimal Reactivity	no sensitiser
Positive Control	67.80	High Reactivity	sensitiser	69.51	Moderate Reactivity	sensitiser

Interpretation of results:

other: results must be considered in the context of integrated approached 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”.
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 262 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.

Experiment 1:

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. Precipitation was observed for the samples of the test item including the co-elution control. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control of the positive control. Precipitation at the bottom was also observed for the samples of the test item 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 insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitisingpotential 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_acetonitrile).

The 100 mM stock solution of the test item showedlowreactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (10.05%). Due to observed precipitation in both peptide samples 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 66.17%.

Experiment 2:

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. 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 solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control of the positive control. No precipitation, turbidity or phase separation was observed for the test item samples. 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 insignificant. 

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_acetonitrile).

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% (2.36%). Based on the prediction model 1 the test item can be considered as non-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 67.80%.

.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-11-14 to 2018-05-07

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 (6.34 (experiment 1); 3.67 (experiment 2) and 2.36 (experiment 3)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

4.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

52.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

1.37

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:

9.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 62.5 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

17.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Key result

Run / experiment:

other: 3

Parameter:

other: luciferase activity

Value:

2.61

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 10 µM

Key result

Run / experiment:

other: 3

Parameter:

other: cell viability [%]

Value:

72.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: EC 1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Conc. [µM]	Cell Viability [%]				Experiment 3
		Experiment 1	Experiment 2	Mean	SD	Conc. [µM]	Cell Viability [%]
Solvent Control	-	100	100	100	0.0	-	100
Positive Control	4.00	94.4	101.8	98.1	5.2	4.00	94.3
	8.00	97.6	111.0	104.3	9.5	8.00	97.7
	16.00	107.1	107.4	107.2	0.2	16.00	102.7
	32.00	112.4	118.5	115.4	4.3	32.00	102.7
	64.00	116.5	125.8	121.1	6.6	64.00	101.6
Test Item	0.98	82.3	93.9	88.1	8.2	2.15	91.1
	1.95	73.0	85.2	79.1	8.6	2.47	94.2
	3.91	72.7	81.5	77.1	6.2	2.84	96.8
	7.81	64.2	72.5	68.4	5.9	3.27	91.7
	15.63	59.7	71.7	65.7	8.5	3.76	82.9
	31.25	52.7	76.3	64.5	16.7	4.32	78.0
	62.50	0.2	17.2	8.7	12.0	4.97	79.8
	125.00	-0.1	0.0	0.0	0.1	5.72	76.0
	250.00	-0.1	-0.2	-0.1	0.1	6.58	77.2
	500.00	0.2	0.1	0.1	0.1	7.56	74.6
	1000.00	0.5	0.4	0.5	0.1	8.70	70.6
	2000.00	0.5	0.4	0.4	0.1	10.00	72.4

 

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	1.00	1.15	1.17	1.11	0.09
	8.00	0.94	0.96	1.12	1.00	0.10
	16.00	1.25	1.30	1.31	1.28	0.03
	32.00	1.50	1.68	1.94	1.71	0.22	*
	64.00	5.44	5.09	8.50	6.34	1.88	*
Test Item	0.98	1.25	1.40	1.49	1.38	0.12
	1.95	1.49	1.71	1.83	1.68	0.17	*
	3.91	1.62	1.79	1.68	1.70	0.09	*
	7.81	1.93	1.88	2.45	2.09	0.32	*
	15.63	1.95	2.18	2.58	2.24	0.32	*
	31.25	3.98	4.23	4.64	4.28	0.33	*
	62.50	0.81	2.13	1.27	1.41	0.67
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.05	1.23	1.08	1.12	0.10
	8.00	1.20	1.18	1.17	1.18	0.02
	16.00	1.42	1.38	1.49	1.43	0.06
	32.00	2.20	1.65	2.07	1.97	0.29	*
	64.00	3.91	3.04	4.08	3.67	0.56	*
Test Item	0.98	1.08	1.40	1.32	1.27	0.17
	1.95	1.42	1.72	1.58	1.57	0.15	*
	3.91	1.58	1.70	2.42	1.90	0.45	*
	7.81	1.76	2.12	2.22	2.03	0.24	*
	15.63	2.01	1.85	2.17	2.01	0.16	*
	31.25	5.71	5.53	6.10	5.78	0.29	*
	62.50	9.31	8.06	10.98	9.45	1.46	*
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

Induction of Luciferase Activity Experiment 3

Experiment 3	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.12	1.11	1.15	1.13	0.02
	8.00	1.10	1.13	1.07	1.10	0.03
	16.00	1.31	1.17	1.39	1.29	0.11
	32.00	1.51	1.75	1.78	1.68	0.15	*
	64.00	2.33	2.54	2.22	2.36	0.16	*
Test Item	2.15	2.01	1.99	1.72	1.91	0.16	*
	2.47	1.86	2.08	1.77	1.90	0.16	*
	2.84	1.83	2.16	1.52	1.84	0.32	*
	3.27	1.98	1.95	1.87	1.93	0.06	*
	3.76	2.23	2.43	1.99	2.22	0.22	*
	4.32	2.20	2.45	2.13	2.26	0.17	*
	4.97	2.22	1.94	2.03	2.06	0.14	*
	5.72	2.48	2.70	2.24	2.47	0.23	*
	6.58	2.02	2.28	2.01	2.10	0.15	*
	7.56	2.21	2.38	2.33	2.31	0.09	*
	8.70	2.27	2.61	2.29	2.39	0.19	*
	10.00	2.79	2.48	2.55	2.61	0.16	*

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

 

Induction of Luciferase Activity – Overall Induction

 

Overall Induction	Conc. [µM]	Fold Induction				Sign.	Experiment 3
		Exp. 1	Exp. 2	Mean	SD		Conc. [µM]	Fold Induction	Sign.
Solvent Control	-	1.00	1.00	1.00	0.00		-	1.00
Positive Control	4.00	1.11	1.12	1.11	0.01		4.00	1.13
	8.00	1.00	1.18	1.09	0.13		8.00	1.10
	16.00	1.28	1.43	1.36	0.10		16.00	1.29
	32.00	1.71	1.97	1.84	0.19	*	32.00	1.68	*
	64.00	6.34	3.67	5.01	1.89		64.00	2.36	*
Test Item	0.98	1.38	1.27	1.32	0.08		2.15	1.91	*
	1.95	1.68	1.57	1.63	0.08	*	2.47	1.90	*
	3.91	1.70	1.90	1.80	0.15	*	2.84	1.84	*
	7.81	2.09	2.03	2.06	0.04	*	3.27	1.93	*
	15.63	2.24	2.01	2.12	0.16	*	3.76	2.22	*
	31.25	4.28	5.78	5.03	1.06	*	4.32	2.26	*
	62.50	1.41	9.45	5.43	5.69		4.97	2.06	*
	125.00	0.00	0.00	0.00	0.00		5.72	2.47	*
	250.00	0.00	0.00	0.00	0.00		6.58	2.10	*
	500.00	0.00	0.00	0.00	0.00		7.56	2.31	*
	1000.00	0.00	0.00	0.00	0.00		8.70	2.39	*
	2000.00	0.00	0.00	0.00	0.00		10.00	2.61	*

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

Exp. = Experiment, Sign. = Significance

Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5	1.37	1.72	n.a.	1.55	0.25
Imax	4.28	9.45	2.61	5.45	3.57
IC30	5.15	34.59	n.a.	19.87	20.82
IC50	32.89	45.15	n.a.	39.02	8.67

n.a.: not applicable

Acceptance Criteria

Criterion	Range	Exp. 1	pass/fail	Exp. 2	pass/fail	Exp. 3	pass/fail
CV Solvent Control	< 20%	17.5	pass	12.5	pass	14.2	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	24.15	pass	18.10	pass	24.63	pass
Induction PC at 64 µM	2.00 < x < 8.00	6.34	pass	3.67	pass	2.36	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: results must 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 material was dissolved in DMSO. Based on a molecular weight of 262 g/mol a stock solution of 200 mM was prepared for experiment 1 and experiment 2. In order to verify the first and second experiment, a third experiment with adapted concentrations was performed. For experiment 3 a stock solution of 1.0 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2 for experiment 1 and experiment 2 and a constant dilution factor of 1:1.15 for experiment 3. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

Experiment 1 and 2:

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

Experiment 3:

10.0, 8.70, 7.56, 6.58, 5.72, 4.97, 4.32, 3.76, 3.27, 2.84, 2.47, 2.15 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 4.28 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was < 70% (52.7%). The lowest tested concentration with a significant luciferase induction >1.5 (1.68) was found to be 1.95 µM. The corresponding cell viability was >70% (73.0%).The calculated EC_1.5 was < 1000 µM (1.37 µM).

In the second experiment, a max luciferase activity (I_max) induction of 9.45 was determined at a test item concentration of 62.50 µM. The corresponding cell viability was <70% (17.2%). The lowest tested concentration with a significant luciferase induction >1.5 (1.57) was found to be 1.95 µM. The corresponding cell viability was >70% (85.2%).The calculated EC_1.5 was < 1000 µM (1.72 µM).

Due to the strong cytotoxicity in experiment 1 and 2 a third experiment with an adapted concentration range and a lower dilution factor was performed.

In the third experiment, a max luciferase activity (I_max) induction of 2.61 was determined at a test item concentration of 10 µM. The corresponding cell viability was >70% (72.4%). A significant luciferase induction >1.5 was found in the whole tested concentration range.Therefore, no EC_1.5value could be calculated.

A dose response for luciferase activity induction was observed for experiment 1 and experiment 2 and during experiment 3 a significant luciferase induction >1.5 was found in the whole tested concentration range.

Under the condition of this study the test item is therefore considered as sensitiser

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-10-02 to 2018-12-13

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 (362% experiment 1; 402% experiment 2; 166% experiment 3) and
200% for CD54 (279% experiment 1; 236% experiment 2; 272 experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

180

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 30.27 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 17.52 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

126

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 43.59 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 21.02 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 36.33 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

116

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 12.17 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		Experiment 2		Experiment 3
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	96.70	--	95.40	--	97.50
Solvent Control	DMSO	--	96.60	--	95.50	--	97.30
Test item	C8	7.81	95.00	7.81	91.00	7.81	92.80
	C7	15.63	94.10	15.63	86.10	15.63	88.00
	C6	31.25	93.00	31.25	75.00	31.25	68.10
	C5	62.50	69.50	62.50	51.90	62.50	40.20
	C4	125.00	8.00	125.00	15.20	125.00	13.40
	C3	250.00	5.70	250.00	7.00	250.00	18.00
	C2	500.00	0.00	500.00	0.00	500.00	0.00
	C1	1000.00	0.00	1000.00	0.00	1000.00	0.00
Calculated CV75 [µg/mL]		53.14		31.25		24.57
Mean CV75 [µg/mL]		36.32
SD CV 75 [µg/mL]		14.94

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	-	94.5	92.9	92.6	2218	1382	761	1457	621	84	99	291	182
Solvent Control	0.20%	94.3	94.7	94.2	2369	1268	640	1729	628	100	100	370	198
DNCB	4.00	84.8	85.4	84.1	7004	2499	745	6259	1754	362	279	940	335
Test item	43.59	52.9	54.5	50.7	1735	1210	783	952	427	55	68	222	155
	36.33	61.0	62.8	62.2	3752	1191	773	2979	418	172	67	485	154
	30.27	65.9	66.9	66.6	3876	1245	757	3119	488	180	78	512	164
	25.23	73.2	75.0	74.0	3324	1312	734	2590	578	150	92	453	179
	21.02	78.0	77.9	78.5	2110	1219	730	1380	489	80	78	289	167
	17.52	82.4	83.2	82.6	2186	1310	720	1466	590	85	94	304	182
	14.60	81.9	81.9	81.9	2950	1302	713	2237	589	129	94	414	183
	12.17	80.9	82.3	82.1	2972	1282	704	2268	578	131	92	422	182

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.9	96.1	96.0	2215	1197	541	1674	656	100	96	409	221
Solvent Control	0.20%	96.4	96.3	96.2	2202	1212	532	1670	680	100	100	414	228
DNCB	4.0	85.1	84.5	84.4	7465	2364	759	6706	1605	402	236	984	311
Test item	43.59	61.1	60.7	60.8	2806	1172	707	2099	465	126	68	397	166
	36.33	74.0	73.3	73.3	2444	1215	701	1743	514	104	76	349	173
	30.27	79.6	80.7	81.2	2178	1257	670	1508	587	90	86	325	188
	25.23	86.9	87.8	86.5	1925	1339	739	1186	600	71	88	260	181
	21.02	89.3	89.0	89.2	1858	1280	651	1207	629	72	93	285	197
	17.52	90.7	91.3	90.8	1800	1237	633	1167	604	70	89	284	195
	14.60	90.5	91.2	90.4	1891	1241	617	1274	624	76	92	306	201
	12.17	93.5	93.4	93.1	1807	1226	617	1190	609	71	90	293	199

 

CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	93.4	94.4	95.1	2534	1111	692	1842	419	73	80	366	161
Solvent Control	0.20%	94.5	93.2	93.6	3186	1183	659	2527	524	100	100	483	180
DNCB	4.0	86.1	84.6	84.7	4864	2092	667	4197	1425	166	272	729	314
Test item	43.59	52.7	53.4	50.6	2818	1358	823	1995	535	79	102	342	165
	36.33	72.3	71.8	73.4	2863	1295	767	2096	528	83	101	373	169
	30.27	77.0	77.7	77.1	2571	1383	795	1776	588	70	112	323	174
	25.23	82.6	82.1	82.6	2484	1351	752	1732	599	69	114	330	180
	21.02	85.4	83.5	84.7	2340	1321	737	1603	584	63	111	318	179
	17.52	84.8	84.7	85.2	2407	1291	730	1677	561	66	107	330	177
	14.60	85.1	84.9	84.6	2504	1276	699	1805	577	71	110	358	183
	12.17	85.9	85.4	85.9	2718	1341	733	1985	608	79	116	371	183

Acceptance Criteria

Acceptance Criterion

Range

Exp. 1

pass/ fail

Exp. 2

pass/ fail

Exp. 3

pass/ fail

cell viability solvent controls [%]

>90

92.6

-

94.7

pass

96.0

-

96.9

pass

93.2

-

95.1

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

362

pass

402

pass

166

pass

RFI of positive control of CD54

≥200

279

pass

236

pass

272

pass

RFI of solvent control of CD86

<150

119

pass

100

pass

137

pass

RFI of solvent control of CD54

<200

101

pass

104

pass

125

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

291

pass

409

pass

366

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

370

pass

414

pass

483

pass

MFI ratio CD54/IgG1for medium control [%]

>105

182

pass

221

pass

161

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

198

pass

228

pass

180

pass

Exp.: Experiment

Historical Data

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

Interpretation of results:

GHS criteria not met

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 might 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 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 36.32 ± 14.94 µg/mL was derived in the dose finding assay.

In all three dose finding assays precipitation of the test item was observed for the highest four concentrations when mixing the test item stock solutions with cell culture medium. Sonication was used to aid solubility.

Based on the CV75, the main experiment was performed covering the following concentration steps:

43.59, 36.32, 30.27, 25.22, 21.02, 17.52, 14.60, 12.16 µg/mL

In all three main experiments no precipitation of the test item was observed anymore.

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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 52.9% (CD86), 54.5% (CD54) and 50.7% (isotype IgG1 control) in the first experiment, to 61.1% (CD86), 60.7% (CD54) and 60.8% (isotype IgG1 control) in the second experiment and to 52.7% (CD86), 53.4% (CD54) and 50.6% (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 172% (36.33 µg/mL), 180% (30.27 µg/mL) and 150% (25.23 µg/mL) only in the first experiment. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since the cell surface marker CD86 exceeded the threshold only in one of three independent experiments the test item is considered as non-sensitiser.

Reference 4

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 in the attached pdf documents.

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

Key result

Parameter:

other: Structural Alert for skin sensitisaton

Value:

0

Interpretation of results:

GHS criteria not met

Conclusions:

Using Derek and Meteor Nexus , the skin sensitising potential of the test item and of the metabolites therefor was estimated to be absent (Nothing to report). The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

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:

other: WoE related information

Reason / purpose for cross-reference:

other: WoE related information

Reason / purpose for cross-reference:

other: Woe related information

Reason / purpose for cross-reference:

other: Woe related information

Key result

Parameter:

other:

Value:

0

Interpretation of results:

GHS criteria not met

Conclusions:

The results in the in vitro assays performed along with the absence of any structural alert for skin sensitisation in the test compound and in the metabolites generated provide enough arguments to conclude that there is no potential for skin sensitisation associated with the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided data, which are considered to be suitable and reliable for classification, the test item is not classified for skin sensitisation according to Regulation (EC) No 1272/2008.