3-Butoxy-7-ethoxy-4,6-difluoro-dibenzothiophene

Endpoint summary

Description of key information

Skin sensitisation was studied using GLP in vitro studies performed according to OECD TG 442C-E. The following results have been obtained:

• OECD 442C: negative
• OECD 442D: positive
• OECD 442E: negative but inconclusive due to logP > 3.5

The conflicting in vitro data are insufficient to conclude on the endpoint of skin sensitisation. Therefore, an additional in vivo study was performed.

The GLP comliant study according to OECD TG 429 showed no evidence of SI values increasing over 3 thus no potential for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 18, 2019 - Apr 02, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Inbred, SPF-Quality
- Age at study initiation: 10 weeks
- Weight at study initiation: Pre-test and Main test: 19.9 to 25.9 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): 42 - 50 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

methyl ethyl ketone

Concentration:

5, 10, and 35% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Two test item concentrations were tested; a 25% and 35% concentration.

- Compound solubility: 35 % in MEK
- Irritation:yes (At a 35% test item concentration No signs of systemic toxicity were noted and only very slight irritation of the ears was observed in animals treated with 35% test item concentration.)
- Lymph node proliferation response: -


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Standard statistical methods have been applied for data processing.

Positive control results:

Conc. SI
0%: 1.0
5% 1.3
10% 1.4
25% 5.2

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

Test Group: 5% in MEK

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test Group: 10% in MEK

Key result

Parameter:

SI

Value:

0.4

Test group / Remarks:

Test Group: 35% in MEK

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)	SD	S.I.
MEK (Vehicle Control)	744	145	1.0
5 % Test Item in MEK	1084	158	1.5
10 % Test Item in MEK	974	219	1.3
35 % Test Item in MEK	321	103	0.4

Interpretation of results:

GHS criteria not met

Conclusions:

The test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

Objective

The objective of this study was to evaluate whether the test material induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

Study Design

The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010).

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 35% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methylethylketone). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Results

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 35% were 1084, 974 and 321 DPM, respectively. The mean DPM/animal value for the vehicle control group was 744 DPM. The SI values calculated for the test item concentrations 5, 10 and 35% were 1.5, 1.3 and 0.4, respectively. Since there was no indication that the test item elicits a SI = 3 when tested up to 35%, the test material was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 35%.

Conclusion

Based on these results, the test material would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-07 to 2019-02-14

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 64.95%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

1.13

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:

0.15

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]	PeakArea at 220 nm	Peptide Concentration [mM]
STD1	17.4940	0.5340	15.0310	0.5340
STD2	8.7090	0.2670	7.5160	0.2670
STD3	4.3730	0.1335	3.7790	0.1335
STD4	2.1750	0.0667	1.8940	0.0667
STD5	1.0820	0.0334	0.9520	0.0334
STD6	0.5670	0.0167	0.4950	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 Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.8780	0.1491	70.09	69.94	0.43	0.62
	4.8480	0.1482	70.27
	4.9820	0.1523	69.45
Test Item	16.2480	0.4964	0.53	1.13	0.52	45.89
	16.1020	0.4919	1.42
	16.1010	0.4919	1.43

Depletion of the Lysine Peptide

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	5.5200	0.1958	61.01	59.97	2.04	3.41
	5.4810	0.1944	61.28
	6.0000	0.2128	57.61
Test Item	14.2380	0.5058	0.00	0.15	0.26	173.21
	14.1410	0.5024	0.45
	14.2880	0.5076	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 / 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	0.64	Minimal Reactivity	(negative)	1.13	Minimal Reactivity	(negative)
Positive Control	64.95	High Reactivity	positive	69.94	Moderate Reactivity	positive

Interpretation of results:

other: Data should 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. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study the test material was dissolved in DMF, based on the results of the pre-experiments, but DMF did not pass the validity criteria for the Reference Control C and was therefore excluded from the evaluation.

The solubility experiment was carried on and ethanol was found as solvent, too. Based on a molecular weight of 336 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM 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 of the test item (including the co-elution control) were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item turbidity and precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item, including the co-elution control. Samples of the test item (including the co-elution control) were centrifuged prior to the HPLC analysis. Phase separation (small droplets) was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis

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

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.64%). Since a precipitation was observed, 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, therefore, no prediction can be made.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-19 to 2019-02-25

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 (3.85 (experiment 1); 4.59 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

2.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 3.91 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

95.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 3.91 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

130.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

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

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	79.5	94.8	87.1	10.8
	8.00	77.7	103.7	90.7	18.4
	16.00	67.6	101.1	84.4	23.7
	32.00	50.1	88.2	69.2	27.0
	64.00	48.9	107.5	78.2	41.4
Test Item	0.98	125.8	119.0	122.4	4.8
	1.95	109.9	95.3	102.6	10.4
	3.91	95.9	130.8	113.3	24.6
	7.81	54.2	74.9	64.6	14.6
	15.63	40.2	49.5	44.8	6.6
	31.25	25.3	40.5	32.9	10.8
	62.50	21.9	36.3	29.1	10.1
	125.00	23.8	36.3	30.1	8.9
	250.00	25.9	37.9	31.9	8.5
	500.00	33.1	46.0	39.6	9.1
	1000.00	27.2	53.6	40.4	18.7
	2000.00	30.3	82.3	56.3	36.8

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	1.09	0.95	1.26	1.10	0.16
	8.00	1.23	1.05	1.22	1.17	0.10
	16.00	1.29	1.24	1.39	1.31	0.08
	32.00	1.89	1.80	1.78	1.82	0.06	*
	64.00	4.43	3.75	3.37	3.85	0.54	*
Test Item	0.98	1.40	1.82	1.55	1.59	0.21	*
	1.95	1.68	1.98	1.77	1.81	0.15	*
	3.91	1.98	2.30	2.02	2.10	0.18	*
	7.81	2.28	2.43	2.28	2.33	0.09	*
	15.63	2.18	2.28	2.06	2.17	0.11	*
	31.25	2.32	2.33	2.05	2.23	0.16	*
	62.50	2.01	2.13	1.99	2.04	0.07	*
	125.00	2.07	2.52	2.18	2.25	0.23	*
	250.00	2.24	2.01	2.11	2.12	0.12	*
	500.00	2.43	2.68	2.27	2.46	0.21	*
	1000.00	2.03	2.08	1.96	2.03	0.06	*
	2000.00	2.03	2.25	1.88	2.05	0.18	*

* = 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	1.21	1.16	1.17	0.04
	8.00	1.21	1.22	1.31	1.25	0.05
	16.00	1.41	1.58	1.71	1.57	0.15	*
	32.00	2.29	2.21	2.17	2.23	0.06	*
	64.00	4.24	4.60	4.91	4.59	0.34	*
Test Item	0.98	2.01	1.71	1.81	1.85	0.15	*
	1.95	2.14	1.89	2.10	2.04	0.13	*
	3.91	2.41	2.40	2.47	2.43	0.04	*
	7.81	2.48	2.52	2.53	2.51	0.02	*
	15.63	2.53	2.45	2.44	2.47	0.05	*
	31.25	2.27	2.52	2.32	2.37	0.13	*
	62.50	2.40	2.44	2.38	2.41	0.03	*
	125.00	2.28	2.43	2.39	2.37	0.08	*
	250.00	2.24	2.51	2.37	2.37	0.14	*
	500.00	2.51	2.60	2.28	2.46	0.16	*
	1000.00	2.50	2.67	2.43	2.54	0.12	*
	2000.00	2.57	2.79	2.46	2.60	0.17	*

* = 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	1.10	1.17	1.13	0.05
	8.00	1.17	1.25	1.21	0.06
	16.00	1.31	1.57	1.44	0.18
	32.00	1.82	2.23	2.02	0.29	*
	64.00	3.85	4.59	4.22	0.52	*
Test Item	0.98	1.59	1.85	1.72	0.18	*
	1.95	1.81	2.04	1.93	0.16	*
	3.91	2.10	2.43	2.26	0.23	*
	7.81	2.33	2.51	2.42	0.13	*
	15.63	2.17	2.47	2.32	0.21	*
	31.25	2.23	2.37	2.30	0.10	*
	62.50	2.04	2.41	2.23	0.26	*
	125.00	2.25	2.37	2.31	0.08	*
	250.00	2.12	2.37	2.25	0.18	*
	500.00	2.46	2.46	2.46	0.00	*
	1000.00	2.03	2.54	2.28	0.36	*
	2000.00	2.05	2.60	2.33	0.39	*

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	n.a.	n.a.
Imax	2.46	2.60	2.53	0.10
IC30[µM]	6.33	15.48	10.91	6.47
IC50[µM]	10.17	9.32	9.75	0.60

n.a.: not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	11.3	pass	8.2	pass
CV Solvent Control TI (1% THF)	<20%	13.7	pass	10.2	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	3.0	pass
EC1.5 PC	± 2 x SD of historical mean	21.99	pass	14.31	pass
Induction PC at 64 µM	2 .00 < x < 8.00	3.85	pass	4.59	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: Data should be considered in the context of an integrated approach 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 may 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 THF. Based on a molecular weight of 336 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 2.46 was determined at a test item concentration of 500 µM but the corresponding cell viability was 33.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.59) was found to be 0.98 µM, the corresponding cell viability was >70% (125.8%). The luciferase activity induction rises up to 2.10 at 3.91 µM, from then on the substance is cytotoxic. No EC_1.5 value could be calculated.

In the second experiment, a max luciferase activity (I_max) induction of 2.6 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 82.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.85) was found to be 0.98 µM, the corresponding cell viability was >70% (119%). The luciferase activity induction rises up to 2.51 at 7.81 µM, from then on the substance is cytotoxic. The last concentration is viable again but that seems to be an artefact. No EC_1.5 value could be calculated.

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 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-29 to 2019-07-04

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 (334% experiment 1; 312% experiment 2) and 200% for CD54 (374% experiment 1; 1360% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 416.67 µg/mL

Key result

Run / experiment:

other: 1

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: 289.35 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

134

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 416.67 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 347.22 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 6 Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	85.4	327	>150	85.6	287	>200	yes	pass
NiSO4	100 µg/mL	86.7	251	>150	86.5	269	>200	yes	pass
LA	1000 µg/mL	96.5	76	≤150	96.6	77	≤200	no	pass

Results of the Cell Batch 7 Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	86.9	422	>150	85.4	647	>200	yes	pass
NiSO4	100 µg/mL	89.1	254	>150	90.0	454	>200	yes	pass
LA	1000 µg/mL	97.9	53	≤150	98.0	70	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	97.20
Solvent Control	THF	--	96.90
Test Material	C8	3.91	96.00
	C7	7.81	95.80
	C6	15.63	96.00
	C5	31.25	95.10
	C4	62.50	96.20
	C3	125.00	95.20
	C2	250.00	94.50
	C1	500.00	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	-	96.3	96.5	96.7	2363	1253	855	1508	398	102	94	276	147
Solvent Control 2 (THF)	0.20%	96.6	96.4	96.4	2202	1353	866	1336	487	100	100	254	156
Solvent Control 1 (DMSO)	0.20%	96.1	96.7	96.8	2307	1248	825	1482	423	100	100	280	151
DNCB	4.00	72.9	70.7	69.0	5846	2477	893	4953	1584	334	374	655	277
Test Material	500	92.6	93.7	97.0	1797	1223	861	936	362	70	74	209	142
	416.67	93.1	94.6	96.9	2148	1180	834	1314	346	98	71	258	141
	347.22	92.8	94.3	94.1	1955	1176	810	1145	366	86	75	241	145
	289.35	94.0	92.8	94.4	1938	1264	810	1128	454	84	93	239	156
	241.13	93.1	92.6	92.3	1888	1177	822	1066	355	80	73	230	143
	200.94	92.7	92.7	92.1	2127	1148	825	1302	323	97	66	258	139
	167.45	94.9	92.2	92.2	1922	1152	778	1144	374	86	77	247	148
	139.54	94.3	93.5	91.4	1952	1189	768	1184	421	89	86	254	155

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	-	97.5	97.0	97.3	1632	1001	795	837	206	92	127	205	126
Solvent Control 2 (THF)	0.20%	97.0	96.9	97.1	1581	1051	874	707	177	100	100	181	120
Solvent Control 1 (DMSO)	0.20%	96.3	96.6	97.4	1779	1035	873	906	162	100	100	204	119
DNCB	4.0	71.5	73.8	75.2	3760	3139	935	2825	2204	312	1360	402	336
Test Material	500.00	96.2	92.5	96.4	1840	1026	1097	743	-71	105	-40	168	94
	416.67	95.8	95.6	96.7	1942	1043	998	944	45	134	25	195	105
	347.22	95.5	95.8	95.9	1578	1023	867	711	156	101	88	182	118
	289.35	96.3	96.1	96.2	1417	980	859	558	121	79	68	165	114
	241.13	96.4	96.6	96.0	1713	1060	926	787	134	111	76	185	114
	200.94	96.3	96.0	96.2	1618	1096	968	650	128	92	72	167	113
	167.45	95.5	96.6	96.1	1563	1019	951	612	68	87	38	164	107
	139.54	95.9	96.3	96.8	1348	1029	1001	347	28	49	16	135	103

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	96.3	-	96.7	pass	96.9	-	97.5	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	334			pass	312			pass
RFI of positive control of CD54	≥200	374			pass	1360			pass
RFI of solvent control of CD86	<150	98			pass	108			pass
RFI of solvent control of CD54	<200	106			pass	51			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	89			pass	84			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	122			pass	56			pass
MFI ratio CD54/IgG1for medium control [%]	>105	276			pass	205			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	280			pass	204			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 THF control of CD86	102.9	17.4	36
RFI of THF control 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/CD86 for DMSO control [%]	221.6	58.5	112
MFI ratio IgG1/CD54 for medium control [%]	308.6	137.1	36
MFI ratio IgG1/CD54 for THF control [%]	158.2	28.4	36
MFI ratio IgG1/CD54 for DMSO control [%]	147.7	25.6	112

Interpretation of results:

other: Data should be considered in the context of an integrated approach such as IATA.

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Since the Log KOW is >3.5 the result 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 250 mg/mL (maximum solubility). For the dose finding assay stock solutions with concentrations ranging from 250 mg/mL to 1.96 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:

500.00, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL

In experiment 1 precipitation of the test item was observed for concentration steps 500.00 µg/mL to 289.35 µg/mL and in experiment 2 precipitation of the test item was observed for concentration step 500.00 µg/mL to 200.94 µg/mL 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 92.6% (CD86), 93.7% (CD54) and 97.0% (isotype IgG1 control) in the first experiment and to 96.2% (CD86), 92.5% (CD54) and 96.4% (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 cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item would be considered as non-sensitiser, however, since the Log K_OWis >3.5 (Log K_OW> 6.7) the result has to be considered as inconclusive.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.