3-diazo-3,4-dihydro-4-oxonaphthalene-1-sulfonyl chloride

Endpoint summary

Description of key information

The skin sensitisation potential was investigated in vitro in three assays addressing key events of the mode of action for skin sensitisation. The results obtained in thaese assays are listed below:

OECD 442C: positive

OECD 442D: negative

OECD 442E: positive

Two of the three key events showed positive results indicating that the compound must be considered as positive for skin sensitisation.

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:

2017-12-14 to 2017-12-21

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 66.21%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

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 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	4895.3867	0.5340	4663.5073	0.5340
STD2	2320.1248	0.2670	2361.9275	0.2670
STD3	1141.6589	0.1335	1192.6749	0.1335
STD4	567.6173	0.0667	602.5394	0.0667
STD5	292.4744	0.0334	302.3090	0.0334
STD6	148.1837	0.0167	151.3044	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	1436.3897	0.1610	68.81	69.31	0.45	0.65
	1406.1344	0.1577	69.46
	1396.6488	0.1566	69.67
Test Item	0.0000	0.0037	100.00	100.00	0.00	0.00
	0.0000	0.0037	100.00
	0.0000	0.0037	100.00

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	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*	0.0000	0.0000	0.00	0.00	0.00	n/a
	0.0000	0.0000	0.00
	0.0000	0.0000	0.00

n/a:        not applicable

* A major peak in the co-elution control of the test item was observed at the retention time of the lysine peptide. Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of lysine peptide present in the test item samples. The values can only be considered as an estimation of the peptide depletion and will not be used for evaluation.

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				100.00	High Reactivity	sensitiser
Positive Control	66.21	High Reactivity	sensitiser	69.31	Moderate Reactivity	sensitiser

Interpretation of results:

other: should be considered in the context of integrated approached such as IATA

Conclusions:

In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. The test item is considered as “sensitiser”.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study the test material was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 268.5 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 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 (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM 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. Phase separation (small droplets) was observed for the samples of the positive control (including the co-elution control). Precipitation was also observed for the samples of the test item (including the co-elution control). Samples of the test item were centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide 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 high reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (100.00%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 2 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.21%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-19 to 2018-03-20

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 (7.72 (experiment 1); 4.70 (experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

10.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 2000 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

67.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

2.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 250 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

2.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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	100.4	87.0	93.7	9.5
	8.00	98.5	66.4	82.5	22.7
	16.00	97.0	76.3	86.6	14.6
	32.00	95.4	59.3	77.4	25.5
	64.00	95.8	48.8	72.3	33.2
Test Item	0.98	45.8	91.3	68.6	32.2
	1.95	90.5	95.4	93.0	3.5
	3.91	87.3	93.1	90.2	4.1
	7.81	91.6	86.7	89.1	3.4
	15.63	95.7	84.0	89.9	8.2
	31.25	95.7	88.8	92.3	4.8
	62.50	93.6	89.2	91.4	3.1
	125.00	97.8	84.4	91.1	9.5
	250.00	96.7	1.1	48.9	67.6
	500.00	102.5	0.0	51.3	72.5
	1000.00	97.9	0.1	49.0	69.2
	2000.00	67.3	0.1	33.7	47.5

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.28	1.23	1.23	1.25	0.03
	8.00	1.44	1.35	1.29	1.36	0.07
	16.00	1.82	1.66	1.50	1.66	0.16	*
	32.00	2.52	2.75	1.68	2.32	0.57	*
	64.00	7.62	(12.54^#)	7.82	7.72	0.10	*
Test Item	0.98	0.87	0.85	0.81	0.84	0.03
	1.95	1.28	1.02	1.04	1.11	0.15
	3.91	1.16	1.19	1.05	1.13	0.07
	7.81	0.92	0.83	0.88	0.87	0.05
	15.63	1.05	0.85	0.90	0.93	0.10
	31.25	0.92	1.17	0.90	1.00	0.15
	62.50	0.94	1.04	1.02	1.00	0.05
	125.00	1.16	1.11	1.09	1.12	0.04
	250.00	1.61	1.50	1.30	1.47	0.15
	500.00	1.30	1.45	1.17	1.31	0.14
	1000.00	2.40	2.54	1.74	2.23	0.43	*
	2000.00	10.24	12.28	8.66	10.39	1.81	*

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

^#= outlier according to Grubbs, Nalimov and Dixon. Excluded from evaluation.

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	1.16	1.18	1.10	1.15	0.04
	8.00	1.16	1.10	1.09	1.12	0.04
	16.00	1.65	1.69	1.49	1.61	0.11	*
	32.00	2.23	1.88	1.93	2.01	0.19	*
	64.00	4.84	4.51	4.75	4.70	0.17	*
Test Item	0.98	1.47	1.56	1.44	1.49	0.06
	1.95	1.26	1.26	1.13	1.22	0.07
	3.91	1.04	1.04	0.93	1.00	0.07
	7.81	1.11	1.07	0.97	1.05	0.07
	15.63	1.14	1.18	1.01	1.11	0.09
	31.25	0.93	0.89	0.83	0.88	0.05
	62.50	1.02	0.94	0.88	0.95	0.07
	125.00	1.15	1.07	1.24	1.15	0.08
	250.00	2.36	2.54	1.47	2.13	0.57	*
	500.00	1.23	1.43	0.40	1.02	0.55
	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 – 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	1.25	1.15	1.20	0.07
	8.00	1.36	1.12	1.24	0.17
	16.00	1.66	1.61	1.63	0.04	*
	32.00	2.32	2.01	2.16	0.22	*
	64.00	9.33	4.70	7.01	3.27
Test Item	0.98	0.84	1.49	1.17	0.46
	1.95	1.11	1.22	1.16	0.07
	3.91	1.13	1.00	1.07	0.09
	7.81	0.87	1.05	0.96	0.12
	15.63	0.93	1.11	1.02	0.13
	31.25	1.00	0.88	0.94	0.08
	62.50	1.00	0.95	0.97	0.04
	125.00	1.12	1.15	1.14	0.02
	250.00	1.47	2.13	1.80	0.46
	500.00	1.31	1.02	1.16	0.20
	1000.00	2.23	0.00	1.11	1.58
	2000.00	10.39	0.00	5.20	7.35

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	604.89	169.69	387.29	307.73
Imax	10.39	2.13	6.26	5.85
IC30[µM]	n/a	146.59	n/a	n/a
IC50[µM]	1911.80	206.60	1059.20	1205.76

n/a not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	13.1	pass	9.6	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	3.0	pass
EC1.5 PC	7 < x < 34 µM	11.72	pass	14.23	pass
Induction PC at 64 µM	2.00 < x < 8.00	7.72*	pass	4.70	pass

* = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.

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: should be considered in the context of integrated approach such as IATA

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

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

Based on a molecular weight of 268.5 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 10.39 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 67.3%. The lowest tested concentration with a significant luciferase induction >1.5 (2.23) was found to be 1000 µM. The corresponding cell viability was >70% (97.9%).The calculated EC_1.5 was < 1000 µM (604.89).

In the second experiment, a max luciferase activity (I_max) induction of 2.13 was determined at a test item concentration of 250 µM. The corresponding cell viability was 1.1%. The lowest tested concentration with a significant luciferase induction >1.5 (2.13) was found to be 250 µM. The corresponding cell viability was <70% (1.1%).The calculated EC_1.5was < 1000 µM (169.69).

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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

The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment one of the technical replicate exceeded the threshold with a fold induction of 12.54. On the one hand, the positive control clearly induces the luciferase induction in the KerationSens™ cell line, showing the capability of the test system to predict sensitisation. On the other hand, the results in this first experiment are clearly negative, indicating no oversensitivity of the test system. The single value of the positive control exceeding the upper threshold was therefore considered to be an outliner and not biological relevant. Furthermore all other controls confirmed the validity of the study

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-09-03 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 (423% experiment 1; 179% experiment 2) and 200% for CD54 (373% experiment 1; 202% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 900 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

497

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 900 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

109

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 520.83 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

329

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 520.83 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

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	83.4	364	>150	82.3	419	>200	yes	pass
NiSO4	100 µg/mL	79.1	351	>150	77.9	688	>200	yes	pass
LA	1000 µg/mL	96.2	77	≤150	95.9	105	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	95.60
Solvent Control	DMSO	--	95.30
Test material	C8	7.03	95.10
	C7	14.06	95.40
	C6	28.13	94.90
	C5	56.25	94.60
	C4	112.50	94.40
	C3	225.00	92.70
	C2	450.00	88.80
	C1	900.00	77.20
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	-	92.9	93.8	94.3	1816	916	623	1193	293	112	129	291	147
Solvent Control	0.20%	93.9	94.1	93.7	1687	850	622	1065	228	100	100	271	137
DNCB	4.0	80.7	80.7	80.7	5119	1467	617	4502	850	423	373	830	238
Test material	900.00	77.1	76.8	76.8	3360	2367	1233	2127	1134	200	497	273	192
	750.00	80.6	81.1	80.8	3217	2211	1227	1990	984	187	432	262	180
	625.00	83.7	84.0	83.9	3146	1891	1134	2012	757	189	332	277	167
	520.83	86.3	85.9	85.5	3063	1884	1091	1972	793	185	348	281	173
	434.03	89.0	88.4	88.2	2626	1595	1050	1576	545	148	239	250	152
	361.69	88.9	89.5	89.2	2656	1579	992	1664	587	156	257	268	159
	301.41	91.1	91.3	90.8	2642	1510	1007	1635	503	154	221	262	150
	251.17	90.6	90.7	89.6	2740	1509	1002	1738	507	163	222	273	151

 

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	-	95.1	94.8	93.6	2966	1186	731	2235	455	89	83	406	162
Solvent Control	0.20%	94.8	93.5	94.7	3152	1185	636	2516	549	100	100	496	186
DNCB	4.00	87.2	84.9	86.7	5173	1774	664	4509	1110	179	202	779	267
Test material	900	11.0	9.0	8.6	1875	3289	2045	-170	1244	-7	227	92	161
	750.00	7.6	8.1	6.7	6689	2530	2758	3931	-228	156	-42	243	92
	625.00	23.9	24.1	23.9	5952	6422	2922	3030	3500	120	638	204	220
	520.83	75.0	74.2	74.0	4793	3847	2040	2753	1807	109	329	235	189
	434.03	76.4	76.3	75.3	4072	3077	1674	2398	1403	95	256	243	184
	361.69	86.5	85.9	86.5	3581	2107	1233	2348	874	93	159	290	171
	301.41	89.9	89.5	89.2	3207	1821	1070	2137	751	85	137	300	170
	251.17	90.6	90.0	90.3	3221	1802	1036	2185	766	87	140	311	174

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	92.9	-	94.3	pass	93.5	-	95.1	pass
number of test dosed with viability >50% CD86	≥4	8			pass	5			pass
number of test dosed with viability >50% CD54	≥4	8			pass	5			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	5			pass
RFI of positive control of CD86	≥150	423			pass	179			pass
RFI of positive control of CD54	≥200	373			pass	202			pass
RFI of solvent control of CD86	<150	89			pass	113			pass
RFI of solvent control of CD54	<200	78			pass	121			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	291			pass	406			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	271			pass	496			pass
MFI ratio CD54/IgG1for medium control [%]	>105	147			pass	162			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	137			pass	186			pass

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:

other: should be considered in the context of integrated approached such as IATA

Conclusions:

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

Executive summary:

In the present study the test material was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 450 mg/mL to 3.52 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

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

Before adding the test item to the cells, it was stirred for 30 minutes. In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Cytotoxic effects were observed for the cells treated with the test item only in the second experiment. Relative cell viability at the highest test item concentration was reduced to 77.1% (CD86), 76.8% (CD54) and 76.8% (isotype IgG1 control) in the first experiment and to 11.0% (CD86), 9.0% (CD54) and 8.6% (isotype IgG1 control) in the second experiment. The first viable cells were observed at a concentration of 520.83 µg/mL with 75.0% (CD86), 74.2% (CD54) and 74.0% (isotype IgG1 control).

The expression of the cell surface marker CD86 was upregulated to 200% in one independent experiment. The upregulation above the threshold of 150% was observed starting from a concentration of 251.17 µg/mL. The expression of the cell surface marker CD54 was upregulated to 497% and 329% both independent experiments. The upregulation above the threshold of 200% was observed starting from a concentration of 251.17 µg/mL and at the concentrations of 434.03 µg/mL and 520.83 µg/mL.

Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test material is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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