6-diazo-5,6-dihydro-5-oxonaphthalene-2-sulphonyl chloride

Endpoint summary

Description of key information

The skin sensitisation potential was investigated in vitro:

OECD 442C: positive

OECD 442D: negative

OECD 442E: inconclusive

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:

2017-11-16 to 2017-11-25

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 64.96%.

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	4523.9644	0.5340	4311.3857	0.5340
STD2	2255.1311	0.2670	2143.2170	0.2670
STD3	1081.2599	0.1335	1043.3549	0.1335
STD4	538.3588	0.0667	519.3511	0.0667
STD5	264.6839	0.0334	259.5146	0.0334
STD6	131.3584	0.0167	130.4601	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	1320.4471	0.1577	70.95	71.10	0.18	0.25
	1315.4908	0.1571	71.06
	1304.7782	0.1559	71.29
Test Item	0.0000	0.0023	100.00	100.00	0.00	0.00
	0.0000	0.0023	100.00
	0.0000	0.0023	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	1589.2585	0.1982	59.95	58.82	0.99	1.68
	1662.4030	0.2073	58.11
	1650.5492	0.2058	58.41
Test Item*	-	-	-	-	-	n/a
	-	-	-
	-	-	-

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	-	n/a	n/a	100.00	High Reactivity	sensitiser
Positive Control	64.96	High Reactivity	sensitiser	71.10	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 method may be not sufficient to conclude on the 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 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.

As the test item undergoes hydrolysis in water, testing might have been performed with reaction products of the test item.

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 positive control (excluding the co-elution control of the positive control), as well as for the samples of the test item (excluding the co-elution control of the test item). 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 (excluding the co-elution control of the positive 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 precipitations and phase separation were regarded as insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore 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%). Based on the prediction model 2 the test item can be considered as sensitiser.

Although precipitation was observed prior to the HPLC analysis with the cysteine peptide, the obtained positive result can still be used to support the identification of the test chemical as a skin 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 64.96%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-07 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:

29.27

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:

3.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

526.22

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

16.99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1000 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

6.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

294.94

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	94.1	107.1	100.6	9.2
	1.95	94.0	101.5	97.8	5.3
	3.91	90.9	101.1	96.0	7.2
	7.81	89.8	100.8	95.3	7.8
	15.63	88.4	100.0	94.2	8.3
	31.25	90.4	93.8	92.1	2.4
	62.50	79.7	85.6	82.7	4.2
	125.00	53.5	63.2	58.3	6.9
	250.00	40.8	47.8	44.3	5.0
	500.00	37.2	53.4	45.3	11.5
	1000.00	34.8	6.2	20.5	20.2
	2000.00	3.8	2.9	3.4	0.7

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	1.03	1.04	1.07	1.05	0.02
	1.95	0.99	1.01	0.92	0.97	0.05
	3.91	1.08	1.06	0.95	1.03	0.07
	7.81	1.16	1.27	1.14	1.19	0.07
	15.63	1.00	1.08	1.01	1.03	0.04
	31.25	1.03	1.07	0.93	1.01	0.07
	62.50	1.04	1.16	0.95	1.05	0.10
	125.00	1.09	0.95	0.88	0.97	0.11
	250.00	1.03	1.15	0.80	0.99	0.18
	500.00	1.30	1.51	1.29	1.37	0.13
	1000.00	3.56	5.19	3.02	3.92	1.13	*
	2000.00	38.70	33.84	15.27	29.27	12.37	*

* = 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	1.17	1.20	1.00	1.13	0.11
	1.95	0.97	1.03	0.88	0.96	0.08
	3.91	0.97	0.91	0.99	0.96	0.04
	7.81	0.87	0.92	0.82	0.87	0.05
	15.63	0.88	0.98	1.07	0.98	0.09
	31.25	0.87	0.81	0.81	0.83	0.04
	62.50	0.93	0.95	0.78	0.89	0.09
	125.00	1.09	1.06	0.85	1.00	0.13
	250.00	1.27	1.04	0.96	1.09	0.16
	500.00	6.05	2.42	1.64	3.37	2.35
	1000.00	9.30	29.65	12.01	16.99	11.05	*
	2000.00	0.01	0.01	1.52	0.51	0.87

* = 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	1.05	1.13	1.09	0.06
	1.95	0.97	0.96	0.97	0.01
	3.91	1.03	0.96	0.99	0.05
	7.81	1.19	0.87	1.03	0.22
	15.63	1.03	0.98	1.00	0.04
	31.25	1.01	0.83	0.92	0.13
	62.50	1.05	0.89	0.97	0.12
	125.00	0.97	1.00	0.99	0.02
	250.00	0.99	1.09	1.04	0.07
	500.00	1.37	3.37	2.37	1.42
	1000.00	3.92	16.99	10.45	9.24
	2000.00	29.27	0.51	14.89	20.33

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	526.22	294.94	410.58	163.54
Imax	29.27	16.99	23.13	8.69
IC30[µM]	85.61	106.04	95.82	14.45
IC50[µM]	159.32	231.00	195.16	50.69

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 approached 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 29.27 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 3.8%. The lowest tested concentration with a significant luciferase induction >1.5 (3.92) was found to be 1000 µM. The corresponding cell viability was <70% (34.8%).The calculated EC_1.5 was <1000 µM (526.22)

In the second experiment, a max luciferase activity (I_max) induction of 16.99 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 6.2%. The lowest tested concentration with a significant luciferase induction >1.5 (3.37) was found to be 500 µM. The corresponding cell viability was <70% (53.4%).The calculated EC_1.5 was < 1000 µM (294.94).

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:

key study

Study period:

2018-07-20 to 2018-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 (328% experiment 1; 267% experiment 2, 491% experiment 3, 363% experiment 4, 382% experiment 5) and
200% for CD54 (241% experiment 1; 266% experiment 2, 689% experiment 3, 551% experiment 4 and 297% experiment 5) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86

Value:

84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 40.06 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: 99.67 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 40.06 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 83.06 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

144

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 196.14 µg/mL

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

129

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 338.93 µg/mL

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD86

Value:

113

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 136.21 µg/mL

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD54

Value:

148

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 338.93 µg/mL

Key result

Run / experiment:

other: 5

Parameter:

other: relative fluorescence intensity CD86

Value:

281

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 167.45 µg/mL

Key result

Run / experiment:

other: 5

Parameter:

other: relative fluorescence intensity CD54

Value:

725

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 167.45 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 21)

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	85.5	251	>150	84.7	287	>200	yes	pass
NiSO4	100 µg/mL	74.9	249	>150	75.5	518	>200	yes	pass
LA	1000 µg/mL	94.8	67	≤150	95.0	108	≤200	no	pass

Results of the Cell Batch Activation Test (Batch 22)

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	85.3	329	>150	85.1	434	>200	yes	pass
NiSO4old	100 µg/mL	88.6	294	>150	89.4	447	>200	yes	pass
NiSO4new	100 µg/mL	88.7	298	>150	88.7	456	>200	yes	pass
LA	1000 µg/mL	96.7	84	≤150	96.6	107	≤200	no	pass

 Results of the Cell Batch Activation Test (Batch 01)

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 02)

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 Assays

Sample		Cell Viability
		Concentration applied [µg/mL]	Assay 1	Assay 2	Assay 3	Assay 4	Assay 5
Medium Control	--	--	94.60	93.90	95.60	95.50	96.50
Solvent Control	DMSO	--	94.30	93.90	95.20	96.20	93.90
F6-Cl	C8	7.81	94.20	92.10	96.30	95.70	94.60
	C7	15.63	94.50	92.60	95.70	96.00	94.30
	C6	31.25	94.40	93.30	95.10	96.50	95.30
	C5	62.50	94.10	91.40	87.90	96.00	94.40
	C4	125.00	93.70	65.10	7.20	95.20	93.50
	C3	250.00	91.10	16.10	4.80	85.80	68.30
	C2	500.00	10.60	19.50	26.20	14.60	23.20
	C1	1000.00	11.20	26.40	10.20	9.00	14.40
Calculated CV75 [µg/mL]		--	287.17	96.29	69.82	277.72	207.92

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.8	95.5	95.7	2078	1010	583	1495	427	98	87	356	173
Solvent Control	0.20%	96.2	96.0	96.1	2087	1057	565	1522	492	100	100	369	187
DNCB	4.00	86.3	86.9	86.0	5604	1790	606	4998	1184	328	241	925	295
F6-Cl	99.67	94.1	94.7	94.4	1900	1089	633	1267	456	83	93	300	172
	83.06	95.6	95.1	95.8	1780	1013	672	1108	341	73	69	265	151
	69.22	95.6	95.7	95.2	1735	1040	631	1104	409	73	83	275	165
	57.68	95.1	95.2	95.1	1802	1011	606	1196	405	79	82	297	167
	48.07	96.7	96.2	95.7	1697	1014	611	1086	403	71	82	278	166
	40.06	95.5	95.4	95.5	1882	1005	611	1271	394	84	80	308	164
	33.38	95.2	95.2	95.3	1856	977	626	1230	351	81	71	296	156
	27.82	95.4	95.3	95.3	1749	983	577	1172	406	77	83	303	170

  CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.4	97.0	97.2	1920	864	516	1404	348	79	96	372	167
Solvent Control	0.20%	96.8	96.8	96.3	2286	878	514	1772	364	100	100	445	171
DNCB	4.0	87.8	86.9	87.8	5309	1539	572	4737	967	267	266	928	269
F6-Cl	99.67	96.1	96.2	95.6	2001	917	571	1430	346	81	95	350	161
	83.06	95.9	95.8	96.2	1996	934	564	1432	370	81	102	354	166
	69.22	96.7	96.7	95.9	1896	928	574	1322	354	75	97	330	162
	57.68	96.3	95.7	95.8	2087	920	566	1521	354	86	97	369	163
	48.07	96.4	96.1	96.0	2006	918	555	1451	363	82	100	361	165
	40.06	95.7	96.2	95.6	2223	899	537	1686	362	95	99	414	167
	33.38	96.2	96.1	95.7	1819	849	514	1305	335	74	92	354	165
	27.82	95.7	95.7	95.8	1794	816	501	1293	315	73	87	358	163

CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.4	96.2	96.3	1374	832	502	872	330	91	97	274	166
Solvent Control	0.20%	96.1	95.5	96.1	1452	832	493	959	339	100	100	295	169
DNCB	4.00	82.5	82.9	82.2	5346	2974	639	4707	2335	491	689	837	465
F6-Cl	338.93	95.6	95.6	95.5	1551	1113	676	875	437	91	129	229	165
	282.44	96.3	95.7	95.9	1619	1110	688	931	422	97	124	235	161
	235.37	96.3	96.0	95.9	1609	1046	629	980	417	102	123	256	166
	196.14	96.5	96.2	95.9	2012	1058	627	1385	431	144	127	321	169
	163.45	96.5	96.0	96.1	1392	977	613	779	364	81	107	227	159
	136.21	96.5	96.6	96.3	1606	966	603	1003	363	105	107	266	160
	113.51	96.4	96.4	96.2	1791	991	596	1195	395	125	117	301	166
	94.59	96.6	95.1	95.9	1601	964	597	1004	367	105	108	268	161

CD54 and CD86 Expression Experiment 4

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.7	96.7	96.3	1301	751	537	764	214	81	83	242	140
Solvent Control	0.20%	96.2	96.6	96.5	1450	767	508	942	259	100	100	285	151
DNCB	4.0	87.5	87.7	87.3	4043	2054	628	3415	1426	363	551	644	327
F6-Cl	338.93	94.1	94.2	93.8	1760	1105	721	1039	384	110	148	244	153
	282.44	94.6	95.2	94.6	1709	1054	698	1011	356	107	137	245	151
	235.37	94.7	95.1	95.4	1706	1020	682	1024	338	109	131	250	150
	196.14	96.2	96.3	96.6	1575	1035	671	904	364	96	141	235	154
	163.45	95.3	94.9	95.8	1672	946	629	1043	317	111	122	266	150
	136.21	96.0	96.0	94.9	1702	986	641	1061	345	113	133	266	154
	113.51	95.8	95.8	96.1	1550	895	599	951	296	101	114	259	149
	94.59	96.4	96.4	96.4	1411	673	554	857	119	91	46	255	121

           CD54 and CD86 Expression Experiment 5

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.7	96.1	95.2	1773	992	548	1225	444	100	93	324	181
Solvent Control	0.20%	95.7	95.6	95.0	1756	1007	531	1225	476	100	100	331	190
DNCB	4.0	85.2	85.2	84.5	5286	2023	611	4675	1412	382	297	865	331
F6-Cl	500.0	22.8	20.5	20.2	4933	4133	3818	1115	315	91	66	129	108
	416.67	22.2	20.7	20.5	5153	4198	3927	1226	271	100	57	131	107
	347.22	24.2	22.7	22.5	4713	2707	1958	2755	749	225	157	241	138
	289.35	19.4	18.1	17.5	5687	4435	4060	1627	375	133	79	140	109
	241.13	18.6	18.2	17.3	5745	4400	3971	1774	429	145	90	145	111
	200.94	21.6	20.3	18.6	5466	4055	3606	1860	449	152	94	152	112
	167.45	54.1	52.8	52.3	4473	4480	1031	3442	3449	281	725	434	435
	139.54	85.1	82.1	84.1	2740	1655	673	2067	982	169	206	407	246

Acceptance Criteria experiment 1 and 2

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.5	-	96.8	pass	96.3	-	97.4	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	328			pass	267			pass
RFI of positive control of CD54	≥200	241			pass	266			pass
RFI of solvent control of CD86	<150	102			pass	126			pass
RFI of solvent control of CD54	<200	115			pass	105			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	356			pass	372			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	369			pass	445			pass
MFI ratio CD54/IgG1for medium control [%]	>105	173			pass	167			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	187			pass	171			pass

 

Acceptance Criteria experiment 3, 4 and 5

Acceptance Criterion

Range

Experiment 3

pass/fail

Experiment 4

pass/fail

Experiment 5

pass/fail

cell viability solvent controls [%]

>90

95.5

-

96.4

pass

95.7

-

96.7

pass

95.0

-

96.7

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

2

fail

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

2

fail

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

2

fail

RFI of positive control of CD86

≥150

491

pass

363

pass

382

pass

RFI of positive control of CD54

≥200

689

pass

551

pass

297

pass

RFI of solvent control of CD86

<150

110

pass

123

pass

100

pass

RFI of solvent control of CD54

<200

103

pass

121

pass

107

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

274

pass

242

pass

324

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

295

pass

285

pass

331

pass

MFI ratio CD54/IgG1for medium control [%]

>105

166

pass

140

pass

181

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

169

pass

151

pass

190

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

 

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of both cell surface markers which came along with no cytotoxic effects up to the 1.2 x CV75 concentration. Since a negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90% and the highest tested concentration was 1000 µg/mL, this study has to be regarded as inconclusive.

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.

Since the CV75 of the dose-finding assay 1 and 2 clearly differed, a third dose-finding assay was performed. Hereby, based on the dose finding assays 2 and 3 a mean CV75 was calculated with 83.06 ± 18.72 µg/mL. Based on the mean CV75, the main experiments 1 and 2 were performed covering the following concentration steps:

99.67, 83.06, 69.22, 57.68, 48.07, 40.06, 33.38, 27.82 µg/mL

Since in the main experiments 1 and 2 no cytotoxic effect and no sensitizing effect was observed, a fourth dose-finding assay was performed in order to verify the CV75. As the CV75 of dose-finding assay 1 and 4 are very comparable, amean CV75 based ondose-finding assay 1 and 4 was calculated with 282.45 ± 6.69 µg/mL. Based on the mean CV75, the main experiments 3 and 4 were performed covering the following concentration steps:

338.93, 282.45, 235.37, 196.14, 163.45, 136.21, 113.51, 94.59 µg/mL

As also for these main experiments 3 and 4 no cytotoxic effect and no sensitizing effect was observed, a fifth dose-finding assay was performed. In this assay, the CV75 ofdose-finding assay 1 and 4 was confirmed.

Therefore, a main experiment 5 using 500 µg/mL as starting concentration was performed covering the following concentration steps:

500, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL

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.

No cytotoxic effects were observed for the cells treated with the test item in the main experiments 1, 2, 3 and 4. Relative cell viability at the highest test item concentration was 94.1% (CD86), 94.7% (CD54) and 94.4% (isotype IgG1 control) in the first experiment, 96.1% (CD86), 96.2% (CD54) and 95.6% (isotype IgG1 control) in the second experiment, 95.6% (CD86), 95.6% (CD54) and 95.5% (isotype IgG1 control) in the third experiment and 94.1% (CD86), 94.2% (CD54) and 93.8% (isotype IgG1 control) in the fourth experiment. Cytotoxic effects were observed for the cells treated with the test item in main experiment 5. Relative cell viability at the highest test item concentration was reduced to 22.8% (CD86), 20.5% (CD54) and 20.2% (isotype IgG1 control) in the fifth experiment.

In the experiments 1, 2, 3 and 4 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. However, since a negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90% and the highest tested concentration was 1000 µg/mL, the experiments1, 2, 3 and 4 have to be regarded as inconclusive.

In experiment 5 the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 281% at a concentration of 167.45 µg/mL.The expression of cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 725% at a concentration of 167.45 µg/mL. However, as only two concentrations showed a viability >50%, this experiment 5 was not valid.

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 data, a clear positive response in the DPRA assay was observed. No conclusion can be made regarding the classification for skin sensitization based on the results of the OECD 442E assay. The OECD 442D assay was negative. The compound is highly reactive when exposed to light or in aqueous environment. Therefore, binding to skin proteins can be assumed leading to hapten formation as initial step of skin sensitisation. Taken together the high reactivity of the compound and the results from the DPRA assay suggest that a classification for skin sensitisation class 1 is fully justified.