Sodium 3-diazo-3,4-dihydro-4-oxonaphthalene-1-sulphonate

Endpoint summary

Description of key information

The skin sensitisation potential was investigated in vitro in two key event related studies:

OECD 442C: positive

OECD 442D: positive

Based on the positive results in two mechanistic endpoints (DPRA and Keratinosens), the compound is considered as skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-05 to 2018-01-26

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 (2.46 (experiment 1); 5.40 (experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

13.94

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:

143.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

1.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

14.62

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 2000 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

171.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1.99

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	113.9	104.0	108.9	7.0
	8.00	104.8	108.0	106.4	2.3
	16.00	119.1	115.1	117.1	2.8
	32.00	123.0	125.4	124.2	1.7
	64.00	127.6	120.4	124.0	5.1
Test Item	0.98	117.3	59.0	88.2	41.2
	1.95	94.0	84.9	89.4	6.4
	3.91	92.7	87.2	89.9	3.9
	7.81	102.6	90.3	96.5	8.7
	15.63	94.5	87.9	91.2	4.7
	31.25	101.8	108.9	105.3	5.0
	62.50	112.9	106.3	109.6	4.7
	125.00	119.4	116.8	118.1	1.9
	250.00	148.2	131.6	139.9	11.7
	500.00	171.9	154.2	163.1	12.6
	1000.00	188.8	185.1	186.9	2.6
	2000.00	143.1	171.7	157.4	20.3

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	0.98	1.09	0.84	0.97	0.12
	8.00	1.12	1.11	0.98	1.07	0.08
	16.00	1.42	1.54	1.27	1.41	0.14
	32.00	1.46	1.98	1.44	1.63	0.31	*
	64.00	2.08	3.16	2.13	2.46	0.61	*
Test Item	0.98	1.02	1.19	1.07	1.09	0.09
	1.95	1.06	1.05	0.97	1.03	0.05
	3.91	1.12	1.06	1.01	1.06	0.05
	7.81	1.01	1.22	1.19	1.14	0.11
	15.63	1.02	1.26	0.93	1.07	0.17
	31.25	1.00	1.30	0.97	1.09	0.18
	62.50	1.06	1.29	0.93	1.09	0.18
	125.00	1.33	1.59	1.14	1.35	0.22
	250.00	1.66	1.94	1.58	1.72	0.19	*
	500.00	2.48	2.89	1.90	2.42	0.50	*
	1000.00	3.65	4.64	3.01	3.77	0.82	*
	2000.00	11.39	18.96	11.46	13.94	4.35	*

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

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.05	1.23	1.01	1.09	0.12
	8.00	1.31	1.18	1.19	1.23	0.07
	16.00	1.52	1.49	1.39	1.47	0.07
	32.00	2.18	2.02	2.41	2.20	0.20	*
	64.00	6.28	4.02	5.91	5.40	1.21	*
Test Item	0.98	1.05	1.13	0.87	1.02	0.13
	1.95	0.92	1.00	0.98	0.97	0.04
	3.91	1.06	1.08	0.87	1.00	0.12
	7.81	1.20	0.93	0.89	1.01	0.17
	15.63	0.94	1.11	0.92	0.99	0.10
	31.25	1.03	0.99	0.95	0.99	0.04
	62.50	1.11	1.16	0.93	1.07	0.12
	125.00	1.33	1.26	1.12	1.24	0.11
	250.00	1.52	1.54	1.35	1.47	0.11
	500.00	2.09	2.03	1.85	1.99	0.12	*
	1000.00	4.55	4.22	3.66	4.14	0.45	*
	2000.00	15.53	13.61	14.72	14.62	0.97	*

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

 

Induction of Luciferase Activity – Overall Induction

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	0.97	1.09	1.03	0.08
	8.00	1.07	1.23	1.15	0.11
	16.00	1.41	1.47	1.44	0.04
	32.00	1.63	2.20	1.92	0.41
	64.00	2.46	5.40	3.93	2.08
Test Item	0.98	1.09	1.02	1.05	0.05
	1.95	1.03	0.97	1.00	0.04
	3.91	1.06	1.00	1.03	0.04
	7.81	1.14	1.01	1.07	0.09
	15.63	1.07	0.99	1.03	0.06
	31.25	1.09	0.99	1.04	0.07
	62.50	1.09	1.07	1.08	0.02
	125.00	1.35	1.24	1.29	0.08
	250.00	1.72	1.47	1.60	0.18	*
	500.00	2.42	1.99	2.21	0.31	*
	1000.00	3.77	4.14	3.95	0.26	*
	2000.00	13.94	14.62	14.28	0.48	*

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

 

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	175.30	264.90	220.10	63.36
Imax	13.94	14.62	14.28	0.48
IC30[µM]	n/a	n/a	-	-
IC50[µM]	n/a	n/a	-	-

n/a not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	19.7	pass	12.6	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	22.53	pass	16.72	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.46	pass	5.40	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

 

Interpretation of results:

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

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

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

Based on a molecular weight of 290 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 and a stable suspension was formed. 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.

A colour change of the MTT medium from yellow to blue/purple was observed resulting in viabilities >100% for the high test item concentrations from 125 µM onwards. Microscopically, no cytotoxic effect was observed.

In the first experiment, a max luciferase activity (I_max) induction of 13.94 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 143.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.72) was found to be 250 µM. The corresponding cell viability was >70% (148.2%). The calculated EC_1.5was < 1000 µM (175.30)

In the second experiment, a max luciferase activity (I_max) induction of 14.62 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 171.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.99) was found to be 500 µM. The corresponding cell viability was >70% (154.2%).The calculated EC_1.5 was < 1000 µM (264.90).

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 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-22 to 2018-03-01

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 61.59%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

99.55

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:

23.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.336	0.5340	4254.6284	0.5340
STD2	8.789	0.2670	2161.7087	0.2670
STD3	4.398	0.1335	1051.8925	0.1335
STD4	2.166	0.0667	525.2503	0.0667
STD5	1.075	0.0334	260.5259	0.0334
STD6	0.504	0.0167	130.8325	0.0167
STD7	0.000	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	3.710	0.1138	77.40	77.79	0.34	0.44
	3.620	0.1110	77.95
	3.608	0.1106	78.02
Test Item	0.222	0.0066	98.64	99.55	0.79	0.79
	0.000	-0.0002	100.00
	0.000	-0.0002	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	1675.9452	0.2100	57.93	56.62	1.13	2.00
	1753.5808	0.2197	55.98
	1754.5223	0.2198	55.95
Test Item*	3125.4258	0.3913	22.44	23.64	1.11	4.72
	3070.0613	0.3844	23.82
	3036.4434	0.3802	24.65

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	-	-	-	99.55	High Reactivity	sensitiser
Positive Control	67.21	High Reactivity	sensitiser	77.79	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 DMF, based on the results of the pre-experiments. Based on a molecular weight of

290 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 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 (excluding the co-elution control). Samples were centrifuged at 400g for 5 min.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the co-elution control of the positive control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

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

Co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

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_DMF).

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was> 13.89% (99.55%). 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 61.59%.

The controls confirmed the validity of the study for both, the cysteine and lysine run, as shown in Table 10 and Table 11.

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 the substance is considerd to be classified for skin sensitisation cat. 1 under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.