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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the experimental conditions described, the test item induced relevant, concentration-dependent increases in revertant numbers exceeding a threshold of 3-fold of the negative control value in the absence and presence of S9-mix in strain TA1537. In TA100, a reproducible increase in revertant numbers (exceeding a threshold of 2-fold of the negative control value) was observed at the maximum concentrations tested in the absence of S9-mix in 2 independent experiments.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Aug - 07 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon, trp operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by ß-Naphthoflavone/phenobarbital

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 158, 500, 1580, 2810, 5000 µg/plate (with and without S9 mix)

Vehicle / solvent:

- Solvent used: DMSO, final concentration 10 µL per plate (1st series), 100 µl per plate (2nd series)
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid AND
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls is observed.
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a 2nd independent experiment.
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is considered negative or non-mutagenic in this assay if
- the assay is considered valid AND
- non of the above criteria are met

Statistics:

Not performed as not mandatory for this test system.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Following treatment with the test item, precipitation on the agar plates occurred at a concentration of 5000 µg/plate. No toxicity to the bacteria was observed. However, a slight decrease in the mutagenic response was observed in TA 1537 in the presence of metabolic activation in one experiment at the highest concetration tested.
Under the experimental conditions described, the test item induced relevant, concentration-dependent increases in revertant numbers exceeding a threshold of 3-fold of the negative control value in the absence and presence of S9-mix in strain TA1537. In TA100, a reproducible increase in revertant numbers (exceeding a threshold of 2-fold of the negative control value) was observed at the maximum concentrations tested in the absence of S9-mix in 2 independent experiments.
According to the criteria for negative and positive results as predetermined in the study plan, the test item was mutagenic under the described experimental conditions.

Table 1: Summary of Experiment 1

Metabolic Activation	Test material	Concentr. (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		37 +/- 8	123 +/- 11	26 +/- 9	6 +/- 2	38 +/- 9
	Test item	5	30 +/- 6	116 +/- 11	29 +/- 7	4 +/- 4	44 +/- 3
		15.8	27 +/- 3	127 +/- 10	23 +/- 2	4 +/- 3	39 +/- 2
		50	26 +/- 4	124 +/- 11	32 +/- 10	9 +/- 5	34 +/- 10
		158	28 +/- 8	135 +/- 9	27 +/- 5	12 +/- 6	37 +/- 5
		500	39 +/- 5	155 +/- 14	19 +/- 4	22 +/- 4	39 +/- 8
		1580	45 +/- 1	191 +/- 14	24 +/- 3	30 +/- 2	54 +/- 7
		5000	64 +/- 8	281 +/- 39 S	25 +/- 6 S	44 +/- 3 S	66 +/- 12 S
	DAUN	1	131 +/- 2
	NaN3	2		1634 +/- 17	869 +/- 21
	9-AA	50				614 +/- 173
	NQO	2					1908 +/- 88
With Activation	DMSO		44 +/- 7	127 +/- 19	18 +/- 3	8 +/- 2	41 +/- 10
	Test item	5	32 +/- 8	131 +/- 14	19 +/- 5	12 +/- 3	44 +/- 2
		15.8	34 +/- 5	131 +/- 8	23 +/- 6	12 +/- 3	43 +/- 2
		50	36 +/- 4	123 +/- 14	20 +/- 4	14 +/- 6	49 +/- 10
		158	48 +/- 8	147 +/- 13	17 +/- 4	26 +/- 2	47 +/- 12
		500	43 +/- 1	159 +/- 17	13 +/- 6	68 +/- 16	53 +/- 4
		1580	36 +/- 8	201 +/- 22	17 +/- 0	123 +/- 14	61 +/- 10
		5000	56 +/- 15	275 +/- 20 S	24 +/- 8 S	140 +/- 22 S	76 +/- 11 S
	2-AA	2	568 +/- 74	1274 +/- 92
	2-AA	5			193 +/- 19	438 +/- 5
	2-AA	10					374 +/- 37

Table 1: Summary of Experiment 2

Metabolic Activation	Test material	Concentr. (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		22 +/- 5	125 +/- 12	27 +/- 6	7 +/- 2	30 +/- 4
	Test item	5	--	--	--	--	--
		15.8	--	--	--	--	--
		158	29 +/- 6	144 +/- 4	15 +/- 6	9 +/- 1	30 +/- 9
		500	25 +/- 6	145 +/- 20	17 +/- 3	19 +/- 10	26 +/- 2
		1580	26 +/- 3	196 +/- 19	18 +/- 5	23 +/- 7	45 +/- 6
		2810	37 +/- 9	214 +/- 4	18 +/- 5	38 +/- 10	54 +/- 13
		5000	38 +/- 9	274 +/- 25	20 +/- 9	70 +/- 6	54 +/- 13
	DAUN	1	160 +/- 30
	NaN3	2		1581 +/- 54	716 +/- 47
	9-AA	50				607 +/- 206
	NQO	2					2086 +/- 98
With Activation	DMSO		32 +/- 13	119 +/- 13	11 +/- 2	13 +/- 5	36 +/- 7
	Test item	5	--	--	--	--	--
		15.8	--	--	--	--	--
		158	26 +/- 5	138 +/- 8	L	27 +/- 8	41 +/- 4
		500	32 +/- 14	135 +/- 4	7 +/- 3	39 +/- 19	39 +/- 5
		1580	29 +/- 6	157 +/- 20	10 +/- 5	114 +/- 18	45 +/- 6
		2810	31 +/- 4	202 +/- 18	8 +/- 3	141 +/- 11	71 +/- 7
		5000	39 +/- 6	191 +/- 37	8 +/- 4	85 +/- 23	64 +/- 2
	2-AA	2	336 +/- 103	844 +/- 104
	2-AA	5			82 +/- 19	198 +/- 61
	2-AA	10					254 +/- 16

Key to Positive Controls

NaN₃                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

S plated as a suspension

L lost due to experimental error

Conclusions:

The test item is considered mutagenic under the test conditions described.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information no conclusion can be made on mutagenicity according to the EU Regulatio n (EC) No 1272/2008 on Classification,Lab elling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.