Sodium 6-amino-5-[[2-[(ethylphenylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, T41535, TA1537 and TA1538

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolic activation system

Test concentrations with justification for top dose:

Range Finder Experiment and Experiment 1: 1.6, 8, 40, 200, 1000 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: sterile purified water.

Untreated negative controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

EVALUATION AND COUNT
- Count: the top one or two dose treatment plates in the Range-Finder Experiment and Experiment 1 were counted manually due to the dark colour of the test article affecting automated counting.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the top dose, in strain TA1535 only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

When data from the Range-Finder Experiment was analysed at the 1 % level using Dunnett's test, a statistically significant increase in revertant numbers was observed following TA100 treatments in the presence of S-9. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no treatments of any of the test strains, either in the absence or presence of S-9, induced statistically significant increases in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced on a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

PRECIPITATION
No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

TOXICITY
Evidence of toxicity, as shown by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9.

Summary of mean revertant colonies (-S9) - Experiment I

Substance	Dose level (μg/plate)	TA98 Mean ± SD	TA100 Mean ± SD	TA1535 Mean ± SD	TA1537 Mean ± SD	TA1538 Mean ± SD
Water	100 μl	51 ± 8	132 ± 13	29 ± 9	14 ± 3	21 ± 5
Test item	1.6	50 ± 3	115 ± 11	26 ± 5	17 ± 2	21 ± 6
	8	49 ± 11	107 ± 11	29 ± 7	13 ± 5	20 ± 2
	40	57 ± 7	118 ± 12	28 ± 3	17 ± 5	28 ± 5
	200	62 ± 2	116 ± 14	19 ± 8	14 ± 3	29 ± 8
	1000	43 ± 9	94 ± 32	11 ± 6	12 ± 2	22 ± 2
	5000	50 ± 3 (M)	87 ± 4 (M)	14 ± 2 (M)	17 ± 4 (M)	21 ± 5 (M)
Positive controls
2-Nitrofluorene	5 μg	861 ± 85	-	-	-	364 ± 39
Sodium azide	2 μg	-	674 ± 8	623 ± 48	-	-
9-Aminoacridine	50 μg	-	-	-	178 ± 15	-

SD: Standard deviation; (M): Plates counted manually

Summary of mean revertant colonies (+S9) - Experiment I

Substance	Dose level (μg/plate)	TA98 Mean ± SD	TA100 Mean ± SD	TA1535 Mean ± SD	TA1537 Mean ± SD	TA1538 Mean ± SD
Water	100 μl	55 ± 6	119 ± 11	20 ± 2	24 ± 2	32 ± 9
Test item	1.6	43 ± 20	128 ± 3	29 ± 4	15 ± 3	27 ± 6
	8	61 ± 6	113 ± 12	24 ± 10	18 ± 2	29 ± 7
	40	58 ± 5	117 ± 15	24 ± 1	15 ± 8	33 ± 6
	200	64 ± 9	106 ± 28	21 ± 5	32 ± 8	36 ± 14
	1000	49 ± 5	77 ± 4	18 ± 5	18 ± 8	33 ± 6
	5000	52 ± 7 (M)	104 ± 11 (M)	13 ± 7 (M)	17 ± 3 (M)	23 ± 1 (M)
Positive controls
Benzo[a]pyrene	10 μg	253 ± 8	-	-	-	-
2-Aminoanthracene	5 μg	-	1783 ± 131	191 ± 11	331 ± 31	1329 ± 60

SD: Standard deviation; (M): Plates counted manually

Summary of mean revertant colonies Range-Finder Experiment

Substance	Dose level (μg/plate)	TA100 (-S9) Mean ± SD	TA100 (+S9)  Mean ± SD
Water	100 μl	110 ± 7	99 ± 8
Test item	1.6	105 ± 1	108 ± 9
	8	98 ± 11	107 ± 6
	40	103 ± 8	116 ± 14
	200	90 ± 5	119 ± 12
	1000	106 ± 9 (M)	138 ± 13 (M)
	5000	106 ± 26 (M)	103 ± 12 (M)
Positive controls
Sodium azide	2 μg	833 ± 9	-
2-Aminoanthracene	5 μg	-	2286 ± 102

SD: Standard deviation; (M): Plates counted manually

Conclusions:

The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.

Executive summary:

The substance was tested for mutagenicity effects in vitro in strains of Salmonella typhimurium TA98, TA100, TA1535 TA1537 and TA1538, with and without metabolic activation. The test consists in two experiments, i.e. range-finding experiment and experiment 1, conducted at the following concentrions: 1.6, 8, 40, 200, 1000 and 5000 μg/plate.

No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

Evidence of toxicity, as shown by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9.

Analysed at the 1 % level using Dunnett's test a statistically showed a significant increase in revertant numbers in TA100 strain upon treatment in presence of S-9, in the range-finding experiment. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no test item treatments of any of the test strains, either in absence or presence of S-9, induced statistically significant increases in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced on a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

Conclusion

The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance was tested for mutagenicity effects in vitro in strains of Salmonella typhimurium TA98, TA100, TA1535 TA1537 and TA1538, with and without metabolic activation. The test consists in two experiments, i.e. range-finding experiment and experiment 1, conducted at the following concentrions: 1.6, 8, 40, 200, 1000 and 5000 μg/plate.

No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

Evidence of toxicity, as manifest by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9. When the data from the Range-Finder Experiment was analysed at the 1 % level using Dunnett's test, a statistically significant increase in revertant numbers was observed following TA100 treatments in the presence of S-9. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response.

In Experiment I, either in the absence or presence of S-9, no statistically significant increases in revertant numbers were seen, upon analysis at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced in a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern for genotoxicity.