Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[(2,2,4-trimethylcyclohexyl)amino]anthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance resulted to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

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:

guideline study without detailed documentation

Qualifier:

according 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: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vhicle: 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:

Maximum solubility of the test article was achieved at 25 mg/ml in sterile purified water, therefore volume additions were increased to 200 µl in order to achieve the required treatment doses.
The top dose treatment plates in the Range-Finder Experiment and Experiment I were counted manually due to the dark colour of the test article affecting automated counting.

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a slight thinning of the background bacterial at two highest doses in TA 98, without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

When the data from the Range-Finder Experiment were analysed at the 1 % level using Dunnett's test a statistically significant increase in revertant numbers was observed following TA 100 treatments in the presence of S9. TA 100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no increases in revertant number were observed following test item treatments of TA 100 in the presence of S9. As the increase in revertant numbers observed in the Range-Finder Experiment was small in magnitude, and showed little evidence of a dose-response it was considered to be due to a chance event.
A statistically significant increase in revertant numbers was also observed following TA 98 treatments in the absence of S9 in Experiment I, but this was observed at one of the lowest treatment doses and was not considered to be indicative of test item mutagenic activity in this strain under this treatment condition. No other treatments in this study induced statistically significant increases in revertant numbers, when the data were analvsed at the 1 % level usins Dunnett's test.

PRECIPITATION
No precipitation of test agent was observed following any of the treatments in either the presence or absence of S9.

TOXICITY
Evidence of toxicity, as manifest by a slight thinning of the background bacterial lawn was observed following the top two dose treatments of strain TA98 in the absence of S9 only.

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

Subsance	Dose Level µg/plate	Mean ± SD
		TA 98	TA 100	TA 1535	TA 1537	TA 1538
Water	200	43 ± 3	87 ± 6	17 ± 4	16 ± 5	19 ± 7
Test item	16	49 ± 5	77 ± 14	17 ± 5	12 ± 3	14 ± 3
Test item	8	59 ± 3	91 ± 6	12 ± 3	7 ± 2	19 ± 2
Test item	40	46 ± 11	86 ± 4	17 ± 4	13 ± 5	19 ± 1
Test item	200	48 ± 8	83 ± 12	16 ± 3	9 ± 3	23 ± 6
Test item	100	30 ± 3 (S)	87 ± 10	10 ± 1	9 ± 2	18 ± 6
Test item	5000	33 ± 4 (S+M)	72 ± 7 (M)	10 ± 1 (M)	8 ± 1 (M)	19 ± 2 (M)
2-Nitrofluorene	5	535 ± 26	-	-	-	101 ± 17
Sodium azide	2	-	392 ± 23	531 ± 8	-	-
9-Aminoacridine	50	-	-	-	300 ± 26	-

(S) Slight thinning of background lawn

(M) Plates counted manually

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

Subsance	Dose Level µg/plate	Mean ± SD
		TA 98	TA 100	TA 1535	TA 1537	TA 1538
Water	200	38 ± 9	88 ± 12	19 ± 5	15 ± 4	21 ± 2
Test item	16	35 ± 4	91 ± 9	16 ± 2	11 ± 2	23 ± 5
Test item	8	40 ± 10	88 ± 1	18 ± 4	12 ± 4	19 ± 5
Test item	40	42 ± 2	88 ± 10	20 ± 2	11 ± 5	19 ± 1
Test item	200	43 ± 4	99 ± 8	14 ± 2	18 ± 4	17 ± 4
Test item	100	38 ± 7	111 ± 33	13 ± 2	13 ± 5	21 ± 2
Test item	5000	39 ± 10 (M)	89 ± 2 (M)	13 ± 4 (M)	12 ± 1 (M)	14 ± 3 (M)
Benzo[a]pyrene	10	252 ± 10	-	-	-	-
2-Aminoanthracene	5	-	1903 ± 62	127 ± 20	431 ± 98	1335 ± 93

(M) Plates counted manually

Summary of mean revertant colonies - Range Finder Experiment

Subsance	Dose Level µg/plate	Mean ± SD
		TA 100 -S9	TA 100 +S9
Water	200	100 ± 8	102 ± 12
Test item	16	100 ± 8	112 ± 11
Test item	8	107 ± 14	106 ± 3
Test item	40	105 ± 5	110 ± 8
Test item	200	103 ± 7	127 ± 7
Test item	100	91 ± 10	116 ± 11
Test item	5000	75 ± 6 (M)	83 ± 3 (M)
Sodium azide	2	849 ± 17	-
2-Aminoanthracene	5	-	2230 ± 110

(M) plates counted manually

Conclusions:

Non-mutagenic

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations using the Salmonella typhimurium strains TA 3538, TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in a range finding test, involving only strain TA 100, and in a complete main Experiment I. The test article was assessed at the following concentrations: 1.6, 8, 40, 200, 1000 and 5000 µg/plate

When the data from the Range-Finder Experiment were analysed significant increase in revertant numbers was observed following TA 100 treatments in the presence of S9. TA 100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no increases in revertant number were observed following test item treatments of TA 100 in the presence of S9. As the increase in revertant numbers observed in the Range-Finder Experiment was small in magnitude, and showed little evidence of a dose-response it was considered to be due to a chance event.

A statistically significant increase in revertant numbers was also observed following TA 98 treatments in the absence of S9 in Experiment I, but this was observed at one of the lowest treatment doses and was not considered to be indicative of test item mutagenic activity in this strain under this treatment condition. No other treatments in this study induced statistically significant increases in revertant numbers

No precipitation of test agent was observed following any of the treatments in either the presence or absence of S9.

Evidence of toxicity, as manifest by a slight thinning of the background bacterial lawn was observed following the top two dose treatments of strain TA98 in the absence of S9 only.

Conclusion

In conclusion, under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The study was performed to investigate the potential of test item to induce gene mutations using the Salmonella typhimurium strains TA 3538, TA 1535, TA 1537, TA 98 and TA 100. When the data from the Range-Finder Experiment were analysed, significant increase in revertant numbers was observed following TA 100 treatments in the presence of S9. TA 100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no increases in revertant number were observed following test item treatments of TA 100 in the presence of S9. As the increase in revertant numbers observed in the Range-Finder Experiment was small in magnitude, and showed little evidence of a dose-response, it was considered to be due to a chance event. A statistically significant increase in revertant numbers was also observed following TA 98 treatments in the absence of S9 in Experiment I, but this was observed at one of the lowest treatment doses and was not considered to be indicative of test item mutagenic activity in this strain under this treatment condition. No other treatments in this study induced statistically significant increases in revertant numbers. No precipitation of test agent was observed following any of the treatments in either the presence or absence of S9.

Evidence of toxicity, as manifest by a slight thinning of the background bacterial lawn, was observed following the top two dose treatments of strain TA98 in the absence of S9 only.

It was concluded that, under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 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 from the genotoxicity point of view.