Chromate(1-), bis[4-[[4-(ethylsulfonyl)-2-hydroxyphenyl]azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]-, compd. with 1,6-hexanediamine (2:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reverse gene mutation assay in bacteria (Ames Test), no mutagenicity was observed with or without metabolic activation.

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9 mix (Aroclor 1254 induced rat liver)

Test concentrations with justification for top dose:

10, 33, 100, 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Each strain was tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the micro colonies and the reduction of the revertant colonies was observed

POSITIVE CONTROLS
Without S9:
TA1535: sodium azide, 1µg/plate
TA1537: 9-aminoacridine, 60 µg/plate
TA98: daunomycine, 4 µg/plate
TA100: methylmethanesulfonate, 650 µg/plate
WP2UvrA: 4-nitroquinoline N-oxide, 10 µg/plate

With S9
TA1535, TA1537, TA98: 2-aminoanthracene, 2.5 µg/plate
TA100: 2-aminoanthracene, 1µg/plate
WP2UvrA: 2-aminoanthracene, 5 µg/plate

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the top agar at concentrations of 100 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at a concentration of 333 µg/plate in all tester strains.

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9 mix. The test substance precipitated in the top agar at concentrations of 100 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 333 µg/plate and upwards in tester strain TA100 and WP2uvrA. No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls and positive controls were within the historical control values.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The bacterial background lawn was not reduced in the main study at all concentrations tested and no decrease in the number of revertants was observed.

Table 1Experiment 1/Range finding test

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
	TA1535	TA1537	TA98	TA100	WP2 uvr A
	Results without S9
Spontaneous Reversion	12	10	24	66	7
Positive control	287	372	261	652	106
3	15	9	19	64	9
10	16	8	22	62	6
33	12	10	24	64	7
100	10	10	22	65	6
333 (SP)	11	8	17	61	10
1000 (SP)	-	-	-	60	6
2500 (SP)	-	-	-	60	7
5000 (MP)	-	-	-	60	8
	Results with S9
Spontaneous Reversion	13	7	32	64	6
Positive control	342	645	1289	1273	108
3	12	7	31	63	7
10	13	6	28	60	7
33	11	8	33	68	10
100	13	7	28	60	8
333(SP)	12	5	26	61	6
1000 (SP)	-	-	-	62	6
2500 (SP)	-	-	-	66	8
5000 (MP)	-	-	-	66	7

SP slight precipitate

MP moderate precipitate

Table 2Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
	TA1535	TA1537	TA98	TA100	WP2 uvr A
	Results without S9
Spontaneous Reversion	9	6	14	65	7
Positive control	183	291	329	619	335
3	9	5	12	62	5
10	13	7	16	63	5
33	9	7	18	62	7
100	10	6	13	63	6
333 (SP)	13	4	13	61	6
	Results with S9
Spontaneous Reversion	10	7	18	63	6
Positive control	336	342	677	1320	112
3	14	5	21	63	4
10	10	6	22	61	5
33	13	7	19	63	5
100	12	8	22	61	7
333(SP)	12	9	19	62	5

Conclusions:

Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2UvrA in two independent experiments. In the dose range finding test, the test article was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TAl00 and WP2UvrA. At this dose level no toxicity was observed. Precipitation was observed on the plates at dose levels of 333 µg/plate and upwards. In the mutation assays, the test item was tested up to concentrations of 333 µg/plate in the absence and presence of S9 mix. Precipitated was observed at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test article did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2UvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Test

The test substance was tested according to OECD guideline 471 using strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli WP2uvrA in two independent experiments. In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA100 and WP2uvrA. At this dose level no toxicity was observed. The test substance precipitated on the plates at dose levels of 333 µg/plate and upwards. In the main mutation assays, the test substance was tested up to concentrations of 333 µg/plate in the absence and presence of S9 mix. The test substance precipitated on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Notox, 1999).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Regulation (EC) No 1272/2008.