5-[(4-chloro-2-nitrophenyl)azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity potential of FAT 36091 was tested on batch A and D. Both experiments were performed according to a method equivalent or similar to the OECD 471 (Bacterial reverse mutation assay). FAT 36091 was found to be negative in both cases.

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:

Study completion date: 17 April 1980

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)

Deviations:

yes

Remarks:

only 4 strains tested; no tester strain included to detect cross-linking mutagens; maximum test concentration was lower than recommended by the guideline

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 36091/D
Purity: 50.2 %

Target gene:

FAT 36091/D was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg/0.1 mL

Vehicle / solvent:

Dimethylsulfoxyde (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA 98 (at 5 and 10 µg/0.1 ml)

Positive control substance:

other: daunorubicin-HCL

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA 100 (at 0.125 and 0.25 microgr/0.1 ml)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA 1535 (at 3 and 5 microgr/0.1 ml)

Positive control substance:

other: N-methyl-N '-nitro-Nnitrosoguanidine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA 1537 (at 50 and 100 microgr/0.1 ml)

Positive control substance:

other: 9(5)aminoacridine hydrochloride monohydrate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

for strain TA 1535 (at 250 microgr/0.1 ml)

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The tests were carried out in accordance with the method described by AMES et al.
The bacteria on which the tests were performed were the histidineauxotrophic TA 98, TA 100, TA 15 35 and TA 15 37, strains of Salmonella typhimurium.
The tests were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 mL. The substance was dissolved in DMSO. DMSO alone was used for the negative controls . In the experiments in which the substance was metabolically activated. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37° C in darkness.
When the colonies had been counted, the arithmetic mean was calculated. A test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentrations.

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 36091/D revealed no marked differences. The slight increase in the number of back-mutant colonies in the experiment on Strain TA 1537 with microsomal activation is attributed to fluctuations in the number of spontaneously occurring back-mutants.

Salmonella/Mammalian-Microsome Mutagenicity Test

Experiments without microsomal activation

Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants

		Strain of S. typhimurium used
		TA 98	TA 100	TA 1535	TA 1537
Test substance
FAT 36091/D	Control	15	112	11	3
	25 µg/0.1 ml	13	132	8	4
	75 µg/0.1 ml	24	124	10	5
	225 µg/0.1 ml	17	16	7	4
	675 µg/0.1 ml	26	118	8	4
	2025 µg/0.1 ml	25	115	10	6

Positive controls (number of colonies)

*Daunorubicin-HCl (TA 98)

- Control: 21

- 5 µg/0.1 ml: 674

- 10 µg/0.1 ml: 1120

*N-Methyl-N'-nitri- N-nitroso-guanidine (TA 1535)

- Control: 10

- 0.125 µg/0.1 ml: ca. 1000

- 0.25 µg/0.1 ml: >1550

*4-Nitroquinoline-Noxide (TA 100)

- Control: 165

- 0.125 µg/0.1 ml: 807

- 0.25 µg/0.1 ml: 1173

*9(5)Aminoacridine hydrochloride (TA 1537)

- Control: 5

- 0.125 µg/0.1 ml: 211

- 0.25 µg/0.1 ml: >400

Salmonella/Mammalian-Microsome Mutagenicity Test

Experiments with microsomal activation

Number (arithmetic mean) of colonies of

histidine-prototrophic back-mutants

		Strain of S. typhimurium used
		TA 98	TA 100	TA 1535	TA 1537
Test substance
FAT 36091/D	Control	31	136	12	4
	25 µg/0.1 ml	33	124	7	6
	75 µg/0.1 ml	30	131	9	6
	225 µg/0.1 ml	51	143	10	8
	675 µg/0.1 ml	50	163	13	10
	2025 µg/0.1 ml	35	137	8	5

Positive control of the microsomal activation:

*Cyclophosphamide

- Control: 8

- 250 µg/0.1 ml: 478

Conclusions:

No evidence of the induction of point mutations by FAT 36 091/D or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium in these experiments.

Executive summary:

FAT 36 091/D was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium according to Ames et al. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 36 091/D revealed no marked deviations. No evidence of the induction of point mutations by FAT 36 091/D or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium in these experiments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A key study, performed on FAT 36091/D was done to determine test the mutagenic effects on Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). The test was performed in accordance with a guideline equivalent or similar to the OECD 471 (Bacterial reverse mutation assay). Bacteria were incubated with the test item at different concentrations in the presence and in the absence of metabolic activation (rat liver microsomes and co-factors). No evidence of the induction of point mutations by FAT 36 091/D or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium in these experiments. One supporting study was carried out on FAT 36091/A. The test was also performed in accordance with a guideline equivalent or similar to the OECD 471. Under the experimental conditions, FAT 36091/A was not mutagenic for S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 in the presence and absence of metabolic activation (S9 mix).

Justification for selection of genetic toxicity endpoint: This study is the most recent available.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified as mutagenic.