2-[[4-[bis(2-hydroxyethyl)amino]-o-tolyl]azo]-5-nitrobenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of mono-nitrobenzene derivatives in the Ames test and rec assay

Author:

Makoto Shimizu and Eiji Yan

Year:

1986

Bibliographic source:

Mutation Research, 170 (1986) 11-22

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Gene mutation toxicity study was performed to evaluate the mutagenic nature of p-Nitrobenzonitrile

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-Nitrobenzonitrile

Cas Number:

619-72-7

Molecular formula:

C7H4N2O2

IUPAC Name:

4-Nitrobenzonitrile

Details on test material:

- Name of test material: p-Nitrobenzonitrile
- IUPAC name: 4-Nitrobenzonitrile
- Molecular formula: C7H4N2O2
- Molecular weight: 148.121 g/mol
- Substance type: Organic

Specific details on test material used for the study:

- Name of test material: p-Nitrobenzonitrile
- IUPAC name: 4-Nitrobenzonitrile
- Molecular formula: C7H4N2O2
- Molecular weight: 148.121 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 98% min
- Impurities (identity and concentrations): 2%

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction was obtained from PCB-induced hamster or rat liver

Test concentrations with justification for top dose:

0, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AA) (S9 mix added only)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 15 mins
- Exposure duration: 70 hrs in dark
- Expression time (cells in growth medium): 70 hrs in dark
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: All tests were performed in duplicate and repeated at least 3 times separately

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, The background bacterial lawn was routinely checked by microscopy, as high doses of the complexes proved toxic to all strains resulting in a thinning out of the bacterial lawn.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: First the tests were carried out without metabolic activation and were terminated if the mutagenicity was positive. In case of negative results, tests with metabolic activation were carried out in addition.

Two strains (TA98 and TA100) were checked routinely for the presence of the ampicillin resistance for the R factor.

Rationale for test conditions:

No data

Evaluation criteria:

Chemicals inducing more than twice the number of revertant colonies on the control plate were considered as mutagenic.

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 10 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Mutagenicity of p-Nitrobenzonitrile

Dose	Revertants/plate
	TA98		TA1538		TA1537		TA100		TA1535
	-	+	-	+	-	+	-	+	-	+
0.01	28±4	28±3	24±3	26±3	9±2	10±2	206±19	189±17	35±6	30±5
0.05	26±3	30±4	28±3	24±3	10±2	8±1	180±18	201±23	30±5	34±3
0.1	40±5	30±4	26±3	25±3	11±3	10±1	199±21	224±32	30±4	32±4
0.5	35±3	33±4	30±3	26±4	8±2	11±2	198±23	216±28	21±3	29±5
1	8±7	20±3	0*	12±6*	0*	0*	0*	190±3	0*	9±8*
5	0*	0*	0*	0*	0*	0*	0*	0*	0*	0*
*: Indicates toxic effects

 

Applicant's summary and conclusion

Conclusions:

p-Nitrobenzonitrile did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1538 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of p-Nitrobenzonitrile. The study was performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1538 and TA1537 in the presence and absence of S9 metabolic activation system using the preincubation protocol. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mg/plate by the preincubation for 15 mins. The plates were incubated for 70 hrs in dark and observed for the presence of colonies. Concurrent solvent and positive control chemicals were also included in the study. p-Nitrobenzonitriledid not induce mutation inSalmonella typhimurium strains TA98, TA100, TA1535, TA1538 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.