N'-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]BENZOHYDRAZIDE

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th March 2020 to 24th March 2020

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

N'-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]BENZOHYDRAZIDE

Cas Number:

1453473-00-1

IUPAC Name:

N'-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]BENZOHYDRAZIDE

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Lot No 19092707 and were prepared from 7 week old male Sprague Dawley rats that had been injected intraperitoneally with Phenobarbitol (PB) and 5,6-benzoflavone (BF). PB was injected intraperitoneally 4 times (Day 1: 30 mg/kg body weight & Day 2-4:30 mg/kg body weight). BF was injected intraperitoneally once on Day 3 (80 mg/kg body weight). The S9 was stored at -80 oC

- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept on an ice bath until use. S9-mix contained per 1 mL: MgCl2 8μmol, KCL 33μmol, 4μmol NADH, 5μmol NADPH, 5μmol glucose-6-phosphate in 100μmol sodium phosphate buffer pH 7.4;

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-determination test. Eight concentrations, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate were tested. The number of revertant colonies were counted. Cytotoxicty to bacteria by the test substance and precipitation of the test substance were examined. The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate. Six dose levels separated by a factor of 2 were used for the mutagenicity test.

Vehicle / solvent:

DMSO = dimethyl sulfoxide
Lot No. 009H1642

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: ~ 10 9 cells/mL
- Test substance added in top agar tubes

The test substance solution (0.1ml) was dispensed into a test tube followed by either 0.5 mL S9-mix (in the presence of metabolic activation) or 0.1 M phosphate buffer (in the absence of metabolic activation) and 0.1ml of each bacterial culture. The contents of each test tube was pre-incubated at 37oC for 20mins with shaking, mixed with 2.0ml of the top agar and evenly distributed onto the surface of minimal agar plates. The plates were incubated for 48 hours.

After this period, all plates were assessed for the numbers of revertant colonies and were observed for the presence or absence of cytotoxicty to bacteria by the test substance under a stereomicroscope.

Evaluation criteria:

When the test substance induces a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the negative control and the increase is reproducible, the test substance is judged positive in this test system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test substance was concluded to be non-mutagenic.

Executive summary:

This test was designed to assess the mutagenic potential of N'-(1,1,1-trifluoropropan-2-ylidene)benzohydrazide using a bacterial reverse mutation test system using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli WP2uvrA. Throughout the tests N'-(1,1,1-trifluoropropan-2-ylidene)benzohydrazide did not induce any increases in the number of revertant colonies to at least twice as many as that of the negative control for any of the bacterial strains. 

The test substance was concluded to be non-mutagenic under the conditions of the test.