2-Pyridinecarbonitrile, 5-amino-3-(trifluoromethyl)-

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:

From 2015-12-10 to 2016-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile

EC Number:

815-137-9

Cas Number:

573762-62-6

Molecular formula:

C7H4F3N3

IUPAC Name:

5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile

Details on test material:

- Appearence : Slightly yellowish brown fine powder
- CAS Number: 573762-62-6
- Molecular formula: C7H4F3N3
- Molecular weight: 187.12

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: M15BD0674
- Expiration date of the lot/batch: 2017-02-10
- Physical Description: Slightly yellowish brown fine powder
- Purity: 99.9% (w/w)
- Purity test date: 2015-09-11

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the vehicle: not indicated/A solubility test was performed based on visual assessment. Analysis of test item in vehicle for stability were not performed


OTHER SPECIFICS
- Correction factor: 1
- Test item concentrations were used within 2 hours of preparation.

Method

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment, respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% (v/v) S9-mix (0.5 mL) in the first experiment and 10% S9-mix (v/v) (1.0 mL) in the second experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): - Each S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

- Dose-range finding test: Selection of an adequate range of doses was based on a dose range finding test with tester strain TA100 with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation experiment was 5000 µg/plate.

- The above mentioned dose range finding study with tester strain TA100 is reported as a part of the first mutation experiment. In the second part of the first mutation experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and TA102. Five concentrations, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
- In a second mutation experiment, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. Six concentrations, 275, 492, 878, 1568, 2800 and 5000 µg/plate were tested in triplicate

- The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; 5 μg/plate (TA1535)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix; 10 μg/plate (TA98)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 μg/plate (TA100)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide

Remarks:

Without S9-mix; 250 μg/plate (TA102)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix; 2.5 μg/plate (TA1535 at 5 and 10% S9; TA1537 at 5% S9), 5 μg/plate (TA1537 at 10% S9), 1 μg/plate (TA98 at 5 and 10% S9; TA100 at 5% S9), 2 μg/plate (TA100 at 10% S9), 10 μ g/plate (TA102 at 5 and 10% S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added; in agar (plate incorporation)
- Method: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar:
- 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains,
- 0.1 mL to 0.3 mL of a dilution of the test item in DMSO or Milli-Q water and
- either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. . After this period revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 µg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 µg/plate was selected as the maximum final concentration for the dose range finding test

RANGE-FINDING/SCREENING STUDIES: The test item was tested in tester strain TA100 at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment with the tester strains, TA1535, TA1537, TA98 and TA102 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, the test item was tested up to the dose level of 5000 µg/plate in strains TA1535, TA1537, TA98, TA100 and TA102


STUDY RESULTS
- Precipitate:
- Mutation Experiment 1: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
- Mutation Experiment 2: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

- Signs of toxicity:
- Mutation Experiment 1: See section Overall remarks, attachments for the data tables with results.
- Mutation Experiment 2: See section Overall remarks, attachments for the data tables with results.

Mutagenicity:
- Mutation Experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
- Mutation Experiment 2: No increase in the number of revertants was observed upon treatment with he test item under all conditions tested.

- Discussion:
The negative control values were within the laboratory historical control data ranges, except the response for TA100 (second experiment, absence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (59 revertant colonies) when compared against relevant historical control data (64 relevant colonies), the validity of the test was considered to be not affected.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA102 (first experiment, absence of S9-mix). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was 2.1 times greater than the concurrent solvent control value, this deviation in the mean plate count of the positive control had no effect on the validity of the test results.

Applicant's summary and conclusion

Conclusions:

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