2-fluoro-6-trifluoromethylpyridine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2A and S2B

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-100-3

EC Name:

-

Cas Number:

94239-04-0

Molecular formula:

C6H3NF4

IUPAC Name:

2-fluoro-6-(trifluoromethyl)pyridine

Details on test material:

- Purity: 99.54%

Specific details on test material used for the study:

Substance ID: F6TF
Lot #: P2
Purity: 99.54%

Test animals

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 287 - 383 g
- Housing: housed on mobile rat cage racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light followed by 12 hours darkness

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

dried filtered air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2 tiered nose-only exposure chamber
- Source and rate of air: dry filtered air
- System of generating vapour: atmospheres were generated by metering appropriate amounts of liquid test substance on to the top of a water bath heated condenser column (45°C) using a Hamilton Microlab pump. A counterflow of clean dry compressed air (25 L/min) was used to pass the resultant vapour to a 2 tiered nose-only exposure chamber

TEST ATMOSPHERE
- Brief description of analytical method used: Test atmospheres were sampled by trapping the test substance on Carbotrap C 20/40 mesh in a desorption tube, followed by thermal desorption and quantification using gas chromatography.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

4 hours

Frequency of treatment:

single

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 ppm

Doses / concentrations 2

Dose / conc.:

400 ppm

Doses / concentrations 3

Dose / conc.:

800 ppm

No. of animals per sex per dose:

200 and 400 ppm - 5/dose
800 ppm - 10/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- positive control: cyclophosphamide

- Route of administration: The positive control substance was prepared as a solution in double deionised water and was dosed at a volume of l0 mL/kg bodyweight. A group of rats was given a single oral dose of the positive control substance to coincide with the end of the inhalation exposure period.
- Doses / concentrations: l0 mL/kg bodyweight

Examinations

Tissues and cell types examined:

immature erythrocytes from rat bone marrow taken from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest concentration represents the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities observed in a previous rat toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Groups of male rats were exposed by the inhalation route for a single 4 hour period to either dried filtered air or the test substance at nominal concentrations of 200, 400 and 800 ppm. Bone marrow samples were taken approximately 24 hours after the end of the exposure period for the vehicle control and the test substance at dose levels of 800, 400 and 200 ppm and 48 hours after the end of the exposure period for the vehicle control and the test substance at 800ppm. Bone marrow samples were taken 24 after dosing for the positive control.

DETAILS OF SLIDE PREPARATION: Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide. The slides were allowed to air dry and were stained with acridine orange.

METHOD OF ANALYSIS: Slides were coded and scored blind. Two thousand immature erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of immature to mature erythrocytes in a sample of 1000 erythrocytes.

Evaluation criteria:

The data have been interpreted as follows:
a) No statistically significant increase in the incidence of micronucleated immature erythrocytes above concurrent vehicle control incidences -NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated immature erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range - NEGATIVE.
c) A statistically and biological significant increase in the incidence of micronucleated immature erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated immature erythrocytes which is statistically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.

Statistics:

The data for the incidence of micronucleated immature erythrocytes were transformed using a square root transformation, prior to analysis. The data for the percentages of immature erythrocytes were transformed using the double arcsine transformation of Freeman and Tukey, prior to analysis.
Analyses were carried out using the MIXED procedure in SAS. Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Clinical signs: stains around nose, decreased activity, tiptoe gait, abnormal breathing, reduced stability and reduced response to sound

Mortality: one male found dead before sampling time, macroscopic abnormalities included mesenteric/renal lymph nodes, mottled/pale areas on liver, pelvic dilatation of the kidney, distended bladder, gas-filled caecum and a diaphragmatic hernia on the liver

Genotoxicity: No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes were observed. No statistically significant differences in the percentage of immature erythrocytes between vehicle control and test substance were observed

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test substance is neither clastogenic nor aneugenic in the rat bone marrow micronucleus test.

Executive summary:

An inhalation micronucleus study was conducted in rats to determine whether the test material induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow. In this study, groups of male rats were placed in exposure chambers and exposed single nose-only to target concentrations of 200, 400, and 800 ppm for 4 hours. Bone marrow smears were prepared approximately 24 and 48 hours after exposure and 2000 immature erythrocytes per animal were evaluated for the presence of micronuclei. 

 

No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes were observed. No statistically significant differences in the percentage of immature erythrocytes between vehicle control and test substance were observed. Under the conditions of the test, the test substance is neither clastogenic nor aneugenic in the rat bone marrow micronucleus test.