Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse bone marrow micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
EC Number:
601-478-9
Cas Number:
117428-22-5
Molecular formula:
C18H16F3NO4
IUPAC Name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
Test material form:
solid
Details on test material:
Purity: 93.3% w/w

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Housing: Housed by sex with up to 5 per cage on mobile mouse cage racks
- Diet: ad libitum
- Water: ad libitum
- Age range when supplied: 5-6 weeks for phase-I and 4-7 weeks for phase-II

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Volume: 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An individual stock suspension of the test substance was prepared in corn oil for each group of animals. The positive control substance was prepared as a solution in physiological saline. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 20 mL/kg bw.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Single dose
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
3 200 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 65 mg/kg

Examinations

Tissues and cell types examined:
Tissue: Bone marrow
Cell type: Polychromatic erythrocytes
Details of tissue and slide preparation:
Slide preparation: The animals were killed by asphyxiation in halothane Ph. Eur. followed by cervical dislocation 24 and 48 hours after receiving a single oral dose of the test substance. 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 polychrome methylene blue and eosin using an automatic staining machine.

Slide analysis: Slides were coded and scored blind. Initially two thousand (2 x 1000) polychromatic erythrocytes were examined for the presence of micronuclei for each animal. Following completion of this analysis an additional 2000 polychromatic erythrocytes per female were analyzed for the presence of micronuclei at the 48 hour sampling time. 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 polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes.
Evaluation criteria:
The data have been interpreted as follows:
The assay is considered as negative, if there is no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above concurrent vehicle control incidences or if a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range.

The assay is considered positive, if there is a statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences.

An incidence of micronucleated polychromatic erythrocytes which is statically 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:
Analyses were carried out using the GLM procedure in SAS (1989). 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/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs: eyes diminished, signs of diarrhoea, subdued nature, hunched posture, ungroomed appearance and distended abdomen
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Mean Incidence of Micronucleated Polychromatic Erythrocytes/1000 Polychromatic Erythrocytes ± Standard Deviation (SD) At Two Sampling Times

Group Mean Animal Data – Males

Group

Treatment

Dose

Mean incidence of MPE/1000 PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

2.1 ± 0.8

1.7 ± 0.3

12

Cyclophosphamide

65 mg/kg

26.0 ± 9.4*

 

13

Test substance

2000 mg/kg

2.7 ± 1.2

 

14

Test substance

3200 mg/kg

2.4 ± 1.5

 

15

Test substance

5000 mg/kg

2.3 ± 2.2

2.3 ± 1.4

** Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.01 in the Student’s t-test (one-sided) on transformed data

All means based on 10 observations (2 counts of 1000 PE per animal).

Group Mean Animal Data – Females

Group

Treatment

Dose

Mean incidence of MPE/1000 PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

1.6 ± 0.8

0.8 ± 0.8

12

Cyclophosphamide

65 mg/kg

31.0 ± 12.4**

 

13

Test substance

2000 mg/kg

2.3 ± 0.8

 

14

Test substance

3200 mg/kg

2.9 ± 2.2

 

15

Test substance

5000 mg/kg

2.5 ± 1.4

2.7 ± 1.7*

* Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

** Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.01 in the Student’s t-test (one-sided) on transformed data

All means based on 10 observations (2 counts of 1000 PE per animal).

Table 2: Mean Percentage of Polychromatic Erythrocytes ± Standard Deviation (SD) At Two Sampling Times

Group Mean Animal Data – Males

Group

Treatment

Dose

Mean % of PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

42.6 ± 9.3

51.2 ± 8.5

12

Cyclophosphamide

65 mg/kg

34.9 ± 7.8

 

13

Test substance

2000 mg/kg

35.5 ± 6.8

 

14

Test substance

3200 mg/kg

36.7 ± 4.5

 

15

Test substance

5000 mg/kg

41.7 ± 7.9

38.7 ± 7.6*

* Statistically significant decrease in the percentage of polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

All mean based on 5 observations (1 count of 1000 erythrocytes per animal).

Group Mean Animal Data – Females

Group

Treatment

Dose

Mean % of PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

39.8 ± 8.8

50.4 ± 9.7

12

Cyclophosphamide

65 mg/kg

43.4 ± 10.1

 

13

Test substance

2000 mg/kg

30.5 ± 8.7*

 

14

Test substance

3200 mg/kg

40.5 ± 2.5

 

15

Test substance

5000 mg/kg

36.2 ± 9.4

48.5 ± 7.1

* Statistically significant decrease in the percentage of polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

All mean based on 5 observations (1 count of 1000 erythrocytes per animal).

Applicant's summary and conclusion

Conclusions:
The test substance was not clastogenic in the mouse bone marrow micronucleus test.
Executive summary:

The test substance has been evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of 5 male and 5 female mice at dose levels of 2000, 3200 and 5000 mg/kg. The latter dose level used represents the limit dose for the assay. Bone marrow samples were taken 24 and 48 hours after dosing.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over vehicle control values, were seen at the 24 hour sampling time in either male or female mice dosed with test substance or at the 48 hour sampling time in the male mice dosed with test substance.

Although a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control value, was seen at the 48 hour sampling time in the female mice dosed with test substance at 5000 mg/kg, extended analysis of a further 2000 polychromatic erythrocytes per female showed no such increase thus confirming that the small increase observed in the original counts is of no biological significance.

Comparison of the percentage of polychromatic erythrocytes showed no biologically significant differences between the vehicle control and test substance treated mice at any of the dose levels or either of the sampling times investigated.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the conditions of this test, the test substance was not clastogenic in the mouse bone marrow micronucleus test.