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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
Pharmaco LSR Toxicology Services Worldwide, Eye Suffolk,England
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6 DCPTFMA = RPA099325 = MB039608
- Analytical purity: 94.3%
- Lot/batch No.: OPE7/PCL
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK), Margate, Kent, England
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 14.8 - 22.3 g
- Assigned to test groups randomly: yes, under following basis: using computer-generated random numbers
- Fasting period before study: no
- Housing: single sex groups of 2 or five
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-60
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The test material was found to be freely miscible with corn oil.

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was warmed to approximately 37°C before use to facilitate formulation. The test material was found to be freely miscible with corn oil. Consequently, dosing solutions were freshly prepared in corn oil on the day of dosing; each concentration being individually formulated.

Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
once
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
other: nominal dose
No. of animals per sex per dose:
15 in control group and 2000 mg/kg
5 in 500 and 1000 mg/kg
Control animals:
yes, concurrent vehicle
Positive control(s):
Chlorambucil
- Route of administration: oral, gavage
- Doses / concentrations: 30 mg/kg in aqueous 10% ethanol

Examinations

Tissues and cell types examined:
polychromatic erythrocytes from bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Single drops of the cell suspension were transferred to clean, dry slides, three smears prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using 5% Giemsa stain (prepared in Sorensen's buffer: pH 6.8) for 20 minutes. After staining, slides were washed in buffer, allowed to air-dry, cleared for five minutes in xylene, and made permanent using DPX mountant.

METHOD OF ANALYSIS: examination under light mircoscope


Statistics:
The frequencies of micronucleated cells per 1000 polychromatic erythrocytes scored were subjected to statistical analysis by the Mann-Whitney U procedure (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1.
Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24,48 or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 50, 100, 200, 500, 2000, 5000 mg/kg
- Clinical signs of toxicity in test animals: Initially, a group of mice was dosed at 500 mg/kg; these mice showed hunched posture (transient) with an hour of dosing. Further groups of mice were then dosed at 50, 100 and 200 mg/kg; these mice showed no adverse reactions to treatment. On the next day, two further groups of mice were dosed at 2000 and 5000 mg/kg. Three mice dosed at 2000 mg/kg showed adverse reactions to treatment including piloerection (3 mice), underactivity (3) and hunched posture (2). One mouse dosed at 5000 mg/kg was found dead approximately 1.5 hours after dosing and the other three mice were found dead approximately 18.5 hours after dosing; they had shown underactivity (3 mice) and hunched posture (1) 1.5 hours after dosing.
- Evidence of cytotoxicity in tissue analyzed: There was some evidence of toxicity of 2,6 DCPTFMA to the bone marrow (as evidenced by depression in bone marrow proliferation) in mice dosed at 2000 and 5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically or statistically significant differences in the frequencies of micronucleated polychromatic cells were seen between sexes, in any group (p>0.05 in each case).
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to mature erythrocytes for all groups treated with 2,6 DCPTFMA were closely similar to corresponding vehicle control group values at each sacrifice time.

Any other information on results incl. tables

Data summary and statistical analysis

Sacrifice Time

Group

Treatment (mg/kg)

MnP cells / 1000

Statistically Analysis

P:M

Mean ± SD

Range

M vs F

Treated vs Control

 

24 hours

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

Vehicle (-)

 

 

M

1.4 ± 1.7

0.0-4.0

ns

 

-

0.6

F

0.0 ± 0.0

0.0-0.0

-

0.6

Group

0.7 ± 1.3

0.0-4.0

-

-

0.6

2

 

 

Test material (500)

 

M

1.1 ± 1.0

0.0-2.0

ns

 

-

0.7

F

0.4 ± 0.5

0.0-1.0

-

0.7

Group

0.8 ± 0.9

0.0-2.0

-

ns

0.7

3

 

 

Test material (1000)

 

M

1.6 ± 1.7

0.0-4.0

ns

 

-

0.6

F

1.4 ± 0.9

0.0-2.0

-

0.7

Group

1.5 ± 1.3

0.0-4.0

-

ns

0.6

4

 

 

Test material (2000)

 

M

1.5 ± 2.1

0.0-3.9

ns

 

-

0.6

F

0.4 ± 0.9

0.0-2.0

-

0.7

Group

1.0 ± 1.6

0.0-3.9

-

ns

0.7

5

 

 

CBC (30)

 

 

M

62.4 ± 10.6

48.9-74.1

ns

 

-

0.6

F

51.8 ± 22.7

33.2-85.0

-

0.6

Group

57.1 ± 17.6

33.2-85.0

-

**

0.6

48 hours

 

 

 

 

 

1

 

 

Vehicle (-)

 

 

M

0.2 ± 0.4

0.0-1.0

ns

 

-

0.6

F

1.0 ± 1.2

0.0-2.0

-

0.6

Group

0.6 ± 0.9

0.0-2.0

-

-

0.6

4

 

 

Test material (2000)

 

M

0.2 ± 0.4

0.0-1.0

ns

 

-

0.7

F

0.7 ± 1.2

0.0-2.0

-

0.6

Group

0.4 ± 0.7

0.0-2.0

-

ns

0.6

72 hours

 

 

 

 

 

1

 

 

Vehicle (-)

 

 

M

0.3 ± 0.5

0.0-1.0

ns

 

-

0.7

F

0.0 ± 0.0

0.0-0.0

-

0.7

Group

0.1 ± 0.3

0.0-1.0

-

ns

0.7

4

 

 

Test material (2000)

 

M

0.4 ± 0.5

0.0-1.0

ns

 

-

0.6

F

0.3 ± 0.5

0.0-1.0

-

0.6

Group

0.3 ± 0.5

0.0-1.0

-

ns

0.6

MnP: Micronucleated polychromatic erythrocytes

P:M: Ratio of polychromatic to mature erythrocytes

Vehicle: corn oil

Test material: 2,6 DCPTFMA

CBC: Chlorambucil

statistical analysis: statistical significance of the frequency of micronucleated polychromatic erythrocytes (Males vs Females, and Treated vs Vehicle Controls): ns: not significant, p>0.05; ** highly significant, p<0.01

Applicant's summary and conclusion

Conclusions:
It is concluded that, under the conditions of the test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of 2,6-dichloro-4-trifluoromethylaniline.