Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep 1995 to 22 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
Tif:MAGf
Remarks:
(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 24 - 38 g
- Housing: Housed individually or 2 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5-21.0
- Humidity (%): : 44-76
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
26 Sep 1995 to 22 Nov 1995

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Bistilled water
- Amount of vehicle: 10 mL/kg
Frequency of treatment:
Single dose
Post exposure period:
16, 24 and 48h. An overview of the terminations times for the different treatment and control groups can be found in Table 1 in 'Any other information on materials and methods incl. tables'.
Doses / concentrationsopen allclose all
Dose / conc.:
312.5 mg/kg bw/day (actual dose received)
Dose / conc.:
625 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Vehicle control: 15 animals/sex
Positive control: 5 animals/sex
312.5 and 625 mg/kg bw: 5 animals/sex
1000 mg/kg bw: 15 males and 5 females (+3 reserve males)
1250 mg/kg bw: 10 females (+3 reserve females)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral (gavage)
- Doses / concentrations: 64 mg/kg

Examinations

Tissues and cell types examined:
Femoral bone marrow / polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary MTD study was performed in mice by the fixed dose procedure at dose levels up to 2000 mg/kg, to determine the highest dose level producing significant toxicity but not death. A solubility test was conducted to determine the highest applicable dose level.

DETAILS OF SLIDE PREPARATION: Bone marrows were harvested from the femurs with foetal calf serum. The bone marrow suspension was centrifuged and the cells re-suspended in foetal calf serum and smears made. The slides were air-dried and stained with May-Grűnwald/ Giemsa solution.

METHOD OF ANALYSIS: Slides were coded and scored blind. Slides of 5 animals/ sex/ dose, showing good differentiation between mature and polychromatic erythrocytes were scored. For each animal the ratio of polychromatic to normochromatic erythrocytes was determined and 2000 polychromatic erythrocytes were scored for micronuclei.
Evaluation criteria:
- Negative effect – if there was no statistically significant difference (Chi-Square ≤ 3.84; p ≥ 0.05) between the mean number of micronucleated PCEs in the groups treated with the test substance and that of the respective negative control and the former did not exceed the range accepted for the negative control (≤ 0.20%).
- Positive Effect - if, in any group with the test substance, the mean number of micronucleated PCEs exceeds the value of 0.20% and if there was a statistically significant difference (Chi-Square > 3.84; p < 0.05) of the number of micronucleated PCEs in comparison with the negative control.
Statistics:
The significance of differences was assessed by the Chi-Square-Test (p<0.5).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0 - 2000 mg/kg bw
- Solubility: Bidistilled water was found to be the most appropriate vehicle giving a suspension at the highest dose of 3750 mg/kg.
- Clinical signs of toxicity in test animals: Both animals treated with 2000 mg/kg died within 1-3 hours of treatment. Animals dosed with 1250 mg/kg showed clinical signs. Clinical signs were also recorded at the lower doses. In the confirmatory experiment both animals survived but showed clinical signs. 1250 mg/ was selected as the high dose for the micronucleus test.


RESULTS OF DEFINITIVE STUDY
- Toxicity: The dose level of 1250mg/kg bw caused death in 7/13 females and this dose was reduced to 1000 mg/kg. Treatment at 1000mg/kg bw produced signs of toxicity including lethargy, hunched posture and ataxia. Some animals treated at 625mg/kg bw also showed reduced locomotor activity.
- Ratio of PCE/NCE: The ratios of polychromatic to normochromatic erythrocytes after treatment with the test substance indicated no effects on erythropoiesis at any dose level (Table 1 in 'Any other information on results, incl. tables').

Any other information on results incl. tables

Table 1. Percentage of MNPCE (PCE/NCE ratio) at different preparation times

Treatment

Dose

 

% MNPCE (PCE/NCE ratio)

 

16 hours

24 hours

48 hours

Vehicle Control

10 mL/kg

males

females

combined

0.03 (0.68)

0.03 (1.27)

0.03 (0.98)

0.07 (0.63)

0.02 (1.20)

0.05 (0.92)

0.04 (0.63)

0.08 (1.02)

0.06 (0.83)

test substance1

1000 mg/kg

males

females

combined

0.04 (0.74)

0.06 (0.91)

0.05 (0.83)

0.09 (0.87)

0.05 (0.75)

test substance

2

1250 mg/kg

female

-

0.04 (0.81)

0.02 (1.16)

test substance

625 mg/kg

males

females

combined

-

0.01 (0.68)

0.01 (0.88)

0.01 (0.78)

-

 

test substance

312.5 mg/kg

males

females

combined

-

0.02 (0.84)

0.00 (0.86)

0.01 (0.85)

-

Positive control

64 mg/kg

males

females

combined

 

3.28* (0.91)

1.10* (0.95)

2.19* (0.93)

 

* p < 0.05 (Chi-Square Test)

10000 PCEs counted per group

1Due to symptoms of toxicity the dose level was reduced to 1000 mg/kg.

2Females for 24 and 48 hours sampling were treated with 1250 mg/kg, which proved highly toxic.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, performed accoring to OECD 474 under GLP, the test substance was not clastogenic or aneugenic in the in vivo mouse bone marrow micronucleus test.
Executive summary:

In an in vivo bone marrow micronucleus assay, performed accoring to OECD 474 under GLP, five male and five female Tif:MAGf young adult mice per group were given a single oral dose of 1000 (or 1250 females 24 and 48 hours), 625.0 or 312.5 mg/kg bw test substance. In addition, five male and five female mice were dosed with10 mL/kg bw bidistilled water (vehicle control) and five male and five female mice with 64 mg/kg bw cyclophosphamide (positive control). From the high dose and negative control groups the animals were killed 16, 24 or 48 hours after dosing. From the intermediate and low dose and positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

The highest dose of 1250 mg/kg applied to females (24 and 48 hours) caused significant toxicity, manifested as premature death of several animals 4 to 5 hours after treatment. The high dose of 1000 mg/kg applied to males (all sampling times) and to females (16 hour sampling time) caused clinical signs in several animals. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was observed when compared to the respective negative control group. The test system positive control, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes at the 24 hour sampling time.

In conclusion, under the conditions of the test, the test substance was neither clastogenic nor aneugenic in the in vivo mouse bone marrow micronucleus test.