Registration Dossier

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:
key study
Study period:
12 Nov. 1997 to 12 Feb. 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Method and EU method in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Range finding

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 51-58 days
- Weight at study initiation: 31-36 males; 24-26 females
- Assigned to test groups randomly: [no/yes, under following basis: ] random
- Fasting period before study:
- Housing: stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess tap water
- Acclimation period: 22 days

first study

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 35-42 days
- Weight at study initiation: 25-30 males; 21-26 females
- Assigned to test groups randomly: [no/yes, under following basis: ] random
- Fasting period before study:
- Housing: stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess tap water
- Acclimation period: 6 days

second study

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 34-41 days
- Weight at study initiation: 24-30 males; 20-26 females
- Assigned to test groups randomly: [no/yes, under following basis: ] random
- Fasting period before study:
- Housing: stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess tap water
- Acclimation period: 5 days

third study

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 36-43 days
- Weight at study initiation: 25-31 males; 19-27 females
- Assigned to test groups randomly: [no/yes, under following basis: ] random
- Fasting period before study:
- Housing: stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess tap water
- Acclimation period: 7 days

All studies

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 52-69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/24

IN-LIFE DATES: From: 12 Nov. 1997 To: 12 Feb. 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
1%(w/v) methyl cellulose
Details on exposure:
range finder: 2000 mg/kg/day
first study Doses of test material: 500, 1000 and 2000 mg/kg were selected based on a range finding toxicity study.
second study: doses 500, 1000, 1500,1750 and 2000 mg/kg were selected based on a fact all animals died in first study at top dose before sampling could occur.
third study: doses: 500, 1000, 1500 mg/kg due to 2 higer doses being to toxic.
Duration of treatment / exposure:
The mice were dosed once daily for 2 consecutive days and one group of mice from each dose level) were killed 24 hours following treatment and a second group were killed after 48 hours. In first study all mice dosed at higher group either died or were killed before the 48 hour sampling could be completed.
Frequency of treatment:
Groups, each of five mice, were dosed once daily for 2 days only via the oral gavage route with test material.
Post exposure period:
One group of mice from each dose level were killed at 24 hours following treatment and a second group were killed after 48 hours. All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
all three main studies
Remarks:
Doses / Concentrations:
1000mg/kg
Basis:
nominal conc.
all three main studies
Remarks:
Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.
all three main studies
Remarks:
Doses / Concentrations:
1750 mg/kg
Basis:
nominal conc.
first study only
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
nominal conc.
range finder, first and second study
No. of animals per sex per dose:
range finder
Male: 2000 mg/kg; No. of animals: 3; Sacrifice time: 4 days observation Female: 2000 mg/kg; No. of animals: 3; Sacrifice time: 4 days observation
first study
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
and 48 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
first study
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
and 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours

second study
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 1500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 1500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
fMale: 1750 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 1750 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours

third study
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Male: 1500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Female: 1500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours and 48 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
A positve control group of five animals male and female were dosed at 80 mg/kg using cyclophosphamide and terminated 24 hours.
Two groups of five animals male and female were used as a vehicle control group with one group terminated 24 hours and the second 48 hours after dosing.

Examinations

Tissues and cell types examined:
Bone marrow from the femurs of each animal used for smears. Cell types examined for the presence of micronuclei in Polychromatic (PCE) and normochromatic (NCE) erythrocytes.
Details of tissue and slide preparation:
Immediately following termination, bone marrow smears were prepared from sections of the femurs from each animal and stained with May-Griinwald/Giemsa.Stained bone marrow smears were coded and examined blind using light microscopy at xlOOO magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE- blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of rnicronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group
Statistics:
Group means and standard deviations for the PCE/NCE ratios were calculated for the data. The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
1750 and 2000 mg/kg
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Range finding study: The test material showed no only minimal clinical signs and the top dose was slected for the main study. Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. The maximum tolerated dose (MTD) of the test material, 2000 mgkg, was selected for use in the main study.

first study:

All animals at top dose died or were a sacrifised prior to sampling. Therefore a second study was conducted.

second study:

Three animals in the top three doses exhibited clinical signs including lethargy, prostration,eye closure and abnormal breathing. One animal died receiving the 1500 mg/kg/day and several animals from the higher 2 doses ( 1750 and 2000 mg/kg) died or were killed. These 2 higher dose groups were not included in the slides preprared. However, due to technical reasons with the staining the 48 hour sampling time treatments has to be repeated.

third study:

Repeat at three lower doses. slides from all dose groups were analyzed.
No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. All doses exhibited group mean PCE/NCE ratios and frequencies of micronucleated PCE which were simialr to the values of the vehicle groups for both sampling times. These frequencies fell within historical control range.

 The positive control produced a marked increase in the frequency of micronucleated polychromatic erythrocytes and were considered acceptable and valid.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test up to 1500 mg/kg/day, a dose at which clinical signs and limited mortalities were seen. The test material was considered to be non-genotoxic under the conditions of the test.