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:
14 Jul - 22 Oct 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 2000
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
adopted in 1998
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus test in mice

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: Hsd/Win:NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 27 - 32 g (females), 37 - 43 g (males)
- Assigned to test groups randomly: yes
- Housing: individual in type I cages with bedding of soft wood granules, type BK 8/15
- Diet: 3883, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5
- Humidity (%): 40 - 70%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Cremophor
- Lot/batch no. (if required): 398261/1 34099
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a microdismembrator for 5 minutes, and formed turbid brownish suspensions. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.
The positive control cyclophosphamide was dissolved in deionized water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg bw
Duration of treatment / exposure:
not applicable
Frequency of treatment:
two administrations separated by 24 h
Post exposure period:
24 h after last treatment
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw (total dose)
Remarks:
males
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
males and females
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
males and females
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
females
No. of animals per sex per dose:
5
additional 5 replacement animals at the top dose of 500 (males) or 1000 mg/kg bw (females)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control: cyclophosphamide is a proven cytostatic agent and known clastogen with bifunctional alkylation action
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow erythroblasts
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A pilot toxicity test was performed to determine a suitable dose level for the main study, and whether differences in effects between males and females are observed. Initially, 3 males and 3 females were treated with 1000 mg/kg bw intraeperitoneally with two injection separated by 24 h. Since all males but no female died 3 males recieved 500 mg/kg bw and 3 females were treated with 2000 mg/kg bw. No male died during the treatment but all females died before prior to the second administration. Finally 3 females were treated with 1500 mg/kg. Since 2 of these females died prior to the second treatment, no second treatment was performed for the last one. Thus, substantial differences between the sexes were demonstrated and 500 mg/kg bw was chosen as maximum tolerable dose (MTD) for males and 1000 mg/kg bw for females.

TREATMENT AND SAMPLING TIMES:
Males received 125, 250 and 500 mg/kg bw and females were administered 250, 500 and 1000 mg/kg bw intraperitoneally with two injections separated by 24 h. Males and females of the positive control group were treated with a single injection of 20 mg/kg bw cyclophosphamide. The animals were sacrificed 24 h after the last treatment and femoral marrow was prepared.

DETAILS OF SLIDE PREPARATION:
At least one intact femur was prepared from each sacrificed animal. Following separation from muscular tissue, the femur was opened and bone marrow was flushed with fetal calf serum. After centrifugation a small amount of the bone marrow suspension was spread on a slide and dried overnight. The smears were stained automatically and then "destained" with methanol, rinsed with deionized water, and left to dry. The slides were immersed with xylene for 10 min and smears were covered with covering agent and a cover glass. Based on findings concerning clinical signs during the study, it was concluded, that there are no relevant differences in toxicity between male and female mice. For that reason, slides were prepared for both sexes but only the slides of the males were assessed.

METHOD OF ANALYSIS:
The slides were examined using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal was scored. To establish the ratio of polychromatic to normochromatic erythrocytes the number of normochromatic erythrocytes per 2000 polychromatic erythrocytes was determined.
Evaluation criteria:
A test was considered positive if there was for at least one sex a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the respective negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes for any sex. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.
Statistics:
The highest mean per sex and treatment group and the respective positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the respective negative control using the one-sided chi² test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (Is ranges) were calculated for all the means.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs after two intraperitoneal administrations of 125, 250 and 500 mg/kg bw; relevant change of the ratio of polychromatic to normochromatic erythrocytes at 500 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Sex:
female
Genotoxicity:
not determined
Toxicity:
yes
Remarks:
clinical signs after two intraperitoneal administrations of 250, 500 and 1000 mg/kg bw/day; death of 8/10 animals at 1000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 1000 mg/kg bw (males), 1000, 1500 and 2000 mg/kg bw (females)
- Clinical signs of toxicity in test animals: All male animals (3/3) died at 1000 mg/kg bw but survived following two intraperitoneal injections of 500 mg/kg bw. All females (3/3) died at 2000 mg/kg bw and 2/3 females died at 1500 mg/kg bw. All females (3/3) survived after two intraperitoneal injections of 1000 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no significant increases in the frequency of micronuclei in any of the treatment groups in male and female mice compared with their respective control groups.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE in males was altered by treatment with the test substance (see table 1).
- Appropriateness of dose levels and route: Clinical signs (apathy, roughened fur, loss of weight, staggering gait, spasm, twitching, periodically stretching of body, reduced body temperature, and difficulty in breathing) were observed in 5/5 males in all treatment groups. Accordingly, clinical signs (apathy, roughened fur, loss of weight, spasm, periodically stretching of body, difficulty in breathing and slitted eyes) were observed in 5/5 females in all treatment groups. Based on the systemic effects observed, the dose levels are considered to be appropriate. It was concluded, that there are no relevant differences in toxicity between male and female mice. For that reason only the slides of males were evaluated. However, slides were prepared for females as well and archived without assessment.

Any other information on results incl. tables

Table 1. Results of the in vivo micronucleus assay in male mice

Exposure group

Number of animals

Dose [mg/kg bw]

Number of NCE per 2000 PCE

Micronucleated NCE per 2000 NCE

Micronucleated PCE per 2000 PCE

Vehicle control (cremophor)

5

0

1457 ± 768

4.7 ± 2.1

4.6 ± 3.2

Positive control (cyclophosphamide)

5

20

1241 ± 504

1.2 ± 1.6

16.6 ± 3.8

Test substance

5

125

1426 ± 560

5.7 ± 3.9

4.6 ± 1.1

Test substance

5

250

1472 ± 460

2.5 ± 1.0

4.6 ± 2.9

Test substance

5

500

2147 ± 1175#

4.7 ± 2.6

4.8 ± 3.9

# considered biologically relevant

Applicant's summary and conclusion