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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
begin: 2005-11-04; end: 2006-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was generated according to valid testing guidelines: OECD guidelines 475

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests, 1995
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: rats were obtained from Charles River UK Ltd, Margate, UK
- Age at study initiation: out-bred young adult
- Fasting period before study: Animals were not fasted prior to dosing.
- Housing: They were housed in groups of the same sex. Aspen wood chips were be used for bedding. Additionally, in order to enrich the
environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks.
- Diet (e.g. ad libitum): Diet (Special Diets Services Ltd, RM1.(E).SQC.) were provided ad libitum
- Water (e.g. ad libitum): Bottled water (public supply) were provided ad libitum.
- Acclimation period: Animals were acclimatised for at least 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%.
- Air changes (per hr): at least 15 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): Holding rooms were illuminated continuously by fluorescent light for 12 hours out of each 24 hour cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Dosing preparations were made by suspending antimony trioxide in 0.5% (w/v) Hydroxypropylmethylcellulose + 0.1% (w/v) aqueous polysorbate
(0.5% HPMC + 0.1% polysorbate).
Details on exposure:
Animals were dosed with the vehicle or test article for 21 consecutive days (approximately 24 hours apart). The positive control was given as a single
administration at 20 mg/kg, on the last day of dosing.
Duration of treatment / exposure:
Animals were dosed with vehicle or test article for twenty one consecutive days.
Frequency of treatment:
Animals were dosed with vehicle or test article once daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
6 male and 6 female per dose per day
Control animals:
yes, concurrent vehicle
Positive control(s):
The negative (vehicle) control was 0.5% HPMC + 0.1% polysorbate.
Cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) was freshly dissolved in physiological saline at 2 mg/mL to serve as the positive control at a
final dose of 20 mg/kg.

Examinations

Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately two hours prior to the scheduled sample time, animals were injected intraperitoneally with colchicine (dose volume 10 mL/kg) to give a final concentration of 2 mg/kg, in order to arrest dividing cells in metaphase for the chromosome aberration endpoint. Two hours prior to harvest is considered sufficient time to achieve this without affecting background micronucleus frequencies due to spindle affects.

Test article and vehicle treated rats were killed 24 hours after the final administration. CPA-treated rats were killed 24 hours after the single dose. Rats were killed by asphyxiation with carbon dioxide (subsequently ensured by cervical dislocation) in the same order as they were dosed.

Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shank.
One bone was used for metaphase processing, the other for micronucleus preparations.


DETAILS OF SLIDE PREPARATION:
Mitotic index analysis:
Slides from animals treated with vehicle or test article were examined, uncoded, for mitotic index (MI) or percentage of cells in mitosis, based on 1000 cells scored per animal.



METHOD OF ANALYSIS:
Mitotic index analysis:
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures.

Analysis of results – Metaphase analysis
Treatment of data

After completion of microscopic analysis, data were decoded. Aberrant cells were
categorised as follows:
1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells

The totals for category 2 in vehicle control groups were used to determine whether
the assay was acceptable or not. For each group, inter individual variation in the
proportion of aberrant cells was estimated by means of a heterogeneity chi-square
calculation.

The group mean for each group was calculated, and the frequency of cells with
aberrations (± standard deviation) determined.

The proportion of cells in category 2 for each treated group (males and females
separately and combined) were compared with the proportions in vehicle controls
by using a 2 x 2 chi-square test. Probability values of p ≤ 0.05 were accepted
as significant. The proportions of cells in categories 1 and 2 were examined in
relation to historical control ranges. A further statistical analysis (linear trend test)
was used to evaluate possible dose-response relationships.
Evaluation criteria:
Metaphase analysis:
A test article was considered as positive in this assay if:
1. a statistically significant increase in the proportion of cells with structural aberrations occurred at one or more concentration and/or sample time, and
2. the proportion of cells with structural aberrations at such data points exceeded the normal range.

Statistics:
Analysis of results - Micronucleus
The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity χ² test

Analysis of results – Metaphase analysis
The totals for category 2 in vehicle control groups were used to determine whether the assay was acceptable or not. For each group, inter individual variation in the proportion of aberrant cells was estimated by means of a heterogeneity chi-square calculation

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:
No clinical signs were observed in any control or test article treated groups.
Group mean body weight gains were reduced for test article treated animals as compared to concurrent vehicle controls (males and females) over the dosing period of the assay.

Mitotic index data did not indicate any test article related toxicity to the bone marrow.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that antimony trioxide did not induce chromosome aberrations or micronuclei in the bone marrow cells of male and female rats when
tested at doses of 250, 500 and 1000 mg/kg/day over a continuous 21-day dosing regime.