Antimony

Genetic toxicity: in vivo

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:

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 474

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests, 1995

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Diantimony trioxide

EC Number:

215-175-0

EC Name:

Diantimony trioxide

Cas Number:

1309-64-4

Molecular formula:

Sb2O3

IUPAC Name:

dioxodistiboxane

Test material form:

not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
250 mg/kg
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.

Doses / concentrations 3

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):

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 - Micronucleus:
Treatment of data
After completion of microscopic analysis and decoding of the data, the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes
(NCE) (expressed as %PCE) for each animal and the mean for each group was calculated. The individual and group mean frequency of micronucleated PCE ± standard deviation (%MNPCE) were also determined.
%PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.
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.
The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2
contingency table to determine χ². Probability values of P≤0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Evaluation criteria:

Micronucleus:
The data were evaluated as to whether exposure to the test article was associated with:
1. a statistically significant increase in the frequency of micronucleated PCE occurring at one or more dose levels.
2. an incidence and distribution of micronucleated PCE at such a point that exceeded the laboratory’s historical vehicle control data.
3. a dose-response trend in the proportion of micronucleated PCE (where more than two dose levels were analysed).

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 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.