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Toxicological information

Genetic toxicity: in vivo

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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:
15 February 1989 - 10 March 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to internationally accepted guidelines and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sodium chlorate was received from Kerr McGee Chemical
Apppearance: fine white crystalline solid
Storage: at ambient temperature in an amber glass screw-capped jar.
Purity: 99.9% pure with 1.9 pmm chromium content.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK) , Margate, Kent, England.
- Age at study initiation: 4-5 weeks
- Weight at study initiation: preliminary study: 27-30 g for males and 19-24 g for females. main study: 23-28 g for males and 19-25 g for females.
- Assigned to test groups randomly: Yes, under following basis: All mice were identified by ear-notch number codes, numbers being assigned using a set of computer-generated random numbers. Cage labels, identifying the occupants by experiment, animal numbers, sex and treatment group, were attached to all cages.
- Fasting period before study: no data
- Housing: single-sex groups of two or five in high density polypropylene cages with stainless steel tops.
- Diet (e.g. ad libitum): Laboratory animal diet LAD 1 (Manea, Cambridgeshire, England) was fed ad libitum throughout the study.
- Water (e.g. ad libitum): Drinking water was supplied to each cage via a polythene bottle and sipper-tube.
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 15 February 1989 - 10 March 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: dustilled water
- Justification for choice of solvent/vehicle: sodium chlorate is soluble in water
- Concentration of test material in vehicle: 20 g/L, 100 g/L, 5000 g/L.
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity:not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was found to be soluble in distilled water to a concentration in excess of 700 mg/ml. Consequently, dosing solutions were freshly prepared in distilled water on the day of dosing; the solution of maximum concentration was initially prepared and the lower concentrations required were prepared from this by serial dilution in distilled water.
Duration of treatment / exposure:
Examination 24, 48 and 72h after administration
Frequency of treatment:
Single dose
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 1000 and 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorambucil (Sigma Chemical Company)
- Justification for choice of positive control(s): know positive chemical
- Route of administration: oral
- Doses / concentrations: A suspension of chlorambucil (Sigma Chemical Company) in aqueous 10% ethanol was prepared immediately prior to dosing, for use as a positive control. The positive control agent, chlorambucil, was administered orally, at a dosage of 30 mg/kg.

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary toxixty test was performed.
The toxicity of sodium chlorate to dividing bone marrow erythrocytes was examined : slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group. The highest level tested was the maximum stated in the Study Protocol. Dosing was by the oral route, on one occasion and at a volume-dosage of 10 ml/kg.
animals were killed 72 hours after treatment. Four groups with 2/animals per sex were dosed with 625, 1250, 2500 or 5000 mg/kg bw sodium chlorate.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Preparations of the test compound or dosing vehicle (distilled water) were administered once, by the oral route, to groups of male and female mice. The volume-dosage was 10 ml/kg. The positive control agent, chlorambucil, was administered orally, at a dosage of 30 mg/kg in aqueous 10% ethanol.Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from groups 1 to 4 were kiled 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the
cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique. When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.

METHOD OF ANALYSIS:
Preliminary toxicity test
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature until a minimum of 2000 of counted.

Main micronucleus test
One slide from each animal not subsequently involved erythrocytes, scoring continued the predominant cell type were was randomly coded by a person n the scoring o f the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias. Slides were examined as detailed for the preliminary toxicity test, but in addition each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the slides were decoded. The resultant data were used to calculate the number of micronucleated cells per 1000 erythrocytes. The ratio of polychromatic to mature cells was also determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.
OTHER: Animals were inspected daily throughout the acclimatisation period and the dosing period for signs o f ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded. In addition, the animals in the preliminary toxicity test were weighed immediately prior to dosing and daily thereafter until termination.
Evaluation criteria:
See above and below.
Statistics:
Using the frequency of micronucleated cells per 1000 polychromatic erythrocytes scored, the data were subjected to statistical analysis by the Mann-Whitney procedure. (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1. Data from males and females within each group were compared. As there was no significant difference within each group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
No indication of bone marrow toxicity was noted in any group treated with sodium chlorate. However clinical signs at 2500 and 5000 mg/kg bw in the prelimininary test and at 5000 mg/kg bw in the main test demonstrated systemic exposure to the test item.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Evidence of toxicity of sodium chlorate was noted in both males dosed with sodium chlorate at 5000 mg/kg; signs included hunched posture, rapid respiration and piloerection on days 3 and 4 of the study. Hunched posture and piloerection on days 3 and 4 were also noted in one male dosed with sodium chlorate at 2500 mg/kg.
Bodyweights were recorded at dosing and daily there after until termination; incidences o f weight loss were noted, but these were small and not dose-related. The ratios o f polychromatic: mature erythrocytes as evidenced by depression in bone marrow proliferation) to the bone marrow of the treated mice. There was no evidence of toxicity of sodium chlorate. After consideration of these data, the highest sodium chlorate dosage selected for the main micronucleus test was 5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): No significant differences in the frequencies of micronucleated polychromatic cells were seen between sexes, in any group.
Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.5 for the vehicle control group, with a range of 0.0-2.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were closely similar: 0.5, 0.8 or 0.7, with ranges of 0.0-1.9, 0.0-3.0 and 0.0-2.8 respectively.
Among mice killed 48 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.4 for the vehicle control group, with a range of 0.0-1.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were 0.5, 0.5 or 0.4, with ranges of 0.0-1.8, 0.0-1.0 and 0.0-1.0 respectively.
Among mice killed 72 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 0.2 for the vehicle control group, with a range of 0.0-1.0. Corresponding values for animals given sodium chlorate at 200, 1000 or 5000 mg/kg were 0.6, 0.6 or 1.0, with ranges of 0.0-1.9, 0.0-2.0 and 0.0-2.0 respectively.
Thus, mean values for sodium chlorate treated groups were closely similar to mean control group values at all termination times.

Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 31 .0-62.9 with a mean of 43.4. Statistical analysis showed that animal s treated with chlorambucil had significantly more micronucleated polychromatic cells than vehicle controls (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.

- Ratio of PCE/NCE (for Micronucleus assay):
The recorded incidence of micronuclei per 1000 mature erythrocytes varied between 0.0 and 2.7 throughout all groups. These findings demonstrate the normal status of the animals used in the study: in particular, the low incidence in animals killed 24 hours after treatment shows the absence of any pre-treatment abnormality in the bone marrow.

The ratio of polychromatic to mature erythrocytes was 1.0 in the vehicle control group at 24 hours. Ratios for groups given sodium chlorate at 200, 1000 or 5000 mg/kg and terminated 24 hours post-dose were also 1.0. Forty eight hours after treatment, the ratio of the vehicle control group was 1.0, and in animals given sodium chlorate at 200, 1000 or 5000 mg/kg it was 1.0, 0.9 and 0.9 respectively.
Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes in the vehicle control group was 0.9, and in animals given sodium chlorate at 200, 1000 or 5000 mg/kg it was 0.9, 0.9 and 1.0 respectively. Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups.
In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was reduced to 0.7.

- Appropriateness of dose levels and route: Toxicty to the bone marrow was not observed however e.g. no direct proof of exposure was provided but the presence of clinical signs at 2500 and 5000 mg/kg bw in the prelimininary test and at 5000 mg/kg bw in the main test was considered to demonstrate systemic exposure to the test item.

- Statistical evaluation: Statistical analysis confirmed that there was no significant difference between the vehicle control group and any sodium chlorate treated group, at any termination time (p > 0.05).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of sodium chlorate. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.
Executive summary:

The effect of sodium chlorate on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. The study was performed under GLP and according to OECD474. Chromosome damage was measured indirectly by counting micronuclei. A preliminary toxicity test was first conducted, using dosages of 625, 1250, 2500 and 5000 mg/kg. Subsequently, male and female mice were given a single dose of sodium chlorate at 200, 1000 or 5000 mg/kg. In all cases sodium chlorate was dosed orally, dissolved in distilled water. Concurrent vehicle and positive control groups of mice were similarly dosed with distilled water or chlorambucil (30 mg/kg) respectively. Five males and five

females from each group were killed 24 hours after treatment; further lots of five males and five females, given sodium chlorate at 200, 1000 or 5000 mg/kg or the vehicle control, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic:mature cells was also calculated for each animal, as an indicator of gross toxicity.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with sodium chlorate. Evidence of toxicity was noted in five male animals dosed with sodium chlorate at 5000 mg/kg; signs included hunched posture and slight piloerection on day 4 of the study. No adverse reactions to treatment were recorded for any other group treated with either sodium chlorate or the vehicle control.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of sodium chlorate were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of sodium chlorate. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.