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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:
29 October 2001 - 05 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study consistent with OECD and EPA test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
yes
Remarks:
Housing temperature was maintained at 64-79 deg F, a slightly wider range than the 19-25 deg C recommended by US EPA.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Housing temperature was maintained at 64-79 deg F, a slightly wider range than the 19-25 deg C recommended by OECD.
Principles of method if other than guideline:
Although only the US EPA OPPTS guideline is specifically referred to in the methods section of the study report, the study methodology used was consistent with the 1997 OECD TG 474, and this OECD guideline is included in the study report's list of references (and in the testing laboratory's assigned study number: 23254-0-455OECD).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):
- Physical state: Liquid.
- Storage condition of test material: Ambient conditions.
- Other:

Test animals

Species:
mouse
Strain:
other: Crl:CD-1(ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC.
- Age at study initiation: Animals were approximately 8 weeks old at the time of dosing.
- Weight at study initiation: 20-40 g (within ±20% of the mean weight of each sex).
- Assigned to test groups randomly: Yes.
- Housing: Group-housed in sanitary polycarbonate cages containing hardwood chip bedding.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: At least five days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79ºF (17.7-26.1ºC).
- Humidity (%): 30-70%.
- Air changes (per hr): At least 10.
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: Animals in the main study were dosed on 13-15 November 2001 and observed for a further 24 hours.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: White oil.
- Justification for choice of solvent/vehicle: Not stated.
- Concentration of test material in vehicle: Dose-range finding study: 25, 50, 100 and 200 mg/mL.
Main micronucleus study: 6.25, 12.5 and 25 mg/mL.
- Dosing volume: 10 mL/kg.
Details on exposure:
The test substance was administered by intraperitoneal injection in both the dose-range-finding assay and the main micronucleus assay.
Duration of treatment / exposure:
Dose-range-finding assay: Three days.
Main micronucleus assay: Three days.
Frequency of treatment:
Dose-range-finding assay: Daily (for three consecutive days).
Main micronucleus assay: Daily (for three consecutive days).
Post exposure period:
Dose-range-finding assay: Two days.
Main micronucleus assay: One day.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 500, 1000 and 2000 mg/kg/day
Basis:
other: Injected dose levels in dose-range-finding assay
Remarks:
Doses / Concentrations:
62.5, 125 and 250 mg/kg/day
Basis:
other: Injected doses levels in main micronucleus assay
No. of animals per sex per dose:
Dose-range-finding assay: Three animals per sex per dose level.
Main micronucleus assay: Six males per dose level.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide.
- Justification for choice of positive control(s):
- Route of administration: Oral gavage.
- Doses / concentrations: 80 mg/kg/day (8 mg/mL at 10 mL/kg).

Examinations

Tissues and cell types examined:
Cytotoxicity was assesssed by scoring the number of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.
At least 2000 PCEs per animal were analysed for the frequency of micronuclei.
Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Approximately 24 hours after the last dose administration, the animals were euthanised by carbond dioxide inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL foetal bovine serum (one tube per animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grunwald solution followed by Giemsa, and protected by permanently mounted coverslips. For control of bias, all slides were coded prior to analysis.


METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from the first five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. the micronucleus frquency (expressed as percent micronucleated cells) was determined by analysing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least the first 500 erythrocytes per animal.
Evaluation criteria:
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
Assay data analysis was performed using an analysis of variance on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p 0.05), a Dunnett’s t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Signs of clinical signs toxicity and mortality were induced, but the test substance was not cytotoxic to the bone marrow
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250-2000 mg/kg/day.
- Clinical signs of toxicity in test animals: All animals in the 1000 and 2000 mg/kg/day dose groups, and one male in the 500 mg/kg/day group, were dead by the one-hour post-dose observation. A further male and two females in the 500 mg/kg/day dose group died before the third dose had been administered, and the remaining male and female in this group were sacrificed due to the excessive mortality. A female in the 250 mg/kg/day group died immediately after the third dose. Other animals in this group displayed various signs of clinical toxicity including hunched posture, rough haircoat, low activity, laboured breathing, and irregular breathing.
- Evidence of cytotoxicity in tissue analyzed: Not assessed.


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: On Day 3, one 250 mg/kg/day animal died prior to dosing and one died following dosing. Clinical signs of toxicity seen in animals within the 250 mg/kg/day group included: urine stains, distended abdomen, hypoactive, rough haircoat, squinted eyes, eye sealed shut, hunched posture, loose faces and faecal stains.
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): There was no statistically significant decrease in the PCE:NCE ratio).
- Statistical evaluation: A statistical significant increase in micronucleated PCEs was not observed at any dose level.

Any other information on results incl. tables

The reported historical background frequency of micronucleated cells in the Crl:CD-1(ICR) BR strain at the test laboratory was about 0.0-0.4%, which was stated to be within the range reported in the published data.

The temperature range of the animal housing was reported as 64–79ºF, equivalent to 17.8–26.1ºC. Recommended animal housing conditions were not included in the June 1996 draft EPA guideline, but the final 1998 guideline (and OECD TG 474) recommends maintaining temperatures within the range of 19–25ºC.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance was negative in the mouse bone marrow micronucleus assay under the conditions of this test.
Executive summary:

The test substance was evaluated for clastogenic potential in a mouse micronucleus test. The GLP study was conducted in accordance with US EPA OPPTS and OECD guidelines. The test substance, in white oil vehicle, was administered by intraperitoneal injection. In the initial dose-range-finding study, doses of 250, 500, 1000 and 2000 mg/kg/day were adminstered to groups of male and female Crl:CD-1(ICR) BR mice causing extensive mortality in the higher dose groups. As there was no indication of sex-dependent effects, doses of 62.5, 125 and 250 mg/kg/day were administered to groups of six males in the main micronucleus study (with two additional males dosed at 250 mg/kg/day in case of mortality in this group). The animals were dosed for three days. 24 hours after the third dose, animals were sacrificed, bone marrow collected and prepared for microscopic analysis of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs). Cytotoxicity to the bone marrow was not seen (i.e. there was no decrease in the PCE:NCE ratio) in any of the test substance treatment groups, but clinical signs of toxicity and two deaths occurred at 250 mg/kg/day. A statistically significant increase in micronuclated PCEs was not seen at any dose level. The positive control, cyclophosphamide administered by gavage, confirmed the sensitivity of the test. It is concluded that the test substance was not clastogenic under the conditions of this test.