Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 16, 2009 to May 15, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Young adult male and female ICR(CD-1) mice. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test substances. The mouse has been routinely utilized as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay.
Sex:
male/female
Details on test animals and environmental conditions:
Dose range-finding study: young adult male and female ICR(CD-1) mice were received on 26 March 2009 from Harlan, Frederick, Maryland.
Micronucleus Assay: young adult male ICR(CD-1) mice were received on 14 April 2009 from Harlan, Indianapolis, IN.

Identification and Acclimation
Animals were randomized into groups using a computer program. Following randomization, each study animal was uniquely identified by ear tag. Animals were acclimated to laboratory conditions for at least 5 days.

Housing
The animals were housed in sanitary polycarbonate cages containing Sani-Chips® Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization. Each batch of wood chips was analyzed by the manufacturer for specific microorganisms and contaminants.

Environmental Conditions
Environmental controls were set to maintain the following animal room conditions: temperature range of 18 to 26°C, relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour dark cycle. Actual temperature and humidity readings were monitored continuously and averaged twice daily. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study.

Diet, Water, and Contaminants
Harlan Teklad Certified Rodent Diet® #2016C was available ad libitum. The manufacturer analyzed the diet for nutritional components and environmental contaminants. Tap water was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants. Results of the diet and water analyses are reviewed for acceptability and are on file at Covance-Vienna.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Prior to dosing, the top stock of the test substance was prepared by adding the appropriate volume of the vehicle, RO/DI water, to a pre-weighed quantity of the test substance and mixed, forming a colorless, opaque, non-viscous, homogeneous suspension. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.
Details on exposure:
Dose Range-finding Study

In the dose range-finding study, the test substance was formulated in reverse osmosis / deionized (RO/DI) water and administered once, by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 500, 1000, or 2000 mg/kg. At initiation of treatment, the animals were approximately 8 weeks old, and their body weights ranged from 34.7 to 39.3 g and 23.4 to 28.8 g, for the males and females, respectively.
The daily observations of toxic signs and/or mortality data were used to estimate the highest appropriate dose level (maximum tolerated dose) for the micronucleus assay.

Micronucleus Assay

Since no relevant differences in toxicity between the sexes were observed in the dose range-finding study, only males were used in the micronucleus assay.
In the dose range-finding study, the maximum tolerated dose was estimated to be 800 mg/kg. In the micronucleus assay, the test substance was formulated in RO/DI water and administered once at 200, 400 and 800 mg/kg to 5 males per dose level.
The high dose, unless non-toxic, should have produced some indication of toxicity, e.g., toxic signs, death, or depression of the ratio of PCEs to NCEs. The use of a high dose, as defined above, increased the likelihood that a weak clastogen could be detected.
At initiation of treatment, the animals were approximately 8 weeks old, and their body weights ranged from 32.4 to 39.3 g.
Duration of treatment / exposure:
Observation period: 2 days after dosing
Frequency of treatment:
All animals were examined immediately after dosing, approximately 1 hour after dosing, and at least daily for the duration of this assay for signs of clinical toxicity and/or mortality.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent no treatment

Examinations

Tissues and cell types examined:
Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from up to 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 fetal bovine serum (one tube per animal).
Details of tissue and slide preparation:
Preparation of Slides
Following centrifugation to pellet the marrow, 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-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

Slide Analysis
Slides prepared from the bone marrow collected from up to five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing 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 500 erythrocytes per animal.

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluishgray and red, respectively).
The historical background frequency of micronucleated cells was expressed as percentage micronucleated cells based on the number of PCEs analyzed. The historical background frequency of micronuclei in the mouse strains at this laboratory is about 0.0 to 0.4%, which is within the range of the published data (Salamone and Mavournin, 1994).
Evaluation criteria:
The criteria for a positive response are 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 substance that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.
Statistics:
- Assay data analysis was performed using an analysis of variance (Winer, 1971) 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), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control.
The 200, 400, and 800 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose Range-finding Study

Survival and Clinical Observations
Mortality was observed in 1 of 3 females at 1000 mg/kg, and in 3 of 3 males and 3 of 3 females at 2000 mg/kg. Based on these results, the maximum tolerated dose was estimated to be 800 mg/kg.

Micronucleus Assay

Survival and Clinical Observations
Mortality was observed in 4 of 13 males at 800 mg/kg. Clinical observations were noted in only one male at 800 mg/kg, and consisted of squinted eyes and labored respiration. All other animals appeared normal immediately after dosing and remained normal until the appropriate harvest timepoint. All animals in the vehicle and positive control groups appeared normal after dosing and remained normal until the appropriate harvest timepoint.


- The test substance, induced signs of clinical toxicity in the treated animals at 800 mg/kg, which included squinted eyes and labored respiration.
- The test substance did not induce statistically significant increases in micronucleated PCEs at any test substance dose examined (200, 400, and 800 mg/kg).
- The test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose.
- The vehicle control group had approximately ≤0.3% micronucleated PCEs and the group mean was within the historical control range.
- The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard deviation of 1.11 ± 0.39%.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was evaluated as negative in the mouse bone marrow micronucleus assay.
Executive summary:

A study was conducted to determine the test substance for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei inpolychromatic erythrocytes (PCE) in CD®-1 mouse bone marrow according to OECD Guideline 474 and EPA OPPTS Method 870.5395, in compliance with GLP. In the dose range-finding study, the test substance was formulated in reverse osmosis /deionized (RO/DI) water and administered once, by oral gavage to 3 males and 3 femalesper dose level. The animals were dosed at 500, 1000, or 2000 mg/kg bw and observed for up to 2 d after dosing for toxic signs and/or mortality. Based on the results of the dose range-finding study, the maximum tolerated dose was estimated to be 800 mg/kg bw. In the micronucleus assay, the test substance was formulated in RO/DI water and administered once at 200, 400 and 800 mg/kg bw. Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 total erythrocytes for each animal. Mortality was observed in 4 of 13 males at 800 mg/kg bw. Clinical observations were noted in only one male at 800 mg/kg bw and consisted of squinted eyes and labored respiration. The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined. In addition, the test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose. Under the study conditions, the substance was evaluated as negative in the mouse bone marrow micronucleus assay (Xu, 2009).