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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 Oct, 1999 to 19 Oct, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Light-brown granules
Details on test material:
- Name of test material (as cited in study report): MON 13900
- Substance type: Light-brown granules
- Analytical purity: 99.2%
- Stability under test conditions: Ambient

Test animals

Species:
rat
Strain:
other: Crl:CD”(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
Young adult male and female rats of the Crl:CD”(SD)IGS BR strain were purchased from Charles River Laboratories, Raleigh, NC. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test substances. The animals were acclimated for at least 7 d before being placed on study. The animals were housed in sanitary, stainless-steel, hanging, wire cages. The animals were housed, separated by gender, up to two animals per cage during acclimation, and individually after randomization.
The animals were housed under the following climatic conditions: temperature, 64 to 79°F; humidity, 30 to 70%; light cycle, 12 h light/dark; air changes, at least 10 air per h. A commercial diet, PMI@Feeds, Inc. Certified Rodent Diet # 5002 (chow), and tap water were available ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed biannually, on a retrospective basis, for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. The animals were randomly assigned, by a computer program, to study dose groups. Each animal was uniquely identified by ear tag. Treatment groups were identified by cage label. The animals were weighed prior to dosing and dosed based upon the individual animal weights. The weight variation of the animals did not exceed ±20% of the mean weight of each sex. All animals were dosed on an acute (one-time only) basis. Unscheduled deaths, if any, were discarded without necropsy.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test substance is soluble in corn oil
-- Concentration of test material in vehicle: 7.5, 15 and 30 mg/mL
- Dosing volume: 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the test substance was ground with a mortar and pestle for approx 5 min. Each level of the test substance was prepared by adding the appropriate volume of the vehicle, corn oil, to a premeasured quantity of the test article. The formulations were mixed until in suspension. The stock concentrations of 7.5, 15 and 30 mg/mL were prepared for dose level of dose levels of 150, 300, and 600 mg/kg bw, respectively.

Duration of treatment / exposure:
One time only
Frequency of treatment:
One time only
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 300 and 600 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Dose range-finding study: 3 males and/or females
Main assay: 6 males per dose level at the 24 h harvesting time, and 6 males at the high-dose and control levels at the 48 h harvesting time. 10 replacement males were included at the high-dose level as potential replacements for original high-dose animals.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marow cells

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the findings of the dose rangefinding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Five animals at each dose level, and five vehicle and positive control animals were euthanized approximately 18 hours after dosing for extraction of the bone marrow. At approximately 42 hour after dosing, five animals dosed at the 600 mg/kg dose level and five animals dosed with the vehicle control article were euthanized for extraction of the bone marrow.

DETAILS OF SLIDE PREPARATION: Approx 1.5 to 2.5 h prior to euthanasia, the animals were injected intraperitoneally with 2.0 mg/kg of colchicines (10 mL/kg). The hind limb bones (tibias and femurs) were removed from the first five surviving animals, when possible, for marrow extraction. The marrow was flushed from the bone and transferred to Hanks’ balanced salt solution. The marrow pellet was collected by centrifugation and then resuspended in 0.075M KCl. The centrifugation was repeated and the pellet was resuspended in fixative (methanol:acetic acid, 3:1). The fixative was then changed and the fixed cells were dropped onto glass slides and air dried. The metaphase cells were stained with 5% Giemsa and air dried; the slides were coverslipped. Animals in excess of the first five survivors (according to eat-tag sequence) were euthanized and no marrow was extracted.

METHOD OF ANALYSIS: The first five surviving animals in each treatment and control group were analyzed, when possible. Slides were coded for control of bias and scored for chromosomal aberrations as defined in the following section. Cells were selected for scoring on the basis of good chromosome morphology, and only cells with the number of centromeres equal to the rat modal number 42±2 were analyzed. Normally 100 cells from each animal, if available, were read. At least 25 metaphase cells were analyzed from animals that have 25% of the cells with one or more aberrations. For each cell bearing an aberration, the microscope stage location (vernier readings) was recorded so that the cell may be relocated if necessary. Percent polyploidy and endoreduplication were also analyzed by evaluating 100 metaphases per animal, if available, and tabulated. A mitotic index was calculated by scoring the number of cells in mitosis per 1000 cells observed, and generating a percentage.
Evaluation criteria:
Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage, as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Cells with more than one aberration was considered to indicate more genetic damage than those containing evidence of single events. Frequently, one is unable to locate 100 suitable metaphase spreads for each animal even after preparing additional slides. Possible causes for this may be related to cytotoxic effects which may alter the duration of the cell cycle, kill the cell, or cause clumping of the chromosomes. Animals with less than 25 analyzable cells were not included in the statistical analysis. Additional information was gained from the mitotic index which also appeared to reflect cytotoxic effects. Comparison with a concurrent vehicle control which happens to show an unusually low frequency of aberrations may suggest statistical significance which is not biologically relevant.
In these instances, the treatment data were considered against the historical control database.
Analyses were performed on a per animal basis for the following variables:
1) Number of cells with a least one structural aberration
2) Number of cells with two or more structural aberrations
Statistics:
Analysis of variance (ANOVA) techniques were used to compare positive control to the vehicle control group. Specifically, Levene’s test was performed to test for variance homogeneity. In the case of variance heterogeneity, the data were ranked. Dunnett’s test was performed to compare test article-treated group means to the vehicle control. In this case, additional tests such as linear regression and Terpstra-Jonckheere test for monotone trend were also performed to evaluate any possible dose-response. The type of aberration, its frequency, the statistical significance of any increase and its correlation to dose in a given time period were all considered in evaluating the clastogenic potential of the test article. The criterion for a positive response is generally a statistically significant dose-related increase in the number of structural aberrations for at least one dose level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs in the high-dose animals included, but were not limited to, hypoactivity, hunched posture, fecal staining, and/or squinted eyes.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose range-finding assay:
-Dose selection: Dose levels of 200, 500, 600, 800, 1500, and 2000 mg/kg bw and 500, 800, 1200, 1500, and 2000 mg/kg bw were chosen for males and females, respectively, in the dose range-finding assay.
-Dosing information: Thirty-five animals, approx 8 wk old at the time of dosing, with a weight range of 227 - 252 g and 163 - 200 g, for the males and females, respectively, were assigned to this study. Two animals were replaced due to misdosing caused by backwashing. At the termination of this assay, all surviving animals were euthanized by CO, inhalation followed by incision of the diaphragm. The treatment regimen for this assay is shown in Table 1. Prior to dosing, the test substance was groundwith a mortar and pestle for approx 5 to 10 min. Each level of the test substance was prepared by adding the appropriate volumeof the vehicle, corn oil, to a premeasured quantity of the test substance. The formulations were mixed until in suspension. The stock concentrations and descriptions are shown in the Table 2.
-Results and interpretation: The mortality data for this assay are summarized in Table 3. Based on these results, the maximum tolerated dose was estimated to be 600 mg/kg bw.

Chromosome aberration assay:
-Dose selection: Based on results from the dose range-finding assay, dose levels of 150,300, and 600 mg/kg bw were selected for testing in rats in this assay. Only males were used in the chromosome aberration assay because there were no substantial differences in clinical observations between the sexes in the dose range-finding assay. A replacement dose group consisting of 10 animals was included at the top dose level in anticipation of mortality.
-Dosing information: 52 animals, approx 8 wk old at the time of dosing, with a weight range of 245 - 300 g were used in this assay. An outline of the dosing scheme and harvest timepoints is found in table 4. Cyclophosphamide, the positive control, was dissolved in sterile deionized water. Prior to dosing, the test substance was ground with a mortar and pestle for approx 5 min. Each level of the test substance was prepared by adding the appropriate volume of the vehicle, corn oil, to a premeasured quantity of the test substance. The formulations were mixed until in suspension. The stock concentrations and descriptions are shown in table 5.
-Animal observations: All animals were examined immediately after dosing, about 1 h after dosing, and at least daily for the duration of this assay for toxic signs and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoints. Few signs of toxicity such as fecal stains, convulsions, hypoactive were also observed.
-Results and interpretation: The test substance induced mortality and signs of clinical toxicity in the high-dose (600 mg/kg) animals by the 18 h harvest time point. Clinical signs in the high-dose animals included, but were not limited to, hypoactivity, hunched posture, fecal staining, and/or squinted eyes. One incidence each of fecal staining and rough haircoat was noted in the mid-dose (300 mg/kg) animals. There were no statistically significant increases in either structural chromosome aberrations or numerical chromosome changes. The test substance was therefore concluded to be negative for induction of structural or numerical chromosome damage under the conditions of exposure in this assay.

Any other information on results incl. tables

Analytical results: A 24 h room temperature stability analysis was performed on the 2 mg/mL sample prepared by Analytical Chemistry and yielded a % change of 3.60%. A 24 h room temperature stability analysis was performed on the 30.00 mg/mL Trial 1 sample prepared by Genetic Toxicology and yielded a % change of -1.60%. There were no interfering peaks detected at the retention time of the peak of interest in either control analyzed for stability.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, MON 13900 was not considered to be clastogenic to rat bone marrow cells in vivo at any of the dose levels tested.
Executive summary:

A study was conducted to assess the clastogenic potential of MON 13900 to rat bone marrow cellsin vivoaccording to the OECD Guideline 475 in compliance with GLP.

In a dose range-finding study, the test article was mixed with the vehicle control, corn oil, at dose levels of 200, 500, 600, 800, 1,500 and 2 000 mg/kg bw and 500, 800, 1,200, 1,500 and 2,000 mg/kg bw for males and females, respectively, and administered once via the oral route to three animals per designated dose level. Based on the findings of the dose range-finding study, dose levels of 0, 150, 300 and 600 mg/kg bw were selected for testing in the chromosome aberration assay in males only. Colchicine was administered 1.5 to 2.5 h before sacrifice for the two harvest time points; 18 and 42 h. The positive control was cyclophosphamide dissolved in sterile deionized water (60 mg/kg bw harvest at only 18 h). If available, 100 cells/animal were examined from five of the six rats/dose level. At least 25 metaphase cells were analyzed from animals that had 25% of the cells with one or more aberrations. Numbers and types of chromosomal aberrations, mitotic index and vernier location of each metaphase containing damage were recorded. The mitotic index was recorded as the number of cells in mitosis per 1000 cells counted. Mortality and clinical observations were observed in the 600 mg/kg bw group at the 18 h harvest time point. The clinical signs observed in the high dose rats included hypoactivity, hunched posture, fecal staining and/or squinting eyes. One incidence each of fecal staining and rough haircoat was observed in the 300 mg/kg bw (mid dose) rats. There were no statistically significant increases in structural chromosomal aberrations or numerical chromosome changes. 

Under the conditions of this study, the test substance was not considered to be clastogenic to rat bone marrow cellsin vivoat any of the dose levels tested.