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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

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 animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD. At the initiation of the study, the mice were 6 to 8 weeks old. Animal body weights were recorded within the following ranges:
Pilot study:
Males, 29.5 - 35.5 grams at randomization
Females, 25.5 - 29.8 grams at randomization

Toxicity study:
Males, 28.1 - 33.2 grams at randomization
Females, 27.5 - 30.6 grams at randomization

Micronucleus assay:
Males, 25.5 - 34.7 grams at randomization
Females, 22.1 - 29.5 grams at randomization

Animal Receipt and Quarantine
Mice were obtained from a source monitored for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were quarantined for no less than 5 days after receipt. The mice were observed each working day for signs of illness, unusual food and water consumption, and other conditions of poor health. The animals were judged to be healthy prior to utilization in the assay.

Animal Care
The mice were housed in an AAALAC-accredited facility with a controlled environment of 74±6°F, 50±20% relative humidity, and a 12 hour light/dark cycle. Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material.
Heat-treated hardwood chips were used for bedding. Mice had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants (Harlan TEKLAD certified Rodent 7012C) and to tap water (Washington Suburban Sanitary Commission, Potomac Plant). No contaminants that could be expected to alter the outcome of the study are known to be present in the diet or water.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The vehicle used to deliver alkyl glycidyl ether to the test system was com oil (CAS number 8001-30-1) obtained from Super G. The test article was reported by the Sponsor to be 98% pure.
Details on exposure:
The test article-vehicle mixture (1000, 2000, or 4000 mg test article/kg ), the vehicle alone, or the positive control was administered by intraperitoneal injection at a constant volume of 20 mllkg body weight. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were observed after dose administration and daily thereafter for up to 3 days for clinical signs of chemical effect.
Frequency of treatment:
one
Post exposure period:
24 hr, 48 hr, and 72 hr
No. of animals per sex per dose:
15 mice/sex/dose
Positive control(s):
Cyclophosphamide (CP, CAS 6055-19-2), was obtained from Sigma Chemical Company and was dissolved in sterile distilled water obtained from Life Technologies, at a concentration of 3 mg/ml for use as the positive control.

Examinations

Tissues and cell types examined:
Erythrocytes, in a bone marrow preparation from the femur, were examined.
Details of tissue and slide preparation:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.
The bone marrow cells were transferred to a capped centrifuge tube containing approximately I ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p  0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979). All analyses were performed separately for each sex and sampling time.

Results and discussion

Additional information on results:
Solubility Test
Com oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in com oil at 500 mg/ml, the maximum concentration tested for solubility. Dosing concentrations were delivered to the system as off-white solutions.
Pilot Assay
For the pilot study, alkyl glycidyl ether was administered by intraperitoneal injection to male mice at 1, 10, 100 or 1000 mg test article/kg and to male and female mice at 5000 mg test article/kg which was administered in a total volume of 20 ml test article-vehicle mixture/kg body weight. Mortality occurred within three days of dose administration as follows: 2/5 males and 2/5 females at 5000 mg/kg. Clinical signs, which were noted within four hours or later after dose administration, included: lethargy in male mice at 1000 mg/kg and lethargy and piloerection in male and female mice at 5000 mg/kg. All other animals appeared normal throughout the observation period.
Toxicity Assay
For the toxicity study, alkyl glycidyl ether was administered by intraperitoneal injection to male and female mice at 2500 or 4500 mg test article/kg which was administered in a total volume of 20 ml test article-vehicle mixture/kg body weight. Mortality occurred within three days of dose administration as follows: 1/5 male mice and 1/5 female mice at 4500 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 2500 mg/kg and lethargy, piloerection and crusty eyes in male and female mice at 4500 mg/kg. The LD50/3 was calculated by probit analysis to be approximately 5721.6 mg/kg for male mice and female mice. The high dose for the micronucleus test was set at 4000 mg/kg for male and female mice which was estimated to be approximately 70% of the LD50/3 (50% of mortality occurred within 3 days after test article dose administration).
Micronucleus Assay
For the micronucleus test, male and female mice were dosed with alkyl glycidyl ether by a single intraperitoneal injection of 1000, 2000, or 4000 mg test article/kg which was administered in a total volume of 20 ml test article-vehicle mixture/kg body weight. No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted on the days following dose administration, included: lethargy in male and female mice at all test article dose levels and piloerection in male and female mice at 4000 mg/kg. All other mice treated with test and control articles appeared normal during the study.
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic
erythrocytes scored and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 1. Reductions of up to 11% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in test article- treated mice relative to their respective vehicle controls. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables). The positive control, CP, induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p>0.05, Kastenbaum-Bowman Tables).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
All criteria for a valid test were met. Under the conditions of the assay described in this report, alkyl glycidyl ether did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The purpose of this study was to assess the clastogenic potential of a test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 mllkg body weight by a single intraperitoneal injection.

 

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in com oil at 500 mg/ml, the maximum concentration tested for solubility. Dosing concentrations were delivered to the system as off-white solutions.

 

In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg of test article/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality was observed in 2/5 male mice and 2/5 female mice at 5000 mg/kg. Clinical signs following dose administration included lethargy in male mice at 1000 mg/kg and lethargy and piloerection in male and female mice at 5000 mg/kg.

 

In the toxicity assay, male and female mice were dosed with 2500 or 4500 mg of test article/kg body weight. Mortality was observed in 1/5 male mice and 1/5 female mice at 4500 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 2500 mg/kg and lethargy, piloerection and crusty eyes in male and female mice at 4500 mg/kg. The high dose for the micronucleus test was set at 4000 mg/kg which was estimated to be approximately 70% of the LD50/3(50% of mortality occurred within 3 days after test article dose administration).

 

In the micronucleus assay, male and female mice were dosed with 1000, 2000 or 4000 mg of test article/kg body weight. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy in male and female mice at all test article dose levels and piloerection in male and female mice at 4000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 11%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls.

No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p>0.05, Kastenbaum-Bowman). The results of the assay indicate that under the conditions described in this report, alkyl glycidyl ether did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. Alkyl glycidyl ether was concluded to be negative in the mouse micronucleus assay.