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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, according to accepted guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 1,4-Butanediol diglycidyl ether
- Analytical purity: 50.95% weight/weight
- Lot/batch No.: 9113719/006

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 29.9 to 34.1 g
- Assigned to test groups randomly: Yes
- Housing: One/cage in stainless steel cages (cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for the rodents' feet. The gauze also provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system)
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form ad libitum
- Water (e.g. ad libitum): Municipal water ad libitum
- Acclimation period: At least 1 week prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 to 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
0.5% METHOCEL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions of the test material were prepared on each of the two consecutive days of administration. A frozen stock solution of CP dissolved in distilled water (thawed and brought to room temperature prior to use) served as the positive control. The vehicle used to mix the test material (0.5% METHOCEL) served as the negative control. The concentrations of the test material in the dosing solutions used for the first day of dosing during the micronucleus test were verified using gas chromatography with flame ionization detection or mass spectrometry detection (GC/FID) detection and external standard quantitation.
Duration of treatment / exposure:
Approximately 4 days (2 days of dosing + collection of peripheral blood 48 hours after the last dosing).
Frequency of treatment:
Once/day for 2 consecutive days.
Post exposure period:
48 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 187.5, 375, or 750 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6 mice/dose in all dose levels and controls except for the high-dose group where there were 8 mice.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate
- Route of administration: oral (gavage)
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Approximately 5,000 reticulocytes (RETS) were analyzed per blood sample. The number of normochromatic erythrocytes (NCE), micronucleated (MN)-NCE, RET, and MN-RET were recorded for each sample and the frequency of MN-RET was determined to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoetic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RET, MN-NCE, and MN-RET observed. The analyses were conducted utilizing a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter).
Details of tissue and slide preparation:
Duplicate cell smears were prepared and stored to serve as backups in the event that the flow cytometric analysis was not possible. Blood was collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears were prepared, fixed in methanol, and stored at room temperature.
Evaluation criteria:
A test was considered valid if all of the following conditions are met:
- The range of MN-RET values in the negative controls were within reasonable limits of the recent laboratory background range.
- There was a significant increase in the incidence of MN-RET in the positive control treatment as compared to the concurrent negative controls.
- The mean for percent RET value in one or more of the test material treated groups was >/= 20% of the control value indicating no undue effect on erythropoiesis (toxicity).

A test material was considered positive in this assay if the following criterion was met:
- Statistically significant increase in MN-RET frequency at one or more dose levels accompanied by a dose response.

A test material was considered negative in this assay if the following criterion was met:
- No statistically significant dose-related increase in MN-RET when compared to the negative control.

A test result not meeting the criteria for either the positive or the negative response was considered to be equivocal.
Statistics:
Bartlett's test and one-way ANOVA.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
- 3 mice of the high-dose group had a decreased amount of feces.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Summary of Micronucleated Reticulocytes (MN-RET) Frequencies and Percent Reticulocytes (% RET) – Males

 

Peripheral Blood-Males

Exposure (mg/kg bw/day)

No. animals

‰ MN-RET

% RET

0

6

1.43 ± 0.43

1.60 ± 0.17

187.5

6

1.23 ± 0.34

1.65 ± 0.42

375

6

2.49 ± 0.96

1.94 ± 0.17

750

6

1.96 ± 0.95

1.67 ± 0.56

Positive Control (40 mg/kg CP)

6

9.05 ± 1.71d

0.55 ± 0.22

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative in the mouse peripheral blood micronucleus test