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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:
1990
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethoxymethane
EC Number:
203-714-2
EC Name:
Dimethoxymethane
Cas Number:
109-87-5
Molecular formula:
C3H8O2
IUPAC Name:
dimethoxymethane
Test material form:
other: liquid
Details on test material:
Name of test material (as cited in study report): C-01361 (=test ID of dimethoxymethane)
Methylal min 97.5% wt
Water: max 1.5% wt
Acidity, as acetic: max 0.01% wt
Aldehydes, as formaldehyde: max 0.10% wt
Methanol: max 1.0% wt

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley
- Age at study initiation: 8 weeks
- Weight at study initiation: 27.8-40.6 g for males ans 20.0-28.1g for females
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Animals were group-housed by sex up to five per cage. Sanitary polycarhonate cages and hardwood bedding were used.
- Diet (e.g. ad libitum): A commercial diet (Purina Certified Laboratory Chow #5002) was available ad libitum for the duration or the study. The feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients.
- Water (e.g. ad libitum): Water was available ad libitum for the duration or the study. The water is analyzed on a retrospective basis for specifiedi microorganisms, pesticides, heavy metals, alkalinity and halogens.
- Acclimation period: Animals were quarantined for eight days before being placed on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-26°C
- Humidity (%): 30-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.

IN-LIFE DATES: Not specified

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: sodium chloride(0.9%)
- Justification for choice of solvent/vehicle: This choice is based upon the solubility data from a previously conducted dose range finding assay (HLA Study No.: 12166-0-459).
- Concentration of test material in vehicle: not specified
- Amount of vehicle (if gavage or dermal): 20 ml/kg body weight
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not specified
- Purity: not specified
Details on exposure:
TEST SITE
Single dose intrapeitoneal injection

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 400, 1333, 4000 mg/kg body weight (in 0.9% sodium chloride at 20 ml/kg body weight)
Duration of treatment / exposure:
Single dose intrapeitoneal injection was performed. Animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.
Frequency of treatment:
Single dose intrapeitoneal injection was performed. Animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.
Post exposure period:
Animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
400 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1333 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
cyclohexylamine
- Justification for choice of positive control(s): not specified
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg body weight (in a volume of 10 ml/kg body weight)

Examinations

Tissues and cell types examined:
polychromatic and normochromatic erythrocytes, micronucleated polychromatic erythrocytes
Details of tissue and slide preparation:
At the appropriate harvest time, the animals were euthanatized with CO2 and the Adhering soft tissue and epiphyses of both tibiae were removed. The marrow was aspirated into a syringe and mixed with about 0.1 ml of fetal bovine serum to form a cellular suspension. Portions of the cell suspension were smeared onto clean slides and were allowed to air-dry. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionizell water according (Schmid, 1975). After being air-dried, the slides were coverslipped using Depex mounting medium.

The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to
record these data. One thousand PCEs per animal were scored. The frequecy of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.

The frequency of PCEs versus NCEs was determined by scoririgthe number of NCEs observed in the optic fields while scoring the 1000 PCEs for
micronuclei.
Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, althourh almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. 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 (bluish-grey and red, respectively).
Statistics:
The analysis of the data was performed using an Analysis of Variance (p≤O.O5) on the square root arcsine transformation (Sokal and Rophlf, 1981) which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons (p≤0.05) was used at each harvest time to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test article, Methylal, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times.

The positive control, cyclophosphamide, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.54% ± 0.23% and 2.60% ± 0.75% for the males and females, respectively.

Any other information on results incl. tables

All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. Within one minute of dosing all animals in the 4000 mg/kg dose group became prostrate with dyspnea and all animals in the 1333 mg/kg dose group became ataxic (uncoordinated movement). Approximately one hour after dosing several animals in the 4000 mg/kg dose group were still languid; however, all other animals appeared normal. The following morning, approximately 13 hours after dosing, all animals appeared normal and they remained healthy until the appropriate harvest time.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material, Methylal, did not induce a significant increase in microcronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. These results are supported by the significant increase in micronuclei in bone marrow polychromatic erythrocytes induced by the positive control. These results are supported by the significant increase in micronuclei in bone marrow polychromatic erythrocytes induced by the positive control, cyclophosphamide, in both sexes, with means and standard errors of 1.54% ± 0.23% and 2.60% ± 0.75% for the males and females, respectively.
Executive summary:

An in vivo mouse micronucleus asssay was performed with 120 mouses (60 males and 60 females) of ICR strain, supplied by Harlan Sprague-Dawley. They were quarantined for eight days before being placed on study.

The test item, Methylal, was solubilized in 0.9% sodium chloride (vehicle) and dosed by intraperitoneal injection at 400, 1333, and 4000 mg/kg body weight. Animals were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group.

Vehicle and positive control groups were constituted with 10 animals (five males and five females) per group. Animals from vehicle group were dosed with 0.9% sodium chloride at 20 mL/kg body weight. Cyclophosphamide was administered by oral gavage at 80 mg/kg to animals from positive control group. Animals from vehicle and positive control groups euthanatized 24 hours after dosing for extraction of the bone marrow.

The test material, Mathylal, did not induce a significant increase in microcronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. These results are supported by the significant increase in micronuclei in bone marrow polychromatic erythrocytes induced by the positive control, cyclophosphamide, in both sexes, with means and standard errors of 1.54% ± 0.23% and 2.60% ± 0.75% for the males and females, respectively.