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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: Guideline study conducted in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted 1997)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Butandiolformal
Batch No. Tank Bu 17
Purity 99.7%
Physical state: colorless liquid
Storage: at RT

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
Designation: healthy male NMRI mice
Source: Charles River Deutschland GmbH
Mean body weight at test initiation: ca. 28.9 g
Housing during acclimatization: in Makrolon cages type MIII, in groups of 5 (3 - 5 days)
Housing during test period: individually, in Makrolon cages type Ml
Environmental conditions: air-conditioned rooms with central air conditioning, temperature range of 20 - 24°C, relative humidity range of 30 - 70%
Day/night rhythm:12 hrs/12hrs
Feed: Standardized pelleted feed (Kliba Haltungsdiaet, Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
Drinking: drinking water from bottles, ad libitum
Feed, drinking water and beeding analysis for contaminants: yes

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
purified water
Details on exposure:
Preliminary test for determination of the dosage levels:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed at 2000 mg/kg bw, which was the highest dose level recommended by the OECD Guideline. At 1750 mg/kg bw, all animals survived but suffered from signs of toxicity such as narcotic like state and piloerection which were observed in both males and females. Thus, only male animals were used for the cytogenetic investigations, and the selected dose levels were 437.5, 875.0 and 1750 mg/kg bw.
Main assay, administration way and application volumes:
The substance was administered to the mice by in 2 i.p. injections/dose.
The low dose group (i.e.437.5 mg/kg bw) received 10 ml/kg bw of a solution with a concentration of 4.375 g/100 ml.
The intermediate dose group (i.e. 875 mg/kg bw) received 10 ml/kg bw of a solution with a concentration of 8.75 g/100 ml.
The high dose group (i.e. 1750 mg/kg bw) received 10 ml/kg bw of a solution with a concentration of 17.5 g/100 ml.
After the administration of the vehicle, the test substance and the positiv controls, the animals were examined for any evident clinical signs of toxicity.
Duration of treatment / exposure:
The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment.
Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.
Frequency of treatment:
See above
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
437.5, 875.0 and 1750 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
5 animals/group were used
Control animals:
yes
Positive control(s):
As a positive control, 20 mg of cyclophosphamide (CPP)/kg bw or 0.15 mg of vincristine sulphate (VCR)/kg bw were used. Both control substances were dissolved in purified water, and were administered to male animals once, intraperitoneally, each in a volume of 10 ml/kg bw

Examinations

Tissues and cell types examined:
Following sacrifice, both femurs were removed from each animal; The preparation of bone marrow and staining were carried out according to the procedure described by Schmid W ( Mut. Res. 31: 9-15, 1975);

Microscopic evaluation, parameters:
In general, 2,000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes;
- Number of polychromatic erythrocytes containing micronuclei;
- Number of normochromatic erythrocytes;
- Number of normochromatic erythrocytes containing micronuclei;
- Ratio of polychromatic to normochromatic erythrocytes;
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter);

Microscopic evaluation, interpretation:
- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provided an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance;
- The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals showed the situation before test substance administration and served as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals;
- An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation;
- The size of micronuclei may give an indication on the possible mode of action of the test substance, i .e. a clastogenic or a spindle poison effect.
Details of tissue and slide preparation:
Staining:
The slides were stained in eosin and methylene blue solution for 5 minutes (May Gruenwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
Mounting:
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.
Evaluation criteria:
Acceptance criteria:
- The quality of the slides allows the identification and evaluation of a sufficient number of analyzable cells, i .e . >2000 polychromatic erythrocytes;
- The proportion of cells with micronuclei in negative control animals is within the normal range of the historical control data;
- The two positive control chemicals induce a significant increase in the number of cells containing small and large micronuclei.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes is observed;
- The proportion of cells containing micronuclei exceed both the values of the concurrent negative control range and the negative historical control range;
The test substance is generally considered negative in this test system if:
- There is no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies;
- The frequencies of cells containing micronuclei is within the historical control range.
Statistics:
- The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG);
- The number of micronuclei in polychromatic erythrocytes was analyzed;
- A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
1,3 Dioxepan did not lead to any increased micronuclei rate. The number of normochromatic or polychromatic erythrocytes with small micronuclei was within vehicle control range. No large micronuclei were observed.
Toxicity:
yes
Remarks:
A slight inhibition of erythropoiesis, induced by the treatment of mice with 1,3-dioxepan was detected at 1750 mg/kg bw.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MICROSCOPIC EVALUATION:
Negative/vehicle control:
The two intraperitoneal administrations of water in a volume of 10 ml/kg bw led to 1.9 per mille polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
1,3 Dioxepan, 437.5 mg/kg bw:
A micronuclei rate of about 2.0 per mille was detected after a sacrifice interval of 24 hours.
1,3 Dioxepan, 875 mg/kg bw:
A micronuclei rate of about 1 .4 per mille was detected after a sacrifice interval of 24 hours.
1,3 Dioxepan, 1750 mg/kg bw:
After the two i.p. injections of the highest dose of 1750 mg/kg bw, 2.1 per mille polychromatic erythrocytes containing micronuclei were found after 24 hours.
Positive control (CPP, 20 mg/kg bw):
As expected, cyclophosphamide resulted in 19 .3 per mille of polychromatic erythrocytes containing exclusively small micronuclei.
Positive control (VCR, 0.15 mg/kg bw):
As expected, VCR resulted in 58.7 per mille of polychromatic erythrocytes containing micronuclei, with a distinct amount of large micronuclei (4.6 per mille).

The number of normochromatic erythrocytes containing micronuclei was quite similar in all groups.

A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at the dose level of 1750 mg/kg bw. This observation is supporting the physiological relevance of the study, since bioavailability in the bone marrow could be demonstrated.

CLINICAL EXAMINATIONS
The two intraperitoneal administrations of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.
The administration of the test substance led to evident signs of toxicity (e.g. piloerection, squatting posture, droged state in the high dose) .
Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0 .15 mg/kg body weight caused any evident signs of toxicity.

Applicant's summary and conclusion