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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: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines, and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Only minor deviations, see below
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C-121 (code for 1,3-dioxolane)
- Physical state: liquid
- Analytical purity: 99.9% (statement)
- Lot/batch No.: not reported
- Expiry date: March 28, 1990

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not reported
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 24-36 g, females 21-33 g.
- Fasting period before study: no
- Housing: Group-housed, up to 5 per cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-80°F (equivalent to 20-27°C).
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: not exactly reported, but between October 2, 1989 and December 28, 1989

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil (10 mL/kg bw) for dioxolane, water for triethylenemelamine (positive control).
Details on exposure:
The dose levels of dioxolane in the micronucleus test were 525, 1050 and 2100 mg/kg bw, the highest dose representing 80% of the LD50. In a toxicity pretest (5 M and 5 F per dose, dose levels 1576, 2048, 2663, 3462 and 4500 mg/kg w, 7-day post-exposure observation), the LD50 following intraperitoneal adminstration was established to be 2603 mg/kg bw.
Duration of treatment / exposure:
Single dose by intraperitoneal (IP) injection
Frequency of treatment:
Once
Post exposure period:
Bone marrow was collected from groups of five treated animals af 24, 48 or 72 hours.
No. of animals per sex per dose:
Five males and five females per harvest time
Positive control(s):
Triethylenemelamine (dissolved in water) by IP injection at 0.25 mg/kg bw

Examinations

Tissues and cell types examined:
At each sacrifice time, bone marrow was collected from each animal and bone marrow cells were isolated for microscopic examinations.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the highest dose represented 80% of the LD50

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): see above

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, bone marrow was aspirated from the femurs. The bone marrow was suspended in fetal bovine serum and the bone marrow cells were separated by centrifugation. The bone marrow cells were spread on slides (4/animal) and the slides were fixed in methanol, stained with May-Gruenwald-Giemsa and mounted.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes (PCEs) were scored for the presence of micronuclei. The proportion of PCEs to total erythrocytes and the number of micronucleated normocytes in the field of 1000 PCEs was enumerated.

OTHER: -
Evaluation criteria:
Reported criteria were: “The test article is considered to induce a positive response if a treatment-related increase in micronucleated PCEs is observed relative to the vehicle control (p ≤0.05, Kastenbaum-Bowman tables). The positive response must be dose-dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time only, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.”
Statistics:
Kastenbaum-Bowman tables, which are based on the binomial distribution.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at highest dose only: 23/35 M and 22/35 F died, toxicity also clear by reduced PCE as % of total eryhrocytes. No mortality and signs of toxicity at lower dose levels.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Induction of micronuclei (for micronucleus assay): the numbers of micronucleated PCEs per 1000 PCEs were acceptable and low in the solvent control (0.0-0.4); the numbers of micronucleated PCEs per 1000 PCEs in all dioxolane treatments were comparable to those in the solvent control (0.0-0.6; not statistically significantly different from control); the positive control gave the expected statistically and biologically significant response (18.4 and 20.8 micronucleated PCEs per 1000 PCEs for M and F respectively).
- Ratio of PCE/NCE (for micronucleus assay): a reduction relative to the solvent control was apparent at the highest dose level only, indicating the required toxicity.
- Appropriateness of dose levels and route: The route is appropriate in case internal exposure to 1,3-dioxolane is relevant (which will be the case for inhalation exposure). The highest dose level caused clear toxicity, hence the doses were appropriate.
- Statistical evaluation: none of the effects (micronucleated PCEs/1000PCEs) in dioxolane treated groups were statistically significantly different from the control, the response of the positive control was statistically significant (all tested at the 5% level).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In an in vivo mouse micronucleus test, performed under GLP and according to OECD 474, 1,3-dioxolane did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male and female mice and was therefore concluded to be negative . The positive and negative controls fulfilled the requirements for a valid test.
Executive summary:

An in vivo mouse micronucleus test was performed under GLP and according to OECD 474 on 1,3-dioxolane in male and female ICR mice. The test substance was dissolved in corn oil and administered by single intraperitoneal injection at dose levels of 525, 1050 and 2100 mg/kg bw. The positive control triethylenemelamine was dissolved in water and administered by single intraperitoneal injection at 0.25 mg/kg bw. At sacrifice times of 24, 48 and 72 hours, bone marrow was collected from five animal per sex and bone marrow cells were isolated for microscopic examinations (for postive controls 24 hour harvest time only). The highest dose caused clear toxicity (mortality and reduced PCE as percentage of total eryhrocytes. No mortality and signs of toxicity were observed at lower dose levels. The numbers of micronucleated PCEs per 1000 PCEs were acceptable and low in the solvent control (0.0-0.4). The numbers of micronucleated PCEs per 1000 PCEs in all dioxolane treatments were comparable to those in the solvent control (0.0-0.6; not statistically significantly different from solvent control). The positive control gave the expected statistically and biologically significant response (18.4 and 20.8 micronucleated PCEs per 1000 PCEs for M and F respectively). The positive and negative controls fulfilled the requirements for a valid test, and 1,3-dioxolane was concluded to be negative .