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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 5, 1985 - April 10, 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus induction

Test material

Constituent 1
Chemical structure
Reference substance name:
3,6-dioxaoctamethylenediamine
EC Number:
213-203-6
EC Name:
3,6-dioxaoctamethylenediamine
Cas Number:
929-59-9
Molecular formula:
C6H16N2O2
IUPAC Name:
3,6-dioxaoctamethylenediamine
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Appearance: colourless liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor; 5601-49-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in receiving container
- Stability under test conditions: There was no apparent change in the physical state of the test and control articles during the assay.
- Solubility and stability of the test substance in the solvent/vehicle: soluble and stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted to dose concentration with distilled water

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: 7.5 weeks
- Weight at study initiation: Males: 26-32 grams; Females: 23-28 grams
- Assigned to test groups randomly: yes
- Fasting period before study: No data
- Housing: By dose group, by sex, 5 per cage, standard stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum, Purina Certified Lab Blox
- Water (e.g. ad libitum): ad libitum, fresh tap water
- Acclimation period: No data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Solubility
- Concentration of test material in vehicle: 125 mg/kg
- Lot/batch no. (if required): 6-246
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test article was weighed and diluted with distilled H2O. Good solutions were obtained and maintained at all levels evaluated. The positive control was dissolved in distilled water and administered at 10mlkg at a dose of 0.05 mg/kg of body weight. The test article solutions and positive control solutions were prepared fresh and dosed within two hours of preparation.
Duration of treatment / exposure:
Single dose, intraperitoneal
Frequency of treatment:
Once
Post exposure period:
Sampled at 30, 48 and 72 hours
Doses / concentrations
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females per group (3 groups)
Control animals:
yes
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): Not specified
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.05 mg/kg

Examinations

Tissues and cell types examined:
Stained micronuclei of femur bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finding study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 30, 48, and 72 h after dosing

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed with 0.2 ml bovine serum, into 1 ml additional serum, and centrifuged at 1000 rpm for 5 min. The supernatant was drawn off, and a drop of the stirred cell button was pulled across a slide at a 45 degree angle. The slides were quick-dried at approximately 56 degrees C, dipped in absolute methanol, and allowed to air-dry overnight. The slides were stained for 29 minutes in 5% Giemsa working solution, and rinsed twice. The first rinse was distilled water adjusted to pH 4-4.5, and the second was distilled water adjusted to pH 7.0. Slides were then dried on a slide warmer at 56 degrees C, cleared in xylene for 5 minutes, and mounted in Permount with a cover glass.

METHOD OF ANALYSIS: 1000 polychromatic eurythrocytes per animal were counted for the presence of micronuclei. The data were expressed as a ratio of micronucleated polychromatic eurythrocytes to bormal polychromatic eurythrocytes.
Evaluation criteria:
Assessment of the test substance as positive is based upon its ability to induce a statistically significant increase in micronucleated polychromatic eurythrocytes as compared to negative control.
Statistics:
The test substance is considered as positive based upon its ability to induce a statistically significant increase in micronucleated polychromatic eurythrocytes as compared to negative control, via one tailed t test. Significance was judged at p

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 166-5000 mg/kg body weight
- Clinical signs of toxicity in test animals: Mortality observed at all doses by 72 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): Mean 1.66

Applicant's summary and conclusion

Conclusions:
The test substance was found to be negative in the micronucleus test at a dose level of 125 mg/kg in a single intraperitoneal dose, with sacrifice times of 30, 48, and 72 hours. Therefore, the test substacne is not to be classified as mutagenic, according to the CLP Regulation
Executive summary:

In a preliminary dose-rage-finding, the test article ws administred intraperitoneally to 6 groups (2 males and 2 females per group) of CD-1 mice at dose levels of 166 / 300 / 500 / 1666 / 3000 and 5000 mg/kg of body weight..

Total mortality occured at the four highest dose levels. In the lowest two dose groups one male per group died and signs were observed at all levels.

Due to the high mortality and severity of signs in the study, 125 mg/kg was seleceted as the dose for the micronucleus test as an estimate of the maximum tolerated dose.

In the micronucleus test, three groups of ten animals (5 males and 5 females/group) were given single doses by intraperitoneal injection at 125 mg/kg and sacrified at 30 / 48 and 72 hours.

Similar groups, dosed with the posiitve control, triethylenemelanine (TEM) and negatice control, distilled water, were evaluated concurrently.

Slides were prepared from the bone marrow of the femur and stained. Coded slides were scored for the number of polychromatic erythrocytes per 1000 erythrocytes per animal was determined.

The results for the test item were negative in the micronucleus test at a dose level of 125 at all of the time intervals evaluated. These findings are based upon the inability of the test article to produce a statistically significant increase in the number of micronuclei in 1000 polychromatic erythrocytes per animal in the treated groups versus the negactive control group.