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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1973
Report Date:
1973

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
repeated administration, limited information about test item, animals were already killed 2 hours after colcemid application, only fifty metaphase spreads were scored per animal; repeated exposure instead of single dosing
GLP compliance:
not specified
Type of assay:
other: chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from a closed colony (random-bred)
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 280-350 g
- Housing: groups of 5
- Diet: 4 % fat diet
- Water: yes
- Acclimation period: 4-11 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
Duration of treatment / exposure:
5 days
Frequency of treatment:
daily one gastric intubation on 5 consecutive days
Post exposure period:
6 h after the last dose
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (experimental groups), 3 (negative control)
Control animals:
yes, concurrent vehicle
Positive control(s):
A positive control was not included for consecutive dosing, but single administration of Triethylene Melamine (TEM) was performed in the same study, yielding the expected results.

Examinations

Tissues and cell types examined:
cells of the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on acute oral toxicity results

TREATMENT, SAMPLING TIMES and SLIDE PREPARATION:
Four hours after the last compound administration, and two hours prior to killing, each animal was given 4 mg/kg of colcemid intraperitoneal in order to arrest the bone marrow cells in C-mitosis. Animals were killed by using CO2, and the adhering muscle and epiphysis of one femur were removed. The marrow "plug" was removed with a tuberculin syringe and an 18 gauge needle, aspirated into 5 ml of Hanks' balanced salt solution (BSS) in a test tube and capped. The specimens were centrifuged at 1500 RPM in a table-top centrifuge for 5 minutes, decanted, and 2 ml of hypotonic 0.5 % KCl solution was added with gentle agitation to resuspend the cells. The specimens were then placed in a 37 °C water bath for 20 minutes in order to swell the cells. Following centrifugation for 5 minutes at 1500 RPM, the supernatant was decanted and 2 ml of fixative (3:1 absolute methanol:glacial acetic acid) was added. The cells were resuspended in the fixative with gentle agitation, capped, and placed at 4 °C for 30 minutes. The specimens were again centrifuged, decanted, 2 ml of prepared fixative was added, and the cells were resuspended and placed at 4 °C overnight. The following day the specimens were again centrifuged, decanted and 0.3 - 0.6 ml of freshly prepared fixative was added to obtain a suitable density. The cells were resuspended and 2 - 3 drops of the suspension were allowed to drop onto a clean, dry slide held at 15 °C from the horizontal. As the suspension flowed to the edge of the slide, it was ignited by an alcohol burner and allowed to flame. Following ignition, the slides were allowed to dry at room temperature overnight. Duplicate slides were prepared. The slides were stained using a 5 % Giemsa solution (Giemsa buffer pH 7.2) for 20 minutes, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 minutes. The slides were then mounted using permount (Fisher Scientific) and 24 X 50 mm coverglasses. The preparations were examined using microscopes with brightfield optics and xenon light sources. These specimens were scanned and suitable metaphase spreads that were countable were then examined critically using oil immersion flat - field apochromatic objectives. The chromosomes for each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. They were recorded on the currently used forms and expressed as / percentages on the summary sheets. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.
Negative controls consisted of the vehicle in which the compound was administered, a positive control was not included, but single administration of Triethylene Melamine (TEM) was performed in the same study, yielding the expected results.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Mitotic indices were normal. The only aberrations observed were 3 % breaks in the negative controls and 2 % breaks in the high dose level. These were within normal limits.

Applicant's summary and conclusion

Conclusions:
The substance was found to be negative in the in vivo mammalian bone marrow chromosomal aberration test
Executive summary:

The genetic toxicity potential of the substance was evaluated in the in vivo chromosome aberration assay in a test perfomed similar to the OECD Guideline 475. Male rats were orally exposed to the substance (at 50, 500 and 5000 mg/kg bw/day) for 5 concecutive days. Prior to euthanasia, animals were treated with colcemid intraperitoneally in order to arrest the bone marrow cells in C-mitosis. Chromosome preparations were made from bone marrow cells and were stained, and metaphase cells were analysed for chromosomal aberrations. Duplicate slides were prepared. The preparations were examined using microscopes with brightfield optics and xenon light sources. The chromosomes for each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index. Negative controls (vehicle) run in paraller while a single administration of Triethylene Melamine (TEM) was performed in the same study, yielding the expected results.

Mitotic indices were normal. The only aberrations observed were 3 % breaks in the negative controls and  2 % breaks in the high dose level. These were within normal limits.

The substance was found to be negative in the in vivo mammalian bone marrow chromosomal aberration test