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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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-chloropropyne
EC Number:
210-856-9
EC Name:
3-chloropropyne
Cas Number:
624-65-7
Molecular formula:
C3H3Cl
IUPAC Name:
3-chloroprop-1-yne
Details on test material:
- Name of test material (as cited in study report): Propargylchlorid dests. ca. 70% in Toluol
- Test substance No.: 90/741
- Batch No.: Vers. 198/M
- Physical state: colorless to yellowish liquid
- Storage condition of test material: +4°C to +6°C

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, FRG.
- Weight at study initiation: 27.5 g (mean)
- Housing (week before study): Makrolon cages type M III, 5 animals separately according to sex
- Housing (from start of study): Makrolon cages type M I, Single housing
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland) ad libitum
- Water: bottles ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24
- Humidity (%): 30-70
- Air: Fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Concentration of test material in vehicle: 0.33 g/100 mL, 1 g/100 mL, 3 g/100 mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was emulsified in olive oil. All test substance formulations were prepared immediately before administration.
Frequency of treatment:
Single administration
Post exposure period:
Control, low- and mid-dose groups: 24 hours
High-dose group: 16, 24, and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
33, 100, 300 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
29, 87, 231 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, vincristine
- Route of administration: oral gavage (cyclophosphamide), intraperitoneally (vincristine)
- Doses / concentrations: the substances were dissolved in aqua dest: 20 mg/kg bw (cyclophosphamide), 0.15 mg/kg bw (vincristine)

Examinations

Tissues and cell types examined:
Bone marrow of two femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on a pretest for the determination of the acute oral toxicity, a dose of 300 mg/kg bw was selected as the highest dose in the present cytogenetic study. 100 mg/kg bw and 33 mg/kg bw were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes are evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 1000 polychromatic erythrocytes were also registered.
Evaluation criteria:
The following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.

- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve 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.

- Ratio of polychromatic to normochromatic erythrocytes
This ratio indicates an influence of the test substance specifically on the bone marrow.

- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may give an indication an the possible mode of action of the test substance i.e. a clastogenic or a spindle poison effect.
Statistics:
A statistical evaluation was not necessary to perform. The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: the acute oral toxicity deaths were observed down to a dose of 400 mg/kg body weight. 300 mg/kg body weight was survived by all animals but led to signs of toxicity such as irregular respiration and piloerection about 15 minutes after test substance treatment. After about 2 hours closed eyelids and, in few cases, apathy and squatting posture were additionally observed and the general state of the animals was poor.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Doses of 300 mg/kg body weight led to clinical signs such as irregular respiration and piloerection about 15 – 30 minutes after test substance treatment. After about 2-5 hours closed eyelids and, in few cases, apathy, abdominal position, diarrhoea and squatting posture were additionally observed and the general state of the animals was poor. Some of these symptoms could be observed two days after test substance administration. One animal was found dead the day after treatment. After the administration of 100 mg/kg or 33 mg/kg body weight irregular respiration and piloerection were found about 15-30 minutes which were partly be observed the day after treatment.
- Induction of micronuclei: no increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the solvent control value at any of the sacrifice intervals. Nor were large micronuclei (d ≥ D/4) observed either in the negative control group or in the three dose groups.
- Ratio of PCE/NCE (for Micronucleus assay): not determined.
- Statistical evaluation: A statistical evaluation was not performed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative