ethynyl cyclopropane

Genetic toxicity: in vivo

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

Study period:

The study was performed between 22 July 1997 and 9 October 1997 .

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The procedures used were based on the recommendations of the following guidelines:
EEC (29 December 1992) Official Journal of the European Communities No. L 383 A (vol 3 5) : Methods for determination of toxicity, B 12 : Mutagenicity (Micronucleus test), p. 154.
OECD (February 1997) Proposal for updating guideline No. 474. Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test.
US EPA (1997) Toxic Substances Control Act Test Guidelines ; Title 40 Code of Federal Regulations Part 799

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-430-1

EC Name:

-

Cas Number:

6746-94-7

Molecular formula:

C5 H6

IUPAC Name:

ethynylcyclopropane

Details on test material:

- Name of test material (as cited in study report): EthynyI cyclopropane (SD 957)
- Physical state: Colourless liquid
- Analytical purity: 97-100 %
- Lot/batch No.: 3590-129-D4
- Expiration date of the lot/batch: January 1999
- Storage condition of test material: -20°C, under nitrogen and in the dark
- Intended use: Pharmaceutical intermediate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England
- Weight at study initiation: 140 to 149 g
- Fasting period before study: no data
- Housing: sexes separated, in suspended stainless steel cages
- Diet: standard laboratory rodent diet (Special Diet Services RM I (E) SQC expanded pellet) ad libitum
- Water: drinking water ad libitum
- Acclimation period: for a minimum period of five days prior to the start of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 12 to 20
- Photoperiod (hrs dark / hrs light): 12/12

The batch(s) of diet used for the study was analysed for certain nutrients, possible contaminants and microorganisms.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [corn oil]

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:

single oral treatment

Frequency of treatment:

single treatment

Post exposure period:

24 hours and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

five male and five female animals in the negative control, each of the test substance groups and the positive control group (24 hours);
five male and five female animals in the negative control and high level treatment groups (48 hours)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition, bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

Details of tissue and slide preparation:

Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from the negative control each of the test substance groups and the positive control group were sacrificed 24 hours after dosing. In addition five male and five female animals in the negative control and high level treatment groups were sacrificed 48 hours after dosing. (Additional animals not used in the preparation of bone marrow smears were killed at the 48 hour time point.) The animals were killed by cervical dislocation and both femurs were dissected from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was aspirated through a 21 g needle fitted to a plastic syringe and pooled in a total volume of 10 ml Hanks' balanced salts solution. The resulting cell suspensions were spun at 1000 rpm (150 x g) for 5 minutes. Each resulting cell pellet was resuspended in 2 ml of filtered foetal calf serum before being sedimented out using the MSE centrifuge. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976). Several smears were prepared from each femur. Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods, a modified Feulgen staining method is employed for the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

The slides were fixed and stained as described in the following schedule:
1. Fixed for 10 minutes in SLR grade methanol (Fisons M/3950/17)
2. Hydrolysed in Bouin's fluid at room temperature for 30 hours
3 . Washed three times in purified water (5 minutes per wash)
4. Stained in Schiff s reagent (BDH 19120) for one hour at room temperature
5 . Washed three times in purified water (5 minutes per wash)
6. Counter-stained for one minute in very dilute (approximately 0.02 g/1) aqueous Eosin yellowish (BDH 34197)
7. Washed for five minutes in purified water
8. Stained for 30 minutes in Mayer's Haemalum (BDH 35060) diluted I volume: 2 volumes purified water
9. Rinsed in purified water briefly
10. Briefly rinsed in running tap water
11 . Washed for 5 minutes in purified water
12. Air-dried
13 . Slides were mounted with coverslips using DPX mountant
14. The mountant was allowed to harden at approximately 37°C

NB All stains and Bouin's fluid were filtered immediately prior to use to remove particulate material. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear. Immature erythrocytes were identified by their slate-blue staining reaction with Haemalum while mature erythrocytes stain orange. Micronuclei were identified by the following criteria:
Large enough to discern morphological characteristics
Should possess a generally rounded shape with a clearly defined outline; most micronuclei appear spherical although more bizarre forms include crescent, almond and ring shapes
Should be deeply stained and similar in colour to the nuclei of other cells - not black
Should lie in the same focal plane as the cell
Lack internal structure, ie they are pyknotic
There should be no micronucleus-like debris in the area surrounding the cell
Micronuclei should be less than 1/4 the diameter of the erythrocyte but large enough to identify morphological and staining characteristics

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

Evaluation criteria:

see below

Statistics:

see below

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
No mortalities occurred in any group in any part of the study.
No clinical signs of toxicity were observed at any dose level of SD957 at any time after treatment. Results showed that a dose level of 2000 mg/kg, the standard limit dose level for the micronucleus test, was tolerated and this level was considered to be an appropriate maximum for use in the micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for use in the main test.


RESULTS OF DEFINITIVE STUDY
Chemical analysis
Chemical analysis results indicated that formulations of the test substance in the chosen vehicle were expected to be acceptable in terms of homogeneity and stability and that achieved concentrations for formulations used in the main micronucleus test were within acceptable limits.

Clinical signs and mortalities
No adverse clinical signs were obtained in any group.

Micronucleated immature erythrocyte counts (mie )
4 The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time [P>0 .01].
Cyclophosphamide caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes.

Micronucleated mature erythrocytes (mme)
The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Proportion of immature erythrocytes (% ie/ie + me)
The test substance failed to cause any significant decreases in the proportion of immature erythrocytes [P>0.01].
Cyclophosphamide caused slight but statistically significant decreases in the proportion [P<0.01].

Any other information on results incl. tables

The test substance did not cause any significant increase in the incidence of micronucleated immature erythrocytes or any significant decrease in the proportion of immature erythrocytes relative to the vehicle control. Therefore, it is concluded that SD957 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally at levels of up to 2000 mg/kg bodyweight in this in vivo test procedure.