Prop-2-yn-1-ol

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:

02.10.1989 - 05.10.1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Prop-2-yn-1-ol

EC Number:

203-471-2

EC Name:

Prop-2-yn-1-ol

Cas Number:

107-19-7

Molecular formula:

C3H4O

IUPAC Name:

prop-2-yn-1-ol

Details on test material:

Test substance:
- Name of test material (as cited in study report): Propargylalkohol (1.4-Dicyanobutan)
- Molecular formula: C3H4O
- Molecular weight: 56.1
- Physical state: colourless solution (liquid)
- Analytical purity: 99.4 %
- Impurities (identity and concentrations): 0.48 % formaldehyde; 0.03 % water
- Lot/batch No.: Partie B 463; 2111 88/10, Abl.-Nr. 477/88/R
- Storage condition of test material: dark at 20°C
- Other: Certificate of analysis: BASF ZSU dated 12-29-88

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males x= 29.1 g (26 - 32 g); females: x= 23.3 g (20 - 28 g)
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet: rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours


IN-LIFE DATES: From: 02.10.1989 To: 05.10.1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test compound dilutions were prepared fresh each day. 175 mg Propargylalkohol were weight in a 25 mL flask, mixed with deionized water and topped up to the calibration mark. A solution was formed.
For the Endoxan stock solution used as positive control item, 5 mL distilled water were added to 100 mg Endoxan in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 mL of the 2 % stock solution were mixed with 6 mL distilled water.

The test compound was administered orally by gavage to male and female mice.

Duration of treatment / exposure:

once

Frequency of treatment:

single application

Post exposure period:

The animals were killed 24, 48 or 72 hours after application

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 70 mg/kg bw
Basis:
nominal conc.
dose volume: 10 mL/kg bw

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan
- Route of administration: orally by gavage
- Doses / concentrations: 50 mg/kg bw (10 mL/kg)

Examinations

Tissues and cell types examined:

Bone marrow (femoral), erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 100 mg Propargylalkohol/kg bw has caused partial lethality in female mice. The highest sublethal dose of 70 mg/kg bw was selected for the main study.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application.


DETAILS OF SLIDE PREPARATION:
For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded.One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours. The staining procedure was:
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan


METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).

Evaluation criteria:

The test is evaluated as positive if the number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes is statistically significantly increased in comparison with the control and biological relevance (i.e. micronucleus rate not within the historical control rate) is given.

Statistics:

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

at sublethal dose (oral administration of 70 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at sublethal dose (oral administration of 70 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 50, 70, 100 mg/kg bw Propargyl alcohol, Number of animals used per dose: 3 males and 3 females
- Clinical signs of toxicity in test animals:
- 50 mg/kg bw: narrowed palpebral fissures, reduced spontaneous activity and uncoordinated galt
- 70 mg/kg bw: narrowed palpebral fissures, reduced spontaneous activity and uncoordinated galt
- 100 mg/kg bw: lacrimation, narrowed palpebral fissures, reduced spontaneous activity, uncoordinated galt and gasping respiration
- Lethality rate 50 mg/kg bw : 0/3 males, 0/3 females
- Lethality rate 70 mg/kg bw: 0/3 males, 0/3 females
- Lethality rate 100 mg/kg bw: 0/3 males, 1/3 females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Only the female mice of the 24 and 72 hours killing times showed a very small but statistically significant increase in the number of micronucleated polychromatic erythrocytes. The increase was within the normal range of the negative control values and therefore considered as of no toxicological significance.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by Propargyl alcohol.
- Appropriateness of dose levels and route: dose level and route of exposure were appropriate
- Statistical evaluation: Wilcoxon test (paired, one-sided, increase)

Any other information on results incl. tables

For further details on results see attached background material.

Applicant's summary and conclusion

Executive summary:

In a NRMI mouse bone marrow micronucleus assay 5 animals/sex/dose were treated orally by gavage with Propargyl alcohol (99.4% a.i.) as aqueous solution at doses of 0 and 70 mg/kg bw (the vehicle was water). The animals were treated once with the test compound and were killed 24, 48 and 72 hours after administration. Subsequently, bone marrow cells were harvested at 24, 48 and 72 hours post-treatment. Propargyl alcohol was tested at an adequate dose (based on a preliminary study). Only the females of the 24 and 72 hours killing times showed a very small but statistically significant increase in the number of micronucleated polychromatic erythrocytes. The increase was within the normal range of the negative control values and therefore considered as of no toxicological significance. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values. The positive control induced the appropriate response. The reuslts demonstrate that Propargyl alcohol is not mutagenic in the micronucleus test. This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.