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Diss Factsheets

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 - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

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-methylbut-2-en-1-ol
EC Number:
209-141-4
EC Name:
3-methylbut-2-en-1-ol
Cas Number:
556-82-1
Molecular formula:
C5H10O
IUPAC Name:
3-methylbut-2-en-1-ol
Details on test material:
- Name of test material (as cited in study report): Prenol
- Test substance No.: 00/0274-1
- Physical state: Colorless liquid
- Analytical purity: 99.1 area %
- Date of manufacture: 10 Apr 2000
- Lot/batch No.: Ch.00/18, Abl. Nr. 56-1706
- Stability under test conditions: The stability of the test substance throughout the study period was verified by reanalysis. Thereby the stability of the test substance at room temperature in the vehicle olive oil was determined analytically.
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany.
- Age at study initiation: 5 - 8 weeks
- Mean weight at study initiation: 28 g
- Housing:individually
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water from bottles, ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil;
- Justification for choice of solvent/vehicle: Olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 125 mg/kg body weight: 1.25 g/100 ml; 250 mg/kg body weight: 2.5 g/100 ml; 500 mg/kg body weight: 5.0 g/100 ml.
- Amount of vehicle: 10 ml/kg body weight
The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the 1 st administration.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was dissolved in olive oil and prepared immediately before administration.
Duration of treatment / exposure:
- vehicle control and the dose groups: two intraperitoneal injections with a 24-hour interval, samples of bone marrow were taken 24 hours after the last treatment.
- positive control groups: intraperitoneal injection once, samples of bone marrow were taken after 24 hours.
Frequency of treatment:
- vehicle control and the dose groups: twice at a 24 hour interval;
- positive control groups: once
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250 and 500 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP);
- Justification for choice of positive control(s): clastogenic effects
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg cyclophosphamide in 10 ml test solution

vincristine sulphate (VCR);
- Justification for choice of positive control(s): aneugenic effects
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.15 mg vincristine in 10 ml test solution

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Due to a pretest for the determination of the acute-intraperitoneal toxicity (for details see results) a dose of 500 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 250 mg/kg and 125 mg/kg body weight were administered as further doses.

SAMPLING TIMES:
24 hrs after the last treatment samples of bone marrow of the 2 femora were taken and prepared.  Preparation of the bone marrow: according to the method of Schmidt (1976 and 1977) and Salamone et al. (1980).

DETAILS OF SLIDE PREPARATION:
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution),. They were finally stained in 7.5% Giemsa solution for 15 minutes.

METHOD OF ANALYSIS:
- Microscopic evaluation
2,000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
• Ratio of polychromatic to normochromatic erythrocytes
• Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D= cell diameter)

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.

An alteration of the ratio of polychromatic to normochromatic erythrocytes indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.

The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown were rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.

- Clinical examinations
After the administration of the vehicle, the test substance and the positiv controls, the animals were examined for any evident clinical signs of toxicity.

- Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2000 polychromatic erythrocytes.
• The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
• The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of the historical control data.
Evaluation criteria:
The test chemical is considered positive if the following criteria are met:
• A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.
• The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.

A test substance is considered negative if:
• There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
• The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG).
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test (one-sided) for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 750 mg/kg body weight
- Clinical signs of toxicity in test animals:
• deaths were observed down to a dose of 750 mg/kg body weight.
• 500 mg/kg body weight were survived by all animals but led to evident signs of toxicity such as gasping respiration, abdominal position, staggering, squatting posture and the general state of the animals was poor, however, showing no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the cytogenetic investigations.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of Prenol.
- Ratio of PCE/NCE : A slight inhibition of erythropoiesis, determined from the PCE/NCE ratio was detected at 250 and 500 mg/kg bw. The vehicle and the the positive control substances, CPP and VCR, caused no evident signs of toxicity.

- Statistical evaluation: The administration of the test substance did not lead to any statistical significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

• Control: The two intraperitoneal administrations of olive oil in a volume of 10 ml/kg body weight led to 0.8‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
• 500 mg/kg body weight: 1.5‰ polychromatic erythrocytes containing micronuclei were found after 24 hours.
• 250 and 125 mg/kg body weight: rates of micronuclei of about 0.8‰ (250 mg/kg group) and 1.1‰ (125 mg/kg group) were detected.
• Positive control, cyclophosphamide: With 16.7‰ the positive control for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei (p < = 0.01).
• Positive control vincristine: With 89.9‰, the positive control for induction of spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes (p < = 0.01) containing micronuclei with the expected amount of large micronuclei, i.e. 19.0‰.

• The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.

Applicant's summary and conclusion