Sodium butanolate

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting 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:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Butan-1-ol

EC Number:

200-751-6

EC Name:

Butan-1-ol

Cas Number:

71-36-3

Molecular formula:

C4H10O

IUPAC Name:

butan-1-ol

Details on test material:

- Name of test material (as cited in study report): n-butanol
- Substance type:
- Physical state: liquid
- Analytical purity: 99.9 %
- Lot/batch No.: Container 10
- Stability under test conditions: The stability of the test substance throughout the study period has been verified by reanalysis
- Storage condition of test material: Room temperature (N2-conditions)

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
- Age at study initiation: 5-8 weeks
- Weight at study initiation: mean: 26.9 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Fasting period before study:
- Housing: groups of 5 seperately according to sex in Makrolon cages, type MIII before the start of the study (acclimation period) and individually during the study in Makrolon cages, type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ca. one week


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [olive oil]
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, 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: 500, 1000 and 2000 mg butan-1-ol in a total of 10 ml
- Amount of vehicle (if gavage or dermal): 10 ml including test substance

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.
The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.

Duration of treatment / exposure:

single oral application

Frequency of treatment:

single oral application

Post exposure period:

24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 or 10

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide; vincristine sulphate
- Justification for choice of positive control(s): cyclophosphamide induces clastogenicity, vincristine sulphate induces spindle poison effects
- Route of administration: cyclophosphamide: oral; vincristine sulphate: intraperitoneally
- Doses / concentrations: cyclophosphamide: 20 mg/kg bw; vincristine sulphate: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 1000 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also registered.
In addition, the number of normochromatic erythrocytes with micronuclei and the ratio of polychromatic to normochromatic erythrocytes were determined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline No. 474 were survived by all animals, but led to evident signs of toxicity such as abdominal position, irregular respiration, staggering, squatting posture and narcotic like state; the general state of the animals was poor. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The animals were treated once at a 24-hour interval and samples of bone marrow were taken 24 hours and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Preparation of the bone marrow:
- The two femora were prepared from the animals, and all soft tissues were removed.
- 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 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended with 50 pl fresh FCS .
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges; the preparations were dried in the air and subsequently stained.
Staining of the slides:
- The slides were stained in eosin and methylene blue solution for 5 minutes (May Gruenwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 15 minutes.
- After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored.
The following parameters are 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 shows 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.
An alteration of this ratio indicates that the test substance actually reached the target.
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- 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 on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at the 24 hour interval.
- 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 generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- 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 Aktiengesellschaft).
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 for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p<0 .05, ** for p<0 .01) were printed with the group means in the tables. This test was performed one-sided.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The administration of the test substance led to evident signs of toxicity in the highest dose group of 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

According to the results of the present study, the single oral administration of butan-1-ol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance butan-1 -ol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative