Registration Dossier

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 based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
20 March 1997 - 09 Sept 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations For justification of read across please refer to section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
Institute of Toxicology, Merck KGaA
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Methylbutanol-1 (BG-Nr. 95), Isoamylalkohol
- Lot/batch No.: S18054
- Analytical purity: 99.7 % (GC)

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 8 weeks
- Weight at study initiation: mean 35 g (males), 26 g (females)
- Assigned to test groups randomly: yes, under following basis: provided by the SAS program TXS750 developed by Corporate Biometrics, Merck
KGaA, Darmstadt
- Housing: individually in Makrolon cages type 1(floor area: 21 x 10 cm, height: 13 cm) on softwood chippings
- Diet (e.g. ad libitum): Altromin standard diet TPFR N 1324 (10 mm pellets, poor in nitrosamines) from Altromin; ad libitum
- Water (e.g. ad libitum): tap water from Makrolon drinking bottles (water source: Südhessische Gas- und Wasser AG, Darmstadt, Germany and waterworks Merck KGaA, Weiterstadt, Germany); ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 58 - 74
- Atmospheric pressure: 761 - 762.5 mm Hg
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.25% aqueous Methocel K4M premium
- Concentration of test material in vehicle: 150 mg/ml
- Amount of vehicle (if gavage): 10 ml
Details on exposure:
Dose selection rationale:
The highest 3-Methylbutanol-1 (BG-Nr. 95) dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments, 2 male and 2 female mice treated orally with 1500 mg 3-Methylbutanol-1 (BG-Nr. 95) / kg bw, showed clear toxic effects ( dyspnea, prone position, titubation and a loss in body weight) but no mortality. At the next higher dose (2000 mg/kg bw) extremly strong and at the next lower dose (1000 mg/kg bw) only weak toxic symptoms were detected. For these reasons, the dose of 1500 mg 3-Methylbutanol-1 (BG-Nr. 95) / kg bw was selected as the highest dose for both, the male and female mice in the main study of this investigation.
Duration of treatment / exposure:
single
Frequency of treatment:
once
Post exposure period:
24 and 48 hours, respectively
Doses / concentrations
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 19.75 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes from 2 femurs of each animal
Details of tissue and slide preparation:
SAMPLING TIME (in addition to information in specific fields): 24 and 48 hours after administration of the test substance, respectively. For the positive and negative control groups, the preparation time was 24 hours after start of the treatment.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were stained with Giemsa's solution. For microscopic investigation one slide from each animal preparation was coded.

METHOD OF ANALYSIS:
The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micronucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.
Evaluation criteria:
A test material is defined as mutagenic in this system if dose-related or single, reproducible (in independent experiments) positive effects occur. Establishment of dose-dependent effects of the test material is deemed preferable.
Statistics:
Pairwise comparison:
Each treatment group was compared to the negative control. For comparisons the exact Fisher-Pitman permutation test (Leimer, 1991) was used against onesided alternatives.
Multiple test procedure:
As there was more than one treatment group, the p-values were considered jointly to maintain an error rate (multiple level of significance) of a = 5 %. For this purpose the Bonferroni-Holm multiple test procedure was used (Holm, 1979). The positive control (cyclophosphamide) was not included in Holm's procedure. It was compared with the control separately at a level of 5 %.

Leimer I, Peil H and Ellenberger J (1991). Statistical Analysis of the Micronucleus Test with the Fisher-Pitman Permutation Test. In: Hothorn L., ed., Statistical Methods in Toxicology, Springer, 20 - 24.
Holm S (1979). A simple sequentially rejective multiple test procedure. Scand. J. Statist. 6: 65 - 70.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In a preceding range finding test with 2 male and 2 female mice, the dose of 1500 mg/kg bw was severely toxic, however, no mortality occurred.
Vehicle controls validity:
valid
Negative controls validity:
other: The negative control (solvent) values were all in or very close to the expected range predetermined as historical controls of the laboratory.
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No relevant treatment-related variation was observed.
- Clinical signs of toxicity: At 1500 mg/kg bw the following clinical signs were noted: dyspnoea (16/20), prone position (15/20) and titubation (5/20). The animals of the solvent control group showed no abnormalities.
- Body weight: A relevant treatment-related decrease in body weight was observed at the 3-Methylbutanol-1 (BG-Nr. 95) doses tested in male and female animals.

Any other information on results incl. tables

48 hours after administration of the test material, no significant increase in the micronucleus frequency has been detected in the main study as compared to the negative control group. 24 hours after start of the treatment, no increase in the micronucleus frequency was seen in the male group either. Micronucleus frequency of the 24 hour female group was weakly (0.17 % cells with micronuclei, negative control: 0.05 %) and statistically significantly (p = 0.02) increased.

The obtained mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE (MN-PCE) are presented below:

Group

MN-PCE (%) in males

MN-PCE (%) in females

A

0.08

0.05

B

0.15

0.17 (p0.05)

C

0.18

0.08

D

0.99 (p0.01)

0.77 (p0.01)

For the number of normochromatic cells with micronuclei, no increase was observed.

Since the observed positive effect in the female animals (24 hrs) was only minimal, it was considered to be not relevant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative