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Toxicological information

Genetic toxicity: in vivo

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
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): isobutanol
- Physical state: colorless liquid
- Analytical purity: 99.87%
- Purity test date: no data
- Lot/batch No.: date of manufacture: 32nd week number 1999, BASF Aktiengesellschaft
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean weight 26 g
- Assigned to test groups randomly: yes, assignment with an appropriate computer program
- Housing: in groups of 5 of the same sex in Makrolon cages, type MIII
- Diet (e.g. ad libitum): standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 3 - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- 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: insolubility of test substance in water, suitability of olive oil in the vivo micronucleus test as demonstrated by historical data
- Concentration of test material in vehicle: 5 g, 10 g, and 20 g/100 mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in olive oil immediately before administration. Concentrations were 5 g, 10 g, and 20 g TS/100 mL vehicle.
Duration of treatment / exposure:
Test substance was orally administered in one dose
Frequency of treatment:
single dose
Post exposure period:
24 h (all groups) and 48 h (vehicle controls, 2000 mg/kg group)
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
actual ingested
TS concentrations in vehicle were analytically tested and were found to be in the range of 97 - 105% of the nominal value
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
actual ingested
TS concentrations in vehicle were analytically tested and were found to be in the range of 97 - 105% of the nominal value
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
actual ingested
TS concentrations in vehicle were analytically tested and were found to be in the range of 97 - 105% of the nominal value
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substances: cyclophosphamide (CPP) and vincristine (VCR)
- Justification for choice of positive control(s): control substances are proven to induce micronuclei by a chromosome breaking effect and by spindle activity respectively.
- Route of administration: CCP oral, VCR intraperitoneal
- Doses / concentrations: 20 mg CPP/kg bw and 0.15 mg VCR/kg bw each dissolve in 10 mL purified water/kg bw.
- Number of animals for positive controls: 5 males and 5 females, subdivided between CPP (2 males/3 females) and VCR (3 males/2 females)

Examinations

Tissues and cell types examined:
bone marrow, polychromatic erythrocytes with and without micronuclei, normochromatic erythrocytes with and without micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: doses were selected on basis of a preliminary experiment. In a pretest for the determination of the acute oral toxicity, all animals (males and females) survived treatment with 2000 mg/kg bw which is set as limit dose according to OECD TG 474. As signs of toxicity only piloerrection was observed. In addition to 2000 mg/kg bw, doses of 1000 and 500 mg/kg bw were selected.

DETAILS OF SLIDE PREPARATION: bone marrow was prepared according to W. Schmidt (see study report for references). Shortly, bone marrow was prepared from the two femora by flushing it out of the diaphysis into a centrifuge tube with fetal calf serum. After mixing and centrifugation, one drop of the resuspended precipitate in FCS was placed onto clean microscopic slides. Smears were produced, dried in the air, and subsequently stained.
Slides were first stained in eosin and methylen blue solution, rinsed in purified water, and finally stained in 7.5% Giemsa solution. After additional rinsing and clarification in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) from each animal of every test group was investigated for micronuclei. The normochromatic erythrocates (NCE) were also scored. Slides were coded before microscopic analysis.
The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei
Number of monochromatic erythrocytes
Number of monochromatic erythrocytes containing micronuclei
Ratio of polychromatic to normochromatic erythrocytes
Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
Evaluation criteria:
Criteria for positive results in this test:
A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
The proportion of cells containing micronuclei exceeded both the values of the concurrent negative controls 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:
Statistical evaluation of data was carried out using the program system MUKERN (BASF AG).
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test (Lienert, Verteilungsfreie Methoden in der Biostatistic, Tafelband, Verlag Anton Hain, Meisenheim am Glan, 1975) for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used (statistic significance at p ≤ 0.05 and 0.01). The test was performed one-sided.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: single dose 2000 mg/kg bw
- Solubility: no sufficiently soluble in water, soluble in vehicle (olive oil)
- Clinical signs of toxicity in test animals: slight signs of toxicity were observed (piloerection)
- Evidence of cytotoxicity in tissue analyzed: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): ca. 2000/670; for all dose groups in the same range as that of controls
- Appropriateness of dose levels and route: yes

Any other information on results incl. tables

There are no relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control and the 3 dose groups or between the two sacrifice intervals (see attached tables taken from the report, file: Mous Micronucleus Study isobutanol results.pdf).

 

Clinical signs were only observed in the 1000 and 2000 mg/kg bw dose groups. Males and females revealed the same clinical signs. For the 1000 mg/kg bw dose group, piloerection was observed for all animals at 30 min and for some animals again at day one. All animals of the 2000 mg/kg bw dose group showed a narcotic-like state at 30 min after dosing but no symptoms from 90 min until day 1. On day 1, piloerection was noticed in all animals which could not be seen any more on day 2.

 

In feed, water and bedding no components were detected which could have interfered with the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the micronucleus test performed, isobutanol did not lead to any increase in the rate of micronuclei in polychromatic and normochromatic erythrocytes at doses of 500, 1000, and 2000 mg/kg bw..
Executive summary:

In a mouse bone marrow micronucleus test, 5 NMRI-mice per sex/dose were treated orally with isobutanol at doses of 0, 500, 1000, and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post treatment. The test substance was administered orally dissolved in olive oil in single doses.

 

There were signs of toxicity during the study (narcotic-like state shortly after exposure and later (1 day) piloerection in the 2000 mg/kg dose group, piloerection shortly after exposure in the 1000 mg/kg dose group). Isobutanol was tested at adequate doses. The positive controls induced the appropriate responses.

 

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose and any treatment time. Under the experimental conditions of the test, isobutanol had no chromosome damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (CMA/BASF 2000).

 

This study is classified as acceptable. It satisfies the requirement of EU Method B.12 and OECD Test Guideline 474 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) for in vivo cytogenetic mutagenicity data.