Isovaleric acid

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutan-1-ol

EC Number:

204-633-5

EC Name:

3-methylbutan-1-ol

Cas Number:

123-51-3

IUPAC Name:

3-methylbutan-1-ol

Details on test material:

- Name of test material (as cited in study report): 3-methylbutanol-1
- Substance type: aliphatic alcohol
- Physical state: liquid
- Analytical purity: 99.7%
- Purity test date: 1995-08-24
- Lot/batch No.: S18954

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 8 weeks
- Weight at study initiation: males 33-35.4 g; females 25-37 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in Macrolon cages type I
- Diet: Altromin TPF N1324 (pellets, poor in nitrosamines)
- Water: tap water ad libitum


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.25% Methocel K4M premium solution
- Justification for choice of solvent/vehicle: improved water solubility of the TS
- Concentration of test material in vehicle: 150 g/L
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no.: ZDP 33/97; released for toxicological investigations until August 29, 1997

Duration of treatment / exposure:

single dose

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
- Justification for choice of positive controls: significant increase of polychromatic erythrocytes expected as proof of proper function of the test system
- Route of administration: oral gavage
- Doses / concentrations: 19.75 mg/kg bw

Examinations

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on results (toxicity) of pilot study with 1000 and 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES:
24 and 48 hr post treatment

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were prepared from two femura of each animal, suspendend in fetal calf serum, wasshed, resuspended, and bone marrow smears were prepared from the resulting cell suspension. After 3 hours of air drying, the slides were stained according to a modified Giemsa­
staining method described by Gollapudi and Kamra (1979) using Giemsa's solution with Weise buffer solution and mounted in Entellan.

METHOD OF ANALYSIS:
Microscopic evaluation. A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250x). Round particles with about 1/20 - 1/5 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. They were differentiated from granules by thorough examination at different optical levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic.

For determination of the quotient of normochromatic to polychromatic erythrocytes, both erythrocyte stages were screened for micronuclei and counted separately up to a total of 1000 erythrocytes per animaL

Evaluation criteria:

As predetermined in the study protocol, the test materials are classified as mutagenic or non-mutagenic in this system according to the following rules:
A positive effect in this test system is defined by the occurrence of mean MN-PCE values of a treatment group which are statistically significantly higher than those of the actual negative control. A prerequisite for this is, however, that these values are above those predetermined as historical negative controls of our laboratory
An indispensable prerequisite for evaluating the results of such investigations is the occurrence of significant positive effects in the actual positive control group.
A test material showing no positive effect is defined as a non-mutagen in this test system. In this case the study is terminated.
If a positive effect in a single test group occurs (Le. dose-independently), a repeat ex­periment has to be considered. In case that no positive effects occur in that experiment the test material is defined as a non-mutagen. The single positive effect of the first experiment is interpreted as a randomly occurring event of no biological significance.
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 preferable.

Statistics:

Numerical calculations were made usin a softeware package.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: prone position, dyspnea, weight loss, but no mortality

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of NCE/PCE (for Micronucleus assay): (report, table 6)
Solvent, 24 h: 1.1 +/-0.3
Test substance, 24 h: 1.0 +/- 0.2
Test substance, 48 h: 1.1 +/- 0.1
CPA, 24 h: 1.0 +/- 0.2

- Appropriateness of dose levels and route: yes
- Statistical evaluation: yes

Any other information on results incl. tables

Clinical findings

 

Number of animals showing symptoms/number of treated animals per dose group

 

Symptoms	Dose 3-methylbutanol (mg/kg bw)
	0	1500
Dyspnea Prone position Titubation	0/10 0/10 0(10	16/20 15/20 5/20

 

Body weight:

A significant decrease in body weight was observed in animals at 1500 mg 3-methylbutanol/kg bw.

Micronucleated cells:

2000 polychromatic erythrocytes per animal were evaluated, 5 males and 5 females per group. The solvent control group values were all in the historical control range of the laboratory. Statistically increased number of erythrocytes with micronuclei (p<0.01) was established with cyclophosphamide. Of the groups receiving 3.methylbutanol, no significant increase in the micronucleus frequency was observed at 48 hours after treatment. At 24 hours, no increase was seen in males, whereas a weak increase (1.7 per 1000 cells with micronuclei; negative control: 0.5 per 1000 cells; p<0.02) was seen in females.

No increase was seen for the number of normochromatic cells with micronuclei.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
3-methylbutanol was not genotoxic in-vivo (mouse micronucleus test). The result can be interpolated to isovaleric acid because the alcohol is rapidly oxidised to the acid.

Executive summary:

3-methylbutanol was tested for its potential to induce chromosome damage (micronuclei) in mice. The study was conducted according to OECD 474 using NMRI mice (5 per sex and dose) and under GLP conditions. The animals received 1500 mg 3-methylbutanolkg bw by oral gavage; vehicle and positive controls (cyclophosphamide) were included. The dose was selected based on clear toxicity observed in animals at 2000 mg/kg bw, but not at 1000 mg(kg bw.

Clear signs of toxicity were seen (dyspnea, prone position in 75% of the animals; titubation in 25% of the animals) along with a significant body weight decrease. No increase of micronucleated polychromatic was noted in males and females at 24 and 48 hours after treatment, except a weak, yet statistically significant increase in females at 24 hours (1.7 per 1000 cells with micronuclei; negative control: 0.5 per 1000 cells; p<0.02).No increase was seen for the number of normochromatic cells with micronuclei. The vehicle and positive controls performed as expected. It was thus concluded that 3-methylbutanol did not induce micronuclei in-vivo (Utesch and Walter, 1999).

 

The study is considered to be valid and the result can be transferred to isovaleric acid since the alcohol is rapidly oxidised to acid in-vivo.