O-isobutyl ethylthiocarbamate

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:

other: published data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Isobutyl alcohol is reagents used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC). Therefore, the health effects of Isobutyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC) .

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpropan-1-ol

EC Number:

201-148-0

EC Name:

2-methylpropan-1-ol

Cas Number:

78-83-1

Molecular formula:

C4H10O

IUPAC Name:

2-methylpropan-1-ol

Test material form:

liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Germany- Age at study initiation: 5-8 weeks- Weight at study initiation: mean: 26 g- Assigned to test groups randomly: [yes, under following basis: Male and female animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.]- Housing: in groups of 5 during the acclimation period, individually later on- Diet: ad libitum - Water: ad libitum- Acclimation period: 3-5 days ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24 °C- Humidity (%): 30-70 %- 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 insolubility 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: 5 g/100 ml; 10 g/100 ml and 20 g/100 ml- Amount of vehicle (if gavage or dermal): 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The substance to be administered per kg body weight was dissolved in olive oil and prepared immediately before administration.- The 500 mg/kg group was given 500 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 5 g/100 ml.- The 1000 mg/kg group was given 1000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 10 g/100 ml.- The 2000 mg/kg groups were given 2000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 20 g/100 ml.

Duration of treatment / exposure:

single application

Frequency of treatment:

single application

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP); vincristine sulphate (VCR)- Justification for choice of positive control(s): Both positive control articles (CPP and VPR) are well-defined clastogens and aneugens respectively.- Route of administration: orally or intraperitoneally- Doses / concentrations: 20 mg/kg bw (CPP) / 0.15 mg/kg bw (VCR)

Examinations

Tissues and cell types examined:

In general, 2000 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 cells were prepared from the bone marrow of two femora from animals either sacrificed 24 or 48 hours after dosing.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, all animals (male and female) survived treatment with 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline. As clinical signs only piloerection was observed.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 and samples of bone marrow were taken 24 hours and 48 hours after the treatment.DETAILS OF SLIDE PREPARATION:The bone marrow was prepared according to the method described by SCHMID, W.- The two femora were prepared by dissection and removing all soft tissues.-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 in about 50 µl 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. The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald 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 7.5% Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-BalsamMETHOD OF ANALYSIS:In general, 2000 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 micronucleiThe 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 micronucleiThe 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 targetIndividual 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 any of the intervals.- 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) wereprinted 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:

narcotic like state and piloerection

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY- Dose range: 2000 mg/kg- Clinical signs of toxicity in test animals: piloerectionRESULTS OF DEFINITIVE STUDYThe single oral administration of olive oil in a volume of 10 ml/kg body weight led to 1.5 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.1 ‰ after the 48-hour sacrifice interval. After the single administration of the highest dose of 2000 mg/kg body weight, 1.8 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.3‰ after 48 hours.In the two lower dose groups, rates of micronuclei of about 1.6 ‰ (1000 mg/kg group) and 1.5 ‰ (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.With 17.9‰ the positive control substance cyclophosphamide for clastogenicity, led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.With 67.3 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 8.0 ‰.The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.Thus, the test substance Isobutanol did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range.No inhibition of erythropoiesis induced by the treatment of mice with Isobutanol was detected the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results : negative Oral gavage dose of 500, 1,000 or 2,000 mg/kg of isobutanol did not have any chromosome-damaging (clastogenic) effect, and there were noindications of any impairment of chromosome distribution in the course of mitosis.
Isobutyl alcohol is reagents used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC). Therefore, the health effects of Isobutyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC) .