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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
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:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Methylpropan-1-ol
- Analytical purity: > 99 %

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: ca. 24-29 g
- Assigned to test groups randomly: [yes, under following basis: The animals were assigned to the cages according to a random list which had been provided by the SAS program TXS750 developed by Corporate Biometrics, Merck KGaA, Darmstadt.]
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 °C
- Humidity (%): 58-74 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: [0.25% aqueous Methocel® K4M premium]
- Justification for choice of solvent/vehicle: none given
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered orally by gavage in this investigation. Dosing of the animals of different groups was staggerred over one to three day intervals to allow for sufficient sample preparation time.
Duration of treatment / exposure:
single oral application
Frequency of treatment:
single oral application
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males at 500 mg/kg bw; 5 females at 1500 mg/kg bw
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): none given
- Route of administration: oral
- Doses / concentrations: 30 mg/10 ml orally

Examinations

Tissues and cell types examined:
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 difterent optical levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest 2-Methylpropanol-1 (BG-Nr. 96) dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments with the doses of 1500 and 1000 mg/kg bw in a total of seven male mice, mortality occurred in three animals. Application of the dose of 2000 mg/kg to female mice resulted in extremely toxic symptoms. Male and female mice treated orally with 500 and 1500 mg 2-Methylpropanol-1 (BG-Nr. 96) / kg bw, respectively, showed clear toxic effects (dyspnea, incomplete eyelid closure and a weak loss in body weight) but no mortality.
For these reasons, the doses of 500 and 1500 mg 2-Methylpropanol-1 (BG-Nr. 96) / kg bw was selected as the highest dose for the male and female mice in the main study of this investigation, respectively.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
As proposed in the EEC Directive and in the OECD Guideline, the animals were treated with 2-Methylpropanol-1 once. The test material was administered orally by gavage in this investigation. Dosing of the animals of different groups was staggered over one to three day intervals to allow for sufficient sample preparation time. The mice were killed 24 and 48 hours after administration of the test substance for preparation of the bone marrow cells from the two femora.

DETAILS OF SLIDE PREPARATION:
Immediately after the mice had been killed by C02, two femora of each animal were dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum with the aid of a syringe, and suspended in the serum. After centrifugation for 5 min at 150 x g, the sediment was then resuspended in fetal calf serum 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 using Giemsa's solution with Weise buffer solution and mounted in Entellan.

METHOD OF ANALYSIS:
In addition to the randomized distribution of the animal numbers to the groups, one slide of each animal was coded before microscopic evaluation.
The code numbers were supplied by a SAS program TXS750 developed by Corporate Biometrics, Merck KGaA, Darmstadt.
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.
Normochromatic erythrocytes with micronuclei, however, observed during scoring were registered also. Thus, based on this value and on the quotient, the number of micronucleated normochromatic erythrocytes per 1000 could be extrapolated. After termination of the microscopic evaluation, the data were loaded into the DATATOX system (Instem).
Evaluation criteria:
The primary parameter for evaluation of the results of this test system is the number of micronucleated, polychromatic erythrocytes (MN-PCE). This number is increased by treatment of cells with mutagenic test materials as compared to the respective negative controls.
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 (i .e. 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:
Descriptive statistics:
For all groups mean values were calculated of the following parameters:
PCE -number of polychromatic erythrocytes
MN-PCE - number of micronuclei-containing cells/1000 PCE/animal
NCE -number of normochromatic erythrocytes
MN-NCE - number of micronuclei-containing cells/1000 NCE/animal
For the parameter body weight the mean values were calculated for each group, separately for males and females. The relative body weight gain to the preceding mean values was calculated also.
Statistical tests
For further statistical analysis, the absolute numbers of micronuclei-containing cells per animal were used. All calculations were done separately and combined for males and females.
Pairwise comparison
Each treatment group was compared to the negative control. For comparisons the exact Fisher-Pitman permutation test was used against one-sided alternatives.
Multiple test procedure
As there is more than one treatment group, the p-values are considered jointly to maintain an error rate (multiple level of significance) of a=5%. For this purpose the Bonfeffoni-Holm multiple test procedure was used. The positive control (cyclophosphamide) was not included in Holm's procedure. It was compared with the control separately at a level of 5%.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
strong toxic reactions were observed, one female in one high dose group died
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The negative control (solvent) values were all in or very close to the expected range predetermined as historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.

2-Methylpropanol-1 has been assessed in this limit test in mice (5 males or 5 females in one group; 500 and 1500 mg/kg bw, oral application) for its potential to induce micronuclei. In a preceding range finding test with 7 male mice, the dose of 1500 mg/kg bdw was strongly toxic and mortality occurred in 3 animals. When females were treated with this dose in the main study, one animal died in the 48 hour group. All other 4 females showed strong toxic reactions. Males have only been tested up to 500 mg/kg bw. Also this dose caused strong toxic reactions but no mortality. 48 Hours after administration of both doses of the test material, no significant increase in the micronucleus frequency, as compared to the negative control group, has been detected in males or females. 24 Hours after start of the treatment with 1500 mg/kg bdw, no increase in the micronucleus frequency was seen in the female group. In the 4th group (males, 500 mg/kg bw; 24 hours), micronucleus frequency was weakly (2 ‰ cells with micronuclei, negative control: 0.8 ‰) and statistically significantly (p = 0.02) increased. As there was no dose dependency and as this value was within the range of historical controls (0.4 - 2.8 ‰), the test substance is not mutagenic under the selected experimental conditions.

Applicant's summary and conclusion