Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria
S. typhimurium TA 1535, TA 1537, TA 97, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (standardized test protocol; Zeiger et al.1988)
S. typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538; E. coli WP2 uvrA, with and without metabolic activation (Ames test): negative (Shimizu et al. 1985)

Gene mutation in mammalian cells
CHL V79 cells (HPRT test), with and without metabolic activation: negative (Kreja and Seidel, 2002).
Mouse L5178Y cells with and without metabolic activation (Mouse lymphoma assay): negative (TSCATS OTS 0513186, 1987)

Cytogenicity in mammalian cells
CHL V79 cells (in vitro micronucleus test), without metabolic activation: negative (Kreja and Seidel, 2002).

Gene mutation in bacteria

Isobutanol was not mutagenic in a pre-incubation Ames test with and without metabolic activation (tested up to 10000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 97; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters; (Zeiger et.al., 1988)). Cytotoxicity (reduction of the background lawn) was observed at the highest concentration tested in TA100, TA1535, TA97 and TA98 without S-9 mix.

In another pre-incubation Ames test with and without metabolic activation (tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538 as well as Escherichia coli WP2 uvrA; metabolic activation: liver S-9 mix),  isobutanol was also not mutagenic (Shimizu et al. 1985). No cytotoxicity was observed.

 

Gene mutation in mammalian cells

A HPRT-gene mutation assay (with and without metabolic activation; V79 Chinese hamster fibroblasts) found the test substance to be negative for causing gene mutations at dose levels of up to 107 mM, respectively (Kreja and Seidel, 2002). Cytotoxicity was not observed.

The test substance was also negative for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 10 mg/ml (without metabolic activation) and 5 mg/ml (with metabolic activation) (TSCATS OTS 0513186, 1987). Cytotoxicity was observed at the highest dose without metabolic activation.

 

Cytogenicity in mammalian cells

A more recent set of experiments using a micronucleus assay (without metabolic activation; V79 Chinese hamster fibroblasts) also found the test substance to be negative for causing cytogenicity at dose levels of up to 53 (Kreja and Seidel, 2002). Cytotoxicity was not observed in the test. Additionally, a comet assay with human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells was performed by Kreja and Seidel (2002). Again, the assays provided negative results for isobutanol.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The test substance was evaluated for mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test).
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Methylpropan-1-ol
- Analytical purity: Highest commercially available (typically >99%), from Riedel de Haen
Target gene:
hypoxanthine-guanine-phosphoribosyl transferase gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM Eagle medium (V79 cells) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine,
100 IU/ml penicillin and streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
up to 107 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [no data]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 14 days


SELECTION AGENT (mutation assays): 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test compound was classified as a mutagen when it was able to enhance in a concentration-depended manner the spontaneous HPRT frequency by a factor of three or more.
Statistics:
no data
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no cytotoxicity observed up to 107 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Clastogenic effects were studied by the micronucleus test using the Chinese hamster V79 cell line as a target.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Methylpropan-1-ol
- Analytical purity: Highest commercially available (typically >99%), from Riedel de Haen
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM Eagle medium (V79 cells) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine,
100 IU/ml penicillin and streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
without
Test concentrations with justification for top dose:
11 and 53 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [no data]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours at 37 °C
- Expression time (cells in growth medium): additional 24 hours


NUMBER OF CELLS EVALUATED: 1000 cells/culture


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test compound was classified as a clastogen when it was able to enhance the spontaneous MN frequency at least three-fold or higher over that of the control for at least one dose tested.
Statistics:
no data
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The mutagenicity of the test chemical was examined in Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) and Escherichia coli (WP2uvrA) using the preincubation method with and without S9 mix.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutyl alcohol
- Analytical purity: 99.7 %
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
5; 10; 50; 100; 500; 1000; 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: none given
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA98, TA100 and E.coli WP2 uvrA:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; TA1535: N-ethyl-N-nitro-N-nitrosoguanidine; TA1537: 9-aminoacridine; TA1538: 4-nitroquinoline-N-oxide
Remarks:
without S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA100, TA98, TA1537 and TA1538: benzo(a)pyrene; TA1535 and E.coli WP2 uvrA: 2-aminoanthracene
Remarks:
with S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 °c
- Exposure duration: 2 days at 37 °C


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The test substance was tested for mutagenicity in Salmonella typhimurium, using a preincubation protocol. The test was performed in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutyl alcohol
- Analytical purity: > 99 %
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA97 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
100 - 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: none given
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), 4-nitro-o-phenylenediamine (TA98). With S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 2 days at 37 °C

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic: (+), weakly mutagenic (+W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of "+ W", if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic.
A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a "+W" response, or if only single doses produced increases in his' revertants in repeat trials.
Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.
Statistics:
no data
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA97, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction of the background lawn at the highest concentration tested in some strains without S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduction of the background lawn at the highest concentration tested in TA100, TA1535, TA97 and TA98 without S-9 mix
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

NMRI mouse (micronucleus test), up to 2000 mg/kg: negative (CMA 2000)
NMRI mouse (micronucleus test), up to 1500 mg/kg: negative (BG Chemie 1999).

The most robust data for genetic toxicity in vivo were the oral in vivo mouse micronucleus tests conducted by two different laboratories according to GLP requirements and OECD 474 (CMA 2000, BG Chemie 1999). The test substance was administered once orally to male and female NMRI mice at doses up to 2000 or 1500 mg/kg body weight, respectively.

Positive and negative controls all produced appropriate responses. The test substance did not produce any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (spindle poison effect). A weakly and statistically increased micronucleus frequency in one study at 500 mg/kg body weight (BG Chemie 1999) was judged to be incidental as there was no dose dependency and as this value was within the range of historical controls. PCE/NCE ratios were not affected, but systemic toxicity (narcotic like state or mortality) was observed in both studies.

Link to relevant study records

Referenceopen allclose all

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

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.

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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutanol
- Physical state: liquid
- Analytical purity: 99.87 %
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals 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
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
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
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)
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-Balsam.

METHOD 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 micronuclei
The 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 micronuclei
The 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 target
Individual 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.
Key result
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: piloerection


RESULTS OF DEFINITIVE STUDY
The 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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro and in vivo.

 

Gene mutation in bacteria

Isobutanol was not mutagenic in a pre-incubation Ames test with and without metabolic activation (tested up to 10000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 97; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters; (Zeiger et.al., 1988)). Cytotoxicity (reduction of the background lawn) was observed at the highest concentration tested in TA100, TA1535, TA97 and TA98 without S-9 mix.

In another pre-incubation Ames test with and without metabolic activation (tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538 as well as Escherichia coli WP2 uvrA; metabolic activation: liver S-9 mix),  isobutanol was also not mutagenic (Shimizu et al. 1985). No cytotoxicity was observed.

 

Gene mutation in mammalian cells

A HPRT-gene mutation assay (with and without metabolic activation; V79 Chinese hamster fibroblasts) found the test substance to be negative for causing gene mutations at dose levels of up to 107 mM, respectively (Kreja and Seidel, 2002). Cytotoxicity was not observed.

The test substance was also negative for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 10 mg/ml (without metabolic activation) and 5 mg/ml (with metabolic activation) (TSCATS OTS 0513186, 1987). Cytotoxicity was observed at the highest dose without metabolic activation.

 

Cytogenicity in mammalian cells

A more recent set of experiments using a micronucleus assay (without metabolic activation; V79 Chinese hamster fibroblasts) also found the test substance to be negative for causing cytogenicity at dose levels of up to 53 (Kreja and Seidel, 2002). Cytotoxicity was not observed in the test. Additionally, a comet assay with human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells was performed by Kreja and Seidel (2002). Again, the assays provided negative results for isobutanol.

 

In vivo

The most robust data for genetic toxicity in vivo were the oral in vivo mouse micronucleus tests conducted by two different laboratories according to GLP requirements and OECD 474 (CMA 2000, BG Chemie 1999). The test substance was administered once orally to male and female NMRI mice at doses up to 2000 or 1500 mg/kg body weight, respectively.

Positive and negative controls all produced appropriate responses. The test substance did not produce any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (spindle poison effect). A weakly and statistically increased micronucleus frequency in one study at 500 mg/kg body weight (BG Chemie 1999) was judged to be incidental as there was no dose dependency and as this value was within the range of historical controls. PCE/NCE ratios were not affected, but systemic toxicity (narcotic like state or mortality) was observed in both studies.


Short description of key information:
Gene mutation in bacteria
S. typhimurium TA 1535, TA 1537, TA 97, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (standardized test protocol; Zeiger et al.1988)
S. typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538; E. coli WP2 uvrA, with and without metabolic activation (Ames test): negative (Shimizu et al. 1985)

Gene mutation in mammalian cells
CHL V79 cells (HPRT test), with and without metabolic activation: negative (Kreja and Seidel, 2002).
Mouse L5178Y cells with and without metabolic activation (Mouse lymphoma assay): negative (TSCATS OTS 0513186, 1987)

Cytogenicity in mammalian cells
CHL V79 cells (in vitro micronucleus test), without metabolic activation: negative (Kreja and Seidel, 2002).

Cytogenicity in vivo
NMRI mouse (micronucleus test), up to 2000 mg/kg: negative (CMA 2000)
NMRI mouse (micronucleus test), up to 1500 mg/kg: negative (BG Chemie 1999).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was not genotoxic in in vitro experiments using human, rodent, and bacterial cells or in vivo experiments in mice. For isobutanol, there is therefore no need for classification for mutagenic effects according to 1272/2008/EC (CLP) requirements.