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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro
Gene mutation in bacteria
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (NTP 1995)
S. typhimurium TA 102, with and without metabolic activation (Ames test): negative (Jung et al. 1992)


Gene mutation in mammalian cells
V79 cells, HPRT Test, with and without metabolic activation: negative (GLP, OECD 476; BASF SE 2010)
L5178 cells, mouse lymphoma assay, with and without metabolic activation: negative (Celanese 1978)

Cytogenicity in mammalian cells
CHL V79 cells, in vitro micronucleus test, without metabolic activation: negative (Lasne et al. 1984)
CHO cells, SCE assay, without metabolic activation: negative (Obe et al. 1977)
S. thyphimurium TA1 535/pSK1 002, umu test: negative (Nakamura et al. 1987)


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from phenobarbital/β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Experiment I (4 h treatment)
23.1 - 740 µg/mL (with and without metabolic activation)

Experiment II
23.1 - 740 µg/mL (24 h; without metabolic activation)
23.1 - 740 µg/mL (4 h; with metabolic activation)
(740 μg/mL equals to approximately 10 mM, the recommended limit dose of OECD TG 476)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with metabolic activation: DMBA (1.1 μg/mL = 4.3 μM); without metabolic activation: EMS (0.150 mg/mL = 1.2 mM)
Details on test system and experimental conditions:
DURATION
- Exposure duration: Experiment I: 4 h; Experiment II: 24 h

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 50

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity.
Cloning efficiency II (viability): cloning efficiency determined after the expression period to measure viability of the cells without selective agent.

cloning efficiency I (survival, absolute): mean number of colonies per flask divided by the number of cells seeded
cloning efficiency I (survival, relative): (mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding control) × 100
cell density % of control: (cell density at 1st subcultivation divided by the cell density at 1st subcultivation of the corresponding control) × 100
cloning efficiency II (viability, absolute): mean number of colonies per flask divided by the number of cells seeded
cloning efficiency II (viability, relative): cloning efficiency II absolute divided by the cloning efficiency II absolute of the corresponding control × 100
cells survived (after plating in TG containing medium): number of cells seeded × cloning efficiency II absolute
mutant colonies / 10e6 cells: mean number of mutant colonies per flask found after plating in TG medium × 10e6 divided by the number of cells survived
induction factor: mutant colonies per 10e6 cells / mutant colonies per 10e6 cells of the corresponding solvent control
Evaluation criteria:
The gene mutation assay is considered acceptable if:
- the numbers of mutant colonies per 10e6 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon-ding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.0 – 33.2 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

see attached document for details

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
Remarks:
tested only without metabolic activation with one concentration
Principles of method if other than guideline:
Butan-1-ol was evaluated in the in vitro mammalian cell micronucelus test for its potential to induce chromosomal aberrations.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM supplemented with 10 % foetal calf serum and antibiotics
- 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
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
50 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [no data]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 1 hour
- Expression time (cells in growth medium): 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): giemsa stain

NUMBER OF CELLS EVALUATED: 4000 or 7000 cells
Evaluation criteria:
(1) staining intensity equal to that of the nucleus, (2) diameter less than one-fifth that of the nucleus, (3) location in cytoplasm, and (4) no contact with nucleus (to distinguish from nuclear blebs)
Statistics:
The dose response of V79 cells to chemical mutagens has been estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested

control (medium): 4.00±0 .71 (mean number of cells with MN per 1000 cells ± S .E)

butan-1 -ol: 2.75 ± 0.48 (mean number of cells with MN per 1000 cells ± S .E)

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
missing strain to detect oxidising/ cross-linking agents
Principles of method if other than guideline:
Standard NTP protocol.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
10% and 30% hamster liver, 10% and 30% rat liver
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 3333, 6666 and 10000 µg/plate

Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see "details on test system"
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

Positive control chemicals used in NTP Ames tests:

For strains tested in the absence of S9
TA98, 2-nitrofluorene or alternatively, TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine

For strains tested with S9
All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity was observed in TA98 and TA100 at the highest tested dose with metabolic activation (30%)
Additional information on results:
In none of the treatments, the maximum revertant factor exceeded a value of 1.5, indicating that the test substance is not mutagenic in the Ames test under the tested conditions.
Remarks on result:
other: all strains/cell types tested
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented
Principles of method if other than guideline:
Ames test with S. typhimurium TA 102 as described by Maron and Ames (1983) and as specified for TA102 by D. Levin et al . (1982).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of Aroclor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
up to 5000 µg/plate
Details on test system and experimental conditions:
The compounds were tested in at least 2 independent experiments using 5 doses and 3 plates per dose.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: up to limit dose or to precipitating or cytotoxic doses
Remarks on result:
other: all strains/cell types tested
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo
Cytogenicity in mammals
mouse (micronucleus test): negative (OECD 474; BASF 1998)

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: mean: 26.9 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Fasting period before study:
- Housing: groups of 5 seperately according to sex in Makrolon cages, type MIII before the start of the study (acclimation period) and individually during the study in Makrolon cages, type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ca. one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 °C
- 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 limited solubility 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: 500, 1000 and 2000 mg butan-1-ol in a total of 10 ml
- Amount of vehicle (if gavage or dermal): 10 ml including test substance
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.
The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.
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.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 or 10
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide; vincristine sulphate
- Justification for choice of positive control(s): cyclophosphamide induces clastogenicity, vincristine sulphate induces spindle poison effects
- Route of administration: cyclophosphamide: oral; vincristine sulphate: intraperitoneally
- Doses / concentrations: cyclophosphamide: 20 mg/kg bw; vincristine sulphate: 0.15 mg/kg bw
Tissues and cell types examined:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 1000 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also registered.
In addition, the number of normochromatic erythrocytes with micronuclei and the ratio of polychromatic to normochromatic erythrocytes were determined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline No. 474 were survived by all animals, but led to evident signs of toxicity such as abdominal position, irregular respiration, staggering, squatting posture and narcotic like state; the general state of the animals was poor. 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 at a 24-hour interval and samples of bone marrow were taken 24 hours and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Preparation of the bone marrow:
- The two femora were prepared from the animals, and all soft tissues were removed.
- 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 with 50 pl 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.
Staining of the slides:
- The slides were stained in eosin and methylene blue solution for 5 minutes (May Gruenwald 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 Giemsa solution for 15 minutes.
- After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 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 the 24 hour interval.
- 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) were printed with the group means in the tables. This test was performed one-sided.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The administration of the test substance led to evident signs of toxicity in the highest dose group of 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

According to the results of the present study, the single oral administration of butan-1-ol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance butan-1 -ol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

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

Butan-1-ol was tested in the Ames test with and without metabolic activation for its ability to induce mutations in the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 in doses up to 10000 µg/plate following a standardized protocol (NTP 1995). All tested strains were negative with and without metabolic activation.

Jung et al. (1992) performed an Ames test with the S. typhimurium strain TA102, which is reliable to detect crosslinking agents. Butan-1-ol was negative with and without metabolic activation in doses up to 5000 µg/plate.

Additionally, a few other tests were also negative (i.e. McCann et al. 1975).

 

Gene mutation in mammalian cells

In a GLP conform HPRT test according to OECD TG 476, the potential of butan-1 -ol to induce gene mutations in the HPRT locus in V79 cells was studied (BASF SE 2010). After exposure to 4 or 24 hours, Butan-1 -ol did not induce gene mutations either with or without metabolic activation in doses up to 740 mg/mL (= ca. 10 mM, limit dose).

Butan-1 -ol did not induce relevant increases in TK mutations in a reliable mouse lymphoma assay with L5178Y cells with or without metabolic activation in noncytotoxic doses (6.25 and 5.0 µL/mL without and with metabolic activation, respectively; Celanese 1978).

 

Cytogenicity in mammalian cells

Butan-1-ol was evaluated in the in vitro mammalian cell micronucleus test of its potential to induce chromosomal aberrations (Lasne et al. 1984). CHL V79 cells were treated with 50 µL/mL (=ca. 41 mg/mL) without metabolic activation and subsequently 4000 to 7000 cells were scored for micronuclei. Butan-1-ol did not cause an increase in micronuclei.

Obe et al. (1977) tested butan-1-ol for its potential to induce DNA damage/repair in a sister chromatide exchange assay with CHO cells without metabolic activation. In doses up to 1% (v/v), Butan-1-ol did not cause an increase in sister chromatide exchanges.

No increase, above negative control, in DNA repair was seen in the umu test with S. typhimurium TA1 535/pSK1 002, with and without metabolic activation in doses up to 27 µg/mL (Nakamura et al. 1987).

 

Other studies

Aneuploidy induction was observed during early germination of Aspergillus nidulans at concentrations up to 1% (v/v), the highest, cytotoxic concentration tested (Crebelli et al. 1989, Val. 3 adopted from the ECETOC JACC dossier). This effect on funghi is considered as not relevant for humans, since micronucleus tests in vitro and in vivo (reflecting aneuploidy and clastogenicity) were negative.

 

Cytogenicity in vivo

A micronucleus test was performed following GLP requirements and OECD guideline 474 with male and female NMRI mice orally dosed with 500, 1000 and 2000 mg/kg bw butan-1-ol (BASF 1998). Groups of 5 or 10 animals per sex per dose were sacrificed after 24 or 48 h and 2000 stained erythrocytes were scored per animal. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. Thus, under the experimental conditions chosen here, the test substance butan-1 -ol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.