3-methoxybutan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

other: This is a contemporary study, performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-methoxybutan-1-ol

EC Number:

219-741-8

EC Name:

3-methoxybutan-1-ol

Cas Number:

2517-43-3

Molecular formula:

C5H12O2

IUPAC Name:

3-methoxybutan-1-ol

Constituent 2

Reference substance name:

1-butanol, 3-methoxy-

IUPAC Name:

1-butanol, 3-methoxy-

Test material form:

other: clear, colourless liquid

Details on test material:

- Name of test material (as cited in study report): 3-Methoxybutanol
- Physical state: Clear, colourless liquid
- Analytical purity: 99.7%
- Lot/batch No.: Batch# 120000267542
- Expiration date of the lot/batch: 24 March 2011 (allocated by testing lab as 1 year after receipt of test substance)
- Stability under test conditions: Not indicated
- Storage condition of test material: At room temperature, in the dark

Method

Species / strain

Species / strain / cell type:

primary culture, other:

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 20% (v:v) heat-inactivated fetal calf serum, L-glutamine, penicillin/streptomycin, and heparin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

Rat liver microsomal enzymes, prepared at the study lab from adult male Wistar rats.

Test concentrations with justification for top dose:

Control (medium only)
10 microgram/microL
33 microgram/microL
100 microgram/microL
333 microgram/microL
1042 microgram/microL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test substance was dissolved in RPMI 1640 culture medium
- Justification for choice of solvent/vehicle: test article was soluble in culture medium, allowing introduction of test article without affecting growth/maintenance conditions of the cells.

Controls

Untreated negative controls:

yes

Remarks:

RPMI 1640 culture medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C was positive control for "-S9" experiments; Cyclophosphamide was the positive control for the "+S9" experiments

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were exposed in suspension to test article dissolved in culture medium.

DURATION
- Exposure duration: Cells were exposed to test article for 48 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): After 2 rinses in HBSS, cells were re-suspended in culture media, and incubated for 20-22 hours for fixation.

CHROMOSOME PREPARATION: During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

NUMBER OF REPLICATIONS: 3-Methoxybutanol was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.

DETERMINATION OF CYTOTOXICITY: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. 3-Methoxybutanol was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of 3-Methoxybutanol for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

The highest tested concentration was 1042 μg/ml (= 0.01 M).

After 3 h exposure to 3-Methoxybutanol in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).

Cytotoxicity of 3-Methoxybutanol in the lymphocyte cultures was determined using the mitotic index.

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M.

ANALYSIS OF SLIDES: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were
examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:

a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

(N-1) (ad-bc)^2
X^2 = --------------------------------
(a+b) (c+d) (a+c) (b+d)

where:
b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
n0 = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
n1 = the total number of cells scored in the treated cultures.
N = sum of n0 and n1


[ (N-1) (ad-bc)^2 ]
If P | X^2 > ------------------------------- | (one-tailed) is small (p< 0.05) the hypothesis that the
[ (a+b) (c+d) (a+c) (b+d) ]

incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

3-Methoxybutanol is not clastogenic in human lymphocytes under the described experimental conditions.

Executive summary:

The study evaluated the effect of 3-Methoxybutanol on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of 3-Methoxybutanol was tested in two independent experiments.

The described study procedures were based on the most recent OECD and EC guidelines.

Batch 120000267542 of 3-Methoxybutanol was a clear colourless liquid with a purity of 99.7%(m). 3-Methoxybutanol was dissolved in RPMI 1640 medium. In the first cytogenetic assay, 3-Methoxybutanol was tested up to 1042 μg/ml (= 0.01 M) for a

3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration which should be tested according to the guidelines.

In the second cytogenetic assay, 3-Methoxybutanol was tested up to 1042 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix 3-Methoxybutanol was also tested up to 1042 μg/ml for a 3 h exposure time with a 48 h fixation time.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

3-Methoxybutanol did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of 3-Methoxybutanol on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 3-Methoxybutanol does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

The final conclusion was that the test was valid and that 3-Methoxybutanol is not clastogenic in human lymphocytes under the described experimental conditions.