Butane-1,4-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,4 -Butanediol was not genotoxic in a bacterial reverse mutation assays, an in-vitro mammalian chromosome aberration test or a mammalian cell gene mutation assay (HRPT Test), all with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Method according to GLP, but not all details of the study were available for review in English

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

Method: other: Guidelines for Screening Mutagenicity of Testing of Chemicals (Japan)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

Vehicle
DMSO for positive controls, except water was used for sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5 nitro-2-furyl)acrylamide

Remarks:

for TA98, TA100, and WP2 uvrA; without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains tested with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: for TA1537 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: for TA1535 without S9 mix

Evaluation criteria:

A two-fold increase in the average number of colonies on the plate containing the test substance, as compared to that of the solvent control or a reproducible dose-dependent increase, the test substance shall be mutagenic in test systems.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

This chemical did not induce mutations in the S. typhimurium and E. coli strains. Toxicity was not observed at 5000 ug/plate in five strains with or without an S9 mix.

Reference 2

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

Study period:

1993-Apr-13 to 1993-June-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Chinese hamster Ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells were used. These were originally derived from the ovary of an adult Chinese hamster. Cells were obtained from Flow Laboratories, 5309 Meckenheim, Germany and a stock culture is stored under liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix, induced with Aroclor 1254

Test concentrations with justification for top dose:

20, 60, 200, 600, 2000 (5000) ug/ml

Vehicle / solvent:

Ham's F12 cell culture medium as follows:

- H10: Ham's F12 medium with 10 % fetal calf serum (FCS), 2 mM L-Glutamine, 100 IU/ml Penicillin, 100 Ag/m1 Streptomycin.

- HO: Ham's F12 medium with 2 mM L-Glutamine, 100 IU/ml Penicillin, 100 Ag/m1 Streptomycin

- H6TG: Ham's F12 medium with 10 % FCS, 2 mM L-Glutamine, 100 IU/ml Penicillin, 100 Ag/m1 Streptomycin, 10 Ag/m1 6-thioguanine. H6TG does not contain hypoxanthine.

- HAT: Ham's F12 medium with 10 % FCS, 2 mM L-Glutamine, 100 IU/ml Penicillin, 100 Ag/m1 Streptomycin, 200 AM glycin, 5 AM thymidine, 10 jiM hypoxan-thine, 3,2 gM aminopterin.

Untreated negative controls:

yes

Remarks:

H0 culture medium plus S9 mix in the assay with metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

H0 culture medium plus S9 mix in the assay with metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

To reduce the number of cells harboring spontaneous mutations of the HPRT locus, the cells for the main study were treated in HAT medium for approx. 1 week. Then the HAT medium was replaced by HO-medium containing the following concentrations of 1,4-Butanediol:

- HPRT #1 and 2: 0, 20, 60, 200, 600, and 2000 pg/ml (±59)
- HPRT #3: 0 and 5000 pg/ml (±S9)

Positive controls demonstrating the sensitivity of the test system were ethyl methane sulfonate (EMS, without S9) and 3-methylcholanthrene (MCA, with S9). Duplicate cultures with a total of 1xE6 cells were seeded for each treatment group. After an incubation time of 4 hrs the cells were washed 3 times with phosphate buffered saline (calcium and magnesium free) and trypsinized. Duplicate cultures were pooled and cells were counted. The cells of each treatment group were divided into two subgroups. One subgroup was used to determine the cloning efficiency (in HPRT tests #1 and #2, however, 6 instead of 3 dishes were plated for the determination of the cloning efficiency). The cells of the other subgroup were cultured in H10 medium for 7 days to allow for expression of the mutated phenotype (cells were subcultured after 3 days at an approx. cell density of 2xE6 cells/flask). At the end of the expression period the cloning efficiency was determined. The selection of mutants was performed using five cultures for each treatment group. 2xE5 cells/75 cm2 flask were seeded in H6TG medium. After incubation for 6 days at 37 °C the colonies were fixed with methanol, stained with Giemsa and counted.

Evaluation criteria:

The validity of an experiment is determined by the following criteria: (1) The cloning efficiency of the negative control at the end of the expression period must be at least 20 %, (2) The mutation frequency of the negative control should not exceed the maximum spontaneous mutation frequency of approx . 20/10-6 cells (Gupta, R.S., 1984), (3) The mutation frequencies of the positive controls must be significantly elevated, (4) At least 4 dose levels must be examined, the highest dose being either significantly toxic (cloning efficiency approx. 20 %) or being the solubility limit of the test substance. Concentrations higher than 5 mg/ml will not be tested, and (5) Positive results must be reproducible in a second independent experiment.

A test compound will be reported as being mutagenic in the HPRT test with CHO cells if it causes a statistically significant, dose related increase in mutant frequency at concentrations of the test substance resulting in greater than 20 % cell survival. In addition, a positive response is claimed only, if the mean mutant frequency in treated cultures reaches a value significantly above the maximum spontaneous mutant frequency (of approx. 20/106 viable cells).

Statistics:

Statistical significance is determined on the basis of a t-test ("Two-sample analysis").

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

HPRT Test without Metabolic Activation: The negative controls in tests #1 and #2 were clearly within the range (0-20 mutants/10xE6 viable cells) normally obtained in the absence of exogenous metabolic activation, while the mutant frequency of the negative control in test #3 (30±8) was above this range. The reason for this increased mutant frequency is unknown but may have its origin in the variability of the CHO cell line (in the 22 HO controls which have so far been performed in the laboratory, 20 were in the range of 0-12 mutants/1 million viable cells, in two trials 36 or 40 mutants/1 million viable cells were observed). The validity of the study is considered not to be influenced by this increase. The positive control, EMS, in all 3 trials induced mutant frequencies being significantly higher than the negative controls thus demonstrating the sensitivity of the test system. In trials #1, #2 and #3, 1,4-Butanediol did not induce mutant frequencies significantly higher than the concurrent or historical negative controls. The test substance was therefore considered to be non mutagenic in the HPRT test without metabolic activation.

HPRT Test with Metabolic Activation by S9 Mix: As in the without S9 experiment, the negative controls in tests #1 and #2 were within the range (0-20 mutants/10xE6 viable cells) normally obtained in the presence of exogenous metabolic activation, while the mutant frequency of the negative control in test #3 (28±13) was above this range. The reason for this increased mutant frequency is unknown but may have its origin in the variability of the CHO cell line (in the 22 HO controls which have so far been performed in the laboratory, 21 were in the range of 0-18, in 1 trial 29 mutants/1 million viable cells were observed. A background mutant frequency in one trial of 28/10xE6 is therefore considered not to influence the validity of this study. The positive control, MCA, in all 3 trials induced mutant frequencies being significantly higher than the negative controls thus demonstrating the sensitivity of the test system. In trials #1, #2 and #3, 1,4-Butanediol did not induce mutant frequencies significantly higher than the concurrent or historical negative controls. At a concentration of 60 µg/ml, 1,4-Butanediol in trial #2, the number of mutants was statistically different from the concurrent negative control (16±5 versus 4±4) but was still within the range of historical controls. As this increase was not observed in trial #1 and there were no indications of a dose dependency (at the adjacent concentrations of 20 and 200 µg/ml the mutant frequencies were slightly lower than the concurrent negative control) it is considered to be of no biological relevance. It is therefore concluded, that 1,4-Butanediol has no mutagenic potential in the HPRT test with metabolic activation.

Conclusions:

It is concluded, that 1,4-Butanediol, in the presence as well as in the absence of Aroclor 1254-induced rat liver S9 mix, has no mutagenic potential in this in vitro mammalian cell assay.

Reference 3

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

Study period:

1993-Feb to 1993-Apr

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chinese hamster lung V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cloned V79 cells with a modal chromosome number of 22 and a population doubling time of approx. 20 hrs were used. These were originally derived from the lung tissue of a male Chinese hamster. Cells were obtained from Dr. Engel¬hardt, BASF, D-6700 Ludwigshafen and the stock culture was maintained under liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

400, 3000, 5000 ug/ml

Vehicle / solvent:

MEM cell culture medium as follows:

MEM5: MEM (Eagle) medium with 5 % fetal calf serum (FCS), 2 mM L-Glutamine, 100 £U/ml Penicillin, 100 pg/ml Streptomycin.
MEMO: MEM (Eagle) medium with 2 mM L-Glutamine, 100 IU/m7 Penicillin, 100 gg/ml Streptomycin (no FCS).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Served as solvent/negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cells were removed from the liquid nitrogen container, thawed at 37 °C, and transferred into MEM5 medium. The cells were cultured at 37 °C, 5 % CO2 and approx. 95 % rel. humidity with a first culture medium exchange after approx. 24 hrs. Cells were subcultured 4 and 7 days after seeding. During the second subculture, cells were seeded onto microscopic slides. 2.5 x 104 cells were seeded onto each slide and incubated for approx. 24 hrs at 37 °C ( 5 % CO2, 95 % rel. humidity). After 24 hrs culture on microscopic slides, V79 cells were treated with 10 different 1,4-butanediol concentrations (25, 50, 100, 200, 400, 600, 1000, 1800, 3000, 5000 itg/m1 +/- S9 mix), Cyclophosphamide and Mitomycin C. MEM5 medium (MEMO medium in the test with S9 mix) served as the solvent control. To limit the cytotoxic effects of the S9 mix, the medium of the with S9 mix exposure was replaced by MEM5 medium after 3 hours. During the without S9 mix exposure, an exchange of the cell culture medium was not necessary. To arrest cells in metaphase, 2 hours prior to the cell preparation Colcemid (0.2 lig/m7 final conc.) was added to the cultures. Treatment was terminated 28 hrs after the start of exposure in experiment #1 and 28 hrs and 43 hrs after the beginning of treatment in experiment #2 by addition of 50 mM (prewarmed to 37 °C). Hypotonic treatment was terminated after 20 min by fixation in methanol/glacial acetic acid (3 parts methanol, 1 part acetic acid; precooled to 4 °C). 3 consecutive fixation steps of 15, 10 and 5 min were performed. After air drying for 15-20 min, the slides were dried at 60 °C for approx. 1 hr and stained with Giemsa.

Evaluation criteria:

The test chemical was considered clastogenic if chromosomal aberrations (excluding gaps) were induced in a statistically significant and reproducible manner in one or more concentrations; the induced proportion of aberrant cells at such test chemical concentrations exceeds the normal range. Increases in the proportion of cells with gaps or increases in the numbers of cells with structural aberrations not exceeding the normal range are discussed on a case by case basis.

Statistics:

After completion of scoring, slides were decoded. The aberrant cells from each culture were categorized as follows: (A) cells with structural aberrations incl. gaps, (B) Cells with structural aberrations excl. gaps, and (C) Polyploid cells or cells with endoreduplications. On the basis of the category totals for the negative controls the acceptabili-ty of the assay was determined (see section "Acceptance criteria"). The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant. The aberration frequencies of the positive controls were > 5 % (excl. gaps) and were therefore not tested for statistical significance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The solvent controls revealed chromosomal aberration frequencies (without gaps) of 0 to 1.5 % without, and a maximum of 2.5 % aberrant cells with metabolic activation by S9-mix which is consistent with spontaneous aberration frequencies for the V79 cell line in this laboratory (the maximum acceptable spontaneous aberration frequency should be < 5%).

The positive controls, Cyclophosphamide (2 μg/ml with S9 mix) and Mitomycin C (0.02 μg/ml without S9 mix), led to biologically significant increases in the frequency of aberrations, indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.

Treatment with 1,4-Butanediol at 400, 3000 and 5000 ug/ml at both sampling times, either with or without metabolic activation did not induce frequencies of chromosomal aberrations (excluding gaps) being significantly higher than the concurrent negative controls. In addition, all observed aberration frequencies of 1,4-Butanediol treated cells were well within the range of historical aberration frequencies (< 5%). In addition, the frequency of polyploid cells was within the expected rahge (< 10 %).

This result was clearly reproducible in the second, independent experiment.

See illustration (picture/graph) below.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded, that 1,4-Butanediol does not induce chromosomal aberrations in this in vitro test system and is therefore considered to be non-clastogenic to V79 cells is vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1,4 -Butanediol was not genotoxic in a bacterial reverse mutation assays with and without metabolic activation. S. typhimurium strains TA97, TA98, TA100, TA1535, TA1537, and TA1538 have been used, as well as Escherichia coli WP2 uvrA. Among the various studies, test concentrations ranged from 0-10000 ug/plate. In an in vitro mammalian chromosome aberration test using Chinese hamster lung fibroblasts (V79), the authors concluded that 1,4-Butanediol did not induce chromosomal aberrations and was therefore considered to be non-clastogenic with and without metabolic activation. Concentrations used were 400, 3000 and 5000 ug/ml with and without metabolic activation. In a mammalian cell gene mutation assay (HRPT Test) using Chinese Hamster Overy (CHO) cells, the authors concluded that 1,4-Butanediol was not mutagenic. Test concentrations ranged from 20 - 5000 ug/ml. In vivo data are not available for 1,4-Butanediol. However, gamma-butyrolactone (CASRN: 96-48-0), which like 1,4-butanediol rapidly metabolizes to gamma-hydroxybutryic acid, showed a negative result in an in-vivo mouse micronucleus test.

Short description of key information:

Not genotoxic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

1,4-butanediol was negative when tested for mutagenicity/cytogenicity in in vitro assays including a bacterial reverse mutation assays, an in vitro mammalian chromosome aberration assay, and a mammalian cell gene mutation assay. 1,4-butandiol has not been evaluated in an in vivo assay. However, gamma-butyrolactone, which like 1,4-butandiol rapidly metabolizes to gamma-hydroxybutyric acid, showed negative results in an in vivo mouse micronucleus test. Based on this evidence, 1,4-butanediol would not be rated as a mutagen under the EU CLP classification system (EU Regulation 1272/2008).