Octane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of octane-1,2-diol was tested in three in vitro Ames (OECD 471 or other adequate references), one in vitro Chromosome aberration (Notification 1604 MHW Japan 1999, similar to OECD 473) and one in vitro gene mutation (OECD 476) tests, each with and without metabolic activation (+/- S9 mix). In each of these studies, consistent, reproducible and toxicologically relevant indications of genotoxicity were not evident.

Cytotoxicity was evident at the upper test concentrations in all genotoxicity studies, although it was inconsistent in the in vitro chromosome aberration assay in that it was evident in the initial growth inhibition test but was not reflected by relevant reduction in cell proliferation rates concurrent with the chromsome aberration tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

of 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: essential amino acid requiring strain

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated by oral route on 3 consecutive days with Phenobarbital (80 mg/kg bw) and β-Naphtoflavone (100 mg/kg bw) for enzyme induction

Test concentrations with justification for top dose:

Pre-Experiment (Plate-incorporation Test):
0 (vehicle control), 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate TA 98 and TA100 without and with metabolic activation (S9).

Experiments 1 (Plate-incorporation Test, including some of the assays from the pre-experiment) and 2 (Pre-incubation Test):
0 (vehicle control), 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA100, TA1535, TA1537, WP2 uvrA without and with metabolic activation (S9).

Vehicle / solvent:

Dimethylsulfoxide (DMSO)
Justification for choice of solvent/vehicle:
DMSO was a suitable vehicle for exposure to the test material up to the maximum guideline recommended test material concentration of 5000 μg/plate. It was compatible with bacterial survival and S9 activity.

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

Positive control substances for tests without metabolic activation (S9 mix): Methylmethanesulfonate for E. coli WP2 uvrA, sodium azide for S. typhimurium TA100 & TA 1535, 4-NOPD for S. typhimurium TA98 & TA 1537.

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used for all strains in the tests with metabolic activation (S9 mix).

Details on test system and experimental conditions:

Plate-incoroporation tests were performed in the Pre-Experiment and Experiment 1, and pre-incubation tests in Experiment 2.
Precipitation of the test material was not evident at any test concentration and tester strain used in Experiments 1 and 2 (without and with metabolic activation).

Positive Control Substances:
sodium azide: positive control for TA 100 and TA 1535 without metabolic activation; vehicle = distilled water.
4-nitro-o-phenylene-diamine: positive control for TA 98 and TA 1537 without metabolic activation; vehicle = dimethylsulfoxide (DMSO).
methylmethanesulfonate: positive control for WP2 uvrA without metabolic activation; vehicle = distilled water.
2-aminoanthracene: positive control for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with metabolic activation; vehicle = DMSO.

Evaluation criteria:

Evaluation Criteria
Degree of increase in the number of revertant colonies in any tester strain. Dose dependency of any effects. Confirmation in independent repeat experiment.

An increase in number of revertant colonies is considered to be biologically relevant:
- if in tester strains TA 100 and/or WP2 uvrA the number of revertant colonies is at least twice higher,
- if in tester strains TA 98, TA 1535 and/or TA 1537 the number of revertant colonies is at least three times higher,
than the number of revertant colonies in the solvent/vehicle control.

A test substance producing neither a dose related increase in the number of revertant colonies nor a reproducible biologically relevant positive response in any of the dose groups is considered to be non-mutagenic in this system.

Validity Criteria
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- mean values of the the control plates without and with S9 mix are within specified historical control ranges:
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates compared with the control plates.

In addition, the properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as
normal spontaneous mutation rates were checked regularly according to Ames et al. 1973. In this way it was ensured that the experimental conditions set up by Ames were fulfilled.

Reference:
Ames BN, Durston WE, Yamasaki E, Lee FD 1973.
Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection.
Proc. Natl. Acad. Sci. (USA) 70, 2281-2285

Statistics:

Statistical analysis of the results attained was not considered to be necessary.

Species / strain:

S. typhimurium, other: TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains tested at 2500 and/or 5000 µg/plate with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains tested at 2500 and/or 5000 µg/plate with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains tested at 1000, 2500 and/or 5000 µg/plate with and without S9

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'. Remarks: in Experiment 2 (Pre-incubation Test)

Conclusions:

Interpretation of results : negative without and with metabolic activation (S9)

In the present study, the test material, octane-1,2-diol, was tested for mutagenicity, both at non-toxic and cytotoxic test material concentrations, in a reverse mutation assay using four different strains of S. typhimurium and one strain of E. coli in the absence and presence of metabolic activation with S9. Mutagenicity of the test material was not evident, as it did not induce any relevant increase in the number of revertant colonies in the five bacteria strains tested. Hence, there was no evidence of point mutations by base pair changes or frameshifts attributable to treatment with the test material.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to a Japanese technical guideline and similar to OECD 473 in compliance with GLP in a recognized contract research organization. Reliability restrictions comprise the present robust study summary being based only on a translation of the original study report. In addition, the negative result attained in the presence of S9 was not confirmed by a repeat experiment and in the translation any justification for not repeating was not given. Nevertheless, these restrictions are acceptable and the study is adequate for assessment. The attained study results are considered to be unequivocal.

Qualifier:

according to guideline

Guideline:

other: Guideline on Genotoxicity Tests, Notification No.1604 of Pharmaceutical Affairs Bureau, MHW, Japan, Nov. 1, 1999

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 1997

Deviations:

yes

Remarks:

Negative result in the presence of S9 was not confirmed by repeat experiment nor any justification for not repeating was given in the available report translation

GLP compliance:

yes

Remarks:

but the growth inhibition test was conducted non-GLP

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung (CHL/IU) cell line

Details on mammalian cell type (if applicable):

- Supplier of cell line: American Type Culture Collection (ATCC)
- Cell line properties: 25 chromosomes, cell cycle 15 to 17 hours
- Date received: 15 NOV 2002
- Type and identity of media: EMEM (Eagle's minimum essential medium) supplemented with 10% foetal bovine serum
and antibiotics (mixture of 10000 units/mL penicillin G & 10000 µg/mL streptomycin sulfate at 100 to 1).
- Maintenance of cell line / culturing
Storage: In liquid nitrogen tank
Culturing: In CO2 incubator at 95% r.h., 37°C & 5% CO2. Addition of 0.25% Trypsin-EDTA solution for cell separation
and of medium for cell suspensions. Subculturing up to 16 passages.
Properly maintained: No further data.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated i.p. on 4 days with phenobarbital (PB) and/or 5,6-benzoflavone (BF) [PB: 30 mg/kg on Day 1 & 60 mg/kg/day on Days 2 to 4; BF: 80 mg/kg on Day 3) for enzyme induction.

Test concentrations with justification for top dose:

PRE-EXPERIMENT FOR TOXICITY TESTING, i.e. Cell Growth Inhibition Test without and with metabolic activation (-/+S9)
(harvest at 24 h: ---> - S9/+ S9: 6 h treatment followed by 18 h recovery and ---> - S9: 24 h treatment):
0 (vehicle control), 5, 10, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL

EXPERIMENT 1 (harvest at 24 h: ---> - S9/+ S9: 6 h treatment followed by 18 h recovery)
Evaluation of cell proliferation and microscopic examination (metaphase analysis) -/+S9: 0*, 175, 350 and 700 µg/mL

EXPERIMENT 2 (harvest at 24 h: ---> - S9: 24 h treatment)
Evaluation of cell proliferation and microscopic examination (metaphase analysis) -/+S9: 0*, 45, 90 and 180 µg/mL

* 0 μg/mL = vehicle control (dimethyl sulfoxide, DMSO)

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
see field "Any other information on materials and methods incl. tables"

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Justification for choice of solvent/vehicle:
The test material was soluble in DMSO at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C at 0.05 μg/mL for the 6 h treatment (followed by 18 h recovery) and 24-hour treatment cultures Migrated to IUCLID6: without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation (S9 mix)

Positive control substance:

benzo(a)pyrene

Remarks:

benzo(a)pyrene at 20 μg/mL (6 h treatment followed by 18 h recovery)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Dissolved in the vehicle and dosed:
- In the growth inhibition test, dosed into cell culture in the wells (with medium present; total 200 µL/well) of the 96 well plate, 4 wells per dose;
- In the chromosome aberration assay, dosed into 60 mm culture dishes (with medium present; total 5 mL/dish), 2 dishes per dose;

DURATION
Treatment durations, durations of subsequent recovery in fresh culture medium (free from test material) and the harvest time point are specified in the field "Test concentrations"

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® (Invitrogen USA) was added to the cultures (0.2 µg/mL culture medium) 2 hours before the harvest time.

The cells were trypsinised to detach them from the tissue culture flask.
The cells were further processed with hypotonic solution (0.075 mol/L KCl).
After incubation in the hypotonic solution, the supernatant was removed and the cells were fixed with 3 + 1 methanol + glacial acetic acid (v+v).

STAIN (for cytogenetic assays):
After fixation the cells were stained with 5% Giemsa solution in 0.01 mol/l Sörenson phosphate buffer (pH 6.8) for ca. 20 minutes.

NUMBER OF REPLICATIONS:
- growth inhibition test: 4 wells per dose
- chromosome aberration assay: duplicate cultures at each dose

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (excluding gaps),
- Number of gaps (chromosome and chromatid gaps),
- Types of aberrations
Chromatid break, Chromosome break, Chromatid exchange, Chromosome exchange,
Cells with several gaps or cuttings were recorded as fragments
In addition, a category "other" was included (which was 0 at all doses and test conditions).

Polyploidy was recorded. For numerical aberration, any cell with one or more polyploidy aberrations was counted as one aberrant cell.


DETERMINATION OF CYTOTOXICITY

Growth Inhibition Test (non-GLP, prior to dose selection for metaphase analysis):
Cell count with hemocytometer for subculturing in 96 well plate (200 µL/well, 4 wells/dose). Treatment on the wells for 6 h (-/+ S9) followed by 18 h culturing on fresh cell medium, or continuous treatment for 24 h (-S9). At 24 hours addition of 50 µL·3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (5 mg/mL PBS) and culturing for a further 4 hours. Then the wells were dried and DMSO (150 µL/well) was added for dissolution. Optical density of the DMSO solution samples were spectrophotometrically measured at 540 nm using an ELISA reader (VERSA mat™, Molecular Devices, USA). Growth inhibition (IC50) values were calculated for each condition [6 h treatment (-S9) + 18 h recovery, 6 h treatment (+S9) + 18 h recovery and for 24 h continuous treatment (-S9) + 0 h recovery].

Determination of Cell Proliferation Rate (GLP, concurrent with chromosme aberration test):
Conducted in the same manner as chromosme aberration test using 6 well plates (Nunc, USA). After culturing, the cell numbers were counted at each concentration and condition and cell proliferation rate (%) values calculated.

Evaluation criteria:

Clastogenicity of a test material was evaluated and categorized according to Sofuni 1998 as follows.

- percentage of chromosome aberrations (excluding gaps) at all test conditions and doses < 5 %: negative,
- percentage of chromosome aberrations (excluding gaps) at any test condition or dose ≥ 5% ≤ 10% equivocal,
- percentage of chromosome aberrations (excluding gaps) at any test condition or dose > 10% positive

Reference: Toshio Sofuni (Ed.)(1998): Data book of chromosomal aberration test in vitro, Revised edition 1998.

Statistics:

Statistical analysis of the chromosome aberration results was not performed. The study result was unequivocal.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung (CHL/IU) cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments at all concentrations and test conditions

Cytotoxicity / choice of top concentrations:

other: inconsistent: cytotoxicity was evident in the initial growth inhibition test, but at such "cytotoxic" concentrations cell proliferation rates concurrent with the chromosome aberration tests were not reduced

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitate or changes in pH or osmolality when the test material was dosed into culture media were not mentioned in the available report translation
- Growth inhibition (IC50) values attained in the growth inhibition test:
= 697.4 µg/mL after 6 h treatment (-S9) + 18 h recovery
= 704.6 µg/mL after 6 h treatment (+S9) + 18 h recovery
= 182.7 µg/mL after 24 h continuous treatment (-S9) + 0 h recovery.
Based on these IC50 values high doses of 700, 700 and 180 µg/mL, respectively were selected for metaphase analysis. However, concurrent with
the chromosome aberration tests cell proliferation rates were close to 100% at each dose and test condition selected for metaphase analysis.

Remarks on result:

other: strain/cell type: CHL/IU from ATCC

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results : negative without and with metabolic activation (S9)

During the described in vitro chromosomal aberration test and under the experimental conditions reported, the test material, Octane-1,2-diol, did not induce structural chromosomal aberrations in the Chinese hamster lung (CHL/IU) cell line. Chromosome aberration rates (excluding gaps) were consistently low and close to the vehicle controls at all test material doses and test conditions. In addition, at 6 h treatment without and with metabolic activation (followed by 18 h recovery), the maximum test concentrations were rather high, i.e. about 0.005 mol/L, which is about half the maximum OECD 473 recommended test concentration. Therefore, the present study is considered to be conclusive and adequate for evaluation of clastogenicity of the test material, despite the inconsistency of cytotoxicity being evident in the initial growth inhibition tests, but not being reflected by relevant reduction in cell proliferation rates concurrent with the chromsome aberration tests.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Types and identity of media and culturing conditions:
The V79 cell line is stored over liquid nitrogen in the cell bank of the testing facility as stock cultures allowing the repeated use of the same
cell culture batch in experiments.
Subculturing in MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum) and 1% neomycin.
Routine cell culturing at 37°C with 4.5% CO2 in air.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT mediumas described by Bradley et al. 1981.
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from male Wistar rats treated on 3 consecutive days by i.p. injection with phenobarbital and by oral gavage with β-naphthoflavone (80 mg/kg/day, each) for enzyme induction. The concentration of S9 mix in final test medium was 5% (v/v).

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (cloning efficiency relative to that of vehicle controls)
Test concentrations at 4 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
0 (vehicle control), 11.6, 23.1, 46.3, 92.5, 185.0, 370.0, 740.0 & 1480.0 µg/mL

MUTATION TESTS
Experiment 1, 4 h exposure (–S9):
Exposure concentrations: 11.6, 23.1, 46.3, 92.5, 185.0 & 370.0 µg/mL
Mutant phenotype determination at: 11.6, 23.1, 46.3, 92.5 & 185.0 µg/mL

Experiment 1, 4 h exposure (+S9):
Exposure concentrations: 92.5, 185.0, 370.0, 740.0, 1110.0 & 1480.0 µg/mL
Mutant phenotype determination at: 92.5, 185.0, 370.0 & 740.0 µg/mL

Experiment 2, 24 h exposure (-S9):
Exposure concentrations: 46.3, 92.5, 185.0, 370.0, 555.0 & 740.0 µg/mL
Mutant phenotype determination at: 92.5, 185.0, 370.0, 555.0 & 740.0 µg/mL

Experiment 2, 4 h exposure (+S9):
Exposure concentrations: 46.3, 92.5, 185.0, 370.0, 555.0 & 740.0 µg/mL
Mutant phenotype determination at: 92.5, 185.0, 370.0, 555.0 & 740.0 µg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
In the absence of confounding factors, 4 analysable concentrations up to 5000 µg/mL or 10 mM (whichever is lowest) for freely soluble test materials are the guideline requirement for mutant frequency determination. Therefore, in the preliminary tox. test, the maximum selected concentration represented 10 mM including a correction for test material purity. Precipitate was not evident up to the highest test concentration. Variations in osmolality and pH between vehicle control and high dose culture media (without metabolic activation) were within acceptable limits. Hence, the choice of test concentrations for determination of mutant frequency was only limited by cytotoxicity.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Justification for choice of solvent/vehicle:
The use of DMSO was not explicitly justified in the report. However, the report states that the test material is adequately stable in DMSO and that there was no precipitation in the present study. Therefore, it can be concluded that the test material was adequately miscible with DMSO thus facilitating maximum exposure to the test material. In addition, DMSO is a vehicle commonly known to be compatible with the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (0.5% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control substance for tests without metabolic activation (-S9): Experiment 1, 4 h exposure, concentration in final medium 150 µg/mL; Experiment 2, 24 h exposure, concentration in final medium 75 µg/mL, vehicle nutrient medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (0.5% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Positive control substance for tests with metabolic activation (+S9): Experiments 1 & 2, 4 h exposure, concentration in final medium 1.1 µg/mL, vehicle DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: DMSO test material dilution applied to the medium

DURATION
- Exposure duration: Experiment 1: 4 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 4 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9)

- Selection time: 3 days after treatment subcultivation. Following a 7 day expression time seeding in medium containing the
selection agent thioguanine (6TG), then allowing 8 days for cells to grow with 6TG.

SELECTION AGENT: Thioguanine (6TG)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
- from each test concentration, negative, vehicle or positive control:
2 flasks for assessment of survival, i.e. cloning efficiency I immediately after treatment.
2 flasks for assessment of viability, i.e. cloning efficiency II after the expression period measuring viability
in the absence of 6TG.
and 5 flasks for assessment of mutant potential.

DETERMINATION OF CYTOTOXICITY:
Preliminary toxicity test: Colony forming ability in vehicle controls & at each test material concentration (duplicate cultures)
Main tests: Cloning efficiency I immediately after treatment;
Cloning efficiency II after the expression period measuring viability in the absence of 6TG.

Evaluation criteria:

Mutagenic response of test material was considered to be evident if the test material induced a concentration-related increase in mutant frequency or a reproducible positive response at one of the test conditions.

A response was considered to be positive if the mutation frequency of at least one test condition reproducibly was three times higher than the spontaneous mutation frequency of the vehicle control. In case of a reproducibly concentration-related increase in mutant frequency a test material may be regarded as mutagenic also if the increase is less than threefold.

However, the level of spontaneous mutant frequency in the present study compared with the historical range and variability of mutation frequency in solvent controls was duely accounted for when evaluating the data of the present study.

A final decision on mutagenicity was taken by the study director case by case accounting for reproducibility, dose-relationship and the degree of mutant frequency increase.

Statistics:

The data were analysed by linear regression (least squares) to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 (SYSTAT Software, Inc., Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for test material treated groups was compared with the vehicle control groups. p-values < 0.05 were considered to indicate a statistically significant trend. However, both, biological relevance and statistical significance were given due consideration.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 4h exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 24 h exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiments 1 & 2, 4 h exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment 2, 24 h exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In the present study, precipitate was not evident up to the highest test concentration and variations in osmolality and pH between vehicle control and high dose culture media (without metabolic activation) were within acceptable limits. However, cytotoxicity of the test material was a confounding factor. Hence, the choice of test concentrations for determination of mutant frequency was only limited by cytotoxicity.

DOSE DEPENDENCE
Linear regression (least square) analysis produced p-values > 0.05 in both experiments, in both replicate cultures and at all test conditions indicating that there was no statistically significant dose dependence in mutant frequency. In addition, there were also no biologically relevant or reproducible increases in mutant frequency.

Conclusions:

Interpretation of results : negative without and with metabolic activation (-/+S9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies octane-1,2 -diol is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].