3-ethyloxetane-3-methanol

Genetic toxicity: in vitro

Administrative data

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:

29th May 2012 - 8th October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-ethyloxetane-3-methanol

EC Number:

221-254-0

EC Name:

3-ethyloxetane-3-methanol

Cas Number:

3047-32-3

Molecular formula:

C6H12O2

IUPAC Name:

(3-ethyloxetan-3-yl)methanol

Specific details on test material used for the study:

Name of test material (as cited in study report): Trimethylolpropane Oxetane
- Physical state: Colourless liquid
- Analytical purity: 99.5%
- Lot/batch No.: 4277708
- Expiration date of the lot/batch: 01 May 2013
- Storage condition of test material: Stored at ambient temperature, protected from light

Method

Target gene:

TK locus of mouse lymphoma L5178Y cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells used were from the tk+tk- -3.7.2C mouse lymphoma L5178Y cell line. The cells grow in suspension culture, have a generation time of
about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentrations used in the toxicity test: 0.12, 0.39, 1.16, 3.87, 11.60, 38.72, 116.16, 387.2 and 1161.6 μg/mL
Concentrations used in the four mutation assays (in μg/mL):
Assay 1 (4 hours in the absence of S9 mix): 72.6, 145.2, 290.4, 580.8 and 1161.6
Assay 2 (4 hours exposure in the presence of S9 mix): 72.6, 145.2, 290.4, 580.8 and 1161.6
Assay 3 (24 hours exposure in the absence of S9 mix): 72.6, 145.2, 290.4, 580.8 and 1161.6
Assay 4 (4 hours exposure in the presence of S9 mix): 72.6, 145.2, 290.4, 580.8 and 1161.6

Vehicle / solvent:

Water

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Remarks:

250 μg/mL for 4 hour exposure and 100μg/mL for 24 hour exposure

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of S9 mix

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 μg/mL for 4 hour exposure and 5μg/mL for 24 hour exposure

Positive control substance:

methylmethanesulfonate

Remarks:

In the absence of S9 mix

Controls 3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

2.5 and 10μg/ml for 4 and 24 hour exposure

Positive control substance:

3-methylcholanthrene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The test item formulations were prepared immediately prior to dosing (within 1 hour). All experimental procedures were conducted using aseptic technique and under amber light.

In the toxicity test, only one culture was prepared for each treatment. The cell population densities were recorded over 2 days (following treatment) using a haemocytometer, then the total suspension growths were expressed as percentages of the vehicle control group (= relative suspension growth, or RSG).
The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 24 h exposure to Trimethylolpropane Oxetane, as a contingency against the later requirement for a full experiment using this extended exposure period.
Observations on the precipitation of Trimethylolpropane Oxetane were made after dosing and at the end of the exposure period. Observations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.

In the mutation test with 4 hour exposure period, samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes containing R0P (3.9 mL). Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by the test formulation (0.1 mL). Vehicle control cultures received water (0.1 mL). Positive control cultures received the appropriate solution (0.1 mL). The final reaction mixture in all cultures contained 10 mL of cells, at a population density estimated at 6.0 x 10E5 cells/mL, in R5P medium. All tubes were placed on a 10 r.p.m. rotating drum, inside an incubator set to maintain a temperature of 37°C, for 4 h. After this, the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density estimated at 3 x 10E5/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on Day 1.

A mutation test with a 24 hour exposure period was conducted when the results of the first experiment in the absence of S9 mix were negative. On the day of the test (Day 0), samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes containing R0P (7.8 mL). R50P (R0P:serum, 50:50, v/v) (2 mL) was added to each tube followed by the test formulation (0.2 mL). Vehicle control cultures received water (0.2 mL). Positive control cultures received the appropriate solution (0.2 mL). The final reaction mixture in all cultures contained 20 mL of cells, at a population density estimated at 3 x 10E5 cells/mL, in R10P medium.
All tubes were incubated on the rotating drum (as described above) for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and were then resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give an estimated 3 x 10E5 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on Day 2.

On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined, which provided a measure of suspension growth. the treated cultures from the 4 highest concentrations of Trimethylolpropane Oxetane were selected for final assessment in all 4 assays.
The cultures were then assessed for expression of genetic damage. This was determined by performing parallel cloning assays for cloning efficiency and mutant selection.
For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.
For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 10E4 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well.
All dishes were placed in an incubator set to maintain a humid atmosphere of 5% CO2:95% air (v/v) at 37°C until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).

The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies.

NUMBER OF REPLICATIONS: Duplicate cultures were tested.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth and relative total growth, mutant fractions and colony size fractions.

Evaluation criteria:

Criteria for a Positive Result:
An experiment was considered positive if one or more concentrations showed a biologically significant increase in mutant fraction and there was a significant linear trend. Additional comparisons that can aid interpretation of results include:
a comparison of the induced mutant fraction with the historical maximum for differencebetween vehicle controls
b comparison of the mutant fraction of a treated group with the historical range of vehicle control values

A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a negative response in the standard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment with the extended exposure.

Criteria for a Negative Result:
A test item was defined as non-mutagenic, provided data were obtained in both the absence and the presence of S9 mix that accompanied one or more of the
following:
• the predetermined maximum concentration of 5000 μg/mL or 10 mM, whichever is lower
• the highest practicable concentration limited by the solubility or pH of the test item
• RTG in the range 10-20%

Statistics:

The results for each experiment were subjected to a test for linear trend in mutant fraction with concentration of Trimethylolpropane Oxetane by the recommended UKEMS method. Analysis included:
1. Determination of the heterogeneity factor for each dose level.
2. Comparison of the heterogeneity factor with historical control. Any dose levels with heterogeneity factor statistically higher than historical control were excluded from all statistical analysis.
3. Determination of the heterogeneity factor for the experiment.
4. Calculation of a new historical control heterogeneity factor.
5. Calculation of the log mutant fraction.
6. Test for linear increasing trend of mutant fraction with increasing dose of test item (at P<0.05).

All assays were conducted in chronological order. The dose-specific heterogeneity factors were compared with the historical controls for consistency using a one-sided F-test at the 0.1% level. Any dose levels where either the mutant or the survival heterogeneity factors were significantly higher than their respective historical controls were excluded.

The heterogeneity factors were compared with the historical controls for consistency using a one-sided F-test at the 1% level. If either the mutant or the survival heterogeneity factors were significantly higher than their respective historical controls the assay was discarded and the old historical controls remained in place. Otherwise, new historical control heterogeneity factors were calculated, using weighting of 1/20 of the current estimate and 19/20 of the historical estimate.
For each assay not discarded under the above, the following were performed:
The log mutant fraction and its variance were calculated for each dose level,
A one sided χ2 test (Pearson and Hartley (1989)) with 1 degree of freedom was performed at the 5 % level to test for linear increasing trend of mutant fraction with increasing dose of test item, if and only if the direction of the slope parameter was positive.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity tests:
The results of the toxicity tests show that Trimethylolpropane Oxetane demonstrated minimal toxicity towards the cells. The highest concentration of 1161.6 μg/mL (10 mM) resulted in the following relative suspension growth levels:
In the absence of S9 mix (4 h exposure): 79.0%
In the presence of S9 mix: 58.0%
In the absence of S9 mix (24 h exposure): 45.3%
No precipitation of Trimethylolpropane Oxetane occurred.

Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value.

Mutation Assays - 4 hour exposure in the absence and presence of S9 mix:
Trimethylolpropane Oxetane was assessed for mutagenic activity at concentrations of: 145.2, 290.4, 580.8 and 1161.6 μg/mL in the absence (4 h exposure) and presence of S9 mix. In the absence of S9 mix the test for linear trend in mutant fraction with concentration of Trimethylolpropane Oxetane was not reported as the slope was negative. In the presence of S9 mix, the test for linear trend in mutant fraction with concentration of Trimethylolpropane Oxetane was not significant (P=0.80). There was no apparent reduction in relative total growth at the highest concentration of
1161.6 μg/mL (10 mM) in either assay. It was determined that the test substance was considered to be not mutagenic for both assays. As a consequence, the second experiment in the absence of S9 mix was conducted with the extended, 24 h exposure period.

Mutation Assays - 24 hour exposure in the absence and presence of S9 mix:
Trimethylolpropane Oxetane was assessed for mutagenic activity at concentrations of: 145.2, 290.4, 580.8 and 1161.6 μg/mL in the absence (24 h exposure) and presence of S9 mix. The tests for linear trend in mutant fraction with concentration of Trimethylolpropane Oxetane was not significant for either assay (P=0.55 in the absence of S9 mix; P=0.42 in the presence of S9 mix). There was no apparent reduction in relative total growth at the highest concentration of 1161.6 μg/mL (10 mM) in either assay. It was determined that the test substance was considered to be not mutagenic for both assays.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation when tested to the predetermined maximum concentration of 1161.6 μg/mL (10 mM).

Under the conditions of this study, Trimethylolpropane Oxetane was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the predetermined maximum concentration of 1161.6 μg/mL (10 mM).

Executive summary:

A study was conducted in accordance with GLP, OECD test guideline 476 and EU Method B.17 to determine the mutagenic potential of Trimethylolpropane Oxetane in a mouse lymphoma L5178Y cell line. Trimethylolpropane Oxetane was formulated in water and tests were conducted both in the absence and in the presence of metabolic activation (S9 mix).

Preliminary cytotoxicity tests showed that Trimethylolpropane Oxetane induced minimal toxicity to the cells at the predetermined maximum concentration of 1161.6 μg/mL (10 mM). Four independent mutation assays were conducted at concentrations of 145.2, 290.4, 580.8 and 1161.6 μg/mL. Positive control cultures were also included (methylmethanesulfonate, ethylmethanesulphonate and 3-methylcholanthrene). Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate.

Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, all experiments were tested for dose-related trends in mutant fraction.

No evidence of mutagenic activity was obtained in any experiment. In conclusion, Trimethylolpropane Oxetane was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the predetermined maximum concentration of 1161.6 μg/mL (10 mM).