3-ethyloxetane-3-methanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies of bacterial reverse mutation (Ames test), mammalian cell clastogenicity and mutagenicity are available for the submission substance.

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:

weight of evidence

Study period:

19th May 1998 - 3rd August 1998.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Nonclinical Studies of Drugs Manual

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): TMP oxetane
- Physical state: Colourless liquid.
- Analytical purity: No information provided
- Lot/batch No.: JNS 980121 F 1-3
- Expiration date of the lot/batch: No information provided
- Stability under test conditions: No information provided
- Storage condition of test material: Stored in a refrigerator at 2-8°C in the dark.

Target gene:

Histidine and Trytophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: 12.5 g Oxoid Nutrient Broth No. 2 per 500 ml H2O for the Salmonella strains.
- Properly maintained: No information provided.
- Periodically checked for Mycoplasma contamination: No information provided.
- Periodically checked for karyotype stability: No information provided.
- Periodically "cleansed" against high spontaneous background: No information provided.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

- Type and identity of media: 12.5 g Oxoid Nutrient Broth No. 2 per 500 ml H2O mixed (1:1) with the following medium: 50ml Davis-Mingiolis salt solution, 4 ml 20% D-glucose solution, 5 mL 10% Difco cassamino acids solution, 8 mL 0.025% L-tryptophan solution and 150 mL distilled water.
- Properly maintained: No information provided.
- Periodically checked for Mycoplasma contamination: No information provided.
- Periodically checked for karyotype stability: No information provided.
- Periodically "cleansed" against high spontaneous background: No information provided.

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

3.1, 6.3, 12.5 and 25 mg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation. Migrated to IUCLID6: 1µg/plate for TA 100 and TA 1535

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation. Migrated to IUCLID6: 1µg/plate for TA 98 and TA 1537

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation. Migrated to IUCLID6: 2µg/plate for WP2 uvrA pKM101

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthrazene ; 2µg/plate for TA 98, TA 100, TA 1535 and TA 1537

Remarks:

with metabolic activation

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthrazene ; 15µg/plate for WP2 uvrA pKM101

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation were both performed.

DURATION
In the plate incorporation method, a series of test tubes was prepared containing the test material solution (0.1 ml), S-9 mix (0.5ml) when required, bacterial suspension (0.1ml approximately 10E9 bacteria/ml) and top agar (2ml). Afer vortex mixing, the mixtures were spread on selective agar plates. After incubation for 48 to 72 hours at 37°C, the number of colonies on each plate was counted.

In the preincubation test, a series of test tubes was prepared containing the test material solution (0.1ml), S-9 mix or 0.02M phosphate buffer pH 7.4 (0.5ml) and bacterial suspension (0.1 ml, approximately 10E9 bacteria/ml). Each test tube was incubated for 1 hour at 37°C with gentle shaking, then top agar (2ml) was added and after vortex mixing, the mixtures were spread on selective agar plates. After incubation for 48 to 72 hours at 37°C, the number of colonies on each plate was counted.

NUMBER OF REPLICATIONS: Triplicate plates were used at each test point.

Evaluation criteria:

The test article was considered to have shown evidence of mutagenic activity in this study if the following criteria were met:
- dose related increases in the numbers of revertant colonies were observed at one or more test points.
- the increases were reproducible between replicate plates and between tests.
- the increases were statistically significant.
- the increases were more than twice the corresponding negative value.

The test article would have been considered to have shown no evidence of mutagenic activity if no increases in the numbers of revertant colonies which exceed 1.5 tiems the negative control value were observed at any test point. Sporadically occurring statistically significant increases in revertant numbers which were not dose related were usually considered incidental and not relevant for evaluation.

Increases between 1.5 and 2-fold greater than the negative control values which met the other criteria for a positive result would have been considered to demonstrate weak mutagenic activity.

Statistics:

The number of revertant colonies at each treatment test point were compared to the corresponding negative control using the Analysis of Variance test. The statistical analyses was performed with SAS procedures (version 6.12).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Small statistically significant decreases in the numbers of revertant colonies were observed after treatment at all doses tested in the plate incorporation test without S-9 mix in the TA 100, while the same observation was recorded for WP2 uvrA pKM101 strain with S-9 mix at 310 and 5000 µg/plate. In the TA 100 group, a small statistically significant increase in the numbers of revertant colonies was observed after treatment at 5000 µg/plate in the plate incorporation test with S-9 mix. These effects are not considered to be biologically significant because they were small, did not show a concentration relationship and they were not reproduced in the preincubation test.

In the TA 98, TA 1537 and TA 1535 strains, there were no increases in the number of revertant colonies in either plate incorporation or preincubation tests that were considered to be related to treatment, nor was there any effect of the S-9 mix.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity study, the test material was shown to be non-toxic to TA 98 at doses above 5000 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A further plate incorporation test and preincubation test were performed in error using test material doses in the range 19 - 300 µl/plate. These doses were above the maximum limit required by the regulatory guidelines. The results reflected those obtained in the main test, with no biologically significant increases in the numbers of revertant colonies observed.

No additional information provided.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of the study, the test material, TMP oxetane as found to be non-mutagenic in the Ames test.

Executive summary:

The genotoxicity of TMP oxetane in bacterial test systems was investigated in the Ames test using Salmonella typhimurium strains TA 100, TA 98, TA 1537 and TA 1535 and Escherichia coli strain WP2 uvrA pKM101. The test material was tested in a plate incorporation and a preincubation assay by addition of 310 - 5000 µg of the test material (mixed with sterile water) to each plate. The highest dose tested (5000 µg/plate) is the highest required by the OECD, EU and Japanese Guidelines for materials of low toxicity. Negative control cultures were treated by the addition of 100µl of sterile water.

The sensitivity of the test system and the efficacy of the S-9 mix were demonstrated by large increases in the numbers of revertant colonies on plates treated with the positive control materials. Based on the results of the study, the test material, TMP oxetane was found to be non-mutagenic in the Ames test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 16th 1997 - March 27th 1997.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: "Notification on Partial Revision of Testing Methods relating to the New Chemical Substances"

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name of test material (as cited in study report): Oxetane (OH)
- Molecular formula (if other than submission substance): C6H12O2
- Molecular weight (if other than submission substance): 116.16
- Physical state: Colourless and transparent Liquid
- Analytical purity: ≥99.9%
- Lot/batch No.: 960501-4
- Storage condition of test material: Store in a cold dark place.
- Other: Melting point: -37°C
Boiling point: 105°C/7mmHg
Readily soluble in water
Soluble in Acetone, methanol and toluene.

Target gene:

No information provided.

Species / strain / cell type:

other: Chinese hamster lung fibroblasts (CHL cells, clone No. 11)

Details on mammalian cell type (if applicable):

Modal number of chromosomes was 25 per cell and the doubling time was about 15 hours. The passage number was 18 at recepit.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

300, 600 and 1200µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: cyclophosphamide monohydrate (CPA)

Remarks:

with S9 mix

Details on test system and experimental conditions:

A preliminary cell growth inhibition test and a cell division inhibition test were performed initially to determine the dose ranges to be used in the chromosomal aberration test. The concentrations used in these preliminary tests were 0, 10, 30, 50, 100, 300, 500, 1000, 3000 and 5000µg/ml. Based on the results achieved in the preliminary testing, the concentrations used for the chromosomal aberration test were determined to be 300, 600 and 1200µg/ml. Two Petri dishes were used at each concentration level in the chromosomal aberration test.

The number of cells with structural aberrations, such as gaps, breaks or exchanges in chromatid or chromosome and numerical aberrations (polyploidy, endo-reduplication) were examined by observing 100 metaphase cells in each dish. An aberrant cell was defined as a cell with any type of structural or numerical aberrations. 200 metaphase cells were observed in each dose to obtain the frequencies of cells with chromosomal aberrations.

The frequencies of structural aberration were calculated for each case with and without gaps that were identified as an achromatic region larger than a width and less than double width of one chromatid located on the same axis.

Evaluation criteria:

Structural aberrations (including gaps) and numerical aberrations were judged according to their incidences as follows:

negative (-) for less than 5%
suspected positive (±) for 5% or more and less than 10%
positive (+-+++) for 10% or more.

The positive criteria were subdivided into (+) for 10% or more and less than 20%, (++) for 20% or more and less than 50% and (+++) for 50% or more.

The test substance was judged to be positive when there was dose-dependency or reproducibility in the induction of chromosomal aberrations.

Statistics:

No information provided.

Key result

Species / strain:

other: Chinese hamster lung fibroblasts (CHL cells, clone No. 11)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary cell growth inhibition and cell division inhibition tests performed at doses of 0, 10, 30, 50, 100, 300, 500, 1000, 3000 and 5000µg/ml, at 24 hours, 50% cell growth inhibition in 100% solvent control was observed at 2900 µg/ml and about 2500 µg/ml in 48 hours when treated by the direct method. When treated using the metabolic activation method, 50% cell growth inhibition concentration was not obtained due to the weak cytotoxicity of the test substance.
In the cell division inhibition test, the available highest doses in the chromosomal aberration test were 3000 µg/ml in both direct methods and 5000 µg/ml in both metabolic activation methods.
Due to the test substance being dissolved in medium, the highest dose in the chromosomal test was determined to be 1200µg/ml.

Chromosomal Aberration Test:
Direct Method:
The frequencies of cells with structural aberrations including gaps were 0, 0 and 0.5% at 300, 600 and 1200µg/ml and 1.0, 0.5 and 1.0%, at 24 and 48 hours respectively. The solvent control was 0.5% which was within the normal range. The frequency in the positive control with mitomycin C was 43.5 % and induction of chromosome aberration was clearly observed. The frequency of numerical aberration were less than 5% in all treatment groups.

Metabolic Activation Method:
In the absence of S9 mix, the frequencies of cells with structural aberrations including gaps were 1.5, 0.5 and 1.0% at 300, 600 and 1200µg/ml, with the solvent control at 0% which was within the normal range. The frequency in the positive control with mitomycin C was 37.0% and induction of chromosome aberration was clearly observed. The frequency of numerical aberration were less than 5% in all treatment groups.

In the presence of S9 mix, the frequencies of cells with structural aberrations including gaps were 0.5, 0 and 0.5% at 300, 600 and 1200µg/ml, with the solvent control at 0.5% which was within the normal range. The frequency in the positive control with cyclophosphamide monohydrate was 42.0% and induction of chromosome aberration was clearly observed. The frequency of numerical aberration were less than 5% in all treatment groups.

No additional information.

Conclusions:

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

In the chromosomal aberration test using Chinese hamster lung cells, oxetane (OH) did not induce any structural or numerical aberrations at the dose levels employed in both direct and metabolic activation methods, therefore, it can be concluded that oxetane (OH) did not induce chromosomal aberrations with or without metabolic activation under the conditions employed in this test.

Executive summary:

In a study conducted to determine the ability of oxetane (OH) to induce chromosomal aberrations, Chinese hamster lung fibroblasts (CHL cells) were examined in the presence and absence of metabolic activation (S9 mix), with Mitomycin C and cyclophosphamide monohydrate used as positive controls. A preliminary cell growth inhibition test and cell division inhibition test was conducted to determine the dose levels to use in the chromosomal aberration testing. Three dose concentrations of 300, 600 and 1200µg/ml were tested in the chromosomal aberration test. The cells were exposed for 24 and 48 hours in the absence of metabolic activation and for 6 hours in the presence and absence of metabolic activation. Oxetane (OH) did not induce any chromosomal aberrations at the dose levels tested, therefore, it was concluded that oxetane (OH) did not induce chromosomal aberrations with or without metabolic activation under the conditions employed in the test.

Reference 3

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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

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

Target gene:

TK locus of mouse lymphoma L5178Y cells

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

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

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

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.

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP/OECD Test Guideline 471 study to determine the mutagenic potential of 3-ethyloxetane-3-methanol in bacterial cells in vitro (Edwards, 1998), negative results were obtained in Salmonella typhimurium strains TA 100, TA 98, TA 1537 and TA 1535 and Escherichia colistrain WP2 uvrA pKM101, in the presence and in the absence of metabolic activation (S9 mix). The potential for 3-ethyloxetane-3-methanol to induce cytogenic effects in mammalian cells in vitro was investigated in a study conducted using methods comparable to OECD Test Guideline 474 (Kikuno, 1997). In the study, the ability of the test substance, oxetane (OH) to induce chromosomal aberrations was investigated using Chinese hamster lung fibroblasts (CHL cells) in the presence and in the absence of metabolic activation (S9 mix). Based on the results of cell growth and cell division inhibition tests, doses of 300, 600 and 1200 mg/mL were used in the chromosome aberration test. The substance did not induce chromosomal aberrations with or without metabolic activation under the conditions of the test. The potential for 3-ethyloxetane-3-methanol to induce gene mutations in mouse lymphoma L5187Y cells in vitro was investigated in a gene mutation study conducted according to OECD Test Guideline 476 (Riach, 2012). In the study, the test substance Trimethylolpropane Oxetane was not mutagenic in mouse lymphoma cells, in either the absence or the presence of metabolic activation (S9 mix), when tested to the predetermined maximum concentration of 1161.6 μg/mL (10 mM).

Justification for classification or non-classification

3-ethyloxetane-3-methanol is not mutagenic in bacterial cells or in mammalian cells in vitro. The substance does not meet the criteria for classification for genetic toxicity according to the CLP Regulation