5-bromo-5-nitro-1,3-dioxane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-Oct-2018 to 08-Jan-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

Due to a technical error, only 451 wells were used instead of 480 wells. Since the fallout was only 6% and the response of the solvent control was within the acceptability criteria, this deviation had no effect on the results of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-5-nitro-1,3-dioxane

EC Number:

250-001-7

EC Name:

5-bromo-5-nitro-1,3-dioxane

Cas Number:

30007-47-7

Molecular formula:

C4H6BrNO4

IUPAC Name:

5-bromo-5-nitro-1,3-dioxane

Details on test material:

- Chemical name: 5-Brom-5-nitro- 1,3-dioxan
- Trade name: Bronidox

Specific details on test material used for the study:

Identification: 5-Bromo-5-nitro-1,3-dioxane
Batch (Lot) Number: 201807021
Expiry date: 30 Jun 2020 (expiry date) (taken from label)
Physical Description: White crystal powder
Storage Conditions: At room temperature

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The concentration of the S9-fraction in the exposure medium was 4% (v/v)

Test concentrations with justification for top dose:

In the first experiment, the test item was tested up to concentrations of 10 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. In the absence of S9-mix, the Relative total growth (RTG) was reduced to 0% at the concentration of 10 µg/mL. Therefore, this concentration could not be used for mutation frequency determination. RTG was reduced to 37% at the concentration of 5 µg/mL. In the presence of S9-mix, RTG was reduced to 6% at the concentration of 10 µg/mL. The test item did not precipitate in the culture medium.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany).

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells. Rationale Recommended test system in international guidelines (e.g. OECD). Source American Type Culture Collection, (ATCC, Manassas, USA) (2001). Stock cultures of the cells were stored in the freezer (-150°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 10 to the power of 6 cells/mL.

Cell Culture Horse serum Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes. Basic medium RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies). Growth medium Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium). Environmental conditions All incubations were carried out in a humid atmosphere (80 - 100%, actual range 54 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.3 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

Metabolic Activation System: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Preparation of S9-Mix S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Cleansing Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Dose-range Finding Test In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix. In the initial experiment, the highest tested concentration was 2000 µg/mL exposure medium. In the second and third experiment, the highest concentrations were 120 and 30 µg/mL, respectively. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium). For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter. The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted.

The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests. The results of the dose-range finding studies with the 24 hours treatment period have not been reported, since the test item already showed clear mutagenic responses after the 3 hours treatment period.

Mutagenicity Test Eight doses of the test item were tested in the mutation assay. The test item was tested in the absence and presence of S9-mix with a 3 hour treatment period. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested. The highest dose level tested in the absence of S9-mix showed a RTG below 10% and was therefore not used for mutagenicity determination.

Treatment of the Cells The test item was tested both in the absence and presence of S9-mix. Per culture 8 x 106 cells (106/mL) were used. Cell cultures were exposed for 3 hours to the test item in exposure medium in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 rpm. Solvent and positive controls were included and the solvent control was tested in duplicate. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).

Expression Period For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10 to the power of 6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the Mutation Frequency For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10 to the power of 5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10 to the power of 5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days.

After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Rationale for test conditions:

L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which is lethal to the cells. Thus, cells that are able to proliferate in culture medium containing TFT are mutated, either spontaneously or by the action of the test item, to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes. A test item, which induces a positive response in this assay, is presumed to be a potential mammalian cell mutagen.

Rationale: Recommended test system in international guidelines (e.g. OECD).

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria: a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation. b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors. c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32. d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
The test item precipitated directly in the exposure medium at concentrations of 1000 μg/mL and above. After 3 hours, the test item did not precipitate in the exposure medium at concentrations of 2000 μg/mL. The highest concentration which did not precipitate directly in the exposure medium was 500 μg/mL. The pH and osmolarity at a concentration of 500 μg/mL were 7.370 and 0.409 Osm/kg respectively (compared to 7.361 and 0.418 Osm/kg in the solvent control).

Dose-range Finding Test
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 125 to 2000 µg/mL in the absence and presence of S9-mix with a 3 hour treatment period. No or hardly any cell survival was observed at all test item concentrations tested.

Dose-range Finding Test 2 (additional)
Since in the first dose range finding test too many dose levels with severe toxicity were observed after 3 hours of treatment, an additional dose range finding test was performed. In the additional dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 10 to 120 µg/ml in the absence and presence of S9-mix with a 3 hour treatment period.

In the absence and presence of S9-mix, the relative suspension growth was respectively 2 and 31% at the test item concentration of 10 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 30 μg/mL and above.

Dose-range Finding Test 3 (additional)
Since in the second dose range finding test too many dose levels with severe toxicity were observed after 3 hours of treatment, an additional dose range finding test was performed. In the second additional dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 0.03 to 30 µg/ml in the absence and presence of S9-mix with a 3 hour treatment period.

In the absence of S9-mix, the relative suspension growth was 42% at the test item concentration of 30 μg/mL compared to the relative suspension growth of the solvent control.

In the presence of S9-mix, the relative suspension growth was 65% at the test item concentration of 10 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at the test item concentration of 30 μg/mL.

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test: Without S9-mix: 0.01, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL exposure medium. With S9-mix: 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5, 10, 15, 20, 25, 30 and 50 μg/mL exposure medium. Evaluation of toxicity In the absence of S9-mix, the dose levels of 0.01 to 2.5 μg/mL showed similar no cytotoxicity. Therefore, the dose level of 0.01 µg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 20 to 60 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 15 to 50 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: Without S9-mix: 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5 and 10 μg/mL exposure medium. With S9-mix: 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5 and 10 μg/mL exposure medium

In the absence of S9-mix, the effect of the test item was evaluated up to the dose level of 5 µg/ml giving a RTG value of 37%. Above this dose level the RTG was below the acceptable limit of 10%. In the presence of S9-mix, the relative total growth of the highest test item concentration was 6% compared to the total growth of the solvent controls. Evaluation of the mutagenicity In the absence of S9-mix, the test item induced increases above the 95% control limits of the distribution of the historical negative control database. However, the increases were not above the GEF + MF(controls) (225 per 10 to the power of 6 survivors) and therefore considered not biologically relevant. In the presence of S9-mix, the test item induced increases above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (203 per 10 to the power of 6 survivors).

Applicant's summary and conclusion

Conclusions:

In conclusion, 5-Bromo-5-nitro-1,3-dioxane is mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of  5-Bromo-5-nitro-1,3-dioxane by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).  The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the presence and absence of S9-mix with a 3 hour treatment period.  The study procedures described in this report were based on the most recent OECD guideline.   Batch 201807021 of the test item was a white crystal powder.  The vehicle of the test item was dimethyl sulfoxide.  In the first experiment, the test item was tested up to concentrations of 10 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours.  In the absence of S9-mix, the Relative total growth (RTG) was reduced to 0% at the concentration of 10 µg/mL. Therefore, this concentration could not be used for mutation frequency determination. RTG was reduced to 37% at the concentration of 5 µg/mL.  In the presence of S9-mix, RTG was reduced to 6% at the concentration of 10 µg/mL. The test item did not precipitate in the culture medium.  In the absence of S9-mix, the test item induced increases above the 95% control limits of the distribution of the historical negative control database.  However, the increases were not above the GEF + MF(controls) (225 per 106 survivors) and therefore considered not biologically relevant. In the presence of S9-mix, the test item induced increases above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (203 per 106 survivors).   The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.   Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency.  In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In conclusion, the test item is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report