4,4-dimethyloxazolidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.11.2018 - 22.11.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4-dimethyloxazolidine

EC Number:

257-048-2

EC Name:

4,4-dimethyloxazolidine

Cas Number:

51200-87-4

Molecular formula:

C5H11NO

IUPAC Name:

4,4-dimethyl-1,3-oxazolidine

impurity 1

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

liquid

Details on test material:

Batch No.: 10043767
Appearance: Light yellow liquid
Storage: At room temperature (15°C - 25°C), away from direct sunlight

Specific details on test material used for the study:

Batch No. : 10043767
Appearance: Light yellow liquid
Storage: At room temperature (15°C - 25°C), away from direct sunlight

Method

Target gene:

histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) rat liver S9 mix

Test concentrations with justification for top dose:

Initial Mutation Test: 1250, 1000, 750, 500, 160, 50, 16 µg/plate
Confirmatory Mutation Test: 750, 600, 500, 300, 160, 50, 16, 5, 1.6 µg/plate
Additional Pre-Incubation Test: 300, 200, 160, 100, 75, 50, 25, 16, 5 µg/plate

Concentration Range Finding Test (Informatory Toxicity Tests):
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 4092, 1309, 409, 131, 41, 13 and 4 µg/plate.
The revertant colony numbers of vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
IIn the performed Informatory Toxicity Test strong inhibitory effect of the test item was observed at the concentration levels of 4092 and 1309 µg/plate.
At the concentration choice the guideline criterion for cytotoxic test compounds was taken into consideration. Accordingly in this test the highest test item concentration used for the initial mutation test was 1250 µg/plate.
Because of the noticed cytotoxic effect in the Initial Mutation Test, revision of the examined concentration range for the Confirmatory Mutation Test was considered.
Based on the results of the Confirmatory Mutation Test a partial repetition (Additional Pre-Incubation Test) was performed with the Salmonella typhimurium TA98 and TA100 strains, with the lower concentration levels
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water (ASTM Type I)
- Justification for choice of solvent/vehicle: In the solubility test the test item behaviour was investigated in the applied test system. The test item was dissolved and further diluted in ultrapure water (ASTM Type I), accordingly. The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension. No precipitation was observed.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

Positive control concentration: 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control concentration: 2 µg/plate for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

positive control concentration: 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

posititve control concentration: 2 µL/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

Positive control with metabolic activation, concentration: 2 µg/plate for S. typhimurium strains; 50 µg/plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: initial mutation test: in agar (plate incorporation); confirmatory test: in agar (plate incorporation) with preincubation

- Bacterial cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37°C Benchtop Incubator Shaker.

- Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The typical content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain 100 µL (containing aprox 10^9 CFU/ml)
phosphate buffer (pH: 7.4) or S9 mix 500 µL
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube.

- Exposure duration: incubated at 37°C for about 48 hours

- Preincubation period (in confirmatory test and partial repetition of confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates.

NUMBER OF REPLICATIONS: Triplicates

METABOLIC ACTIVATION SYSTEM: Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

VALIDITY CRITERIA
The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in
induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest doses tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
- The test has to be included five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non-precipitated dose levels.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Statistics:

Based on the evaluation criteria no statistical analysis was required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Validity criteria: All criteria for the validity of the performed experiments according to the OECD guideline were met.
- Controls : In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the ultrapure water (ASTM Type 1) vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

Initial Mutation Test (Plate Incorporation Test):
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with 4,4-dimethyloxazolidine at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were noticed. These increases did not show a clear dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase observed in this experimental part of the study was noticed in Salmonella typhimurium TA98 strain at 160 µg/plate, in the absence of metabolic activation (-S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.91, which was far below the genotoxicological threshold for being positive.
In the Initial Mutation Test unequivocal cytotoxic effects of the test item were noticed in all examined strains. The cytotoxicity was indicated by affected background lawn development: absent, reduced or slightly reduced background lawn and/or absent or decreased revertant colony counts (below the corresponding historical control data ranges). Because of the noticed cytotoxic effect in the Initial Mutation Test, revision of the examined concentration range for the Confirmatory Mutation Test was considered.
In the performed experiment no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

ConfirmatoryMutation Test (Pre-Incubation Test):
In this test positive results, significant, biologically relevant revertant colony number increases, revertant colony numbers above the vehicle control data and above the historical control data ranges were obtained in S. typhimurium TA98 and TA100 strains in the absence and presence of exogenous metabolic activation (± S9).
The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA98 at the concentration of 50 µg/plate (-S9 Mix) and at 160 µg/plate (+S9 Mix), in TA100 at the concentration of 50 µg/plate (-S9 Mix) and at 160 and 50 µg/plate (+S9 Mix).
No positive results, biologically relevant increases were observed in revertant colony numbers of any of the remaining experimental parts using tester strains S. typhimurium TA1535, TA1537 and in E. coli WP2 uvrA following treatment with 4,4-dimethyloxazolidine at any concentration level, either in the presence or absence of metabolic activation (S9 mix).
Additionally, the increased revertant colony numbers were above the corresponding historical control data range but, below the genotoxicological threshold for being positive for S. typhimurium TA98 at 16 µg/plate (-S9 Mix), at 300 and 50 µg/plate (+S9 Mix); for TA1535 at 300 µg/plate (+S9 Mix); and for E. coli WP2 uvrA at 50 µg/plate (+S9 Mix).
Similarly to the Initial Mutation Test, cytotoxic effects of the test item were noticed in all examined strains in this experimental phase as well. The cytotoxicity was indicated by affected background lawn development: absent, reduced or slightly reduced background lawn and/or absent or decreased revertant colony counts (below the corresponding historical control data ranges).
For unequivocal confirmation of repeatability of the obtained positive results a partial repetition of the Confirmatory Mutation Test, an Additional Pre-Incubation Test was considered necessary with the affected strains.
In the performed Confirmatory Mutation Test no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

Additional Pre-Incubation Test:
In this test positive results, significant, biologically relevant revertant colony number increases, revertant colony numbers above the vehicle control data and above the historical control data ranges were obtained for S. typhimurium TA98 and TA100 strains at the concentrations of 160, 100 and 75 µg/plate, in the presence of exogenous metabolic activation (+S9). Furthermore equivocal, borderline positive results were noticed for S. typhimurium TA100 at 100 µg/plate, in the absence of exogenous metabolic activation (-S9).
The increased revertant colony numbers were above the corresponding historical control data range but below the genotoxicological threshold for being positive for S. typhimurium TA98 at the concentration range of 100-25 µg/plate (-S9), at 200 µg/plate (+S9); and for TA100 at 75 µg/plate (-S9 Mix) and at 200 µg/plate (+S9).
The positive results obtained in the Confirmatory Mutation Test were unequivocally repeated confirmed for both strains at 160 µg/plate (+S9); additionally, biologically relevant revertant colony number increases were noticed in both strains at 100 and 75 µg/plate (+S9). The positive results obtained in the Confirmatory Mutation Test in the absence of exogenous metabolic activation were not confirmed.
In this experimental part of the study slight inhibitory effect of the test item was noticed in both strains at the highest examined concentration levels. The cytotoxicity was indicated by decreased revertant colony counts (below the corresponding historical control data ranges) and/or affected background lawn development: slightly reduced background lawn.
In the performed Additional Pre-Incubation Test no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains used, in the presence of exogenous metabolic activation, while equivocal mutagenicity results were obtained in the absence of metabolic activation.
In conclusion, the test item 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) has mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of exogenous metabolic activation, under the test conditions used in this study.

Executive summary:

The test item 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (±S9) prepared from livers of Phenobarbital/ß-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), a Confirmatory Mutation Test (Pre-Incubation Test) and an Additional Pre-Incubation Test.

Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in ultrapure water (ASTM Type I).

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial Mutation Test:

±S9: 1250, 1000, 750, 500, 160, 50 and 16 µg/plate.

The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the guideline criterion for cytotoxic test compounds was taken into consideration. Accordingly in this test the highest test item concentration was 1250 µg/plate.

Because of the noticed cytotoxic effect in the Initial Mutation Test, revision of the examined concentration range for the Confirmatory Mutation Test was considered and the following concentrations (with lowered top concentration) were investigated in the follow-up experiment for the Salmonella typhimurium TA98 and TA100 strains: 500, 300, 160, 50, 16, 5 and 1.6 µg/plate; for TA1535 and TA1537: 600, 300, 160, 50, 16, 5 and 1.6 µg/plate and for Escherichia coli WP2 uvrA: 750, 500, 300, 160, 50, 16 and 5 µg/plate.

Based on the results of the Confirmatory Mutation Test a partial repetition (Additional Pre-Incubation Test) was performed with the Salmonella typhimurium TA98 and TA100 strains, with the following concentration levels: -S9: 160, 100, 75, 50, 25, 16 and 5 µg/plate; +S9: 300, 200, 160, 100, 75, 50 and 16 µg/plate.

At the preparation of the test item solutions before each experimental phase a correction factor of 1.222 was taken into consideration [based on the water content (i.e. 1/(1-0.182)=1.222) of the test item as the water does not belong to the substance identity].

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In the Initial, Confirmatory Mutation and Additional Pre-Incubation Tests, inhibitory, cytotoxic effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by decreased revertant colony counts (absent revertants or revertants below the corresponding historical control data and/or vehicle control ranges) and/or affected background lawn development (absent, reduced or slightly reduced background lawn).

In general, 160 µg/plate was considered as lowest concentration showing unequivocal cytotoxicity (noticed following the pre-incubation procedures in S. typhimurium TA98 and TA100 in the absence in TA1537 in the absence and presence of exogenous metabolic activation).

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

Biologically relevant increases in revertant colony numbers, indicating positive mutagenicity results were observed in the performed Confirmatory Mutation Test applying the pre-incubation procedure in S. typhimurium TA98 and TA100 strains: in TA98 at the concentration of 50 µg/plate (-S9 Mix) and at 160 µg/plate (+S9 Mix), in TA100 at the concentration of 50 µg/plate (-S9 Mix) and at 160 and 50 µg/plate (+S9 Mix). The unequivocal, biologically relevant increases positive indicating mutagenicity results were confirmed, during the Additional Pre-Incubation Test, in both strains at the concentration of 160 µg/plate in the presence of exogenous metabolic activation (+S9).

The positive results obtained in the Confirmatory Mutation Test in the absence of exogenous metabolic activation were not unequivocally confirmed in the subsequent experiment, remained equivocal, borderline.

The reported data of this mutagenicity assay shows that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains, in the presence of exogenous metabolic activation, while equivocal mutagenicity results were obtained in the absence of metabolic activation.

In conclusion, the test item 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) has mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of exogenous metabolic activation, under the test conditions used in this study.