4,4-dimethyloxazolidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several studies performed according to OECD guidelines or scientifically recognized methods, performed under GLP, with reliability 1 or 2 are available for this endpoint.

1. bacterial mutation tests

a) Ames (OECD 471) mutagenic for S. typhimurium strains TA98 and TA100 in presence of metabolic activation, ambiguous mutagenicity for S. typhimurium strains TA98 and TA100 in the absence of metabolic activation.

b) Ames (OECD 471) positive for S. typhimurium strains TA98 and TA100 in presence of metabolic activation.

c) Ames coupled with bacterial fluctuation assay: Ames (OECD 471) positive with and without metabolic activation for S.typhimurium strain TA100. Bacterial fluctuation assay (according to old test method of Gatehouse and Delow (Mutat. Res. 60, 239. 1979)) negative for all strains with and without metabolic activation. Both studies reliable with restrictions. Low and narrow concentration range applied in both tests.

 

Conclusion bacterial mutation tests: Test substance induces gene mutations with and without metabolic activation in S. typhimurium strains TA98 and TA100.

 

2. mammalian genotoxicity tests

a) Chromosomal aberration tests (guidelines similar to OECD 473):

On human lymphocytes: Positive for all doses with and without metabolic activation.

On CHO cell line: Positive for all doses with and without metabolic activation.

b) Gene mutation test in mammalian cells:

Mouse lymphoma assay according to OECD Guideline 476. Positive: Test substance induced genetic mutation (frameshift or point mutation) in the presence and absence of exogenous metabolic activation.

c) Unscheduled DNA synthesis in mammalian cells:

Study according to OECD 482. Negative: Test substance does not induce DNA repair in a primary culture of rat hepatocytes.

 

Conclusion mammalian mutation tests: Test substance induces gene mutations and chromosomal aberrations in mammalian cells with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.11.2018 - 22.11.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

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

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

Target gene:

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

Species / strain / cell type:

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

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.

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

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

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

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

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.

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

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

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

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

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).

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March and April 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

HP /60/83 identified also as Oxadine A
Storage Conditions: Cool, dry place

Target gene:

histidine locus (Salmonella typhimurium strains)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Liver Microsomal Fraction S-9
S-9 was made according to Toxicol Protocol MU19 from Aroclor 1254-induced Fischer 344 rats. The protein content of the S-9 was determined by the Lowry method (J. Biol. Chem. 193, 265-275), and must not be less than 35mg/ml to be acceptable for use. One batch of S-9 was used in this study which had a protein content of 40.87mg/ml.

Test concentrations with justification for top dose:

0, 15.6, 31.25, 62.5, 125, 250 and 500 µg/plate. This dose range was chosen because the toxicity rangefinder for a separate fluctuation test (Toxicol Report Ref. 36/8303) had shown HP/60/83 produced toxicity at 250µg/ml (equivalent to 500µg/plate) and 2-fold dose intervals were chosen rather than the usual 5-fold so as to parallel exactly some of the doses in the fluctuation test.

Vehicle / solvent:

Sterile water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Two separate experiments were performed using the 6 selected doses in triplicates with and without metabolic activation.
The following components were added sequentially to 2ml of top agar:
0.1 ml of a dilution of test compound (in triplicate)
0.1 ml of sterile water (negative control) .
0.1 ml overnight bacterial culture (approximately 10^8 organisms)
0.5 ml 10% S-9 (where appropriate)
N.B. All manipulations were carried out under an orange photographic safelight.
The ingredients were rapidly mixed on a Whirlimixer, poured on to prepared minimal agar plates and allowed to set. The plates were then inverted and incubated at 37°C for 48 hours, when the number of revertants per plate was counted using a Biotran II automatic colony counter, and the results recorded on forms MU20.F.1.

Statistics:

Means and standard deviations for each treatment were calculated and recorded on forms MU20F.1. An analysis of variance was performed on each set of data to give the F-statistic. If this was not significant with the degrees of freedom shown (form MUSF.1.) or the data fell within expected control ranges no further analysis was carried out. If the F-statistic was significant (p < 0.05) and some of the data, at least, fell outside the control ranges, the correlation coefficient (r) was calculated from the mean revertants per plate for each of the treatments in the apparent response range, the significance of this correlation coefficient was determined from standard tables and the probability was also recorded on form MUSF.1. In this same calculation, the slope, (b) of the regression line in the response range was determined and gave the mutation frequency in revertants/µg. If the molecular weight of the chemical was known the frequency was multiplied by the number of µg in a nanomole (i.e. Mol.Wt./1000) to give the number of revertants/nmole.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HP/60/83 induced increases in TA100 revertants, both with and without S-9, which were not very great but did exceed the normal range for spontaneous revertants and were significantly dose-related giving significant correlation coefficients.
HP/60/83 was toxic to the tester bacteria at the top dose (500µg/plate) as seen by partial or total loss of background lawn and reduced numbers of revertants. The toxicity limit has therefore been achieved in this experiment.

Conclusions:

4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) induced small but significant and dose-related increases in TA100 revertants, both with and without S-9, in both experiments. It is therefore concluded that 4,4-dimethyloxazolidine is a weak bacterial mutagen over a narrow range of doses in the plate incorporation assay.

Executive summary:

S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with HP/60/83 ( 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4)) by the Ames plate incorporation method at 6 doses in triplicate in the range 15.6 to 500 µg/plate in two separate experiments.

Treatment was carried out without metabolic activation, and also with liver extract (S-9) from Aroclor 1254-induced rats at 10% in standard cofactors. The second experiment was carried out on a separate day with fresh cultures and fresh solutions of HP/60/83. This particular range of doses was chosen because it was shown in the toxicity rangefinder for the fluctuation test (Toxicol Report Ref. 36/8303) that HP/60/83 showed toxicity at 250 µg/ml (equivalent to 500 µg/plate), and 2-fold dose intervals were chosen so as to parallel exactly some of the doses in the fluctuation test.

 

All solvent (negative) control plates gave counts of revertant colonies within normal ranges. Slight increases in control counts were seen for most strains with S-9 in both experiments but this was quite normal.

 

All positive control plates gave frequencies of induced revertants within expected ranges. There was some variation in response between the two experiments but this was not significant.

 

HP/60/83 was seen to be toxic at the top dose of 500 µg/plate due to total or partial loss of background lawn and reduced numbers of revertants. As predicted, the toxicity limit has therefore been achieved in this study.

 

HP/60/83 induced small but significant and dose-related increases in TA100 revertants, both with and without S-9, in both experiments. It did not induce increases in revertants of any other strain.

 

It is therefore concluded that HP/60/83 (4,4-dimethyloxazolidine (CAS Nr. 51200-87-4)) is a weak bacterial mutagen over a narrow range of doses in the plate incorporation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 April 1980-5 September 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1980

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

lot numbers 37 and DO63185

Target gene:

histidine locus (Salmonella typhimurium strains)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Preparation and Storage of Liver Microsomal Enzymes
Liver microsomal enzymes will be routinely prepared from male Sprague-Dawley rats that have been injected with Aroclor 1254 at 500 mg/kg. The Aroclor will be diluted in corn oil to a concentration of 200 mg/ml. Five days after their i.p. injection with the Aroclor, the rats will be sacrificed by decapitation, and their livers will be excised. The rats will be denied access to food for 12 hours immediately preceding sacrifice.
The preparation of the microsomal enzyme fraction will be carried out with sterile glassware and solutions at 0-4°C. The liver from each rat will be excised and placed in 20 ml of 0.15M KCl contained ina pre-weighed beaker. After weighing the liver, it will be transferred to another beaker containing 3 volumes of 0.15M KCl (3ml/g of wet liver) where it will be minced with sterile scissors. The minced liver will be homogenized in a Potter-Elvehjen apparatus with a teflon pestle.
The homogenate will be centrifuged at 9000 x g for 10 minutes in the SS-34 rotor of a Sorvall SS-3 centrifuge. The supernatant (referred to by Ames as the S-9 fraction) will be decanted, and small aliquots will be distributed into freezing ampules which will be stored in liquid nitrogen.

Test concentrations with justification for top dose:

0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.1 and 10.0 µl/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-Aminoanthracene and 1,3-propane sultone

Details on test system and experimental conditions:

Eight serial half-log dilutions of the test compound are plated with TA100 on minimal agar plus 10XSA and on minimal agar plus 1XSA. Equal numbers of cells are seeded on each plate in the presence of the test compound. The percent survival of an appropriately diluted TA100 culture on the 10XSA supplemented plates is determined by comparing numbers of colonies on the solvent control with those on the plates containing test compound.
Toxicity on the 1XSA supplemented plates is detectable by a decrease in the number of revertant colonies occurring per plate and by a thinning or disappearance of the background bacterial lawn.

Plating Procedures for Mutagenesis Plate Incorporation Assay
The test article will be prepared immediately before its use in the mutagenesis assay. Five doses of the test article will first be plated will all five tester strains (TA98, TA100, TA1535, TA1537, TA1538) with metabolic activation, after which they will immediately be plated on all tester strains without metabolic activation. All positive controls, solvent controls, and test article dilutions will be plated in triplicate. Without metabolic activation, 50 µl of tester Strain and 50 µl of solvent or test article will be added to 2.5 ml of molten top agar at 45°C. With metabolic activation, 50 µl of tester Strain, 50 µl of solvent or test article, and 0.5 ml of S-9 mix will be added to 2.0 ml of molten top agar at 45°C. To achieve the desired maximum concentration per plate of the test article it will sometimes be necessary to plate 200 µl or 100 µl aliquots instead of the usual 50 µl aliquots. Appropriate solvent controls will be included in these instances. After vortexing, the mixture will be poured onto the surface of 25 ml of bottom agar contained in a 15x100 mm plastic petri dish. After the top agar has solidified, the plates will be inverted and incubated for 48hr at 37°C.

Evaluation criteria:

Evaluation of Mutagenesis Assay Data
For a test article to be considered positive, it must cause at least a doubling in the observed revertants per plate of at least one tester strain. This increase in revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Strains TA98 and TA100 were retested both with and without microsomal activation and additional doses of test article were added to help clarify the increase in revertants seen with these strains.

Conclusions:

The results of the Salmonella/mammalian-microsome mutagenicity assay indicate that the test did not exhibit significant mutagenic activity on any of the tester strains. It should be pointed out, however, that there was a reproducible dose responsive increase in TA98 and TA100 revertants per plate that did not meet EG&G Mason's criteria for a significant mutagenic response. Follow-up studies did confirm that the increase in colonies per plate was due to an increase in true His+ revertants.

Executive summary:

Amine C5-1135 (P-1952, 4,4-dimethyl-1,3-oxazolididine or Oxazolidine A), lot numbers 37 and DO63185 were sent for evaluation. The samples were submitted to EG&G Mason Research Institute Rockville, Maryland, for an Ames mutagenicity assay.

Test Procedure: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were used in the standard Ames test with and without a rat liver metabolic activation fraction. The bacteria in the presence of a mutagen will revert back to histidine independence and will form colonies.

Results: The sample lot numbers 37 and DO63185 did not exhibit significant mutagenic activity on any of the strains with or without metabolic activator. However, there was a reproducible dose response in TA98 and TA100 strains, but the revertants (mutagenic effect) per plate did not meet EG&G Mason's criteria for a significant mutagenic response.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March and April, 1983

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 other study

Guideline:

other: Microtitre fluctuation test method of Gatehouse and Delow (Mutat. Res. 60, 239. 1979)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

HP/60/83 also identified as Oxadine A
Storage Conditions: Cool, dry place

Target gene:

histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 was made according to Toxicol Protocol MU20 from Aroclor 1254-induced Fischer 344 rats. The protein content of the S-9 is routinely determined by the Lowry method, and must not be less than 35 mg/ml to be acceptable for use. The batch of S-9 used in this study had a protein content of 40.87 mg/ml. 0.5ml of a 10% S-9 mix in standard cofactors was added to each of the tubes containing 2.5 ml or 3.42 ml of bacterial culture.

Test concentrations with justification for top dose:

62.5, 31.25, 15.625 and 7.8125 µg/ml.
For TA100 an additional dose of 125 µg/ml was also used

The maximum non-inhibitory concentration both in the presence and absence of S-9 determined was 62.5 µg/ml and therefore this was used as the top dose in the fluctuation test.

Maximum non-inhibitory concentration test:
HP/60/83 was dissolved in distilled water at 50µg/ml, filter sterilised, and 0.8ml was added to one tube containing 3.92 ml of bacteria and one containing 3.42ml of bacteria with 0.5ml S-9 giving a final concentration of 1000 µg/ml.
Fourfold serial dilutions (1ml + 3ml) were made through the other tubes containing 3 ml of bacteria or bacteria with S-9, and 1ml was discarded from the final tube. 0.08ml of sterile water was added to each remaining tube with 3.92ml of bacteria and bacteria with S-9, and, after mixing, 1ml from each of these was discarded. This gave a dilution series, 3ml per tube, as follows: 1000, 250, 125, 62.5, 15.6, 3.9, 0.98, 0.24, 0.06, 0.015 and 0 µg/ml. The tubes were incubated overnight at 37°C and examined for growth.

Vehicle / solvent:

distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (4NOPD)

Remarks:

Positive control substance with S9 all strains: 2-aminoanthracene (2AA), 30µg/ml

Details on test system and experimental conditions:

3.7 aliquots of reaction mixture (0.16% glucose, 2mg/ml histidine and 0.3µg/ml biotin in 0.1M sodium phosphate buffer at pH 4.7 were placed in sterile tubes (1 tube/dose/strain/level of S-9). The following were then added as appropriate:
1.0ml, 0.1M phosphate buffer or 1.0ml 10% S-9
0.1ml of a dilution of HP/60/83 or solvent or positive control solution
0.2ml of a diluted bacterial culture
40 wells of a sterile microtitre tray were incubated at 37°C overnight in a humidified incubator, and then 150 µl of 'topping-up medium' (Vogel/Bonner salts with 0.8% glucose) containing 50µg/ml Bromocresol (BCP) was added to each well. After a further three days incubation at 37°C the wells were scored. Wells which were yellow due to acid production from utilisation of glucose by growth of revertants were scored as positive. Wells in which the BCP had remained purple indicated no growth of mutants and were scored as negative. The results were recorded on form MU47F.1.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Experiment:
The responses of the five strains to positive control chemicals in the absence and presence of S-9 was as expected. In every case all treated wells were yellow and so the positive control responses are acceptable.
With the test material there are slight increases in the numbers of positive wells with some strains at certain doses but none of these are statistically significant.

Second experiment:
The responses of the five strains to positive control chemicals in the absence and presence of S-9 was as expected. In every case all treated wells were yellow and so the positive control responses are acceptable.The response of TA1537 to 2AA is a little lower than seen in Experiment 1, but is nonetheless highly significant when compared with solvent control (X2 = 6.38, p<0.025) and is therefore acceptable.
With the test material there are slight increases in positive wells in some strains at certain doses but these are not statistically significant, and the 125 µg/ml treatment of TA100 apprears to be toxic in this test whereas the equivalent plate test dose of 250 µg/plate was not toxic. This is probably due to the greater accessibility of test substance to bacteria in liquid medium over that in agar medium.

Conclusions:

It is therefore concluded that, although 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is a weak bacterial mutagen for TA100 in the plate incorporation test, it is not mutagenic for the Ames strains of bacteria in the fluctuation test, and that in neither of these bacterial assays is the mutagenic potential seen that was exhibited in the chromosome mutation test in vitro (AMES)

Executive summary:

S.typhimurium TA1535, TA1537, TA1538, TA98 and TA100 were treated with HP/60/83 (4,4-dimethyloxazolidine (CAS Nr. 51200-87-4)) by the 'microtitre' fluctuation test method of Gatehouse and Delow (Mutat. Res. 60, 239. 1979) at 4 doses in the range of 7.8 µg/ml to 62.5 µg/ml, with and without S-9 (equivalent to 15.6 to 125 µg/plate in the plate incorporation test, Toxicol Report Ref. 35/8303). The repeat experiment was carried out on a separate day with fresh bacterial cultures and fresh solutions of HP/60/83.

All solvent (negative) control trays gave acceptably low levels of positive wells (i.e. spontaneous revertants). All positive control treatments resulted in significant increases in the number of positive wells, both with and without S-9, giving acceptable levels of induced mutation.

None of the HP/60/83 treatments produced significant increases in the numbers of positive wells either in the presence or absence of S-9 in either experiment. As HP/60/83 had produced increased TA100 revertants in the plate test (Toxicol Report Ref. 35/8303) at 250 µg/plate (equivalent to 125 µg/ml) and this dose was not used, it was decided to treat TA100with HP/60/83 at 125 µg/ml in the fluctuation test. However, this treatment actually produced decreases in the numbers of positive wells indicating some toxicity.

It is therefore concluded that, although 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is a weak bacterial mutagen for TA100 in the plate incorporation test, it is not mutagenic for the Ames strains of bacteria in the fluctuation test.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 1982 - 21 December 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Lot No. 6178-29
Storage Conditions: Room Temperature

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cryopreserved Lot No. 9-17-82. The Chinese Hamster Ovary (CHO-K1) cell line (cell repository number CCL 61) was originally obtained from American Type Culture Collection, Rockville, Maryland.
The cells were cultured in Ham's F-12 nutrient medium without antibiotics and supplemented with 10% fetal bovine serum. The cultures were harvested before reaching confluency and reseeded 1.4 x 10^6 cells/T-75 tissue culture flask. Approximately 24 hours later, the cells were harvested, resuspended in an appropriate volume of Ham's F-12 medium to yield a final cell density of 5 x 10^6 cells/ml and used in the assay.

Cytokinesis block (if used):

Colcemid 0.1 µg/ml (two hours)

Metabolic activation:

with and without

Metabolic activation system:

Induced livers of Sprague-Dawley rats were used for preparing liver homogenate (S-9). The Aroclor induction was performed by inoculating a mixture of two parts Aroclor 1242 and one part Aroclor 1254 at a combined final concentration of 500 mg of Aroclor per kilogram of rat weight. Approximately one gram of induced liver was used to make 3 ml of sucrose buffered S-9 fraction. The S-9 mix was prepared just before the assay was performed. Solutions of MgCl2 (50 mM), glucose-6-phosphate (50 mM), and NADP (40 mM) were prepared in Ham's F-12 medium without serum. Two ml of each were added to a graduated cylinder and the pH was adjusted to approximately 7.0 with NaOH or HCl. The volume was then adjusted to 14 ml, and the solution was sterilized by passing it through a 0.22 µm filter. The rat liver homogenate (S-9) was then diluted 1:2 with the above solution and the mix was stored on ice until used.

Test concentrations with justification for top dose:

Initial cytotoxicity assay:
In the initial cytotoxicity test, the test article showed a relative cloning efficiency (RCE) of 0% at 0.23 µl/ml and an RCE of 56.25% at 0.07 µl/ml without activation, and a RCE of 0% at 0.75 µl/ml and a RCE of 40.18% at 0.23 µl/ml with activation.
It was therefore decided to perform the chromosome aberrations assay at eight decreasing doses starting at the maximum dose of 0.15 µl/ml in the non-activated system (0.6 - 0.11 µl/ml) and at 0.5 µl/ml in the activated system (0.16 - 0.28 µl/ml).

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

- Dosing Procedure
Dosing was done in sterile 15 ml plastic centrifuge tubes. One ml of either the S-9 mix or Ham's F-12 medium without serum was added to each tube. The test article was added to this and mixed thoroughly. Finally, 0.2 ml of 5 x 10^6 cells/ml suspension was added to each of the tubes. The tubes were gassed with 53% CO2 in balanced air, capped and incubated for 4 hours at 37°C in a shaking incubator.
Following the 4 hour incubation, 9 ml of fresh F-12 medium was added to each tube and the cells were collected by centrifugation (250 xg). The supernatant was discarded and the cells were washed once more with 10 ml of fresh medium. The cells were centrifuged, supernatant discarded and the cell pellet resuspended in 10 ml of fresh F-12 medium. Nine ml portions of this cell suspension were removed and placed in T-25 flasks. The flasks were incubated in a CO2 incubator for the cytogenetics assay.

- Toxicity Assay
From the remaining 5 ml of cell suspension, 2 serial 1:10 dilutions in F-12 medium were made.* From each of these three dilutions, 0.2 ml was removed and added to respective tubes containing 5.0 ml of fresh F-12 medium. The contents of the tubes were mixed and poured into appropriate 60 mm tissue culture plates. The plates were incubated for 7 days at 37°C in a 5% CO2 incubator. The plates were not disturbed during the incubation period to minimize the formation of sattelite colonies. Following the incubation period, the colonies were fixed in methanol, stained with Giemsa stain and counted.

- Cytogenetics Assay
The T-25 flasks from the Dosing Section were incubated for 16 hours and treated with 0.1 ug/ml of colcemid for two hours. The metaphase cells were collected by mitotic shake-off and harvested, treated with hypotonic KCl, fixed in Carnoy's fluid, dropped on coded microslides, air-dried and stained in Giemsa stain.

- Scoring
The slides from the three highest doses in which 90% or less of the cells were killed during dosing**, along with the positive and negative control slides, were coded and scored blind. A maximum of 50 metaphase spreads for each dose was scored. Those cells that appear intact with the chromosomes spread symmetrically were used to obtain the final count. Celis that show chromosomes widely separate or with other metaphase cells in the vicinity were disregarded. The cells selected for scoring were placed under approximately 1000X magnification for scoring. Vernier settings and microscope number were recorded for each cell scored.

*In the initial toxicity assay, 10 ml cell suspension was used instead of the remaining 5 ml for making 2 serial 1:10 dilutions in F-12 medium.
**Even though dose levels 0.15 µl/ml in the nonactivated system and 0.5 µl/ml and 0.38 µl/ml in the activated system showed a relative cloning efficiency of <=50%, chromosome aberrations were not scored at these dose levels due to the absence of dividing cells.

Each metaphase figure was scored for the following items:
A. Number of chromosomes in each metaphase figure.
B. Gaps - Achromatic region in chromatid no greater than the width of the chromatid (tg)
C. Chromatid breaks - Achromatic region in the chromatid greater than the width of the chromatid or where the broken piece is misaligned with the rest of the arm (tb).
D. Chromosome breaks - Achromatic region in both chromatids at the same locus with marked displacement of both distal fragments (sb).
E. Fragments - Chromatid(s) not containing a centromere. May be seen in association or not in association with a parent chromatid (tf).
F. Exchange figure - Chromatid interchange involving two or more chromosomes, with either symmetrical or asymmetrical distortion of the usual chromatid pattern (ef).
G. Ring - Chromosome whose ends have joined to form a double or Single circle, with or without a centromere (r).
H. Polyploid - Increase in chromosome number in excess of the diploid and in multiple of the haploid number (pp).
I. Pulverization - Extreme fragmentation of the chromatid material (pu).
J. Severely damaged cell - Cell with ten or more aberrations of any type or with pulverization (>10).
K. Dicentric - Chromosome with two centromeres

Evaluation criteria:

The test article was assessed for its capability to produce visible structural alterations in the morphology of the chromosomes.
The final assessment of the test article was made by correlating the types of aberrations and their frequencies to their dose levels

Statistics:

positive linear dose response trend using linear regression T-test

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The test article Dimethyl Oxazolidine Solution was tested for its solubility in water and found to be soluble at the maximum stock concentration used in this study.
In the initial cytotoxicity test, the test article showed a relative cloning efficiency (RCE) of 0% at 0.23 µl/ml and a RCE of 56.25% at 0.07 µl/ml without activation and a RCE of 0% at 0.75 µl/ml and a RCE of 40.18% at 0.23 µl/ml with activation.
Compared to the number of cells with chromosome aberrations in the solvent control, the test article caused more than 5 fold increase in the frequency of cells with chromosome aberrations at all test doses in the activated and non-activated systems. When the results were analyzed for. a positive linear dose response trend using linear regression T-test the slope was significant at P<0.05 for both the activated and non-activated system.
In this study the positive control articles caused a significant increase in the number of cells with chromosome aberrations.

Conclusions:

The results of the assay indicate that under the conditions of the test, the test article caused a significant dose dependent increase in the frequencies of chromosome aberrations in the Chinese Hamster Ovary cells with and without S-9 activation

Executive summary:

The test article 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) was tested in vitro in the chromosome aberrations assay ussing Chinese Hamster Ovary cells. The test article was tested at eight dose levels with and without metabolic activation. The chromosome aberrations were scored and evaluated at three dose levels.

Compared to the number of cells with chromosome aberrations in the solvent control, the test article caused more than 5 fold increase in the frequency of cells with chromosome aberrations at all test doses in the activated and non-activated systems. When the results were analyzed for. a positive linear dose response trend using linear regression T-test the slope was significant at P<0.05 for both the activated and non-activated system.

The results of the assay indicate that under the conditions of the test, the test article 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) caused a significant dose dependent increase in the frequencies of chromosome aberrations in the Chinese Hamster Ovary cells with and without S-9 activation.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Lot: HP/60/83 Project SR 364
Storage: Cool dry place

Species / strain / cell type:

lymphocytes: human

Cytokinesis block (if used):

demecolcine 0.5 µg/ml for 1 hour

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Fraction S-9
S-9 was made according to Toxicol Protocol MU19 from Aroclor 1254-induced Fischer 344 rats. The protein content of the S-9 is determined by the Lowry method, and must not be less than 35mg/ml to be acceptable for use. The batch of S-9 used in this study had a protein content of 37.2 mg/ml.

Test concentrations with justification for top dose:

without S-9: 45, 22.5, 11.25, 5.625 and 0 µg/ml
With S-9: 120, 60, 30, 15 and 0 µg/ml

Cytotoxicity Test: After 44 hours of stimulation with phytohaemagglutinin (PHA) whole blood cultures from one donor were treated with 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) in medium at concentrations of 0, 1, 10, 100, 1000 and 10000 µg/ml for 3 hours with and without S-9. Cells were harvested 25 hours after treatment in order to assess the mitotic index. The cytotoxicity test suggested that an appropriate top dose (i.e. 50-80% inhibition of mitosis) in the absence of S-9 would be 45 µg/ml and in the presence of S-9 would be 120 µg/ml.

Vehicle / solvent:

serum-free RPMI medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

- Cells
Peripheral blood was drawn from two young healthy volunteers with no history of chromosome fragility, no recent X-ray exposure and no recent virus infection.
The blood was taken fresh on the day of the experiment, into a sterile lithium heparin tube and 0.4ml aliquots were added to sterile Plastic universal bottles to give 10ml cultures in Hepes buffered RPMI medium containing 20% foetal bovine serum (both media and sera obtained from Gibco), 0.1ml of phytohaemagglutinin (PHA, Burroughs Wellcome) and 100 units/ml penicillin and streptomycin.

- Cytotoxicity Assay
Blood from Donor A was established in culture as described above and incubated at 37°C for 44 hours. The cells were then pelleted by centrifuging at 800 rpm for 5 minutes, the medium was removed, and 5 ml of serum-free medium containing test item added to the cells. The following concentrations of test item were used: 0, 1, 10, 100, 1000 and 10000 µg/ml, and treatments were in duplicate. 0.5ml of 10% S-9 in standard cofactors was added to one out of each pair of duplicate treatments. Cells were resuspended in the treatment medium by mixing on a Whirlimixer, and incubated at 37°C for 3 hours.
After treatment the cells were again pelleted by centrifuging at 800 rpm for 5 minutes, the treatment medium removed, the cells washed with sterile Phosphate buffered saline (PBS), centrifuged again and resuspended in fresh growth medium containing serum.
After a further 25 hours incubation at 37°C cells were arrested in mitosis, harvested, fixed and Slides prepared.
Toxicity was determined by counting 1000 cells and assessing dividing cells as a percentage of total lymphocytes (the mitotic index).

- Chromosome Mutation Assay
11 cultures were established from each donor and incubated at 37°C for 44 hours. Cultures were then treated for 3 hours with test item at 45, 22.5, 11.25, 5.625 and 0 µg/ml in the absence of S-9 and at 120, 60, 30, 15 and 0 µg/ml in the presence of 10% S-9. A positive control culture for each donor was treated with 50µg/ml cyclophosphamide (CPA) plus S-9.
After treatment, cells were washed and resuspended in fresh medium and reincubated for a further 25 hours.

- Metaphase arrest and harvest
24 hours after treatment cells were arrested in mitosis by adding demecolcine to each culture at a final concentration of 0.5 µg/ml for 1 hour. Each culture was then centrifuged at 2000 rpm for 5 minutes. The supernatants were removed and cells swollen in 0.075M KCl for 15 minutes at room temperature. The cells were then fixed by dropping the KCl Suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1), followed by several changes of fixative, centrifugation and resuspension, until the supernatant was clear.

- Slide Preparation:
Fixed cells which had been left in ice-cold fixative for a least 18 hours were pelleted and resuspended in approximately 0.2ml of fresh fixative plus 2 or 3 drops of 45% acetic acid in water. This suspension was then dropped on to scrupulously clean microscope slides which had been dipped in distilled water. 2 or 3 drops of cell suspension were put on each slide and 2 slides were made from each culture. The slides were dried on a heated drying bench, and then cooled to room temperature before staining with 4% Gurrs Giemsa R66 for 5 minutes. The slides were rinsed, blotted dry and mounted in Gurrs Neutral Mounting Medium.

- Scoring of Chromosome Damage
Where possible, 100 cells were scored from each set of 2 slides with one code, thus resulting in up to 200 cells scored for each level of treatment either with or without S-9. Only well-spread metaphases with a chromosome count of 44 or more chromosomes were scored. As analysis was carried out by more than one cytogeneticist, random checks of aberrations were made to ensure consistency of scoring of aberrations liable to more than one interpretation.
Chromatid and chromosome gaps and breaks as well as fragments and rearrangements were all recorded on raw data sheets with the stage coordinates (Vernier readings) of every cell containing any aberrations. Aberrations were listed according to the scheme detailed bellow:
AR = acentric ring
c = chromatid deletion
C = chromosome deletion
c/c = asymmetrical chromatid exchange
c7c = asymmetrical chromatid intrachange
C/C = asymmetrical chromosome exchange
C7C = asymmetrical intrachange
CR = centric ring
CR + F = centric ring and fragment
D = dicentric
D+F = dicentric and fragment
F = double fragment or isolocus deletion
g = chromatid gap
G = chromosome gap
i/c = isochromatid/chromatid interchange
m = Single minute fragment or interstitial deletion
M. = double minute fragment or interstitial deletion
Nud = isochromatid deletion with non-union distally
Nup = isochromatid deletion with non-union proximally
SU = isochromatid deletion with complete sister union

The mitotic index for each culture was determined, based on a total of 500 cells per culture, in order to check that mitosis was being inhibited at the higher doses

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Analysis of Data
Treatment of cells with the test item in the absence of S-9 resulted in increases in chromatid deletions at all doses, and increases in chromosome deletions and chromatid exchanges at the top dose, but donor A showed many more chromatid deletions than donor B at this dose.
These increases meant that total aberrations (excluding gaps) per 100 cells for the combined donors was increased above control at all treatments. However, only the increase at the top dose 45µg/ml was statistically significant. (Chi^2 = 109.82, P < 0.001).

Treatment of cells with the test item in the presence of S-9 resulted in increases in chromosome deletions and exchanges at the top 2 or 3 doses, and increases in chromatid deletions and exchanges at all doses. As in the absence of S-9, donor A shows higher frequencies of aberrations at most doses than does donor B.
These increases meant that total aberrations (excluding gaps) per 100 cells for the combined donors was increased above control at all treatments, and the increases at the top three doses (30, 60 and 120 µg/ml) were statistically siggificant (Chi^2 = 15.26, P< 0.001; Chi^2= 109.49, P < 0.001; and Chi^2 = 161.30, P< 0.001 respectively).
The mitotic indices verify that mitotic inhibition did occur at the higher treatments for both donors.
Treatment of cells with CPA at 50 µg/ml resulted in increases in deletions and exchanges such that the increased aberrations (excluding gaps ) per 100 cells were significantly greater than solvent (Chi^2 = 52.29, P < 0.001), and show firstly that the S-9 are able to metabolise indirect-acting mutagens, and secondly that the lymphocytes of each donor were susceptible to the chromosome damaging effects of a known clastogene

Conclusions:

It is concluded that 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is clearly a chromosome mutagen in human lymphocytes, and that its mutagenic activity relative to toxicity is enhanced by the presence of rat liver S-9.

Executive summary:

Whole blood cultures from one donor were treated with 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) in medium at 0, 1, 10, 100, 1000 and 10000 µg/ml for 3 hours with and without S-9 after 44 hours of stimulation with phytohaemagglutinin (PHA) and cells were harvested 25 hours after treatment in order to assess the mitotic index. This assessment suggested that an appropriate top dose (i.e. 50-80% inhibition of mitoses) in the absence of S-9 would be 45 µg/ml and in the presence of S-9 would be 120 µg/ml.

Whole blood cultures from two donors were then treated with 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) in medium in the absence of S-9 at 45, 22.5, 11.25, 5.625 and 0 µg/ml and in the presence of S-9 at 120, 60, 30, 15 and 0 µg/ml for 3 hours after 44 hours of PHA stimulation. Positive control cultures were similarly treated with cyclophosphamide ( CPA) at 20 µg/ml in the presence of S-9. After treatment, cells were resuspended in fresh growth medium for a further 25 hours, and after arresting cells in mitosis with demecolcine treatment, they were harvested. Metaphase spreads were made from fixed cells, and stained by conventional methods. Slides were coded by an independent observer, and, where possible, 100 cells scored from each culture. Solvent control cultures gave 3 and 4 aberrations per 100 cells in the absence of S-9 and 4 and 0 aberrations per 100 cells in the presence of S-9 for donors A and B respectively, both of which are entirely acceptable.

50 µg/ml CPA gave 332 and 72 aberrations per 100 cells for donors A and B respectively, both of which are significantly greater than the solvent controls, giving Chi-square values of 68.77 and 36.02 respectively (both giving P < 0.001). Treatment of cells with 4,4-dimethyloxazolidine resulted in increases in aberrations at all doses, both with and without S-9, for the two donors combined results although donor A consistently showed increased aberrations at lower doses than did donor B.

The increases in aberrations for the donors combined were statistically significant for the top dose (45µg/ml) in the absence of S-9 (Chi^2 = 109.82, P < 0.001) and for the top three doses (30, 60 and 120 µg/ml) in the presence of S-9 (Chi^2= 15.26, P < 0.001; Chi^2 = 109.49, P< 0.001; and Chi^2 = 161.30, P < 0.001 respectively).

It is therefore concluded that 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is clearly a chromosome mutagen in human lymphocytes, and that its mutagenic activity relative to toxicity is enhanced by the presence of rat liver S-9.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 1982 - 16 January 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Lot No.: 6178-29
Storage Conditions: Room Temperature

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver microsomes (S-9)

Test concentrations with justification for top dose:

Without metabolic activation: 0.032, 0.024, 0.018, 0.013, 0.010, 0.0075, 0.0056, 0.0042, 0.0032 or 0.0024 µl/ml
With metabolic activation: 0.10, 0.075, 0.056, 0.042, 0.032, 0.024, 0.018, 0.013 or 0.010 µl/ml

The Initial Toxicity Test conducted on test article indicated a threshold level of complete toxicity at 0.1 µl/ml for both the nonactivated and the S-9 activated cultures. Based on the data derived from the toxicity test, the test article was prepared so that the highest concentration was 100% toxic (0.1 µl/ml).

Vehicle / solvent:

Acetone and DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

1. Cell Preparation
Prior to use in the assay, L5178Y cells which were actively growing in culture were cleansed as described by Clive, et al. Three ml of THMG stock solution was added to a 100 ml cell suspension containing 0.1 x 10^6 cells per ml. The culture was gassed with 5% CO2 in air and placed on an environmental incubator shaker at 125 rpm and 37°C. After 24 hours the THMG was removed by pelletizing the cells and decanting the supernatant. The cells were rinsed in 20 ml of F10P and reinstated in culture at 3 x 10^4 cells per ml in 100 ml of F10P plus 1 ml of THG stock solution.
The cell population density of the prepared cultures was determined by adding a 1 ml sample of cells to 9.0 ml of 0.1% trypsin, incubating at 37°c for 10 minutes, and making three counts per sample with an electronic cell counter. Based on the determination of the number of cells per ml, a cell suspension containing 1.0 x 10^6 cells per ml was prepared, and 6 ml aliquots were dispensed in Corning polypropylene centrifuge tubes.

2. Test Compound Preparation
Based on the data derived from the toxicity test, the test article was prepared so that the highest concentration was 100% toxic. The test article was solubilized and 15 serial eighth log dilutions were carried out. This produced 16 dose levels decreasing approximately 100-fold from highest to lowest. The test article was added to each tube, labeled with the test article T No., test concentration and NA or S-9, in amounts at which the final solvent concentration was nontoxic to the cells. The compound was tested with and without S-9 activation.
Four ml of S-9 activation mixture was added to half of the tubes and 4 ml of FoP was added to the other half. This yielded a final cell suspension of 0.6 x 10^6 cells per ml.
Two control tubes received solvent only and the positive controls were treated with EMS (1.0 and 0.5 µl/ml) and 7,12-DMBA (7.5 and 5.0 µg/ml). All tubes were gassed with 5% CO2 in air and placed on a roller drum apparatus for 4 hours at 37°C. The preparation and addition of the test article was carried out under amber lighting and the cells were incubated in the dark during the 4-hour exposure period.
At the end of the exposure period, the cells were washed twice in 10 ml of F10P by centrifuging at 1000 xg for 10 minutes and decanting the supernatant. The cells were resuspended in 20 ml of F10P, gassed with 5% CO2 in air, and replaced on the roller drum apparatus at 37°C.

3. Expression Time
After the initial exposure to the test article, the cells were incubated for two days with a cell population adjustment at 24 and 48 hours. The adjustment was made by taking daily cell counts and then replacing a volume of cells with fresh medium which yielded a cell population density of 0.3 x 10^6 cells per ml.

4. Cloning
At the end of the expression period, the cells were placed in cloning medium (C.M.) containing 0.34% Noble agar. TFT at a final concentration of 3 µg/ml was used as the restrictive agent.
- a. General Preparation
Two Florence flasks per culture to be cloned were labeled with the compound concentration and whether or not they received S-9 activation. For each pair of flasks one was labeled TFT and one was labeled V.C. (viable count). Each flask was prewarmed to 37°C, filled with 100 ml of C.M., and placed on an incubator snaker at 37°c until used.
Six 100 mm petri plates per culture were labeled with the concentration, whether or not activation was used, and the experiment number. Three of the six were labeled TFT and three were labeled V.C.
- b. Cell Plating
Cell counts were made for each tube to determine the volume of each cell population which would yield 3 x 10^6 cells. This volume was removed, the remainder of the cells were discarded, and the 3 x 10^6 cells were replaced in the centrifuge tube. The cells were centrifuged at 1000 x g for 10 minutes, and the supernatant, except for 2 ml, was removed by pipetting.
The cells were resuspended in the remaining 2 ml of medium and placed in a TFT flask labeled with the corresponding concentration of the test article.
A2 x 10^-4 dilution was carried out by adding 1.0 ml of the TFT flask suspension to a test tube containing 4 ml of F10P, adding 1.0 ml of this to 9 ml of F10P, and adding 1.0 ml of that dilution to the appropriate V.C. flask containing 100 ml of C.M. After the dilution 1 ml of stock solution of TFT was added to the TFT flask, and both this flask and the V.C. flask were placed on the shaker at 125 rpm and 37°C.
After 15 minutes the flasks were removed one at a time, and 33 ml of the cell suspension was pipetted into each of three appropriately labeled petri plates. To accelerate the gelling process, the plates were placed in cold storage (4°C) for 20 minutes. The plates were removed and incubated at 37°C in a humidified 5% CO2 atmosphere for 10-12 days.

5. Accumulation of Data
After the incubation period, both the TFT plates and the V.C. plates were scored for the total number of colonies per plate. Three counts per plate were made on an automatic colony counter, and the median count was recorded. The mutation frequency was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies x 10^4 in the three corresponding V.C. plates and multiplying the quotient by two. All mutant frequency and
toxicity data calculations were performed using a Texas Instruments TI-59 calculator with programs labeled "IMF" and "Total Compound Toxicity".

Evaluation criteria:

The following criteria were used as guidelines in judging the significance of the activity of a test article in this system:
Positive - if there is a positive dose response and one or more of the three highest doses exhibit a mutant frequency which is two-fold greater than the background level.
Equivocal - if there is no dose response but any one or more doses exhibit a two-fold increase in mutant frequency over background.
Negative - if there is no dose response and none of the test cultures exhibit mutant frequencies which are two-fold greater than background.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Five of the nonactivated cultures (0.032, 0.024, 0.018, 0.013 and 0.010 µl/ml) that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The mutant frequencies ranged from 18 to 2 times the average mutant frequency of the solvent controls. The % Total Growth of these cultures ranged from 1% to 58%. None of the remaining nonactivated cultures exhibited mutant frequencies which were
more than twice the mean mutant frequency of the solvent controls.
The % Total Growth of these cultures ranged from 84% to 102%.
Two of the S-9 activated cultures (0.10 and 0.075 µi/ml) that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The mutant frequencies were 4.3 and 3 times respectively the mean mutant frequency of the solvent controls. The % Total Growth of these cultures was 5% and 26%. None of the remaining S-9 activated cultures that were cloned exhibited mutant frequencies which were Significantly greater than the mean mutant frequency of the solvent controls.
The % Total Growth of these cultures ranged from 49% to 113%.

Conclusions:

ANGUS Chemical Company's test article Dimethyl Oxazolidine Solution was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9. Five nonactivated and two S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. A dose dependent response was noted in both the nonactivated and the S-9 activated cultures. The results indicate that under the conditions of this test, test article Dimethyl Oxazolidine Solution, produced a positive response in the presence and absence of exogenous metabolic activation.

Executive summary:

4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9. The nonactivated cultures that were cloned were treated with a range of concentrations of test article which produced from 1% to 102% Total Growth. The S-9 activated cultures that were cloned were treated with a range of concentrations of test article which produced from 5% to 113% Total Growth.

Five nonactivated and two S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. For both the nonactivated and the S-9 activated cultures, the response was dose dependent.

The results indicate that under the conditions of this test, test article 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4), produced a positive response in the presence and absence of exogenous metabolic activation.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 11 1989 - December 21 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

October 23, 1986

Qualifier:

according to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Version / remarks:

May 30, 1988)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

Version / remarks:

July 1, 1986

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Unscheduled DNA Synthesis in Mammalian Cells in vitro

Specific details on test material used for the study:

Oxaban A
Batch Nr. E-06159
Purity: ca. 78%
test substance storage: At room temperature in the dark

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

Source: Male Wistar rats
Male Wistar rats (age 8-10 weeks), fed ad libitum, were used. The animals had free access to tap water. Feed was withheld overnight before isolation of the liver. The rats were injected intraperitoneally (i.p.) with 0.25 ml Narcovet. Then the liver was isolated and perfused with 400 ml Ca2+/Mg2+ free Hank’s balanced salt solution (HBSS) and subsequently with collagenase /HBSS (final collagenase concentration 0.03%) supplemented with 1.5 mM CaCl2. This latter solution was recirculated for 10-12 min.
The liver was then transferred into a petri dish, the Glisson capsules were removed and the liver was shaken in HBSS supplemented with 2.5 mM CaCl2 and 2.5% bovine serum albumin (BSA). In this way the tissue was desintegrated into single cells. The obtained cell suspension was incubated at 37°C under an atmosphere of 95% O2, 5% CO2 for 10 min and subsequently filtrated over a nylon filter (100 µm) to remove cell aggregates. By centrifugation (50 g) dead cells, endothelial cells and Kupffer cells were removed, whereas living parenchymic liver cells, the hepatocytes, were retained.

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Experiment 1: 10, 33, 100, 333, 1000, 3330 and 5000 µg/ml
Experiment 2: 1.0, 3.3, 10, 33, 100, 333 and 1000 µg/ml.

Selection of dose levels/Cytotoxicity test
Selection of adequate concentrations for the UDS assay was based on a preliminary cytotoxicity test, with a (generally 4-log) range of test substance concentrations in half-log steps. Cytotoxicity was determined as a decrease in trypan blue dye exclusion after exposure to the test compound. About 10^5 cells were seeded in wells of a 24-well dish and treated for 18 h. After exposure the cells were harvested using trypsin and treated with trypan blue. The concentration which produced a 90% decrease in viability as compared to the control (EC10) was determined. If possible the highest dose level used in the UDS assay was the EC10. The other four dose levels were evenly spaced in between the approximate EC10 and a dose level which showed a viability comparable with the control. In
case the test compound was difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium (without serum). Concentrations exceeding 5 mg/ml were not tested.

Vehicle / solvent:

Culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

-CELL CULTURE
Primary cell culture conditions: Viability of the hepatocytes was determined using the trypan blue dye exclusion method. In general, approximately 90% of the cells should be viable. After isolation, the hepatocytes were seeded on 1 cm2 cover slips in wells of a 24-well dish. Per cover slip 10^5 cells were seeded. After attachment of the cells the total volume of culture medium was adjusted to 0.5 ml per well.

Culture medium: The culture medium consisted of Williams E medium supplemented with fetal calf serum (10%), L-glutamine (2 mM) and gentamycin (50 µg/ml).

Environmental conditions: All incubations were carried out in a humid atmosphere (80-95%) containing 5% CO2 in air in the dark at 37°C. The temperature and CO2-percentage were monitored during the experiment.

-Unscheduled DNA-synthesis assay ("Williams" test)
The test was started 90 min after seeding of the cells on the coverslips. At that time the test compound was added together with 3HTdR (10 µCi/ml; specific activity 18-30 Ci/mmol). Every dose level including positive and solvent controls was tested in triplicate. The cells were exposed overnight (18 h). After exposure the cells were rinsed extensively with HBSS and fixed with methanol-acetic acid 3:1 (v/v). The whole procedure was repeated once so that in total two independent experiments were carried out.

-Autoradiographic procedure
After fixation of the cells the coverslips were mounted on microscopic slides. These slides were dipped in Ilford K5D emulsion at 42°C and dried for 2h at room temperature. After drying the slides were placed in light tight boxes in the presence of silica gel. The photographic emulsion was exposed for 7 to 14 days at 4°C. The emulsion was developed for 4 min in Kodak D19 developer at 15°C, rinsed in Milli-RO water (Millipore Corp., Bedford, Mass., USA) and fixed for 5 min in Kodak fixative. The slides were rinsed with running tap water and the cells were stained with haematoxylin/azophloxin.

-Scoring
All slides were randomly coded before examination. The numbers of grains above the nuclei of 50 cells on every coverslip were counted. Averages and standard deviations were calculated. Grain counts over nuclear areas were compared to grain counts over the adjacent cytoplasm (cf. Lonati-Galliani et al., 1983). When cytoplasmic grain counts of cells treated with the various concentrations of test substance did not differ from the control the corrected nuclear grain count was calculated for each cell.

Evaluation criteria:

ACCEPTABILITY OF ASSAY
An UDS assay is considered acceptable if it meets the following criteria:
a)The background counts of grains were below 20 per average nuclear area.
b)The positive control substances should produce significant increases in the number of grains.
c)The selected dose range should include a toxic concentration as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/ml or should extend to the limit of solubility.

DATA EVALUATION AND STATISTICAL PROCEDURES
A test substance was considered positive in the UDS-assay if:
a) It induced a statistically significant (two-way analysis of variance; Casciano and Gaylor, 1983) dose-related increase in the average number of grains per nucleus. At least two consecutive concentrations should produce grain counts which exceed those of the control by at least two standard deviations of the control value.
b) The results were reproducible in an independently repeated assay.

A test substance was considered negative in the UDS-assay if:
a) No positive response, as defined above, was observed.
b) The results were reproducible in an independently repeated experiment.

Key result

Species / strain:

hepatocytes: Rat

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DNA REPAIR ASSAY
The cultures dosed with 5000 µg/ml (experiment 1) and 1000 µg/ml (experiment 2) were not analysed for the presence of UDS because of high cytotoxicity.
No increase in the number of grains per nucleus or cytoplasm was detectable. Also the corrected nuclear grain counts (number of grains over the nucleus minus the number of grains over a nuclear-sized area of the cytoplasm of the same cell) revealed no increase at any test substance concentration.
The positive control substances DMBA and 4-NQO, produced significant, 92- to 121-fold, increases in the number of grains per nucleus. In the scored coverslips the mean nuclear background 0-2 grains per average nuclear area. Therefore, it can be concluded that the test conditions were optimal and that the intrinsic metabolic activation system functioned properly.

Conclusions:

In conclusion, OXABAN A (4,4-dimethyloxazolidine (CAS Nr. 51200-87-4)) was found to respond negatively in the DNA repair assay using a primary cell culture of rat hepatocytes under conditions described in this report.

Executive summary:

This report describes the effect of OXABAN A (4,4-dimethyloxazolidine (CAS Nr. 51200-87-4)) on DNA repair, measured as unscheduled DNA synthesis (UDS), in a primary cell culture of rat hepatocytes. OXABAN A was tested up to a concentration of 3330 µg/ml (experiment 1) and 333 µg/ml (experiment 2).

No significant dose-related increase in the amount of UDS was observed in two independently repeated experiments. Therefore, OXABAN A can be considered to be unable to induce DNA repair in a primary culture of rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Three studies performed according to OECD guidelines or scientifically recognized methods, performed under GLP, with reliability 1 or 2 are available for this endpoint

3. in vivo genotoxicity tests

a) in vivo unscheduled DNA synthesis assay:

Study according to OECD 486 in primary rat hepatocyte cultures after oral gavage. Negative for all doses tested.

b) Comet Assay (In vivo alkaline single cell gel electrophoresis assay for DNA strand breaks):

Study according to OECD 489 in rat testicular cells after i.p. injection of test substance. Negative for all doses tested.

c) In vivo micronucleus test, somatic cells (bone marrow):

Study according to OECD 474. Negative for all three sampling timepoints.

 

Conclusion in vivo mutagenicity tests: Test substance does not induce DNA damage and/or chromosomal aberrations in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 1989 - 22 May 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

September 19, 1984

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch Number E-03099
Purity: not indicated
Storage Conditions: Room temperature in the dark

Species:

mouse

Strain:

Swiss

Remarks:

CD-1 (SPF-quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Date of arrival of animals: March 21, 1989
Number of animals per test: 5 male and 5 female mice per treatment group.
Age at start of treatment: Approximately 8 weeks
Body weight at start of treatment: 20-27 g females, 27-34 g males
Identification: By unique cage number and a mark on the tail (for a unique animal number).
Acclimatisation: At least 6 days under laboratory conditions.
Allocation: Allocated to treatment groups as they came to hand from delivery boxes.

On arrival and prior to final assignment to study, all animals have undergone a detailed clinical examination to ensure selected animals were in a good state of health.

Conditions: Standard Laboratory Conditions. Air-conditioned room, 7.5 air changes per hour, temperature 21±3 °C, relative humidity around 40-70%, 12 hours artificial fluorescent light/12 hours dark.

Accommodation: In groups of 5 per sex in polycarbonate cages.
Bedding: Purified sawdust (Woody Clean, from the Broekmann Institute, Someren, The Netherlands).
Diet: Standard laboratory animal diet RMH-B, pellet diameter 10 mm, Hope Farms, Woerden, The Netherlands. Feed was withheld overnight prior to dosing approximately 3-4 hours after administration the test substance. The feed was analysed for contaminants by the manufacturer, and the resultswere archived.
Water: Tap water, ad libitum. Results of chemical and contaminant analyses were archived.

Route of administration:

oral: gavage

Vehicle:

Milli-RO water (Millipore Corp., Bedford,Mass., USA).

Frequency of treatment:

Single dosing

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Dosing volume: 10ml/kg bw

No. of animals per sex per dose:

5 Female, 5 male

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CAS 50-18-0); Endoxan, Asta-Werke, 50mg/kg body weight dissolved in 0.9% NaCl (Merck) in Milli-RO water.

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation at 24, 48 and 72 hours after dosing of the test substance and the vehicle and at 48 hours after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened untill a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed by aspiration with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the RCC NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of 45°C over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears:
The slides were stained for 3 min in undiluted May-Grünwald solution Followed by 2 min in May-Grünwald solution diluted 1:1 with Sörensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei:
All slides were randomly coded before examination. An adhesive label with RCC NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

The micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.

A test substance is considered positive in the micronucleus test if:
a) It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
a) None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Statistics:

Averages and standard deviations were calculated.
Wilcoxon rank-sum test; two-sided test at p > 0.05.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in the frequency of micronuclei was observed.
The incidence of micronuclei in the control animals was found to be in the range of historical data (0.66 ± 0.93; mean ± standard deviation, N = 910).
The groups that were treated with Cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis . The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei.

Conclusions:

It is concluded that this test is valid and that OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) can be considered as not mutagenic in the Micronucleus Test under the experimental conditions described in this report

Executive summary:

OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) was tested in the Micronucleus Test in mice. Three groups (D to F), each comprising 5 males and 5 females, received a single oral dose of 500 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups (A to C) served as negative controls. Bone marrow from a positive control group (G), treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

It is concluded that OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions described in this report.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 1987 - 5 June 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Lot : 7A 29 DF 28A
Storage Conditions: Room Temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male Sprague-Dawley rats were obtained from Charles River Laboratories, Inc., Raleigh, NC or from Harlan Sprague Dawley Inc., Frederick, MD. The animals were monitored for evidence of parasites, pathogenic respiratory and enteric bacteria, mycoplasmas, and pertinent murine viruses and were quarantined for a minimum of 7 days after receipt. The rats were observed each working day for signs of illness, unusual food and water consumption, and other general conditions of poor health. The animals were judged to be healthy prior to utilization in the study.
The animal rooms were maintained at approximately 68 to 75 °F and 30 to 70% relative humidity, and were on a 12 hour light/12 hour dark cycle. The animals were housed in groups of three in polycarbonate cages, with hardwood bedding, which conformed to the upper weight range recommended in the Guide for Care and Use of Laboratory Animals, DHEW, (NIH) No. 86.23. The animals were transferred to clean cages with fresh bedding twice weekly. All test animals received Purina certified rodent chow and water (Washington Suburban Sanitary Commission, Potomac Plant) ad libitum. The rats were randomly assigned two to a cage for each treatment group before the dosing period.

Route of administration:

intraperitoneal

Vehicle:

Double distilled deionized water

Duration of treatment / exposure:

Single dose administration: 2, 6 and 24 h
Repeated dose administration: 5 days, sacrifice after 2 h on day 5

Frequency of treatment:

Single dose or 5 day of consecutive repeated dose administration

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

5 mg/kg bw/day

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

Methyl methanesulfonate (MMS, CAS 66-27-3) 30 mg/kg

Tissues and cell types examined:

spermatocytes and sperm cells derived from the testes

Details of tissue and slide preparation:

Tissue Harvest
Cell suspensions consisting of rat spermatocytes and sperm cells were derived from the testes of normal adult male Sprague-Dawley rats after the treatment period. The rats were anesthetized by Metofane (Pittman-Moore) inhalation at the appropriate time interval after dosing. An incision was made into the lower abdomen and one testis exposed. The testis was perfused with 3 ml ice-cold PBS, excised and placed in 1 ml ice-cold CMF-HBSS. Rats were then sacrificed by Metofane overdose.

Cell Preparation
To prepare the cells for the assay, the tunica albuginea of the 2 excised testes (one from each rat in the dose group) were removed and discarded.
Next, the tubules were minced and gently pressed through a No. 50 stainless steel mesh fitted in a Cellector tissue sieve. Cells were washed through the screen with six - 5 ml aliquots of ice-cold CMF-HBSS. The cell suspension was then filtered through a nylon mesh to remove extracellular debris. Cells were counted with a hemacytometer to determine an appropriate aliquot volume containing 1.5 - 2.0 million cells.

Alkaline Elution
The alkaline elution procedure of Skare and Schrotel is a modified procedure of Kohn et al. (Handbook of DNA Repair Techniques: 379-402, 1980) and Bradley, M.O. and Erickson L.C. (Biochim. Biophys. Acta 654:135-141, 1981). Duplicate cell aliquots (1.5 - 2.0 million cells / aliquot)(*)(**) of each dose group were diluted in 15 ml of ice-cold PBS and were loaded onto 2 µm pore size PvC filters (Millipore) mounted in specially prepared elution columns. Cells were lysed by adding 2 ml of a warm (37°C) sodium dodecyl sulfate (SDS) lysis solution containing 0.5 mg/ml proteinase K for 30 minutes.
The lysis solution was allowed to drip out after the 30 minute lysis period, and the filters were rinsed with 5 ml 0.02 M EDTA, PH 10.0. Tubing was connected from each column, through a pair of peristaltic pumps set at 0.035 ml/min., and into a pair of manifolds capable of delivering samples into 15 ml centrifuge collection tubes mounted in an ISCO fraction collector. The fraction collector was set to collect five 3-hr fractions over a 15 hr. elution period. Finally, the colums were filled with 35 ml Tetraethyl Ammonium Hydroxide (TEAH) eluting solution, PH 12.1-12.3, and the filters were eluted in absence of light.
After the elution period was completed, the solution remaining in the tubing was pumped into tubes labeled 'line fraction'. The filters were removed and placed into labeled scintillation vials and allowed to air dry for about two hours. The filter holders were reassembled and rinsed by adding 6 ml of a 0.4N NaOH solution and allowing the solution to remain in the filter holders for about 4 hours. The solutions in the filter holders were pumped into tubes labeled 'wash fraction’.
(*)Triplicate aliquots were used in trial three of the 6 hour acute assay and trial 4 of the 24 hour acute assay.
(**) This was a deviation from the protocol. The protocol states that each elution filter will be loaded with 1.0 million cells.

Fluorometric DNA Assay
The amount of DNA eluted and remaining on the filter was determined by a modification of the photofluorometric assay described by Kissane and Robins (J. Biol. Chem. 223:184-188, 1958). The filters were air dried for approximately 2 hrs. Duplicate sets of DNA standards were prepared by adding 10 µl aliquots of DNA ranging from 0 to 10.0 µg of DNA to pre-wet PVC filters in labeled scintillation vials. Four hundred µl of a freshly prepared 40% 3,5-diaminobenzoic acid solution was added to all the filter samples and standards, and the vials incubated at 60°C for 45 minutes. The vials were vortexed several times during the incubation, removed after 45 minutes and allowed to cool to room temperature. To stop the reaction, 3 ml of 1N HCl was added to each vial and the contents were again vortexed. Finally, the samples and standards were transferred to 1 cm cuvettes and their fluorescence measured on an Aminco-Bowman spectrophotofluorometer. The excitation wavelength was set at 420 mm, and the emission wavelength set at 520 mn.
The volumes of solutions in all the fractions including the line and wash fractions were equalized to approximately 7 ml by adding deionized water.
Duplicate sets of DNA standards were prepared by adding 10 µl aliquots of DNA ranging from 0 to 10.0 ug of DNA to the same type of 15 ml centrifuge tubes as used for fraction collection. Six ml of TEAH eluting solution was added to each of the standard tubes. As a carrier, 150 µl of a 2 mg/ml solution of BSA was added to all fraction samples and standard tubes and allowed to precipitate. To aid the precipitation of the BSA and DNA, 1.5 ml of 25% Trichloroacetic acid (TCA) solution was added to all tubes except the wash fractions. The wash fractions received 2.5 ml of the TCA solution. The samples and standards were mixed on a vortex and stored at 2°C overnight to allow precipitation.
On the following morning, all samples were centrifuged at 3000 rpm for 15 minutes at 2°C. The supernatant was removed by aspiration, and the remaining pellets were rinsed with 1 ml ice-cold ethanol containing 0.055 M glacial acetic acid and 0.3 M sodium acetate. The samples and standards were again centrifuged as above and the supernatant aspirated. The samples and standards were left open overnight to air-dry at room temperature. Two hundred µl of a freshly prepared 40% 3,5-diaminobenzoic acid solution was added to all the tubes and incubated in a water bath at 60°C for 45 minutes. The tubes were periodically vortexed to break up the pellets. After incubation, the tubes were allowed to cool to room temperature for approximately 10 minutes and the reaction stopped with 3 ml of 1N HCl. The tubes were mixed on a vortex, centrifuged at 2800 rpm for 15 minutes at room temperature and fluorescence measured using an Aminco-Bowman spectrophotofluorometer

Presentation of Data
Raw fluorescence data from all the samples and standards were entered into an IBM PC or compatible computer running a LOTUS 1-2-3 and/or BASIC program designed to make the appropriate DNA data calculations.
A linear regression analysis is performed on each of the two sets of standards to compute correlation coefficients, y-intercepts and slopes. The validity of the standard fluorescence values can be determined by examining the correlation coefficient. The y-intercept (background fluorescence) or "blank" is assigned a DNA value of 0.0 µg DNA. The filter blank is subtracted from each of the sample filter fluorescence values to obtain relative fluorescence values. The elution solution blank is likewise subtracted from each of the sample elution fluorescence values to obtain relative fluorescence values. DNA values are calculated for all filter and elution samples by the following equation,
DNA = Relative Fluorescence / Slope
The fraction of DNA retained on the filter after 3, 6, 9, 12 and 15 hours elution time is calculated and presented as a percentage of DNA retained on the filter. In this method the DNA present in the line and wash fractions is treated as if it was retained by the filter. The data is plotted as fraction of DNA vs. elution time using semi-log graph paper and assigning elution time to the linear scale (abscissa). An elution rate is calculated from the data by the following equation,
K = -InQ(v) / V
where K is the elution rate, V is the volume of eluted solution after 15 hours, and Q(v) is the fraction of DNA remaining on the filter after elution volume V.

Evaluation criteria:

Criteria for Evaluation of Test Results

The extent of single strand breaks is measured by comparing the elution rate of the testicular DNA derived from cells treated with the test article to the elution rate of DNA from non-treated cells as well as cells treated with a known mutagen. A test article is judged to be positive if it induces a significant increase in the elution rate when compared to the elution rate of the vehicle control.

Criteria of a Valid Test
The alkaline elution test was accepted if the DNA elution profiles of the vehicle control and the positive control for each exposure condition fell in a range consistent with the historical range. Furthermore, a positive control is judged to be valid if it induces a significant increase in the elution rate when compared to the elution rate of the vehicle control.

Statistics:

A one-tailed t-test was employed to compare the elution rates of the controls.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity Tests
The results of the Pilot Toxicity Study indicated that in dose groups of 500 mg/kg and above, all animals died two days after dose administration and that in dose groups of 100 mg/kg and lower, all animals survived two days after dose administration. Based upon these results, 600 mg/kg and 200 mg/kg doses were chosen as upper and lower doses for the Toxicity Study.
As defined by the protocol, the proper maximum tolerated dose (MTD) chosen should have been the LD1; however, by probit analysis, the LD1 = -118 mg/kg and -288 mg/kg respectively. To aid in the selection of an MTD, a Tertiary Toxicity test (performed coincidently with the five-day repeated dose assay) was performed on 9 male rats. The selection of 50 mg/kg as an MTD is based upon the apparent toxic effects exhibited by the rats in the 100 mg/kg dosage group. The weight of at least one of the three rats in the 100 mg/kg dose group was reduced by 20%. The rats in the 100 mg/kg dosage group were sacrificed and eliminated from the assay.
The toxicity of the control articles and the test article to testicular cells was determined by Trypan Blue exclusion. These determinations were made on the cell preparations used for the alkaline elution assay.

Alkaline Elution Assays
Each assay had to be repeated at least once to obtain valid control results. In each initial assay, the vehicle demonstrated an elution rate significantly higher than acceptable historical results. Furthermore, the positive controls failed to demonstrate a significantly higher elution rate than the vehicle controls. The second trial of the 6 hour and 24 hour acute dose assays as well as the third trial of the 2 hour acute dose assay were repeated because the positive control failed to demonstrate a significantly higher elution rate than the vehicle. The second trial of the 2 hour acute dose assay was repeated because the high dose group demonstrated a moderate increase in the elution rate over the vehicle. Although this increase was not statistically significant, the observation did warrant a repeat. The dose level of the positive control was increased from 30 mg/kg MMS to 50 mg/kg MMS in the fourth trial of the twenty-four hour acute dose assay. The dose level was increased in this assay to improve the positive control response.
An elution rate was calculated for each individual replicate in each dose group as well as an elution rate mean. A relative elution rate was also calculated comparing the elution rate means of each dose group to the vehicle control elution rate mean.
In all valid assays, only the positive control demonstrated a significant increase in the elution rate over the vehicle. The test article did not induce a significant increase in the elution rate of testicular DNA over that of the vehicle control in any assay replicate. The repeat of the 2 hour exposure assay showed no increase in the elution rate after treatment with either the high or low dose of Oxaban-A.

Conclusions:

The results of the alkaline elution assays indicate that under the test conditions, the test article 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) did not cause an increase in the elution rate of testicular DNA over that of the vehicle control. Therefore, the test article is considered to be negative for its ability to induce DNA single strand breaks in rat testicular DNA after intraperitoneal injection.

Executive summary:

4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) was tested using the protocol "Detection of Single Strand Breaks in Rat Testicular DNA by Alkaline Elution". The test article was adminstered to animals via intraperitoneal injection (IP) at dose levels of 5.0 and 50 mg/kg. Testicular cells were harvested 2, 6 and 24 hours after Single treatments of the above listed doses and 2 hours after the last dose of a five day repeated dose administration.

The results of the alkaline elution assays indicate that under the test conditions, the test article 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) did not cause a significant increase in the elution rate of testicular DNA over that of the vehicle control. Therefore, the test article is considered to be negative for its ability to induce DNA single strand breaks in rat testicular DNA after intraperitoneal injection.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2001 - 22 October 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

BIOBAN™ CS 1135 (4,4’-dimethyloxazolidine)
Lot number: 1 H25-DF87
Storage Conditions: Ambient temperature

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
The indicator cells for this assay were hepatocytes obtained from healthy young adult Fischer 344 rats, purchased from Harlan, Indianapolis, Indiana (dose rangefinding assay) or Harlan, Frederick, Maryland (UDS assay). Male and female rats were used in the dose rangefinding study, and males were used in the UDS study. The animals were 7 to 9 weeks of age at the initiation of dosing.
Animals were housed in suspended, stainless-steel cages measuring 24.2 cm x 22.0 cm x 17.3 cm (DxWxH). PMI Certified Rodent Diet(R) 5002, and tap water were supplied ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens.
The temperature and relative humidity were maintained at 22 ± 4°C (64.4 - 78.8°F) and 55 ± 15% respectively. Temperature and humidity were recorded at least once daily. The lighting controls were set to maintain a 12-hour light/12-hour dark cycle (lights on approximately 0600 to 1800 hours), which was briefly interrupted during animal dosing of the 14- to 16-hour timepoint. The air handling controls were set for ten or greater air changes/hour in the study room.
Animals were acclimated for at least 7 days prior to the initiation of dosing. They were identified by eartag after computer generated random assignment to treatment groups according to Covance-Vienna SOPs. Treatment groups were identified by cage label. Animals were weighed prior to dosing and were dosed based upon the individual animal weights. Animals used in the UDS study were anesthetized (Ketamine and Xylazine by intraperitoneal injection) prior to surgery to obtain the hepatocytes and exsanguinated during the procedure.

Route of administration:

oral: gavage

Vehicle:

water
The test article, BIOBAN™ CS 1135, formed a transparent colorless solution in water at 200 mg/mL, the highest concentration prepared for the dose rangefinding assay.

Details on exposure:

An acute dosing regimen (single administration) was used. The route of administration for test article and vehicle control groups was oral gavage and the dosing volume was 10 mL/kg. The positive control was prepared fresh for each timepoint, and administered by IP injection at a dosing volume of 1 mL/kg. Delivery volumes were calculated on the basis of the most recent animal weight and the target dose. The animals were observed within 0.5 hours of dosing, and just prior to perfusion for toxic signs and mortality.

Duration of treatment / exposure:

Two timepoints for UDS were employed, one at 2 to 4 hours and another at 14 to 16 hours after administration of a single dose of the test article.

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Positive control: 4 animals
Vehicle control: 5 animals
300 mg/kg bw: 5 animals
600 mg/kg bw: 7 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

N-dimethylnitrosamine
- Justification for choice of positive control(s): known to induce UDS in rat hepatocytes in vivo
- Route of administration: Intraperitoneal injection
- Doses / concentrations: dosing volume of about 1 mL/kg. Administration at 10 mg/kg and 15 mg/kg for the 2- to 4-hour and 14- to 16-hour timepoints, respectively.

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

Media and Cell Culture
Williams’ Medium E supplemented with 2 mM L-glutamine, 100 µg/mL streptomycin sulfate, and 150 µg/mL gentamicin (WMEI) was the base culture medium, and was modified for each specific requirement. The hepatocytes were obtained by perfusion of livers in situ with HBSS/EGTA followed by WMEC: Hanks’ balanced salts (Ca++-and Mg++-free) containing 0.5 mM ethyleneglycol-bis(ß-aminoethyl ether)-N, N-tetra-acetic acid and 50 mM HEPES buffer at pH 7.2 (HBSS/EGTA); and WMEI containing 50 to 100 units/mL of collagenase and 50 mM HEPES buffer (WMEC). The cultures were established in WMEI supplemented with 10% fetal bovine serum (WME+). All cell cultures were maintained as monolayers in a humidified incubator at 35 to 37.5°C in an atmosphere of 4 to 6% CO2 in air. After the establishment period, the culture labeling was initiated using WMEI containing 10 µCi/mL 3H-TdR at 35 to 60 Ci/mmol (WME-treat).

Cell Collection and Culture
This assay was based on the procedures described by Butterworth et al, (1987). The hepatocytes were obtained by perfusion of livers in situ with HBSS/EGTA followed by WMEC. The hepatocytes were obtained by mechanical dispersion of excised liver tissue in a sterile culture dish containing WMEC. The suspended tissues and cells were allowed to settle to remove cell clumps and debris prior to collection, when necessary. The collected cell suspension was centrifuged and the cell pellet resuspended in WME+. After obtaining a viable cell count, a series of culture dishes were inoculated with approximately 0.5 x 10^6 viable cells in 3 mL of WME+. Culture dishes that were used for the UDS assay contained plastic coverslips. Dishes used to assess attachment efficiency had no coverslips. Cultures were identified with the animal eartag number.

An attachment period of 1.5 to 2 hours at 35 to 37.5°C in an atmosphere of 4 to 6% CO2 in air was used to establish the cell cultures as monolayers. Unattached cells were then removed, washed twice, and labeling was initiated by refeeding the cultures with 2.5 mL of WME-treat. Three of the replicate cultures from each animal were used for the UDS assay, and one culture was used to assess attachment. Any remaining cultures were kept for analysis in the event of technical problems. Attachment efficiency, an estimate of the number and viability of cells attaching to the dishes, was determined for one culture from each animal using trypan blue dye exclusion and in situ analysis.
After a labeling period of about 4 hours, labeled cell cultures were washed twice, refed with WMEI containing 0.25 mM thymidine, and returned to the incubator for 16 to 20 hours.

Termination
The nuclei were swollen by addition of 1% sodium citrate to the cultures (containing cell monolayers) for 8 minutes. Next, the cells were fixed in acetic acid:ethanol (1:3) and dried at least overnight. The coverslips were mounted on glass slides, dipped in an emulsion of Kodak NTB2 and water, and dried. The emulsion-coated slides were stored for 8 days at 2 to 8°C in light-tight boxes containing a desiccant. The emulsions were developed in Kodak D19, fixed with Kodak Rapid Fixer, and stained with a modified hematoxylin and eosin procedure.

Slide Analysis
After autoradiography, all slides were reviewed for quality of analysis. The quality of the autoradiography, the number and distribution of cells on the slides, and cellular morphology were considered in the evaluation. Two treatment groups from each timepoint were analyzed for nuclear labeling. Four animals from the vehicle and test article dose groups were analyzed. Three animals from the positive control group were analyzed.
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field displayed on the video screen of an automatic counter. Only normallyappearing nuclei were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (cytoplasmic count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting.
The net nuclear grain count was routinely determined for 50 randomly selected cells on triplicate coverslips (150 total nuclei) for each animal. The average mean net nuclear grain count (± standard deviation) was determined from the triplicate coverslips (150 total nuclei) for each animal and averaged for each treatment condition.

Evaluation criteria:

Assay Acceptance Criteria
Cell Culture Conditions
The viability of the vehicle control hepatocytes collected from the perfusion process must be at least 50%.

Acceptable Controls
The average net nuclear labeling in the vehicle control cultures is typically in the range of -5.00 to 1.00. In addition, no more than 10% of the cells should be in repair (contain five or more net nuclear grains).
The average response to the positive control treatments must exceed either criteria used to indicate UDS.

Acceptable Number of Doses
Two dose levels will be analyzed for nuclear grain counts at each timepoint.
Grain count data obtained per animal will be acceptable as part of the evaluation if obtained from at least two replicate cultures and at least 100 cells per animal. Grain count data should be available from four of the five animals.

Assay Evaluation Criteria
The criteria for a positive response are based on statistical analysis.
Significant group differences are determined using parametric/nonparametric analysis of variance and related statistical techniques (BMPD). In the presence of significant results, appropriate parametric/nonparametric methods to determine dose relationship are performed.
In addition, the test article is considered active in the UDS assay at applied concentrations that also cause:
- An increase in the group average of the mean net nuclear grain count to at least five grains per nucleus, or;
- An increase in the group average of the percent of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is at least 20%.

Statistics:

Significant group differences are determined using parametric/nonparametric analysis of variance and related statistical techniques (BMPD).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING STUDY: A dose rangefinding study was performed to select doses for the UDS study. Five groups of animals were dosed with concentrations of test material in water at 300, 600, 1000, 1500 and 2000 mg/kg. A vehicle control group was also included. Each group consisted of three male and three female rats treated by oral gavage at a dosing volume of 10 mL/kg.
Dose rangefinding animals were observed within 0.5 hours and 2 to 4 hours of dosing, and daily for three days. Signs of toxicity were observed in all animals at 1000 mg/kg and higher concentrations in both sexes, and at 600 mg/kg in one female. All animals with clinical signs were found dead prior to 2 days postdose. Dose selection for the UDS study was based on the results of the dose rangefinding study. Since toxicity was similar in male and female rats, the UDS assay was performed only with male rats.

MAIN STUDY:
For the UDS assay, the test article was administered at 300 and 600 mg/kg in water. The test material was administered as a single oral gavage dose in volumes of 10 mL/kg to 5-7 male rats per dose level at each timepoint. UDS was determined from four of the animals dosed per group. Signs of toxicity were limited to hypoactivity in the 600 mg/kg animals at the 2- to 4-hour timepoint. At the 14- to 16-hour timepoint, signs observed at 600 mg/kg included hypoactivity, fluid out of the mouth and nose, red crust around the nose, audible breathing, and/or red wetness around the nose. All other animals were normal.
At both timepoints, five animals in the vehicle control and treatment groups and four animals from the positive control group were perfused, and hepatocytes from all perfused animals were continued through attachment. Four cultures per group were evaluated for UDS and three were evaluated for the positive control group from both timepoints.
For the early timepoint, perfusions were initiated 2.4 to 2.6 hours after dose administration. The hepatocytes ranged in viability (determined by trypan blue dye exclusion) from 55.1% to 88.3% of the total cells collected in the perfusate. The attachment efficiency varied from 41.2% to 77.7% and the viability of the attached cells was good, ranging from 80.3% to 92.5%.
For the 14- to 16-hour timepoint, perfusions were initiated 15.3 to 15.8 hours after dose administration. The hepatocytes ranged in viability from 48.3% to 87.4% of the total cells collected in the perfusate. The attachment efficiency varied from 38.0% to 75.1% and the viability of the attached cells was good, ranging from 81.0% to 95.7%.

Both treatment groups were selected for analysis of nuclear labeling. Therefore, dose levels of 300 and 600 mg/kg were used to interpret the UDS response at both timepoints.
Parametric/nonparametric analysis of variance was used to determine significant group differences. In addition, a positive response requires an increase in the group average of the mean net nuclear grain count to at least five grains per nucleus or an increase in the group average of the percent of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is at least 20%.
For the 2- to 4-hour timepoint, the test article did not produce any statistically significant effects in either of the treatment groups. In addition, neither of the test article treated groups met the criteria for a positive response for an increase in mean net nuclear grain counts or in the percent of nuclei with five or more net grains.
Heavily-labeled nuclei (blackened with numerous grains) represent cells undergoing DNA replication as opposed to DNA repair. The number observed for each animal in the 2- to 4-hour timepoint was low and did not interfere with the detection of UDS.
The vehicle control results were within the historical control and assay acceptance criteria range, and the DMN treatments induced increases in nuclear labeling that exceeded all criteria used to indicate UDS. None of the treatments with BIOBAN™ CS 1135 caused increases in nuclear labeling that exceeded criteria used to indicate UDS. The test article was therefore evaluated as negative for UDS at the 2- to 4-hour timepoint.

For the 14- to 16-hour timepoint, the test article did not produce any statistically significant effects in either of the treatment groups. In addition, neither of the test article treated groups met the criteria for a positive response for an increase in mean net nuclear grain counts or in the percent of nuclei with five or more net grains.
Heavily-labeled nuclei (blackened with numerous grains) represent cells undergoing DNA replication as opposed to DNA repair. The number observed for each animal in the 14- to 16-hour timepoint was low and did not interfere with the detection of UDS.
The vehicle control net nuclear grain results were within the historical control and assay acceptance criteria range, and the DMN treatments induced increases in nuclear labeling that exceeded all criteria used to indicate UDS. None of the treatments with BIOBAN™ CS 1135 caused increases in nuclear labeling that exceeded criteria used to indicate UDS. Since the positive control animals were responsive, the test results provided conclusive evidence for the lack of UDS induction by BIOBAN™ CS 1135 at the 14- to 16-hour timepoint.

Conclusions:

There was no evidence that BIOBAN™ CS 1135 (4,4 dimethylocazolidine (CAS 51200-87-4)) induced unscheduled DNA synthesis at dose levels of 300 and 600 mg/kg at the 2- to 4-hour or 14- to 16-hour timepoints. The test article, BIOBAN™ CS 1135, was therefore evaluated as inactive in the In Vivo/In Vitro assay for unscheduled DNA synthesis in rat primary hepatocyte cultures at two timepoints.

Executive summary:

The objective of this assay was to detect DNA damage caused by BIOBAN™ CS 1135 4,4 dimethylocazolidine (CAS 51200-87-4)), or an active metabolite, by measuring unscheduled DNA synthesis (UDS) induced in vivo in rat primary hepatocytes cultured in vitro.

A dose rangefinding study was performed to select doses for the UDS study. Five groups of animals were dosed with concentrations of test material in water at 300, 600, 1000, 1500 and 2000 mg/kg. A vehicle control group was also included. Each group consisted of three male and three female rats treated by oral gavage at a dosing volume of 10 mL/kg.

Dose rangefinding animals were observed within 0.5 hours and 2 to 4 hours of dosing, and daily for three days. Signs of toxicity were observed in all animals at 1000 mg/kg and higher concentrations in both sexes, and at 600 mg/kg in one female. All animals with clinical signs were found dead prior to 2 days postdose. Dose selection for the UDS study was based on the results of the dose rangefinding study. Since similar toxicity was observed in male and female rats, the UDS assay was performed with male rats only.

For the UDS assay, the test material was administered as a single oral gavage dose in volumes of 10 mL/kg to male rats at doses of 300 and 600 mg/kg in water; the hepatocytes were then harvested 2 to 4 or 14 to 16 hours after dosing. Vehicle control groups treated with water were

run concurrently at each time point. Positive control groups were treated with single intraperitoneal doses of 10 mg/kg or 15 mg/kg of dimethylnitrosamine (DMN) and the hepatocytes were harvested 2 to 4 or 14 to 16 hours after dosing, respectively. Four to seven male Fischer 344 rats were treated per group.

Signs of toxicity were limited to hypoactivity in the 600 mg/kg animals at the 2 to 4 hour timepoint. At the 14 to 16 hour timepoint, signs observed at 600 mg/kg included hypoactivity, fluid out of the mouth and nose, red crust around the nose, audible breathing, and/or red wetness around the nose. All other animals were normal. At both timepoints, five animals in the vehicle control and treatment groups and four animals from the positive control group were perfused, and hepatocytes from all perfused animals were continued through attachment. Four cultures per group were evaluated for UDS and three were evaluated for the positive control group from both timepoints.

Primary hepatocyte cultures were prepared at the end of each timepoint. After attachment of the cells, the cultures were labeled with 10 µCi/mL3H-TdR for 4 hours. The cultures were prepared for analysis of nuclear labeling after removal of the radioactivity and addition of 0.25 mM thymidine.

After autoradiography, both treatment groups from each timepoint were selected for analysis of nuclear labeling. Dose levels of 300 and 600 mg/kg were therefore used to interpret the UDS response at both the 2- to 4-hour and 14- to 16-hour timepoints.

Parametric/nonparametric analysis of variance was used to determine significant group differences. In addition, a positive response requires an increase in the group average of the mean net nuclear grain count to at least five grains per nucleus or an increase in the group average of the percent of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is at least 20%.

There was no test compound-related increase in net grain mean in any of the treated groups over the vehicle control. Additionally, neither of the test article treated groups for either timepoint met the criteria for a positive response for an increase in mean net nuclear grain counts or in the percent of nuclei with five or more net grains.

BIOBAN™ CS 1135 (4,4 dimethylocazolidine (CAS 51200-87-4)) was therefore evaluated as negative in the in vivo/in vitro assay for UDS in rat primary hepatocyte cultures at two timepoints.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial mutation test (Ames)

Two AMES test (OECD 471) been performed to assess the ability of the substance to induce gene mutations by base pair changes and frameshifts in vitro.
The result of the first AMES test (1980) indicated that the substance (dissolved in acetone and DMSO) does not induce gene mutations in vitro at test item concentrations ranging from 0.003 to 10.0 µl/plate and with and without metabolic activation. However, a dose responsiveness could be observed in the assay for the strains S.typhimurium TA100 and TA98 in presence of metabolic activation.
The second AMES test (2018) indicated that the test item (dissolved in ultapure water) induced (reverse) gene mutations in the S.typhimurium strains TA100 and TA98 in vitro. In the concentration finding test, strong inhibitory effect of the test item was observed at the concentration levels of 4092 and 1309 µg/plate, therefore the test item was applied at 7 test concentrations ranging from 1250 to 16 µg/plate for the initial mutagenicity assay and from 750 to 1.6 µg/plate for the confirmatory Mutation Test in the presence of exogenous metabolic activation. 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.

Given that the second AMES test used higher concentrations of the test substance and because a dose dependency was already noticed in the first AMES test upon metabolic activation, the AMES test is concluded to show mutagenicity (frameshift mutation and point mutations) in the S.typhimurium strains TA100 and TA98 upon metabolic activation. Therefore, further in vitro studies on mammalian cell lines needed to be conducted.

Bacterial Mutagenicity Plate Test (Ames) coupled with bacterial fluctuation test

A valid bacterial mutagenicity plate test was carried out with S. typhimurium strains TA1535, TA1537, JA1538, TA98 and TA100 treated with the test substance (dissolved in sterile water) according to the Ames plate incorporation method (OECD 471). Treatment was done at 6 doses in triplicate in the range 15.6 to 500 µg/plate in two separate experiments and with and without metabolic activation. The test substance induced small but significant and dose-related increases in TA100 revertants, both with and without activation, in both experiments. It did not induce increases in revertants of any other strain. It is therefore concluded that the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is a weak bacterial mutagen over a narrow range of doses in the plate incorporation assay.

A valid Bacterial fluctuation test was carried out on the S.typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 treated with the test substance (dissolved in water) according to the 'microtitre' fluctuation test method of Gatehouse and Delow (Mutat. Res. 60, 239. 1979). The test substance was applied at 4 doses in the range of 7.8µg/ml to 62.5µg/ml in two separate experiments with and without metabolic activation. The repeat experiment was carried out on a separate day with fresh bacterial cultures and fresh solutions of the test substance. None of the test substance treatments produced significant increases in the numbers of positive wells either in the presence or absence of metabolic activation in either experiment. As the test substance had produced increased TA100 revertants in the bacterial mutagenicity plate test at 250µg/plate (equivalent to 125µg/ml), it was decided to also treat TA100 with 125µg/ml of test substance in the fluctuation test. However, this treatment produced decreases in the numbers of positive wells indicating some toxicity. It is therefore concluded that the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4), although it is a weak bacterial mutagen for TA100 in the plate incorporation test, is not mutagenic for the Ames strains of bacteria in the fluctuation test.

 

in vitro tests – mammalian cells

Chromosomal aberration test in mammalian cells

Two valid in vitro chromosomal aberration tests (guidelines similar to OECD 473) were performed for the test item substance on primary human lymphocyte cultures and on Chinese Hamster Ovary (CHO) cell line.

Human lymphocytes were assessed with the test substance (dissolved in serum-free RPMI medium) in presence and absence of metabolic activation. The initial cytotoxicity test showed appropriate top doses (i.e. 50-80% inhibition of mitosis) of 45 µg/ml (non-activated) and of 120 µg/ml (activated). Cultures obtained from fresh peripheral blood of two young healthy volunteers were treated with 5 test item concentrations ranging from 45 to 0 µg/ml (non-activated) and from 120 to 0 µg/ml (activated). Treatment of cells with the test item resulted in increases in aberrations at all doses, both with and without activation, for the two donors combined results. It is therefore concluded that the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) is clearly a chromosome mutagen in human lymphocytes, and that its mutagenic activity relative to toxicity is enhanced by the presence of metabolic activation.

An in vitro cytogenicity study was performed with the Chinese Hamster Ovary (CHO) cell line with the test item substance (dissolved in water) in presence and absence of metabolic activation. The chromosome aberrations assay was performed at eight decreasing doses starting at the maximum doses (found in the initial toxicity test) of 0.15 µl/ml in the non-activated system (0.6 - 0.11 µl/ml) and at 0.5 µl/ml in the activated system (0.16 – 0.28 µl/ml). Treatment of cells with the test item resulted in a dose-dependent, more than 5-fold increase in aberrations at all doses when compared to the solvent control in the activated and non-activated systems. It is therefore concluded that under the conditions of the test, the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) caused a significant dose dependent increase in the frequencies of chromosome aberrations in the Chinese Hamster Ovary cells with and without metabolic activation.

 

Gene mutation test in mammalian cells

A valid in vitro mammalian gene mutation test (mouse lymphoma assay) (1983) was performed according to OECD Guideline 476 with the test substance (dissolved in acetone and DMSO) to assess the forward mutation rate at the TK locus of heterozygous L5178Y mouse lymphoma cells. The test item was applied at concentrations of 10 concentrations ranging from 0.032 to 0.0024 µl/ml (without metabolic activation) and from 0.10 to 0.010 µl/ml (with metabolic activation) (threshold level of complete toxicity: 0.1 µl/ml with and without activation). Five of the non-activated cultures and two of the activated cultures showed mutant frequencies higher than the average mutant frequency of the solvent controls. A dose dependent response was noted in both the non-activated and the activated cultures. Therefore, the results indicate that under the conditions of this test, the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) produced genetic mutation (frameshift or point mutation) in the presence and absence of exogenous metabolic activation in mammalian cells in vitro.

 

Unscheduled DNA synthesis in mammalian cells

A valid in vitro unscheduled DNA synthesis (UDS) assay was performed in primary rat hepatocyte cultures according to OECD 482. The test substance (dissolved in culture medium) was tested with a concentration range from 10 to 3330 µg/ml (experiment 1) and 1 to 333 µg/ml (experiment 2). No increase in the number of grains per nucleus or cytoplasm or significant dose-related increase in the amount of UDS was observed in two independently repeated experiments. Therefore, the test substance 4,4-dimethyloxazolidine (CAS Nr. 51200-87-4) can be considered to be unable to induce DNA repair in a primary culture of rat hepatocytes.

 

In vivo tests

In vivo unscheduled DNA synthesis

A valid in vivo unscheduled DNA Synthesis (UDS) assay was performed in rat primary hepatocyte cultures according to OECD 486. The test material was administered as a single oral gavage dose to male rats at doses of 300 and 600 mg/kg in water. The hepatocytes were harvested 2 to 4 or 14 to 16 hours after dosing.. Four to seven male Fischer 344 rats were treated per group. There was no test compound-related increase in net grain mean in any of the treated groups over the vehicle control. Additionally, neither of the test article treated groups for either timepoint met the criteria for a positive response for an increase in mean net nuclear grain counts or in the percent of nuclei with five or more net grains. The test substance 4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4was therefore evaluated as negative in the in assay for UDS in rat primary hepatocyte cultures at two timepoints.

 

In vivo alkaline single cell gel electrophoresis assay for DNA strand breaks (Comet Assay)

A valid in vivo alkaline single cell gel electrophoresis assay for DNA strand breaks was performed in male rats according to OECD 489. The test substance (dissolved in double distilled deionized water) was administered to 8 male rats via intraperitoneal injection (IP) at dose levels of 5.0 and 50 mg/kg. Testicular cells were harvested 2, 6 and 24 hours after single treatments and 2 hours after the last dose of a five-day repeated dose administration. The results of the alkaline elution assays indicate that under the test conditions, the test article did not cause a significant increase in the elution rate of testicular DNA over that of the vehicle control. Therefore, the test substance 4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4 is considered to be negative for its ability to induce DNA single strand breaks in rat testicular DNA after intraperitoneal injection.

 

In vivo micronucleus test (bone marrow, somatic cells)

A valid in vivo Micronucleus Test was performed in mice according to OECD 474. Three groups, each comprising 5 males and 5 females, received a single oral dose of 500 mg test substance (dissolved in Milli-RO water) per kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes. It is concluded that the test substance 4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4 can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions of the in vivo micronucleus test.

Justification for classification or non-classification

Despite the positive test results for the in vitro mutagenicity assays in both bacteria and mammalian cells and for both genetic mutagenicity and cytogenicity, the test substance has been found not to be mutagenic in vivo. The available key study on in vivo micronucleus test in rat somatic cells (bone marrow) showed no induction of chromosomal aberrations upon exposure to the test substance by oral gavage. In addition, two valid studies detecting DNA strand breaks (Comet assay) in testicular cells upon i.p. injection and unscheduled DNA synthesis (UDS) in primary hepatocytes upon oral gavage of the test substance did not indicate the induction of DNA strand breaks or UDS by the test substance in vivo.

An acute oral toxicity study determined an LC 50 of 956 mg/kg bw, and in this study, bioavailability of the substance after ingestion has been demonstrated. Therefore, it can be concluded that the negative results observed in the in vivo genotoxicity studies, are not due to reduced bioavailability of the substance, and no genotoxicity in vivo of 4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4 can be expected.