2-(3-oxazolidinyl)ethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies are a bacteria reverse mutation assay (OECD 471, GLP) and a CHO/HGPRT Forward Mutation Assay (OECD 476, GLP).

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:

December 4, 2001 - December 27, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A, Federal Register 61:18198-18202, April 24, 1996

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Vehicle / solvent:

Vehicle was sterile distilled water (CAS 7732-18-5), obtained from Invitrogen Corporation (formerly Life Technologies, Inc.). Test article dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.
All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in water.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were received from Dr. Bruce Ames, University of California, Berkeley and E. coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TAl00 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shakerlincubator at room temperature. The shakerlincubator was programmed to begin shaking at approximately 125 rpm
at 37°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately lo9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at -70°C or colder until used (See Appendix m). Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM I3-nicotinamide-adenine dinucleotide phosphate, 8 mM MgC12 and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Solubility Test
A solubility test was conducted to select the vehicle. The test was conducted using water. The test article was tested to determine the vehicle that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL.

Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose-range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. Vehicle controls, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six dose levels of test article along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle controls and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (WN) and 0.5 % NaCl (WN), was melted and supplemented with Lhistidine, D-biotin and L-tryptophan solution to a final concentration of 50 uM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent
Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (WN) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (WN) agar and supplemented with 2.5 % (WN) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (WN) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures.

One-half (0.5) milliliter of S9 or Sham mix, 100 uL of tester strain and 100 uL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45d°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 uL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37d°C. Plates that were not counted immediately following the incubation period were stored at 2-8 degrees C until colony counting could
be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

Rationale for test conditions:

The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Evaluation criteria:

For each replicate plating, the mean and S.D. of the number of revertants per plate were calculated.

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing conc. of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is >/=3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is >/= than 2-times the mean vehicle control value.

The following criteria must be met for the initial toxicity-mutation and the confirmatory mutagenicity assays to be considered valid. All Salmonella tester strain cultures must demonstrate presence of the deep rough mutation and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate deletion in the uvrA gene. All cultures must demonstrate characteristic mean number of spontaneous revertants in the vehicle controls as follows: TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537,3-21; WP2 uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/ml. The mean of each positive control must exhibit at least a 3-fold increase in the number of revertants over the mean value of the respective vehicle control. A min. of 3 non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

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

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

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

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

Species / strain:

E. coli WP2 uvr A

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:

Solubility Test
Water was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble and clear in water at approximately 50 mg/mL, the maximum concentration tested.

Initial Toxicity-Mutation Assay (Table 1)
In the initial toxicity-mutation assay, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 uL plating aliquot. Dose levels tested were 2.5,7.5,25,75,200,600, 1800 and 5000 ug per plate. Toxicity was observed beginning at 1800 or 5000 ug per plate. No precipitate was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 2100 ug per plate for Salmonella and 5000 ug per plate for E. coli. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Confirmatory Mutagenicity Assay (Table 2)
Dose levels tested with Salmonella were 150, 200, 300, 600, 750 and 2100 ug per plate. Dose levels tested with E. coli were 150, 200, 300,750, 2100 and 5000 ug per plate. Toxicity was observed beginning at 2100 and 5000 ug per plate with Salmonella and E. coli, respectively. No precipitate was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Table 1. Initial Toxicity-Mutation Assay.

Average Revertants Per Plate +/- Standard Deviation

Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Liver Microsomes: none
Vehicle	11±0	200±8	14±2	4±1	11±1
2.5	12±1	195±25	15±2	4±0	14±3
7.5	11±6	178±23	13±1	3±1	10±1
25	15±5	203±5	11±1	4±1	10±0
75	9±4	182±16	7±1	4±1	11±3
200	10±0	236±22	10±4	9±1	7±0
600	13±0	298±130	15±3	10±2	12±0
1800	0±0	0±0	0±0	0±0	15±6
5000	0±0	0±0	0±0	0±0	0±0
Positive	63±16	675±37	347±1	631±3	107±4
Liver Microsomes: Rat liver S9
Vehicle	19±2	234±2	14±5	10±6	14±1
2.5	21±5	221±1	15±3	9±1	13±4
7.5	18±2	200±42	11±1	8±2	13±1
25	19±0	219±9	13±1	6±1	14±1
75	22±1	209±13	14±1	8±4	20±2
200	22±7	225±23	14±1	7±4	11±0
600	28±0	407±18	23±0	8±4	9±7
1800	0±0	0±0	0±0	10±3	7±10
5000	0±0	0±0	0±0	0±0	0±0
Positive	545±50	718±65	82±13	77±11	147±24

Table 2: Confirmatory Mutagenicity Assay

Average Revertants Per Plate +/- Standard Deviation

Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Liver Microsomes: none
Vehicle	17±2	211±31	13±2	6±3	12±2
150	15±2	174±24	15±3	5±1	13±3
200	16±5	206±5	14±2	6±1	13±4
300	13±5	202±19	11±2	6±2	13±1
600	21±2	228±19	14±1	6±2
750	21±3	312±19	13±3	7±1	16±1
2100	0±0	0±0	0±0	0±0	18±1
5000					0±0
Positive	175±58	705±49	297±26	908±354	143±2
Liver Microsomes: Rat liver S9
Vehicle	15±1	190±20	11±2	6±1	11±1
150	18±0	210±24	13±2	6±1	11±2
200	15±3	216±2	12±5	6±3	12±2
300	19±5	211±11	13±1	8±4	17±2
600	21±2	267±23	12±4	6±2
750	24±7	315±13	15±1	8±1	15±1
2100	0±0	0±0	0±0	0±0	20±2
5000					0±0
Positive	763±15	1434±33	162±16	112±9	587±136

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, OXEMA did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test article, OXEMA, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Water was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble and clear in water at approximately 50 mg/mL, the maximum concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 uL plating aliquot. Dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 ug per plate. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Toxicity was observed beginning at 1800 or 5000 ug per plate. No precipitate was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay 2100 ug per plate for Salmonella and 5000 ug per plate for E. coli.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. Dose levels tested with Salmonella were 150, 200, 300, 600, 750 and 2100 ug per plate. Dose levels tested with E. coli were 150, 200, 300,750,2100 and 5000 ug per plate. Toxicity was observed beginning at 2100 and 5000 ug per plate with Salmonella and E. coli, respectively. No precipitate was observed.

Under the conditions of this study, OXEMA was concluded to be negative in the Bacterial Reverse Mutation Assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 3, 2016 to September 21, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

other: (CHO/HGPRT) forward gene mutation assay

Specific details on test material used for the study:

Test Material Name: Oxazolidinyl Ethyl Methacrylate
Chemical Name: 2-Methyl-2-propenoic acid 2-(3-oxazolidinyl)ethyl ester
Synonyms: OXEMA, 2-(3-Oxazolidinyl)ethyl methacrylate
Lot/Reference/Batch Number: R03NA03
Purity/Characterization (Method of Analysis and Reference): The test material was determined to have a purity of 94.6% by gas chromatography with identification by nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry (Megregian, 2016).

Target gene:

The gene for Hgprt is located on the mammalian X-chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge
National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4
cell line was selected as the test system for this study because it is sensitive to mutagens,
has a low background mutant frequency, and is readily available. Stock cultures were
stored at approximately -80°C or below. The cultures were periodically checked for
mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture
lab-ware under standard conditions of approximately 5% CO2 in air at 37°C in a
humidified incubator.

MEDIA USED
The cells were routinely maintained in Ham's F-12 nutrient mix supplemented with 5%
(v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum, 25 mM HEPES,
antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml;
fungizone, 0.25 μg/ml), and an additional 2 mM L-glutamine. Treatment medium was
the above-mentioned medium without serum. The selection medium used for the
detection of Hgprt- mutants was Ham's F-12 nutrient mix without hypoxanthine,
supplemented with 10 μM 6-thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine,
and the above-mentioned antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 10 to 240 μg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of the test material.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-methylcholanthrene (20-MCA, CAS No. 56-49-5)

Details on test system and experimental conditions:

Controls:
Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 μg/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 μg/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in DMSO up to 190.2 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in DMSO and further diluted (1:100) in medium to obtain the desired concentrations as recommended in the test guidelines. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in DMSO and further diluted in the treatment medium. All dosing units were expressed in μg/ml.

Treatment Procedure:
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with treatment medium, S9 mix (when applicable), the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by gas chromatography with flame ionization detection (GC-FID). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was not conducted as dosing solutions were not administered as suspensions.

In Vitro Metabolic Activation:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium is 1/5 of the concentrations stated above.

Evaluation criteria:

Evaluation Criteria:
For an assay to be acceptable, the mutant frequency in positive controls should be significantly higher than the vehicle controls. The mutation frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. The test chemical was considered positive if it induced a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was outside the control limit of the laboratory historical vehicle control range. The test chemical was considered negative if it did not induce a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle and positive controls.

Statistics:

Statistical Analysis:
The frequency of mutants per 10^6 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts were assumed to follow a Poisson distribution, therefore, the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9, an RCS value of 11.3 and 17.8% for the replicate cultures was observed. Remaining cultures had RCS values from 30.1 to 105.9%. With S9, an RCS value <10% was observed at the highest dose. Remaining cultures had RCS values from 17.5 to 82.5%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9, an RCS of 26.2 and 20.4% for the replicate cultures was seen at the highest dose. Remaining cultures had values from 42.5 to 109.3. With S9 a range of 10-20% was seen at the highest dose. Remaining cultures had values from 35.7 to 96.9%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 1902.0 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.77, osmolality = 443 mOsm/kgH2O; treatment medium with 1% DMSO, pH = 7.32, osmolality = 469 mOsm/kgH2O).

Remarks on result:

other: Results for Assay B1 – Initial Mutagenicity Assay

Assay A1 – Preliminary Toxicity Assay:

In the preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 7.2, 14.5, 28.9, 57.9, 115.8, 231.5, 463.0, 926.0 and 1852.0 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon the limitations imposed by the 10 mM assay limit. The treated cultures without S9 activation showed excessive toxicity at the five highest concentrations (i.e., 115.8, 231.5, 463.0, 926.0, and 852.0 μg/ml). The remaining cultures had relative cell survival (RCS) values ranging from 23.0 to 110.6% compared to the vehicle control. In the presence of S9 activation, excessive toxicity was observed at the four highest concentrations (i.e., 231.5, 463.0, 926.0, and 1852.0 μg/ml. The remaining cultures had RCS values ranging from 23.3 to 113.3%. Based upon the results of this assay, target concentration of 10.0, 30.0, 40.0, 50.0, 60.0, 80.0, and 120.0 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 10.0, 60.0, 80.0, 100.0, 120.0, 150.0, and 240.0 μg/ml in the presence of S9.

Assay B1 – Initial Mutagenicity Assay:

In the initial mutagenicity assay (Assay B1) at the highest dose (i.e., 120.0 μg/ml), in the absence of S9, a RCS value of 11.3 and 17.8% for the replicate cultures, which hit the target of 10-20% was observed. The remaining cultures had RCS values ranging from 30.1 to 105.9%. In the presence of S9, a RCS value less than 10% (i.e., 6.0 and 7.2%) was observed at the highest dose (i.e., 240.0 μg/ml). The remaining cultures had RCS values ranging from 17.5 to 82.5%. To ensure the guideline required maximum concentration was tested, mutant frequencies at 240.0 μg/ml were analyzed, although this exceeded the guideline recommended RCS range.

The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at all concentration levels were not significantly different from the concurrent vehicle control values. All mutant frequencies were within a reasonable range of historical background values. Mutant frequencies of the vehicle controls were within the control limits of the laboratory historical vehicle control range.

Assay C1 – Confirmatory Mutagenicity Assay:

In the confirmatory assay (Assay C1), the targeted concentrations assayed were identical to concentrations tested in the initial mutagenicity assay, in the presence and absence of S9. In the absence of S9, a RCS of 26.2 and 20.4% for the replicate cultures was observed at the highest concentration (i.e., 120.0 μg/ml). The remaining cultures had RCS values ranging from 42.5 to 109.3. In the presence of S9, one of the vehicle control replicates was excluded in the interpretation of toxicity due to technical error but was evaluated for mutant frequency. A replicate of 80 μg/ml was not plated, due to a technical error, for toxicity and therefore was not available to plate for the expression period or mutant selection. Acceptable RCS range of 10-20% was observed at the highest dose (i.e., 14.3 and 17.8% at 240.0 μg/ml). The remaining cultures had RCS values ranging from 35.7 to 96.9%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were within the control limits of the laboratory historical vehicle control range.

In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations. The mutant frequencies exhibited by the positive control chemicals exceeded control limits of the laboratory historical positive control range, this had no impact on the integrity of the study.

The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 100.0 to 109.0% of target and verified that concentrations used for treatment were within acceptable range.

Conclusions:

It was concluded that under the experimental conditions used, oxazolidinyl ethyl methacrylate was negative in this in vitro CHO/HGPRT forward gene mutation assay.

Executive summary:

Oxazolidinyl ethyl methacrylate (2-Methyl-2-propenoic acid 2-(3-oxazolidinyl)ethyl ester) was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 10 to 240 μg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of the test material. The analytically determined concentrations of oxazolidinyl ethyl methacrylate in the dose preparations ranged from 100.0 to 109.0%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide).

There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. Cultures treated with the positive control chemicals had significantly higher mutant frequencies. Based upon these results, oxazolidinyl ethyl methacrylate was considered to be negative in this in vitro CHO/HGPRT forward gene mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The key study is a mouse bone marrow micronucleus test (OECD 474, GLP).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 22, 2001 - November 9, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female CD-1 mice, approximately 8 weeks old (weighmg approximately 22-30 g) were used for this study. The animals were obtained from Charles River Laboratories (Kingston, N.Y.) and acclimatized in animal facilities at Rohm and Haas Co. for a 7 day quarantine period. Animals were ear tagged prior to the initiation of the study. Purina Certified Rodent Chow 5002 C and water filtered through a reverse osmosis system were available & libitum. The animals were housed in an environmentally controlled room with temperature, relative humidity, and light cycle set according to Rohm and Haas Toxicology Department Standard Operating Procedures (SOP) (section 10). Daily temperatures averaged 23 degrees C with relative humidity averages between 38-49%, which were within the acceptable range as defined in the SOP. Animals were assigned to treatment groups through the use of a computerized randomization procedure according to sex and body weight prior to administration of the test articles. Animals were within the acceptable weight range of plus or minus 20% from the mean. Animals were fasted for approximately 3 hours prior to dose administration.

Route of administration:

other: The test article and the negative control were administered by gavage, in a single oral dose. The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.

Vehicle:

distilled water

Frequency of treatment:

single dose:
The test article and the negative control were administered by gavage, in a single oral dose. The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.

Post exposure period:

Animals from test article and vehicle control groups were euthanized by cervical dislocation at approximately 24'and 48 hours after dosing. Animals from the positive control groups were euthanized 24 hours after dosing. Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice.

Dose / conc.:

820 mg/kg bw (total dose)

Dose / conc.:

410 mg/kg bw (total dose)

Dose / conc.:

82 mg/kg bw (total dose)

No. of animals per sex per dose:

For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point, with a volume of 10 ml/kg. In the high dose group, 4 additional animals per time point were dosed to account for the possibility of unexpected deaths.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control substance was Mitomycin-C (MMC) (Aldrich Chemical Co., Lot No. 10587MI), TD No. 01-111, dissolved in distilled water.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70% ethanol in water. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15-ml centrifuge tube, containing approximately 1 ml of Fetal Bovine Serum (FBS) using a 1-cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x g for 5 minutes, and the supernatant was removed, leaving approximately 0.1 ml above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed. A small drop of the cell suspension (approximately 10 u1) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridine Orange staining solution.

Scoring:
Slides from at least five animals per seddose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain (Hayashi et al., 1983).
Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. Staining was tan to faint grey in normochromatic erythrocytes (NCE) and bright orange in polychromatic erythrocytes (PCE). Micronuclei appeared bright green against an orange background in PCE and generally were round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and were usually between 1/20 and 1/5 the size of the PCE. The end point to be scored was the number of cells containing micronuclei (not the number of micronuclei per cell).
For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCE/NCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCE 2) and were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.
Cell counting was accomplished using the Xybion Path/Tox computer software system, G Module (GICELL program version 4.2.2) which captures data from the cell counter keyboard and provides an audit trail for quality assurance.

Evaluation criteria:

Evaluation Criteria:
The test article is considered positive in this assay if it elicits a dose-response or a statistically significant increase in the number of micronucleated cells over that of the concurrent vehicle control at one or more dose levels. In the event that the test article elicits a significant increase in the number of MN-PCE due to an unusually low number of MN-PCE in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data.
A test article is considered negative in this assay if No indication of a dose-response is observed and the treatment groups do not show a statistically significant increase in the number of MN-PCE when compared to the vehicle control.
The above criteria are to be used as a guideline for evaluating the assay results. The study director may take other appropriate factors into consideration for evaluating the test results.

Statistics:

Data were analyzed separately for male and female animals using a Statistical Analysis System (SAS), version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of 1) an overall effect of OXEMA treatment relative to control, and 2) a linear dose-response trend among the OXEMA, and 3) a quadratic dose-response trend among the OXEMA. Additionally, pair-wise comparisons between each of the three OXEMA groups and the control group were made using Dunnett's t-test (Kirkland, 1989).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical Signs:
Clinical signs included scant feces, passiveness, ataxia, and unkempt. Death occurred in two of the female mice treated with 820 mg/kg of the test article. No clinical signs of toxicity were observed in any of mice treated with 410 or 82 mg/kg of the test article or control articles.

Micronucleus Evaluation:
The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle control values. This was true for both the 24 and 48 hour time points. There was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Table 1. Clinical Signs

Dose Group		No. of Animals Exhibiting Signs
		Day 0	Day 1	Day 2
0 mg/kg – Distilled Water
Male	Normal	10/10	10/10	5/5
Female	Normal	10/10	10/10	5/5
82 mg/kg OXEMA
Male	Normal	10/10	10/10	5/5
Female	Normal	10/10	10/10	5/5
410 mg/kg OXEMA
Male	Normal	10/10	10/10	5/5
Female	Normal	10/10	10/10	5/5
820 mg/kg OXEMA
Male	Normal	16/18	9/18	4/9
	Passive	2/18	2/18	1/9
	Unkempt	1/10	1/18	1/9
	Ataxia	0/18	1/18	0/9
	Scant Feces	0/18	9/18	5/9
Female	Normal	16/18	7/18	4/9
	Passive	2/18	2/18	0/9
	Ataxia	1/18	2/18	0/9
	Scant Feces	0/18	8/18	5/9
	Found Dead	0/18	2/18	0/9
2.0 mg/kg Mitomycin-C
Male	Normal	5/5	5/5	--
Female	Normal	5/5	5/5	--

Table 2a. Mean Summary Data: Male Animals

Group (number)	Day	Dose (mg/kg)	NCE (Mean±SD)	PCE1 (Mean±SD)	MNP (Mean±SD)	PCE2 (Mean±SD)	PCE Total (Mean±SD)	PNR Ratio (Mean±SD)	MNC % (Mean±SD)
Control (n=5)	1	0	512±114	514±124	3±1	1532±107	2046±28	1.08±0.42	0.14±0.06
OXEMA (n=5)	1	82	527±135	532±140	2±1	1538±134	2070±20	1.11±0.48	0.08±0.06
OXEMA (n=5)	1	410	498±160	561±152	2±2	1518±172	2079±22	1.29±0.66	0.10±0.07
OXEMA (n=9)	1	820	539±192	547±198	3±1	1547±212	2093±49	1.26±0.84	0.16±0.07
Control (n=5)	2	0	496±105	578±95	3±2	1524±77	2102±42	1.24±0.41	0.13±0.08
OXEMA (n=5)	2	82	499±121	596±126	1±1	1505±75	2100±79	1.29±0.51	0.06±0.05
OXEMA (n=5)	2	410	450±119	616±105	3±1	1475±108	2091±26	1.51±0.70	0.13±0.07
OXEMA (n=9)	2	820	569±153	530±138	2±2	1564±138	2094±39	1.05±0.52	0.09±0.08
Mitomycin-C (n=5)	1	2	593±247	568±178	99±16	1504±151	2072±41	1.22±0.86	4.78±0.81#

Table 2b. Mean Summary Data: Female Animals

Group (number)	Day	Dose (mg/kg)	NCE (Mean±SD)	PCE1 (Mean±SD)	MNP (Mean±SD)	PCE2 (Mean±SD)	PCE Total (Mean±SD)	PNR Ratio (Mean±SD)	MNC % (Mean±SD)
Control (n=5)	1	0	485±99	607±109	3±2	1518±114	2125±73	1.33±0.49	0.14±0.09
OXEMA (n=5)	1	82	517±158	571±164	3±2	1519±169	2090±45	1.29±0.79	0.16±0.08
OXEMA (n=5)	1	410	480±106	611±92	3±1	1488±149	2099±66	1.36±0.50	0.16±0.04
OXEMA (n=9)	1	820	526±97	575±106	2±1	1516±89	2091±47	1.16±0.39	0.09±0.05
Control (n=5)	2	0	507±127	583±116	2±1	1502±142	2086±38	1.25±0.53	0.09±0.05
OXEMA (n=5)	2	82	486±108	601±103	2±1	1484±120	2085±64	1.33±0.52	0.09±0.06
OXEMA (n=5)	2	410	473±58	598±96	1±1	1507±46	2105±90	1.30±0.35	0.06±0.06
OXEMA (n=7)	2	820	585±177	481±174	3±2	1605±206	2087±68	0.99±0.64	0.13±0.07
Mitomycin-C (n=5)	1	2	723±148	490±128	108±14	1585±111	2076±43	0.72±0.30	5.20±0.62#

NCE=Normochromatic Erythrocytes

PCE=Polychromatic Erythrocytes

PCE 1=Polychromatic Erythrocytes used in combination with Normochromatic

Erythrocytes to total at least 1000 cells and used to calculate the PCE/NCE ratio

PCE 2=The remaining number of Polychromatic Erythrocytes recorded and added to

PCE1to total at least 2000 Polychromatic Erythrocytes

MNP=Micronucleated Polychromatic Erythrocytes

CALCULATIONS:

PCE TOTAL=PCE 1 +PCE2

PNR RATIO=PCE/NCE RATIO=PCE l/NCE

MNC%=MICRONUCLEATED POLYCHROMATIC ERYTHROCYTE PERCENT=

MNP/(PCE 1 +PCE2) X 100

Statistical Methods: Analysis of Variance Followed by Dunnett's T-Test on Least Square Means. For All

Statistical Methods:'*'Indicates a Statistically Significant Difference from Control (p<0.05).

'#'Indicates a Greater Than 2 Fold Increase Over Control Values.

Conclusions:

Under the conditions of this study, OXEMA was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Executive summary:

OXEMA was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. Adult CD-1 male and female mice (5 male and 5 female animals per group, except for the high dose group, which had 4 additional animals per time point) received a single oral dose of the test article at concentrations of 82, 410 or 820 mg/kg. Control animals received a single oral dose of distilled water (vehicle control), or an intraperitoneal injection of 2.0 mg/kg mitomycin-C (positive control) (MMC). Animals from test article and vehicle control groups were euthanized at 24 or 48 hours after treatment. Animals from the positive control group were euthanized 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes was measured as an indicator of cytogenetic damage. For each animal, a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. In addition, the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent.

The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle controls.

An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

Under the conditions of this study, OXEMA was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro

OXEMA was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA 1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. In an initial toxicity-mutation assay, toxicity was observed beginning at 1800 or 5000µg/plate; based on this, doses for the confirmatory mutagenicity assay were 150, 200, 300, 600, 750, and 2100 µg/plate for Salmonella strains and 150, 200, 300, 750, 2100, and 5000 µg/plate for E. coli. No positive mutagenic response was observed at any dose in any tester strain. OXEMA was negative in the Bacterial Reverse Mutation Assay.

 

In a Chinese Hamster Ovary Cell/Hypoxanthine-Guanin-Phosphoribosyl Transferase (CHO/HGPRT) Forward Mutation Assay, OXEMA was tested at concentrations ranging from 10 to 240 µg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of OXEMA. No statistically signficant treatment-related increases in the mutant frequency in the OXEMA-treated cultures compared to the vehicle control cultures in eithe the presence or absence of S9 were observed. Cultures treated with positive control chemicals had significantly higher mutant frequencies. OXEMA was negative in the in vitro CHO/HGPRT Forward Gene Mutation assay.

 

In Vivo

In a mouse bone marrow micronucleus assay, male and female CD-1 mice received a single oral dose of OXEMA at concentration of 82, 410, or 820 mg/kg. Vehicle controls animals received a single oral dose of distilled water and positive control animals received an intraperitoneal injection of 2.0 mg/kg mitomycin-C. OXEMA and vehicle controls groups were euthanized at 24 or 48 hours after treatment and positive control animals were euthanized at 24 hours after treatment. At least 2000 polychromatic erythrocytes were scored for the presence of absence of micronuclei and the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent. OXEMA did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow cells of male or female mice when compared to controls, while an increase in micronucleated polychromatic erythrocytes (> 2 -fold) was observed in the bone marrow cells of male and female mice treated with the positive control. Clear signs of toxicity were seen at 820 mg/kg and included mortality (2 female mice), scant feces, passiveness, ataxia and unkempt appearance. OXEMA was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Justification for classification or non-classification

Based on negative results in in vitro and in vivo genetic toxicity assay, classification for genetic toxicity is not warranted for OXEMA.