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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(3-oxazolidinyl)ethyl methacrylate
EC Number:
256-260-2
EC Name:
2-(3-oxazolidinyl)ethyl methacrylate
Cas Number:
46235-93-2
Molecular formula:
C9H15NO3
IUPAC Name:
2-(1,3-oxazolidin-3-yl)ethyl 2-methylprop-2-enoate
Test material form:
liquid
Remarks:
yellow

Method

Species / strain
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.