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Toxicological information

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals
Author:
Errol Zeiger, Beth Anderson, Steve Haworth, Timothy Lawlor, and Kristien Mortelmans
Year:
1992
Bibliographic source:
Environmental and Molecular Mutagenesis Volume 19, Supplement 21 :2-141 (1992)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to evaluate the mutagenic nature of ethyl acetate
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: Ethyl acetate
- IUPAC name: Ethyl acetate
- Molecular formula: C4H8O2
- Molecular weight: 88.1052 g/mol
- Substance type: Organic
- Physical State: Liquid
Specific details on test material used for the study:
- Name of test material: Ethyl acetate
- IUPAC name: Ethyl acetate
- Molecular formula: C4H8O2
- Molecular weight: 88.1052 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: Vendor’s purity: 99.5% Analyzed purity: 99%
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain:
other: TA100, TA1535, TA1537, TA97, TA98
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333 or 10000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538; Without S9); 2-aminoanthracene (With S9; all strains)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: TA100, TA1535, TA1537, TA97, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Any other information on results incl. tables

Table: Results for the test chemical Ethyl acetate

Dose (µg/plate)

TA100

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

152

0.6

154

9.9

171

9.9

145

2.0

128

1.2

100

135

5.4

151

0.3

173

3.8

151

11.7

122

10.2

300

143

8.3

151

7.2

169

4.4

160

7.6

150

7.1

1000

135

4.9

127

12.2

181

11.9

151

15.1

166

3.2

3333

116

3.5

129

11.1

180

9.8

124

4.5

140

9.6

10000

124

7.3

116

4.6

131

20.5

119

5.7

121

9.1

Positive control

412

8.4

835

37.2

545

10.8

602

17.8

842

33.8

 

Dose (µg/plate)

TA1535

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

20

1.0

11

2.2

14

2.4

9

1.7

15

0.7

100

16

0.7

11

2.2

12

1.8

13

0.6

12

1.0

300

23

3.2

9

0.6

12

2.0

9

1.7

10

2.3

1000

20

2.3

10

0.9

12

3.0

10

1.9

9

2.1

3333

17

2.6

7

0.9

10

0.9

8

0.6

15

2.6

10000

19

5.2

8

0.6

10

0.3

8

0.0

16

1.9

Positive control

562

16.7

255

22.5

403

13.1

168

13.3

96

2.6

 

Dose (µg/plate)

TA1537

-S9

10% HLI

30% HLI

Mean

SEM

Mean

SEM

Mean

SEM

0

8

1.3

10

1.9

8

0.7

100

6

2.0

12

1.6

9

0.9

300

10

2.1

15

1.2

10

0.9

1000

9

2.1

11

2.7

11

1.7

3333

9

0.6

13

1.3

9

1.7

10000

7

0.6

10

1.0

9

0.7

Positive control

330

20.6

26

2.0

37

1.5

 

 

Dose (µg/plate)

TA97

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

149

1.9

180

14.2

191

17.1

166

4.2

216

4.7

100

169

4.5

179

5.2

200

7.7

196

29.8

222

5.0

300

167

1.5

179

12.9

198

12.1

197

9.6

233

1.7

1000

138

3.4

175

15.3

185

8.7

208

0.6

208

2.9

3333

153

2.9

163

12.8

179

17.5

188

11.8

203

8.8

10000

149

6.3

161

5.8

177

5.8

191

11.1

190

24.3

Positive control

1158

52.6

634

44.5

330

13.1

480

9.2

404

1.7

 

Dose (µg/plate)

TA98

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

20

0.3

36

3.8

34

1.3

30

3.2

48

1.7

100

17

0.3

30

1.7

37

4.0

29

1.8

37

2.1

300

13

2.6

29

2.1

35

4.7

25

3.7

31

1.2

1000

15

0.3

33

3.6

29

5.0

23

2.8

38

3.2

3333

17

3.5

5

2.3

34

1.9

23

2.3

35

2.9

10000

19

1.0

34

4.0

41

4.8

27

3.3

40

4.6

Positive control

643

23.8

786

32.0

283

13.6

435

11.0

134

5.9

 

Applicant's summary and conclusion

Conclusions:
Ethyl acetate did not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of ethyl acetate. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 100, 333, 1000, 3333 or 10000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Ethyl acetatedid not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.