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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20. Aug. 2004 - 16. Sep. 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
460-100-9
EC Name:
-
Cas Number:
342573-75-5
Molecular formula:
Hill formula: C8H16N2O4S CAS formula: C6H11N2.C2H5O4S
IUPAC Name:
3-ethyl-1-methyl-1H-imidazol-3-ium ethyl sulfate

Method

Target gene:
his-
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:
rat liver S9, Aroclor 1254 induced
Test concentrations with justification for top dose:
First experiment: 3,10,33, 100, 333, 1000, 3330 and 5000 µg/plate
Second experiment: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
Milli-Q water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene (NF), methylmethanesulfonate, 4-nitroquinoline N-oxide; with S9: 2-aminoanthracene (using strain-specific concentrations)
Details on test system and experimental conditions:
Test System : Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale : Recommended test system in international guidelines (e.g. OECD, EEC).
Source : Salmonella typhimurium strains: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. TA98 received on 21-02-1991, used batch: T A98.050204, TA1535 received on 30-07-2001, used batch: TA1535.050204, TA1537 received on 30-07-2001, used batch: TA1537.050204, Xenometric, Boulder, Co, U.S.A. (obtained from N.V. Organon), TA100 received on 19-09,·2002, used batch: TA100.240604, Escherichia coli strain: Prof. Dr. B.A. Bridges, University of Sussex, Brighton, U.K., WP2uvrA received on 23-10-1987, used batch: EC.160404
The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement,crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDT A treatment (ref. 1 ). The strain was regularly checked to confirm the tryptophan-requirement, UVsensitivity and the number of spontaneous revertants. Stock cultures of the five strains were stored in liquid nitrogen (-196°C).
Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a two-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not biologically relevant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1:

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

Without S9

Positive control

1479±70

317±50

709±47

942±26

349±19

Solvent control

9±1

6±1

31±7

147±8

11±1

3

 

 

 

139±16

11±3

10

 

 

 

124±18

13±2

33

 

 

 

133±8

15±2

100

10±3

5±1

24±6

122±5

12±3

333

8±3

7±4

27±7

135±19

12±3

1000

10±4

7±2

25±2

132±13

9±3

3330

8±4

6±4

29±6

126±8

14±1

5000

6±2

8±3

25±3

139±28

9±5

 

With S9

Positive control

153±12

274±103

578±53

840±37

261±8

Solvent control

9±2

5±1

28±6

124±7

7±3

3

 

 

 

123±5

11±3

10

 

 

 

117±10

14 v 2

33

 

 

 

138±22

9±2

100

8±2

6±1

37±5

116±15

12±3

333

8±2

4±1

28±6

118±8

10±3

1000

9±4

6±2

35±10

131±9

14 v 4

3330

9±2

5±1

25±6

117±7

11±2

5000

10±2

5±2

30±3

139±12

12±4

Solvent control: 0.1 mL Milli-Q water

Experiment 2:

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

Without S9

Positive control

697±17

226±46

695±71

879±71

180±10

Solvent control

11±2

7±3

18±2

134±12

11±2

100

6±2

5±3

15±4

116±13

7±2

333

6±2

5±1

17±2

120±10

10±1

1000

7±2

4±2

17±2

113±17

10±1

3330

9±6

4±0

18±6

106±21

11±1

5000

7±2

4±0

18±4

106±34

10±4

 

With S9

Positive control

99±5

199±15

322±31

968±201

78±5

Solvent control

10±5

6±4

24±3

102±16

12±2

100

7±3

4±2

29±7

103±17

12±3

333

13±5

5±3

22±3

104±13

12±3

1000

9±3

6±2

24±2

89±9

13±1

3330

9±5

5±2

26±4

92±15

9±2

5000

8±2

8±4

26±5

108±23

12±2

Solvent control: 0.1 mL Milli-Q water

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). The study procedures described in this report were based on the most recent OECD and EEC guidelines.

Batch 99/484 of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was a light yellow viscous liquid with a purity of 99%. The test substance was dissolved in Milli-Q water, In the dose range finding test, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA 1535, TA1537 and TA98. In the second mutation assay, 1-ETHYL-3-METHYL IMIDAZOLIUM

ETHYLSULFATE was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain·specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.