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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:
key study
Study period:
30 Oct 2017 - 23 Nov 2017
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
histidine, tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Each tester strain contained: rfa : deep rough mut. gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: 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-EDTA treatment.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range testing: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate. The highest concentration of 3-Methyl Sulfolane used in the subsequent mutation assay was 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: soluble in water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The negative control was Milli-Q water, the vehicle of the test item.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^9 cells/ml

DURATION
- Exposure duration: 48 +/- 4h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
Rationale for test conditions:
According to guideline
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than
two (2) times the concurrent vehicle control, and the total number of revertants in tester
strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent
vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two
(2) times the concurrent vehicle control, or the total number of revertants in tester strains
TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: good
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : refer to table in additional information

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Any other information on results incl. tables

Historical Control Data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 - 27

3 – 20

3 – 23

8 - 41

8 - 55

63 – 176

54 - 160

10 – 59

9 - 69

Mean

10

11

6

7

16

23

108

107

25

32

SD

3

4

2

3

5

7

19

20

7

8

n

2356

2336

2264

2235

2319

2360

2341

2336

2075

2078

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

125 – 1248

73 – 1206

55 – 1353

54 – 1051

365 – 1995

250 – 1977

Mean

846

219

787

353

1406

887

SD

146

119

345

162

258

349

n

2348

2229

2003

2234

2200

2276

 

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1848

408 - 2651

93 – 1951

111 - 1359

Mean

901

1232

1094

437

SD

168

343

477

149

n

2335

2327

2021

2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 


 


 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study 3-Methyl Sulfolane is not considered mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic potential of 3-Methyl Sulfolane and/or its metabolites in a well performed OECD TG 471 study under GLP. Based on the the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In the second mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the absence and presence of 10% (v/v) S9-mix. In all three experiments the test item did not precipitate, neither cause toxicity nor a significant increase revertant colonies at any dose level. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that 3-Methyl Sulfolane is not mutagenic in the Salmonella typhimuriumreverse mutation assay and in the Escherichia coli reverse mutation assay.