Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 04 May 2018 and the experimental completion date was 22 May 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BZA13264
- Expiration date of the lot/batch: 23 June 2018
- Manufacturingt date: 23 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature under nitrogen
- Not stable at higher temperatures

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in DMSO. Test item concentrations were used within 2 hours after preparation.
- Final dilution of a dissolved solid, stock liquid or gel: dilutions of the test item where prepared using DMSO as vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : diluted in DMSO.

Method

Target gene:
Histidine locus and tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH
- Suitability of cells: The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

MEDIA USED
- Type and identity of media: Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma).
Additional strain / cell type characteristics:
other:
Remarks:
deep rough, mutation in the galactose metabolism, mutation in nitrate reductase, defective biotin synthesis, loss of the excision repair system
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
Trinova Biochem GmbH
- Suitability of cells: The Escherichia coli strain was checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants at least every year.

MEDIA USED
- Type and identity of media: Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose-range finding test: Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of Thio Isocyanate Adduct used in the subsequent mutation assays was 5000 μg/plate or the level at which the test item inhibited bacterial growth.

First experiment - direct plate assay: The dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested at 5.4, 17, 52, 164, 512, and 1000 ug/plate. Initially in tester strains TA1535 and TA98 no dose level with precipitation and/or cytotoxicity was tested in the presence of S9-mix. Therefore an additional experiment was performed at 1600 and 5000 ug/plate with these tester strain in presence of S9-mix to complete the data.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 ; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation and preincubation)
- Cell density at seeding (if applicable): 1 mL (10 EXP 9 cells/mL) added to 3.6 mL medium with test item.

DURATION
- Preincubation period: when applicable, 30 +/- 2 minutes
- Exposure duration: 48 +/- 4 h

NUMBER OF REPLICATIONS: 3

- OTHER: Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Rationale for test conditions:
Recommended test system in international guidelines
Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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:
cytotoxicity
Remarks:
At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix
Vehicle controls validity:
valid
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:
cytotoxicity
Remarks:
At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 µg/plate in the absence of S9-mix and at 1600 µ/plate and upwards in the presence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Evidence of test item precipitate on the plates is recorded by addition of the following precipitation definition:
Slight precipitate: Distinguished by noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate.
Moderate precipitate: Distinguished by a marked amount of precipitate on the plate, requiring the plate to be hand counted.
Heavy precipitate: Distinguished by a large amount of precipitate on the plate, making the required hand count difficult.
In the dose-range finding study, the test item precipitated on the plates at the highest dose level tested (1600 µ/plate and upwards at the start of th incubation period and at 5000 µg/plate at the end of the incubation period). In the pre-incubation assay, precipitation of the test substance was observed at the start of the incubation period at 1600 and 5000 µg/plate and no precipitate was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: Thio Isocyanate Adduct was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Based on the results of the dose-range finding test, a dose-range was selected for the first mutation assay with tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512, and 1000 μg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: decrease in the number of revertants and/or reduction of the bacterial background lawn.
Remarks on result:
other:
Remarks:
Precipitation observed at the start of the incubation period at 1600 µ/plate and upwards and at 5000 µg/plate at the end of the incubation period

Any other information on results incl. tables

Historical control data of the solvent control:

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S-9 mix

-

+

-

+

-

+

-

+

-

+

Range

3-29

3-27

3-20

3-23

8-41

8-55

61-176

60-160

10-59

9-67

Mean

10

11

6

6

16

22

110

106

26

33

SD

3

4

2

3

5

7

17

20

6

8

n

2458

2426

2402

2352

2416

2458

2473

2398

2237

2217

  

Historical control data of the positive control items:

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S-9 mix

-

+

-

+

-

+

-

+

-

+

Range

128-1530

73-1206

58-1407

54-1051

365-1978

250-1977

439-1993

408-2379

93-1958

111-1359

Mean

901

239

817

340

1355

903

905

1249

1059

444

SD

174

115

354

160

230

357

163

371

506

144

n

2400

2296

2051

2337

2357

2367

2402

2354

2153

2232

 

 Direct plate assay – Mean number of revertant colonies:

 

TA1535

TA1537

TA98

Dose (µg/plate)

     S9-mix

-

+

-

+

-

+

Positive control

1306

399

830

384

1220

1227

Solvent control

13

12

5

10

13

16

5.4

12

14

4

4

12

23

17

10

12

4

4

12

12

52

6

12

3

5

6

16

164

10

9

4

3

8

13

512

13

11

1

4

12

13

1000

9

10

2

2

13

12

 

Pre-incubation assay – Mean number of revertant colonies:

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Dose (µg/plate)

            S9-mix

-

+

-

+

-

+

-

+

-

+

Positive control

1144

238

175

243

1342

718

784

1931

275

567

Solvent control

18

16

14

13

28

42

107

133

31

45

5.4

18

13

8

8

24

31

124

116

-

-

17

11

15

13

12

35

33

116

130

37

49

52

13

12

9

13

28

32

115

105

38

51

164

14

14

6

10

29

35

103

161

35

39

512

 

13

 

 

 

37

 

106

36

35

1000

 

 

 

 

 

 

 

 

22

36

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that Thio Isocyanate Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of the study was to determine the potential of Thio Isocyanate Adduct and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. Thio Isocyanate Adduct precipitated on the plates at the highest dose level tested. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 1000 μg/plate in the strains TA1535, TA98 and TA1537. Except for tester strains TA1535 and TA98 in the presence of S9-mix where the test item was tested up to concentrations of 1600 μg/plate. Thio Isocyanate Adduct did not precipitate on the plates at this dose level.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 1600 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and up to concentrations of 5000 μg/plate in tester strain WP2uvrA in the pre-incubation assay. Thio Isocyanate Adduct did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related 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 a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Thio Isocyanate Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.