Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 January 2017 - 23 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
Tributyl-2-thiourea
EC Number:
219-350-2
EC Name:
Tributyl-2-thiourea
Cas Number:
2422-88-0
Molecular formula:
C13H28N2S
IUPAC Name:
1,1,3-tributylthiourea
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tri butyl thiourea
- Appearance: Clear pale to medium brown liquid
- Storage conditions: At room temperature
Specific details on test material used for the study:
- No purity correction factor required
- Stability in vehicle: stable
- Solubility in vehicle: soluble

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryprophan gene
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 microsomal enzymes (S9) were 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 (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of experiment 1)
First experiment (TA1535, TA 1537 and TA98, with and without S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without S9): 17, 52, 164, 512 and 1600 μg/plate

The highest test dose for the dose range finding test and experiment 1 were according to OECD 471. The highest test dose for experiment 2 was chosen based on the results of experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the test substance showed to be soluble in dimethyl sulfoxide. The vehicle was according to OECD guideline 471.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See table 1 in 'Any other information on materials and methods incl. tables'
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
The first experiment was a direct plate assay. To obtain more information about the possible mutagenicity of the test item, a pre-incubation assay was performed as a second experiment.

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3 (in both of two independent experiments)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- Experiment 1: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C.
- Experiment 2: The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

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.
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.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to limit concentration in the second experiment (1600 μg/plate)
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the direct plate assay at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the pre-incubation assay at 1600 μg/plate
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
Remarks:
tested up to limit concentration in the second experiment (1600 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to limit concentration in the second experiment (1600 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to limit concentration in the second experiment (1600 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Precipitation: in the dose range finding test: at the start of the incubation period at concentrations of 512 and 1600 μg/plate and upwards in the absence and presence of S9-mix, respectively. At the end of the incubation period the test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate, except in tester strain TA100 in the presence of S9-mix, where precipitation was only observed at 5000 μg/plate. In the first experiment: precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards in the absence and of S9-mix and at 5000 μg/plate in the presence of S9-mix. At the end of the incubation period the test item precipitated on the plates at dose levels of 512 μg/plate and upwards. In the second experiment: precipitation of the test item on the plates was observed at the start of the incubation period at the top concentration of 1600 μg/plate in the absence of S9-mix only. At the end of the incubation period, the test item precipitated on the plates at dose levels of 512 and 1600 μg/plate in the absence of S9-mix and at 1600 μg/plate in the presence of S9-mix.

- Cytotoxicity: cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in both experiments in strain TA1537 at the highest tested doses (5000 μg/plate, with S9 in the direct plate assay and 1600 μg/plate, without S9 in the pre incubation assay). Cytotoxicity was not observed in any of the other strains, with and without S9 at any of the dose levels.

- Mutagenicity: no mutagenicity (an increase in the number of revertants) was observed in both experiments in any of the test strains at any of the dose levels, with and without S9.

HISTORICAL CONTROL DATA (see table 2, 3 and 4 in 'Any other information on results incl tables')
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.
Remarks on result:
other: In both the direct plate assay and the pre-incubation assay

Any other information on results incl. tables

Table 2  Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

 

TA1535

TA1537

TA98

TA100

WP2uvrA

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

Dose rang finding

Positive control

 

 

 

 

 

 

1536 ± 243

1153 ± 51

433 ± 36

1520 ± 105

 

Solvent control

 

 

 

 

 

 

120 ± 7

136 ± 20

31 ± 10

23 ± 7

Direct plate assay

Positive control

268 ± 11

858 ± 37

432 ± 18

1100 ± 33

1334 ± 64

1586 ± 47

 

 

 

 

 

Solvent control

13 ± 2

10 ± 2

4 ± 1

3 ± 2

19 ± 2

15 ± 4

 

 

 

 

Pre incubation assay

Positive control

126 ± 8

835 ± 68

155 ± 7

145 ± 21

393 ± 32

1667 ± 109

1732 ± 77

828 ± 27

560 ± 59

148 ± 50

 

Solvent control

9 ± 5

12 ± 4

6 ± 4

6 ± 4

20 ± 4

22 ± 6

126 ± 8

116 ± 20

38 ± 4

35 ± 4

Table 3 Historical data of the solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

5-36

3-32

3-23

3-23

8-41

9-52

66-156

65-154

10-56

9-69

Mean

12

12

6

8

16

23

100

100

25

31

SD

5

4

3

4

5

7

15

16

6

7

n

1865

1862

1740

1715

1852

1912

1853

1877

1571

1583

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between November 2014 and November 2016.

Table 4 Historical data of the positive control items

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

125-1381

78-1058

55-1311

55-1051

410-1995

250-1907

554-1848

408-2651

112-1951

85-1359

Mean

828

218

686

376

1270

883

892

1352

1165

388

SD

151

109

320

142

338

340

174

342

488

152

n

1875

829

1560

1716

1766

1851

1820

1857

1506

1557

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between November 2014 and November 2016

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and under GLP principles, showed that Tri butyl thiourea is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.