Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A lot of studies are available to evaluate the genotoxic potential of tetramethylthiuram monosulphide. A reliable ames test was performed and TMTM showed a positive result with and without metabolic activation. However, negative results were obtained in the in vitro gene mutation assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2012 - 09 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Without S9: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate or both mutagenicity experiments.
With S9 : 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).
Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no
Species / strain:
S. typhimurium, other: TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA102, TA98 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with TA1537 at 5000 µg/ml (with S9) only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix : The selected treatment-levels for both mutagenicity experiments were: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used, in any experiments. Noteworthy increases in the number of revertants were noted in the TA 100 strain in the first and second experiments. These increases exceeded the threshold of 2-fold the vehicle control (up to 2.3-fold), were dose-related and reproducible. They were therefore considered as biologically significant. The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains

Experiments with S9 mix : The selected treatment-levels were: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. A strong toxicity (thinning of the bacterial lawn) was noted at 5000 µg/plate towards the TA 1537 strain, in the first experiment only. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the other strains used, in any experiments. Increases in the number of revertants were noted in the TA 1535 strain in the first and second experiments. These increases exceeded the threshold of 3-fold the vehicle control (up to 3.2-fold and 6.7-fold the vehicle control in the first and second experiments, respectively). Even if they were not dose-related, the increases observed in the first assay were reproduced in the second experiment performed under the pre-incubation method. Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant. Using the direct plate incorporation method (first experiment), increases in the number of revertants were noted in the TA 100 strain at dose-levels = 1250 µg/plate. These increases did not reach the threshold of 2 -fold the vehicle control but the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control.
Increases in the number of revertants were also noted in the second experiment performed using the pre-incubation method. These increases exceeded the threshold of 2-fold the vehicle control at all tested dose-levels (up to 3.7 -fold). Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant. The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains.
Conclusions:
The test item, Tetramethylthiuram monosulfide, showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the absence and in the presence of a metabolic activation system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Council Regulation No. 440/2008 of 30 May 2008, Part B13/14) and in compliance with the principles of Good Laboratory Practice.

 

A preliminary toxicity test was performed to define the dose-levels of Tetramethylthiuram monosulfide to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item Tetramethylthiuram monosulfide was dissolved in dimethylsulfoxide (DMSO).

 

Experiments without S9 mix : The selected treatment-levels for both mutagenicity experiments were: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used, in any experiments. Noteworthy increases in the number of revertants were noted in the TA 100 strain in the first and second experiments. These increases exceeded the threshold of 2-fold the vehicle control (up to 2.3-fold), were dose-related and reproducible. They were therefore considered as biologically significant. The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains


Experiments with S9 mix : The selected treatment-levels were: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. A strong toxicity (thinning of the bacterial lawn) was noted at 5000 µg/plate towards the TA 1537 strain, in the first experiment only. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the other strains used, in any experiments. Increases in the number of revertants were noted in the TA 1535 strain in the first and second experiments. These increases exceeded the threshold of 3-fold the vehicle control (up to 3.2-fold and 6.7-fold the vehicle control in the first and second experiments, respectively). Even if they were not dose-related, the increases observed in the first assay were reproduced in the second experiment performed under the pre-incubation method. Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant. Using the direct plate incorporation method (first experiment), increases in the number of revertants were noted in the TA 100 strain at dose-levels = 1250 µg/plate. These increases did not reach the threshold of 2 -fold the vehicle control but the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control.

Increases in the number of revertants were also noted in the second experiment performed using the pre-incubation method. These increases exceeded the threshold of 2-fold the vehicle control at all tested dose-levels (up to 3.7 -fold). Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant. The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains.

 

The test item showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the absence and in the presence of a metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January-April 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method: Clive & Spector (1975) Mut. Res., 31, 17-29.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Scoring for mutation was based on selecting cells that have undergone forward mutation from a TK+/- to a TK-/- genotype by cloning them in soft agar with BrdU.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The cells used in this study were derived from Fischer mouse lymphoma cell line L1578Y. The cells are heterozygous for a specific autosomal mutation at the TK locus and are bromodeoxyuridine BrdU sensitive.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mouse liver S-9 preparation.
Test concentrations with justification for top dose:
The test substance was toxicity tested over the range of 5 µg/ml to 2.5 µg/ml. Concentrations greater than 10 µg/ml proved to be highly cytotoxic in the absence of an activation system and even more toxic in the presence of a mouse liver S-9 preparation.
Vehicle / solvent:
DMSO (1%)
Untreated negative controls:
yes
Remarks:
media only
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (1%)
True negative controls:
no
Positive controls:
yes
Remarks:
ethyl methanesulfonate (EMS) and dimethylnitrosamine (DMS)
Details on test system and experimental conditions:
The cells were maintained in Fischer's medium for leukemic cells of mice 10% horse serum and sodium pyruvate. Cloning medium consisted of Fischer's medium with 20% horse serum, sodium pyruvate, and 0.3% noble agar. Selection medium was made from cloning medium by the addition of 7.5 mg BrdU to 100 ml cloning medium.

The test substance was dissolved in DMSO at 250 µg/ml. Working solutions were made from this stock solution by making a series of 2 -fold serial dilutions of the stock solution with DMSO. One tenth milliliter of each of the stock solution or one of the working dilutions was added to 3x10^6 celles in 10 ml of medium to achieve the desired final concentration.
Evaluation criteria:
A compound is considered to be mutagenic in this assay if :
-a dose-reponse relationship is observed over 3 of the 4 dose levels employed.
-the minimum increase at the high level of the dose-response curve is at least 2.5 times greater than the solvent and./or negative control values.
-the solvent and negative control data are within the normal range of the spontaneous background for the TK locus.
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The results of a series of trials with the test substance were negative. The results of the trial 1 showed excessive toxicity under nonactivation conditions and some indication of elevated mutation frequencies. The activation part of the test was not completed. A second test conducted over the same dose range as the initial test proved to be negative and the toxicity was not as high. However, the positive control data was lower than usual and a third trial was conducted. The results from this study were considered to be negative. Again, toxicity was observed at the higher dose levels.

Table 1: Results of First trial

 

TEST

S9

Daily counts

(cells/ml x 10^5)

Relative suspension growth (% of control)

Total mutant clones

Total viable clones

Relative cloning efficiency (% of control)

Percent relative growth*

Mutant frequency ** (x 10^-6)

1

2

3

Solvent control

-

15.4

13.5

15.8

100.0

51.0

269.5

100.0

100.0

18.9

Negative control

-

18.2

9.5

17.0

89.5

35.0

215.0

79.8

71.4

16.3

EMS 0.5 ul/ml

-

12.2

11.4

14.6

61.8

593.0

144.0

53.4

33.0

411.8

TMTM

0.00250 mg/ml

-

10.

14.8

9.0

40.6

53.0

307.0

113.9

46.2

17.3

TMTM

0.00500 mg/ml

-

1.6

2.4

11.4

3.0

66.0

247.0

91.7

2.7

26.7

TMTM

0.01000

mg/ml

-

1.8

4.0

13.0

3.5

78.0

196.0

72.7

2.5

39.8

TMTM

0.02000 mg/ml

-

1.2

3.6

16.4

4.5

99.0

186.0

69

3.1

53.2

TMTM

0.04000 mg/ml

-

0.2

0.4

2.8

0.2

51.0

99.0

36.7

0.1

51.5

* (relative suspension growth x relative cloning efficiency) / 100

** (mutant clones/ viable clones) x 10^-4

 

 

Table 2: Results of Second trial

 

TEST

S9

Daily counts

(cells/ml x 10^5)

Relative suspension growth (% of control)

Total mutant clones

Total viable clones

Relative cloning efficiency (% of control)

Percent relative growth*

Mutant frequency ** (x 10^-6)

1

2

3

Solvent control

-

18.4

13.1

13.8

100.0

35.5

189.5

100.0

100.0

18.0

Negative control

-

19.4

15.8

10.0

92.1

28.0

198.0

104.5

96.3

14.1

EMS 0.5 ul/ml

-

14.2

13.2

16.2

91.3

136.0

177.0

93.4

85.3

76.8

TMTM

0.00250 mg/ml

-

15.0

12.4

24.0

134.3

39.0

184.0

97.1

130.3

21.2

TMTM

0.00500 mg/ml

-

7.2

13.6

12.0

35.3

26.0

199.0

105.0

37.1

13.1

TMTM

0.01000

mg/ml

-

3.6

13.4

11.0

13.5

35.0

171.0

90.2

12.1

20.5

TMTM

0.02000 mg/ml

-

1.0

1.8

8.4

2.5

35.0

222.0

117.2

2.9

15.8

TMTM

0.04000 mg/ml

-

1.0

2.8

3.6

1.0

55.0

262.0

138.3

1.4

21.0

 

 

 

 

 

 

 

 

 

 

 

Solvent control

+

10.1

15.6

13.2

100.0

26.5

209.0

100.0

100.0

12.0

Negative control

+

7.0

14.4

11.8

57.2

43.0

241.0

115.3

65.9

17.8

DMN 0.5 ul/ml

+

4.4

21.0

10.0

44.4

53.0

152.0

72.7

32.3

34.9

TMTM

0.00004

mg/ml

+

9.2

11.4

15.6

78.7

25.0

185.0

88.5

69.6

13.5

TMTM

0.00008 mg/ml

+

8.2

11.6

9.2

42.1

36.0

273.0

130.6

55.0

13.2

TMTM

0.00016 mg/ml

+

0.8

1.6

6.4

2.7

35.0

243.0

116.3

3.1

14.4

* (relative suspension growth x relative cloning efficiency) / 100

** (mutant clones/ viable clones) x 10^-4

 

Table 3: Results of Third trial

 

TEST

S9

Daily counts

(cells/ml x 10^5)

Relative suspension growth (% of control)

Total mutant clones

Total viable clones

Relative cloning efficiency (% of control)

Percent relative growth*

Mutant frequency ** (x 10^-6)

1

2

3

Solvent control

-

11.6

17.5

8.1

100.0

52.5

282.0

100.0

100.0

18.0

Negative control

-

11.6

18.4

9.6

124.6

111.0

342.0

121.3

151.1

32.5

EMS 0.5 ul/ml

-

3.8

26.0

10.4

62.5

339.0

132.0

46.8

29.3

256.8

TMTM

0.00250 mg/ml

-

13.6

14.0

14.6

169.1

67.0

305.0

108.2

182.8

22.0

TMTM

0.00500 mg/ml

-

9.2

20.2

12.4

140.1

92.0

267.0

94.7

132.7

34.5

TMTM

0.01000

mg/ml

-

7.4

9.8

5.8

25.6

7.0

485.0

172.0

44.0

1.4

TMTM

0.02000 mg/ml

-

1.0

1.2

7.2

3.5

10.0

273.0

96.8

3.4

3.7

 

 

 

 

 

 

 

 

 

 

 

Solvent control

+

9.4

17.9

9.4

100.0

34.0

316.0

100.0

100.0

10.0

Negative control

+

6.8

28.0

6.6

79.5

75.0

551.0

174.4

138.5

13.6

DMN 0.3 ul/ml

+

5.0

5.0

7.0

11.1

360.0

67.0

21.2

2.3

537.3

TMTM

0.00002

mg/ml

+

6.6

15.6

20.0

130.2

41.0

166.0

52.5

68.4

24.7

TMTM

0.00004

mg/ml

+

6.8

30.0

8.6

110.9

111.0

374.0

118.4

131.3

29.3

TMTM

0.00008 mg/ml

+

6.2

5.8

11.0

25.0

64.0

258.0

81.6

20.4

24.8

TMTM

0.00016 mg/ml

+

2.2

2.8

6.2

3.6

37.0

376.0

119.0

4.3

9.8

TMTM

0.00032 mg/ml

+

3.2

2.4

5.6

3.2

62.0

280.0

88.6

2.8

22.1

* (relative suspension growth x relative cloning efficiency) / 100

** (mutant clones/ viable clones) x 10^-4

Conclusions:
The test substance was considered to be not active in the L5178Y Mouse lymphoma assay. The mateiral was evaluated in three independent tests. Although some variability in mutant frequencies and toxicity was obtained, the overall set of results appeared negative.
Executive summary:

The test substance was dissolved in DMSO at 250 µg/ml. Working solutions were made from this stock solution by making a series of 2 -fold serial dilutions of the stock solution with DMSO. One tenth milliliter of each of the stock solution or one of the working dilutions was added to 3x10^6 celles in 10 ml of medium to achieve the desired final concentration.

A yellow precipitate formed when the solutions were added to culture medium at final concentrations of 320 µg/ml or higher.

The test substance was toxicity tested over the range of 5 µg/ml to 2.5 µg/ml. Concentrations greater than 10 µg/ml proved to be highly cytotoxic in the absence of an activation system and even more toxic in the presence of a mouse liver S-9 preparation.

DMSO (1%) was used as the sovent control substance and growth medium without the addition of solvent was employed as a negative control. No genetic effects were attributed to the presence of the solvent. EMS and DMN were used as reference mutagens and induced mutation frequencies within the expected range.

The results of a series of trials with the test substance were negative. The results of the trial 1 showed excessive toxicity under nonactivation conditions and some indication of elevated mutation frequencies. The activation part of the test was not completed. A second test conducted over the same dose range as the initial test proved to be negative and the toxicity was not as high. However, the positive control data was lower than usual and a third trial was conducted. The results from this study were considered to be negative. Again, toxicity was observed at the higher dose levels.

The test substance was considered to be not active in the L5178Y Mouse lymphoma assay. The material was evaluated in three independent tests. Although some variability in mutant frequencies and toxicity was obtained, the overall set of results appeared negative.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo cytogenetic assay on rat is available on TMTM and showed negative results.

An in vivo Comet assay will be performed according to the ECHA decision.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Portage, portage, MI
- Age at study initiation: 8-9 week old
- Weight at study initiation: no data
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: one or two per cage prior to dosing and one per cage after dosing; in suspended, stainless steel cages with stainless steel mesh bottom.
- Diet (e.g. ad libitum): certified rodent diet #5002, ad libitum
- Water (e.g. ad libitum): public water system, ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79°F
- Humidity (%):40-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
dose of administration = 10 ml/kg bw
Duration of treatment / exposure:
A single oral gavage
Frequency of treatment:
A single oral gavage
Post exposure period:
During the study, all animals were observed for visible toxic effects and mortality on the day of dosing, and daily thereafter for up to 72 hours.
Animals were weighed at the time of treatment and terminal bodyweights were taken at the time of the colchicine injection (2-3 hours prior to harvest for bone marrow extraction).
Dose / conc.:
750 mg/kg bw/day
Remarks:
(female)
Dose / conc.:
1 300 mg/kg bw/day
Remarks:
(male)
No. of animals per sex per dose:
High dose = 20 males and 20 females
Vehicule control = 15 males and 15 females
Positive control = 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide in 0.9% saline (oral gavage, volume of 10 mg/kg bw)
Tissues and cell types examined:
cells of bone marrow
Details of tissue and slide preparation:
Two to three hours prior to harvest animals were injected intraperitoneally (ip) with colchicine (4 mg/kg) to arrest dividing cells in metaphase. Animals were sacrified by CO2 inhalation. Both femurs were removed and the muscle tissue cleaned away. The bone marrow cells were aspirated from each femur into a centrifuge tube containing approximately 10 ml of phosphate buffered saline solution (PBS) at 37°C. The bone marrow cells were centrifuged at 1000 rpm for 5 minutes. The PBS was decanted and approximately 10 ml of hypotonic KCl (0.075M) prewarmed at 37°C, wa added to each tube. The cells were incubated at 37°C for 12-15 minutes to swell the cells. The bone warrow cells were centrifuged at 1000 rpm for 5 minutes and the supernatant decanted. The bone marrow cells were resuspended by gently tapping the bottom of the tube. While agitating the tube, 0.5 ml of fresh Carnoy's fixative (methanol/acetic acid 3:1, v/v) was slowly added. The cells were resuspended with a Pasteyr pipette to disperse clumps. Fixative was added to each sample to achieve a final volume of 10 ml. The cells were centrifuged, the supernatant decanted, and 5 ml of freash fixative was added to each tube 2-3 more times. Cells were resuspended in an appropriate volume of fixative and dropped onto clean, wet slides. Slides were dried on a slide warmer or over a flame. Slides were stained with 2% Giemsa stain.

Scoring of slides:
The scoring for mitotic index and chromosomal aberrations was performed by Pharmakon USA, Waverly, PA. To eliminate bias, all slides were coded prior to scoring. Whenever possible, 50 metaphases/animal (500 metaphases/treatment group) were scored for the presence of chromosome aberration. Both chromatid- and chromosome-type aberrations were scored. A total of 5000 cells/treatment group were evaluated for mitotic frequency.
Mitotic frequency is expressed as mitotic index, which is the fraction of mitotic cells in the cell population scored.
Evaluation criteria:
no details
Statistics:
Each individual test animal was the unit used for analysis of mean body weight change. A Dunnett’s t-test (one sided) was used for comparison of treatment groups and positive control values with vehicle control values (Dunnett, 1955). Chi-square analysis was performed to compare the proportion of aberrant cells in the cells from animals treated with the test sustance to those from animals treated with vehicle only. Student’s t-test was used to compare structural aberrations per cell in the treatment groups with vehicle controls. Results were considered statistically significant at the probability level of P<005.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the range-finding experiment, male and female rats were treated with Monothiurad at 1000, 2000, 3000, 4000 and 5000 mg/kg body weight. Monothiurad was found to be toxic to male and female rats at 1000 mg/kg and higher as indicated by clinical signs of toxicity and/or death. The approximate male and female LD50 was determined to be 3000 and 1000 mg/kg, respectively, by the binomial method.
Based on these results, target doses of 1300 mg/kg body weight (approximately 43% of the approximate LD50 value) and 750 mg/kg body weight (approximately 75% of the approximate LD50 value) were selected as the maximum dose levels for male and female rats, respectively. These maximum doses were selected to insure a reasonable probability of observing signs of toxicity but allow survival of the treated animals through to the 48 hour time point.

RESULTS OF DEFINITIVE STUDY
In the main cytogenetic experiment. Monothiurad was toxic at the target doses as evidenced by clinical signs of hypoactivity in male rats up to the 24 hour time point and in female rats up to the 48 hour time point. No deaths or clinical signs were observed in the control groups (vehicle and positive control). Statistically significant decreases in mean body weight were observed in the Monothiurad treated male and female rats at 24 and 48 hours after treatment and in the positive control treated male rats 24 hours after treatment.
No statistically significant increases in the proportion of aberrant cells or aberrations/cell were observed at the target doses (1300 mg/kg-males, 750 mg/kg-females) at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression was observed at the 6 hour (24%), 24 hour (33%) and 48 hour (56%) time points.
The positive control group (cyclophosphamide) yielded expected positive responses indicating the adequacy of our experimental conditions for the detection of clastogens.

Table 1: Proportion of cells with one or more aberrations (6 hour harvest), and mean aberrations/cells

compound

Dose (mg/kg)

sex

No.of aberrant cells

No. of normal cells

% aberrant cells/group

Mean proportion of aberrant cells per rat +/-SD

Total aberrations per group

Mean aberrations/cell +/-SD

Vehicle

0

M

2

248

0.8

0.008 +/- 0.018

2

0.008 +/- 0.018

Vehicle

0

F

4

246

1.6

0.016 +/ -0.026

4

0.016 +/ -0.026

Monothiurad

1300

M

1

249

0.4

0.004 +/- 0.009

3

0.012 +/- 0.027

Monothiurad

750

F

2

248

0.8

0.008 +/- 0.018

2

0.008 +/- 0.018

A total of 5 rats per sex dose point were used with 50 metaphases scored per rat (250 metaphases per group).

 

 

Table 2: Proportion of cells with one or more aberrations (24 hour harvest)

compound

Dose (mg/kg)

sex

No.of aberrant cells

No. of normal cells

% aberrant cells/group

Mean proportion of aberrant cells per rat +/-SD

Total aberrations per group

Mean aberrations/cell +/-SD

Vehicle

0

M

0

250

0.0

0.000 +/- 0.000

0

0.000 +/- 0.000

Vehicle

0

F

1

249

0.4

0.004 +/- 0.009

1

0.004 +/- 0.009

Monothiurad

1300

M

1

249

0.4

0.004 +/- 0.009

1

0.004 +/- 0.009

Monothiurad

750

F

1

249

0.4

0.004 +/- 0.009

1

0.004 +/- 0.009

Positive control

25

M

101

149

40.4

0.404 +/- 0.119**

289

1.156 +/- 0.728**

Positive control

25

F

115

135

46.0

0460 +/- 0.225**

353

1.412 +/- 0.953**

A total of 5 rats per sex dose point were used with 50 metaphases scored per rat (250 metaphases per group).

** Statistically significant increase in Chi-Square at p< 0.01

 

 

Table 3: Proportion of cells with one or more aberrations (48 hour harvest)

Compound

Dose (mg/kg)

sex

No.of aberrant cells

No. of normal cells

% aberrant cells/group

Mean proportion of aberrant cells per rat +/-SD

Total aberrations per group

Mean aberrations/cell +/-SD

Vehicle

0

M

0

250

0.0

0.000 +/- 0.000

0

0.000 +/- 0.000

Vehicle

0

F

0

250

0.0

0.000 +/- 0.000

0

0.000 +/- 0.000

Monothiurad

1300

M

2

248

0.8

0.008 +/- 0.018

2

0.008 +/- 0.018

Monothiurad

750

F

4

246

1.6

0.016 +/- 0.017

4

0.016 +/- 0.017

A total of 5 rats per sex dose point were used with 50 metaphases scored per rat (250 metaphases per group).

 

 

Table 4: Summary of Chromatid- and Chromosome-type aberrations in bone marrow cells (6 hour harvest)

Compound

Dose (mg/kg)

sex

Gap

Chromatid-type exchanges

Chromosome-type

total

del

inter

intra

trir

del

ring

dic

misc

Vehicle

0

M

1

2

0

0

0

0

0

0

0

2

Vehicle

0

F

0

4

0

0

0

0

0

0

0

4

Monothiurad

1300

M

0

2

1

0

0

0

0

0

0

3

Monothiurad

750

F

0

2

0

0

0

0

0

0

0

2

 

Table 5: Summary of Chromatid- and Chromosome-type aberrations in bone marrow cells (24 hour harvest)

Compound

Dose (mg/kg)

sex

gaps

Chromatid-type exchanges

Chromosome-type

total

del

inter

intra

trir

del

ring

dic

misc

Vehicle

0

M

0

0

0

0

0

0

0

0

0

0

Vehicle

0

F

1

1

0

0

0

0

0

0

0

1

Monothiurad

1300

M

0

1

0

0

0

0

0

0

0

1

Monothiurad

750

F

0

1

0

0

0

0

0

0

0

1

Positive control

25

M

7

151

73

26

39

0

0

0

0

289

Positive control

25

F

7

199

53

48

48

0

0

0

5

353

 

Table 6: Summary of Chromatid- and Chromosome-type aberrations in bone marrow cells (48 hour harvest)

Compound

Dose (mg/kg)

sex

gaps

Chromatid-type exchanges

Chromosome-type

total

del

inter

intra

trir

del

ring

dic

misc

Vehicle

0

M

0

0

0

0

0

0

0

0

0

0

Vehicle

0

F

0

0

0

0

0

0

0

0

0

0

Monothiurad

1300

M

1

2

0

0

0

0

0

0

0

2

Monothiurad

750

F

3

4

0

0

0

0

0

0

0

4

 

 

Table 7: Mitotic index (MI) (6 hour harvest)

Compound

Dose (mg/kg)

sex

Total of cells scored

Interphase cells

Mitotic cells

%MI

Mean MI/dose /group

% depression in MI #

Vehicle

0

M

5000

4738

262

5.24

4.50

-

Vehicle

0

F

5000

4812

188

3.76

Monothiurad

1300

M

5000

4866

134

2.68

3.44

23.55

Monothiurad

750

F

5000

4790

210

4.20

 # compared to the negative control

 

Table 8: Mitotic index (MI) (24 hour harvest)

Compound

Dose (mg/kg)

sex

Total of cells scored

Interphase cells

Mitotic cells

%MI

Mean MI/dose /group

% depression in MI #

Vehicle

0

M

5000

4699

301

6.02

5.70

-

Vehicle

0

F

5000

4731

269

5.38

Monothiurad

1300

M

5000

4777

223

4.46

3.81

33.16

Monothiurad

750

F

5000

4842

158

3.16

Positive control

25

M

5000

4930

70

1.40

1.34

76.49

Positive control

25

F

5000

4936

64

1.28

 # compared to the negative control

 

Table 9: Mitotic index (MI) (48 hour harvest)

Compound

Dose (mg/kg)

sex

Total of cells scored

Interphase cells

Mitotic cells

%MI

Mean MI/dose /group

% depression in MI #

Vehicle

0

M

5000

4886

114

2.28

3.84

-

Vehicle

0

F

5000

4730

270

5.40

Monothiurad

1300

M

5000

4930

70

1.40

1.70

55.73

Monothiurad

750

F

5000

4900

100

2.00

 # compared to the negative control

Conclusions:
The observations and findings of this study indicate that Monothiurad was non-clastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions utilized in this study.
Executive summary:

The study was designed to evaluate the potential of the test substance, Monothiurad, to induce morphological aberrations in the chromosomes of mammalian bone marrow cells. In this study, the test material was administered by oral gavage to male and female SD rats. At appropriate time intervals, the bone marrow cells were extracted and the morphology of the chromosomes in the cells examined for aberrations.

In the range-finding experiment, male and female rats were treated with Monothiurad at 1000, 2000, 3000, 4000 and 5000 mg/kg body weight. Monothiurad was found to be toxic to male and female rats at 1000 mg/kg and higher as indicated by clinical signs of toxicity and/or death. The approximate male and female LD50 was determined to be 3000 and 1000 mg/kg, respectively, by the binomial method. Based on these results, target doses of 1300 mg/kg body weight (approximately 43% of the approximate LD50 value) and 750 mg/kg body weight (approximately 75% of the approximate LD50 value) were selected as the maximum dose levels for male and female rats, respectively. These maximum doses were selected to insure a reasonable probability of observing signs of toxicity but allow survival of the treated animals through to the 48 hour time point.

In the main cytogenetic experiment. Monothiurad was toxic at the target doses as evidenced by clinical signs of hypoactivity in male rats up to the 24 hour time point and in female rats up to the 48 hour time point. No deaths or clinical signs were observed in the control groups (vehicle and positive control). Statistically significant decreases in mean body weight were observed in the Monothiurad treated male and female rats at 24 and 48 hours after treatment and in the positive control treated male rats 24 hours after treatment.

No statistically significant increases in the proportion of aberrant cells or aberrations/cell were observed at the target doses (1300 mg/kg-males, 750 mg/kg-females) at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression was observed at the 6 hour (24%), 24 hour (33%) and 48 hour (56%) time points.

The positive control group (cyclophosphamide) yielded expected positive responses indicating the adequacy of our experimental conditions for the detection of clastogens.

The observations and findings of this study indicate that Monothiurad was non-clastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions utilized in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation test (Ames Test)

Several Ames tests are available, but only one study is considered to be reliable (Sarlang 2012).

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium (OECD 471).

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item Tetramethylthiuram monosulfide was dissolved in dimethylsulfoxide (DMSO).

In the experiments without S9 mix: No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used, in any experiments.Noteworthy increases in the number of revertants were noted in the TA 100 strain in the first and second experiments. These increases exceeded the threshold of 2-fold the vehicle control (up to 2.3-fold), were dose-related and reproducible. They were therefore considered as biologically significant.The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains

In the experiments with S9 mix: No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in any experiments. A strong toxicity (thinning of the bacterial lawn) was noted at 5000 µg/plate towards the TA 1537 strain, in the first experiment only. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the other strains used, in any experiments.Increases in the number of revertants were noted in the TA 1535 strain in the first and second experiments. These increases exceeded the threshold of 3-fold the vehicle control (up to 3.2-fold and 6.7-fold the vehicle control in the first and second experiments, respectively). Even if they were not dose-related, the increases observed in the first assay were reproduced in the second experiment performed under the pre-incubation method. Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant.Using the direct plate incorporation method (first experiment), increases in the number of revertants were noted in the TA 100 strain at dose-levels = 1250 µg/plate. These increases did not reach the threshold of 2 -fold the vehicle control but the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control.

Increases in the number of revertants were also noted in the second experiment performed using the pre-incubation method. These increases exceeded the threshold of 2-fold the vehicle control at all tested dose-levels (up to 3.7 -fold). Moreover, the corresponding means and individual revertant colony counts were above the historical data range of the vehicle control. Consequently, these increases were considered to be biologically significant.The test item did not induce any other noteworthy or biologically significant increases in the number of revertants in the other tested strains.

The test item showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the absence and in the presence of a metabolic activation system.

In vitro mammalian cell gene mutation assay (Matheson 1978)

In this test, a yellow precipitate formed when the solutions were added to culture medium at final concentrations of 320 µg/ml or higher. The test substance was toxicity tested over the range of 5 µg/ml to 2.5 µg/ml. Concentrations greater than 10 µg/ml proved to be highly cytotoxic in the absence of an activation system and even more toxic in the presence of a mouse liver S-9 preparation. DMSO (1%) was used as the solvent control substance and growth medium without the addition of solvent was employed as a negative control. No genetic effects were attributed to the presence of the solvent. EMS and DMN were used as reference mutagens and induced mutation frequencies within the expected range.

The results of a series of trials with the test substance were negative. The results of the trial 1 showed excessive toxicity under nonactivation conditions and some indication of elevated mutation frequencies. The activation part of the test was not completed. A second test conducted over the same dose range as the initial test proved to be negative and the toxicity was not as high. However, the positive control data was lower than usual and a third trial was conducted. The results from this study were considered to be negative. Again, toxicity was observed at the higher dose levels.

The test substance was considered to be not active in the L5178Y Mouse lymphoma assay. The material was evaluated in three independent tests. Although some variability in mutant frequencies and toxicity was obtained, the overall set of results appeared negative.

In vivo chromosomal aberration test on rats (Kier 1998, OECD 475)

The study was designed to evaluate the potential of the test substance, Monothiurad, to induce morphological aberrations in the chromosomes of mammalian bone marrow cells. In this study, the test material was administered by oral gavage to male and female SD rats. At appropriate time intervals, the bone marrow cells were extracted and the morphology of the chromosomes in the cells examined for aberrations.

Based on these results of the range-finding studies, target doses of 1300 mg/kg body weight (approximately 43% of the approximate LD50 value) and 750 mg/kg body weight (approximately 75% of the approximate LD50 value) were selected as the maximum dose levels for male and female rats, respectively. In the main cytogenetic experiment. Monothiurad was toxic at the target doses as evidenced by clinical signs of hypoactivity in male rats up to the 24 hour time point and in female rats up to the 48 hour time point. No deaths or clinical signs were observed in the control groups (vehicle and positive control). Statistically significant decreases in mean body weight were observed in the Monothiurad treated male and female rats at 24 and 48 hours after treatment and in the positive control treated male rats 24 hours after treatment. No statistically significant increases in the proportion of aberrant cells or aberrations/cell were observed at the target doses (1300 mg/kg-males, 750 mg/kg-females) at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression was observed at the 6 hour (24%), 24 hour (33%) and 48 hour (56%) time points. The observations and findings of this study indicate that Monothiurad was non-clastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions used in this study.

Justification for classification or non-classification

Based on the negative results in the in vitro gene mutation assay and in the in vivo cytogenetic assay, TMTM is considered to be not mutagenic, no classification is required according to the Regulation (EC) No 1272/2008.