Tetramethylthiuram monosulphide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental Start Date: 07 September 2021
Experimental Completion Date: 21 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

Deviations from protocol were recorded, which neither affected the overall interpretation of study findings nor compromised the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetramethylthiuram monosulphide

EC Number:

202-605-7

EC Name:

Tetramethylthiuram monosulphide

Cas Number:

97-74-5

Molecular formula:

C6H12N2S3

IUPAC Name:

N,N-dimethyl[(dimethylcarbamothioyl)sulfanyl]carbothioamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected as there is a large volume of background data in this species and was specifically requested by ECHA for this comet assay.

Sex:

male

Details on test animals or test system and environmental conditions:

Justification for Selection

The comet assay is recommended to assess the genotoxic potential of short-lived reactive mutagens at their site of contact or as a supplementary in vivo test for investigation of genotoxicity (OECD 489). This study has been designed to provide data at the request of the European chemical agency (ECHA), based on Article 41 of the REACH regulation (Regulation (EC) No 1907/2006).

The liver is recommended as the primary site of xenobiotic metabolism and is often highly exposed to both the test article and any metabolites. The stomach and duodenum are recommended tissues to examine for site of contact effects after oral exposure. Male gonadal cells were collected for potential analysis if induction of DNA strand breaks is determined in any somatic tissue. The analysis of gonadal cells may be relevant for the overall assessment of possible germ cell mutagenicity including classification and labelling according to CLP Regulation. As such, this study was performed in male rats only.

Species and Strain

69 male and 9 female young adult out-bred Sprague Dawley rats Crl:CD(SD).

Specification

21 male and 9 female animals were dosed during the Range-Finder Experiment. They were approximately 7 to 10 weeks old and 236-395 g (males) or 7 to 9 weeks old and 173-224 g (females) on the first day of dosing.

27 male animals were dosed during the Main Experiment. They were approximately 8 to 9 weeks old and 291-331 g on the first day of dosing.

Individual animal body weights are detailed in Table 8.2 (please refer to attachment “Range Finder Tables”) and Table 9.2 (please refer to attachment “Main Experiment Tables”).

Environment

Animals were housed in wire topped, solid bottomed cages, with three animals per cage.

The animals were housed in rooms air-conditioned to provide a minimum of 15 air changes/hour and set to maintain temperature and relative humidity in the range 19-25 °C and 40-70%, respectively (see attachment "Protocol Deviations"). Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.

Diet, Water, Bedding and Environmental Enrichment

Throughout the study the animals had ad libitum access to 5LF2 EU Rodent Diet. Each batch of diet was analysed for specific constituents and contaminants.

Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.

Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding. The bedding was analysed for specific contaminants.

In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.

No contaminants were present in any of the above at levels that might interfere with achieving the objective of the study.

Allocation to Treatment Group

On arrival, animals were randomly allocated to cages. Range-Finder animals were allocated to groups of three and Main Experiment animals were randomised to groups of six (three for the positive control).

Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.

Identification of the Test System

The animals were individually identified by uniquely numbered tail mark (Range-Finder) or subcutaneous electronic transponder (Main Experiment). Cages were appropriately identified (using a colour-coded procedure) with study information including study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

All treatments were given via oral gavage as this is a potential route of exposure. ECHA considers that the oral dose route is the most appropriate. Animals were dosed in replicate order i.e. cage 1 of Groups 1-4 dosed in ascending group order then cage 2 of Groups 1-4 in ascending group order. Group 5 was dosed at a time that allowed necropsy of these animals after Group 4 necropsy. Animals were not fasted prior to administration.

The test article and vehicle control were given as two administrations, at 0 and 21 hours; the positive control was administered once only at 21 hours. All animals were sampled at 24 hours.

All doses were administered at a dose volume of 10 mL/kg. Individual dose volumes were based on individual body weight.

Duration of treatment / exposure:

Two administrations at 0 (Day 1) and 21 hours (Day 2)

Frequency of treatment:

Two administrations at 0 (Day 1) and 21 hours (Day 2)

Post exposure period:

For full details on post dosing observations please refer to Post Dosing Observations entry in "Any other information on materials and methods incl. tables" section.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control,
Animal ID Male: R0001-R0006,
Sample Time: Day 2 (24 hours)

Doses / concentrations 2

Dose / conc.:

70 mg/kg bw/day

Remarks:

Tetramethylthiuram monosulfide,
Animal ID Male: R0101-R0106,
Sample Time: Day 2 (24 hours)

Doses / concentrations 3

Dose / conc.:

140 mg/kg bw/day

Remarks:

Tetramethylthiuram monosulfide,
Animal ID Male: R0201-R0206,
Day 2 (24 hours)

Doses / concentrations 4

Dose / conc.:

280 mg/kg bw/day

Remarks:

Tetramethylthiuram monosulfide,
Animal ID Male: R0301-R0306,
Day 2 (24 hours)

Doses / concentrations 5

Dose / conc.:

200 mg/kg bw/day

Remarks:

Positive Control,
Animal Male ID: R0401-R0403,
Day 2 (24 hours),
Administered as a single dose approximately 3 hours prior to sample time.

No. of animals per sex per dose:

Six (three for the positive control group), all males in Main Experiment.

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)

Examinations

Tissues and cell types examined:

The liver, stomach, duodenum and gonad were removed from each control (vehicle and positive) and test article treated animal.

For histopathology, a sample of liver, stomach, duodenum and gonad from vehicle control and test article treated animals only was removed. Liver, stomach and duodenum samples were immediately preserved in neutral buffered formalin and stored at room temperature. Gonad samples were immediately preserved in modified Davidson’s fluid and stored at room temperature. No histopathology samples were preserved for the positive control animals.

For table of comet and histopathological samples please refer to Tissue Samples entry in "Any other information on materials and methods incl. tables" section.

Histopathology

Preserved liver, stomach, duodenum and gonad samples were embedded in wax blocks and sectioned at 5 µm nominal. Liver, stomach and duodenum slides were stained with haematoxylin and eosin and examined by the Study Pathologist. Gonad slides were subsequently examined at the request of the Study Director as the Gonad tissue was required to be analysed in the comet assay.

Details of tissue and slide preparation:

Preparation of Cell Suspensions

The comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in ice cold Merchants solution and incubated on ice for at least 15 minutes prior to processing. The tissue was removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 µL of fresh ice cold Merchants solution to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in ice cold Merchants solution; each sample was vortexed in ice cold Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum three times with the back of a scalpel blade in 150 µL of fresh ice cold Merchants solution to produce single cell suspensions.

The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently pushed through bolting cloth (pore size of 150 µm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

Slide Preparation

Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on a chiller plate with ice packs.

Cell Lysis

Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X 100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis

Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation

At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining

Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

Slide Analysis

Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.

A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code by an individual not connected to scoring of the study.

All animals per group were analysed.

Measurements of tail intensity (%DNA in tail) were obtained from 150 cells per animal per tissue (see attachment "Protocol Deviations"). In general, this was evenly split over three slides.

The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during Comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis.

The following criteria were used for analysis of slides:

1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Comet slides were retained until report finalisation; at this time the slides were discarded with SD approval. Due to the nature of the slides, long term storage is not recommended as Comet integrity cannot be assured.

Evaluation criteria:

Acceptance Criteria

The data were considered valid if the following criteria were met:

1. The vehicle control data were comparable to laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and that produced a statistically significant increase compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analysed
4. The high dose was considered to be the MTD, the maximum recommended dose or the maximum practicable dose.

Acceptance under any other criteria is discussed in the results section.

Evaluation Criteria

For valid data, the test article was considered to induce DNA damage if:

1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue.
3. The increase exceeded the laboratory’s historical control data for that tissue.

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels.

A positive response was based on scientific judgement and included analysis of related, concurrent cytotoxicity information (such as hedgehog assessment, histopathological changes and clinical pathology results) and the historical control data.

Statistics:

For full details on data evaluation please refer to attachment "Data Evaluation".

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

It is concluded that Tetramethylthiuram monosulfide met the criteria for a clear negative response and did not induce DNA strand breaks in the stomach, duodenum and gonad of male Sprague Dawley rats administered up to 280 mg/kg/day

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

In male Sprague Dawley rats administered up to 280 mg/kg/day Tetramethylthiuram monosulfide met the criteria for a positive induction of DNA strand breaks in the Liver.

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main Experiment Results

For all Text Tables referenced in this section please refer to Text Table entry in "Any other information on results incl. tables" section.

For all non-Text Tables referenced in this section please see attachment "Main Experiment Tables".

Formulations Analysis

The formulations analysis results are presented in the attachment "Formulation Analysis Contributory Report".

Analyses demonstrated that test article formulations at 7, 14 and 28 mg/mL were homogenous (0.88-1.86% RSD) and met criteria (100±15% of the nominal test article concentrations) for acceptable achieved concentrations (mean values 92-95%). The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

Post Dose Observations

Post dose observations presented in Table 9.1 in attachment "Main Experiment Tables". 

There were no clinical observations of toxicity for any animal dosed with the vehicle control or the positive control. Clinical observations of toxicity were observed for all animals dosed with the test article.

At 70 mg/kg/day, piloerection was observed from 1-hour post dosing on Day 1.

At 140 mg/kg/day, piloerection and audible respiration or partially closed eyes were observed from 2 hours post dosing on Day 1.

At 280 mg/kg/day, piloerection and partially closed eyes were observed from 2 hours post dosing on Day 1.

However, all animals for all test article dosed groups had returned to a normal state prior to dosing on Day 2 with no further observations post Day 2 dose administration.

Body Weights

Individual body weights and body weight percentage changes are presented in Tables 9.2 and 9.3 respectively in attachment "Main Experiment Tables".

There was a test article-related effect on animal body weight between Day 1 – Day 2 with group mean body weight change values of -5.6%, -4.0% and -5.0% at 70, 140 and 280 mg/kg/day, respectively, compared to +1.8% in the concurrent vehicle control group.

Clinical Pathology

Clinical Chemistry

Clinical chemistry results are presented in Table 9.4 in attachment "Main Experiment Tables".

Increased cholesterol was recorded for animals in all groups administered Tetramethylthiuram monosulfide, with no clear dose relationship.

A small dose-related decrease in chloride was recorded for animals from all groups administered Tetramethylthiuram monosulfide.

Small dose-related increases in phosphate and urea were recorded for animals in all groups administered Tetramethylthiuram monosulfide.

While the clinical chemistry changes were generally very small, based on the acute nature of this study (24 hours) at the maximum-tolerated dose and because the values in Tetramethylthiuram monosulfide-treated animals were outside the control range and often noted with a dose relationship, findings described were considered related to Tetramethylthiuram monosulfide.

Other differences in individual clinical pathology parameters were observed for animals administered the test article; however, they were considered not test article related due to the negligible magnitude of the change, individual animal variability, and overlap of values for test article-treated animals with concurrent control values.

Histopathology

Upon macroscopic examination (incidence of macroscopic observations are summarised in Table 7.1 of the attachment "Pathology Contributory Report Table 7.1"), mottling of the liver was recorded for animals administered Tetramethylthiuram monosulfide, with no dose relationship in incidence, which correlated with centrilobular necrosis in some animals.

Moderate distension of the stomach was recorded for occasional animals administered 140 or 280 mg/kg/day Tetramethylthiuram monosulfide, with no microscopic correlate. Red areas on the mucosal surface of the stomach were recorded for occasional animals administered Tetramethylthiuram monosulfide, which correlated with the inflammation observed microscopically.

Other tissues were considered macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Upon microscopic examination (incidence of microscopic observations are summarised in Table 7.2 of the attachment "Pathology Contributory Report Table 7.2"), decreased hepatocyte glycogen was recorded for animals from all groups administered Tetramethylthiuram monosulfide, with a dose relationship. Centrilobular necrosis was recorded for occasional animals administered 140 or 280 mg/kg/day. Inflammation, primarily in the glandular stomach, was recorded for animals administered Tetramethylthiuram monosulfide. Erosion/ulcer was recorded for one animal (Animal R0203) administered 140 mg/kg/day Tetramethylthiuram monosulfide. In the duodenum, single cell necrosis was recorded for animals from all groups administered Tetramethylthiuram monosulfide.

Glycogen is normally stored in the hepatocytes in large, often perinuclear vacuoles with a granular or feathery appearance. Decreased glycogen vacuolation was noted in animals administered Tetramethylthiuram monosulfide. Animals were not fasted prior to necropsy and were also sacrificed in replicate order (within 1 hour of each other); as such, this decrease in glycogen was attributed directly or indirectly to the effects of Tetramethylthiuram monosulfide. This decrease may have indicated an increased utilization of glycogen, possibly due to the increased metabolism in the liver or decreased food consumption.

Other tissues were considered microscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Comet Analysis

Comet data for liver are presented in Table 9.5 (animal values), Table 9.6 (slide values), and summarised as group means in Text Table 1. Comet data for stomach are presented in Table 9.7 (animal values), Table 9.8 (slide values), and summarised as group means in Text Table 2.

Comet data for duodenum are presented in Table 9.9 (animal values), Table 9.10 (slide values), and summarised as group means in Text Table 3. Comet data for gonad are presented in Table 9.11 (animal values), Table 9.12 (slide values), and summarised as group means in Text Table 4.

For all non-Text Tables referenced above please see attachment "Main Experiment Tables".

The current historical vehicle and positive control data for this laboratory are presented in attachment "Historical Control Ranges".

Validity of Data

The data generated in this study confirm that:

1.The vehicle control data were comparable to laboratory historical control data for each tissue

2.The positive control induced responses that were compatible with the laboratory historical control data and produces are statistically significant compared to the concurrent vehicle control. It was noted that the gonad group mean %tail intensity for the positive control (6.02%) fell below the lower limit of the laboratory’s gonad historical positive control observed range (9.41%). However, there was a clear and unequivocal positive response, the positive control group was statistically significantly higher (p=0.001) than the concurrent vehicle control group and the positive control data for this tissue was based on a limited number of animals. As such, there was no impact on assay validity.

3.Adequate numbers of cells and doses were analysed

4.The high dose was considered to be the MTD

As dosing was via oral gavage, exposure to the stomach and duodenum was assured. Furthermore, there were inflammatory responses observed in the liver, stomach and duodenum tissues such that exposure was considered clear for these tissues (and therefore systemically). It should be noted that histopathological assessment of the gonad tissue was unremarkable for all animals.

The assay data were therefore considered valid.

Data Analysis

There was no dose-related increases in %hedgehogs in liver, stomach, duodenum or gonad, thus demonstrating that treatment with Tetramethylthiuram monosulfide did not cause excessive DNA damage that could have interfered with comet analysis (Table 9.5, Table 9.7 and Table 9.9).

Animals treated with Tetramethylthiuram monosulfide at 140 and 280 mg/kg/day exhibited group mean tail intensities (2.46% and 2.41%, respectively; Text Table 1) in the liver that exceeded the 95% reference range of the laboratory's historical vehicle control data (0.04-1.80%; attachment "Historical Control Ranges"). The tail intensity for all dose groups (70, 140 and 280 mg/kg/day) was statistically significantly (p<0.05) increased compared to the concurrent vehicle control. The linear trend test was also statistically significant (p<0.0001). It is acknowledged that in terms of group mean tail intensity the value for the high dose group (2.41% at 280 mg/kg/day) is equivalent to (marginally lower) the intermediate dose group (2.46% at 140 mg/kg/day). However, the highest individual animal tail intensity values are observed in the high dose group and there is a clear increase in %tail intensity in the liver in response to test article administration to the animals.

The individual animal tail intensity data showed that 4 out of 6 animals at 140 mg/kg/day and 3 out of 6 animals at 280 mg/kg/day exceeded the 95% reference range (Table 9.5 and attachment "Historical Control Ranges"); although not all animals exceeded the 95% reference range, the tail intensity values for all animals in both 140 and 280 mg/kg/day dose groups did exceed all animal values in the concurrent vehicle control group (vehicle control: 0.15-0.69%; 140 mg/kg/day: 1.15-3.84%; 280 mg/kg/day: 1.25-4.14%). The evaluation criteria for a positive response have been met.

It must be noted that inflammatory responses (specifically centrilobular necrosis) were observed in the liver for occasional animals dosed at 140 mg/kg/day (R0202) and 280 mg/kg/day (minimal/slight in R0303, R0304 and R0305). These necrosis observations did not always correlate with the largest tail intensity animal values (e.g. R0203 and R0204 at 140 mg/kg/day; R0301 was noted as the second highest tail intensity value at 280 mg/kg/day). Therefore, this microscopic histopathological observation confirm the test article does induce toxicological effects in the liver. However, toxicity and tail intensity responses were not directly linked.

Animals treated with Tetramethylthiuram monosulfide at 70, 140 and 280 mg/kg/day exhibited group mean (Text Table 2, Text Table 3 and Text Table 4) and individual animal tail intensities (Table 9.7, Table 9.9 and Table 9.11) in the stomach, duodenum and gonad that were similar to the concurrent vehicle control group and that fell within or marginally below the 95% reference range (stomach and duodenum) or observed range (gonad) of the laboratory's historical vehicle control data (attachment "Historical Control Ranges"). There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. These data were considered clearly negative in the stomach, duodenum and gonad.

Any other information on results incl. tables

Range-Finder Results

Post dose observations and body weights are presented in tables included in attachment "Range Finder Tables".

Groups of three male and three female rats were dosed at 0 hours and approximately 21 hours with Tetramethylthiuram monosulfide at 50, 70 and 100 mg/kg/day. These dose levels were considered tolerated under experimental conditions and as there were no substantial differences in toxicity between male and female animals observed, males only were used in further Range-Finder Experiments. Groups of three male rats were dosed at 140, 200, 280 and 350 mg/kg/day.

At 50 mg/kg/day, there were no clinical observations of toxicity on Day 1. On Day 2, all 3 males were recorded with decreased activity from 1 hour post dosing and one male also had piloerection after 2 hours. Two of the three males had recovered to a normal state by approximately 4 hours after the second dose. The females were recorded with decreased activity from 0.5 hours post dosing and piloerection was observed from 1-hour post dosing. At the 2 and 4 hour observation occasions, the females had regressed to a hunched posture. Body weight loss between Day 1 and Day 2 was observed for all animals (male group mean body weight change of -6.1% and female group mean body weight change of -7.0%).

At 70 mg/kg/day, the 3 males showed no clinical observations of toxicity on Day 1. On Day 2, 2 males recorded an isolated instance of piloerection 2 hours post dosing. Of the 3 females, one female showed a number of observations from 1 hour on Day 1 including decreased activity, salivation, mouth rubbing, excessive tears, semi-closed eyes and pale extremities. At 4.5 hours post dose one other female showed semi-closed eyes and decreased activity, and piloerection was observed in all females. The number of observations reduced throughout the day such that all females were in a normal state prior to dosing on Day 2. One female recorded an isolated instance of piloerection, 2 hours post dosing on Day 2. There were no observations for the other 2 females on Day 2. Body weight loss between Day 1 and Day 2 was observed for all 3 males and 2 females with the third female maintain their body weight (male group mean body weight change of -5.4% and female group mean body weight change of -1.6%).

At 100 mg/kg/day, all animals were observed with decreased activity and piloerection on Day 1 and Day 2. However, animals were in a normal state prior to dosing on Day 2 and therefore had recovered from the first dose. Body weight loss between Day 1 and Day 2 was observed for all animals (male group mean body weight change of -5.3% and female group mean body weight change of -2.3%).

At 140 mg/kg/day, clinical observations of toxicity were observed in all animals from 2 hours post the first dose with decreased activity and semi-closed eyes. Although animal condition had improved on Day 2 (piloerection only observed prior to dose), the animal had not fully recovered prior to Day 2. All animals were observed with decreased activity and piloerection on Day 2. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -7.1%).

At 200 mg/kg/day, clinical observations of toxicity were observed in all animals from 0.5 hours post the first dose with decreased activity and piloerection. Observations persisted throughout Day 1 (decreased activity only approximately 4 hours post dose). Animals had recovered to a normal stated prior to dosing on Day 2, however, decreased activity and piloerection were again noted 0.5 hours post dosing, which persisted until the end of the Range-Finder. There were also sporadic instances of diarrhoea. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -7.7%).

At 280 mg/kg/day, clinical observations of toxicity were limited to a sporadic instance of piloerection and semi-closed eyes at the observation occasion 2 hours post dosing on Day 1. On Day 2, observations from 2 hours post dosing included decreased activity, piloerection and diarrhea. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -2.8%).

At 350 mg/kg/day, clinical observations of toxicity on Day 1 included decreased activity, piloerection, excess salivation, excess ‘tears’, semi-closed eyes and irregular breathing. Animals had not recovered overnight, with observations of decreased activity, excess ‘tears’ and semi-closed eyes still observed. Post dosing on Day 2, observations included decreased activity, excess salivation, semi-closed eyes and anogenital soiling. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -6.1%). Due to the persistent observations, it was considered that there was a risk of morbidity and/or mortality such that 350 mg/kg/day exceeded an appropriate MTD for this study.

From these results 280 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 140 and 70 mg/kg/day were also selected (50% and 25% of the MTD).

As no substantial differences in toxicity between the sexes was observed in the Range-Finder, male rats only were used in the Main Experiment as gonadal cells were collected for potential analysis to optimise the use of animals as per regulatory request.

Text Tables

Text Table 1: Tetramethylthiuram monosulfide: Summary of Group Mean Data – Liver

Group/Dose Level (mg/kg/day)		Tail Intensity						Mean % Hedgehogs
		Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)		0.33	0.08	-	-	-	-	0.91
2/ Tetramethylthiuram monosulfide (70)		0.75	0.17	2.48	U	0.0174	*	0.87
3/ Tetramethylthiuram monosulfide (140)		2.46	0.46	8.46	U	<0.0001	***	1.04
4/ Tetramethylthiuram monosulfide (280)		2.41	0.55	8.55	U	<0.0001	***	0.49
5/ EMS (200)		16.69	1.03	71.25	U	<0.0001	***	1.60
Dose response: (groups 1,2,3,4 )					U	<0.0001	***	N/A
0.04-1.80%	Tail Intensity 95% reference range of the liver historical vehicle control data
EMS	Ethyl methanesulfonate
N/A	Not applicable
SEM	Standard Error of Mean
U	Unranked
*	P≤0.05
***	P≤0.001

Text Table 2: Tetramethylthiuram monosulfide: Summary of Group Mean Data – Stomach

Group/Dose Level (mg/kg/day)		Tail Intensity						Mean % Hedgehogs
		Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)		0.64	0.33	-	-	-	-	8.41
2/ Tetramethylthiuram monosulfide (70)		0.47	0.21	1.44	U	0.5671	NS	5.43
3/ Tetramethylthiuram monosulfide (140)		0.19	0.04	0.51	U	0.9463	NS	5.07
4/ Tetramethylthiuram monosulfide (280)		0.26	0.10	0.69	U	0.8817	NS	8.36
5/ EMS (200)		16.87	3.64	80.86	U	0.0051	**	7.53
Dose response: (groups 1,2,3,4 )					U	0.7934	NS	N/A
0.16-8.29%	Tail Intensity 95% reference range of the stomach historical vehicle control data
EMS	Ethyl methanesulfonate
N/A N/S	Not applicable Not significant
SEM	Standard Error of Mean
U	Unranked
**	P≤0.01

Text Table 3: Tetramethylthiuram monosulfide: Summary of Group Mean Data – Duodenum

Group/Dose Level (mg/kg/day)		Tail Intensity						Mean % Hedgehogs
		Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)		0.31	0.06	-	-	-	-	6.79
2/ Tetramethylthiuram monosulfide (70)		0.27	0.08	0.75	U	0.9311	NS	7.20
3/ Tetramethylthiuram monosulfide (140)		0.17	0.02	0.49	U	0.9956	NS	6.88
4/ Tetramethylthiuram monosulfide (280)		0.34	0.10	0.81	U	0.8995	NS	6.96
5/ EMS (200)		6.17	1.13	23.14	U	<0.0001	***	7.17
Dose response: (groups 1,2,3,4 )					U	0.7992	NS	N/A
0.20-3.14%	Tail Intensity 95% reference range of the duodenum historical vehicle control data
EMS	Ethyl methanesulfonate
N/A	Not applicable
NS	Not significant (P>0.05)
SEM	Standard Error of Mean
U	Unranked
***	P≤0.001

Text Table 4: Tetramethylthiuram monosulfide: Summary of Group Mean Data – Gonad

Group/Dose Level		Tail Intensity						Mean % Hedgehogs
(mg/kg/day)		Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
01/ Vehicle (0)		0.18	0.03	-	-	-	-	0.00
02/ Tetramethylthiuram monosulfide (70)		0.27	0.12	0.81	U	0.9008	NS	0.00
03/ Tetramethylthiuram monosulfide (140)		0.18	0.03	1.05	U	0.7025	NS	0.00
04/ Tetramethylthiuram monosulfide (280)		0.10	0.01	0.50	U	0.9956	NS	0.11
05/ EMS (200)		6.02	0.49	41.57	U	<0.0001	***	0.00
Dose response: (groups 1,2,3,4 )					U	0.9274	NS	N/A
0.04-0.45%	Tail Intensity observed range of the gonad historical vehicle control data
EMS	Ethyl methanesulfonate
N/A	Not applicable
NS	Not significant (P>0.05)
SEM	Standard Error of Mean
U	Unranked
***	P≤0.001

Applicant's summary and conclusion

Conclusions:

It is concluded that Tetramethylthiuram monosulfide met the criteria for a clear negative response and did not induce DNA strand breaks in the stomach, duodenum and gonad of male Sprague Dawley rats administered up to 280 mg/kg/day (the maximum tolerated dose determined in this study). In the same animals, Tetramethylthiuram monosulfide met the criteria for a positive induction of DNA strand breaks in the Liver.

Executive summary:

Tetramethylthiuram monosulfide was tested for its potential to induce DNA strand breaks in the liver, stomach or duodenum of treated rats. As strand break induction was observed in the liver, the gonad was also assessed.

Strain / Species:	Sprague Dawley rats
Vehicle:	Corn oil
Administration route:	Oral via gavage
Dosing regimen:	Two administrations at 0 (Day 1) and 21 hours (Day 2)
Sex:	Male rats only due to the potential analysis of gonadal cells. There were no substantial differences in toxicity between the sexes in the Range-Finder Experiment.
Dose levels:	70, 140 and 280 mg/kg/day
Maximum dose:	Maximum tolerated based on Range-Finder data
Positive control:	Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)
Animals per group:	Six (three for the positive control group)
Dose volume:	10 mL/kg
Clinical signs of toxicity:	Clinical observations of toxicity were observed for all animals dosed with the test article. At 70 mg/kg/day, piloerection was observed from 1-hour post dosing on Day 1. At 140 mg/kg/day, piloerection and audible respiration or partially closed eyes were observed from 2 hours post dosing on Day 1. At 280 mg/kg/day, piloerection and partially closed eyes were observed from 2 hours post dosing on Day 1. However, all animals for all test article dosed groups had returned to a normal state prior to dosing on Day 2 with no further observations post Day 2 dose administration. There was a test article-related effect on animal body weight between Day 1 – Day 2 with group mean body weight change values of -5.6%, -4.0% and -5.0% at 70, 140 and 280 mg/kg/day, respectively, compared to +1.8% in the concurrent vehicle control group.
Tissues sampled:	Liver, Stomach, Duodenum and Gonad were sampled on Day 2, equivalent to 24 hours.
Formulation analysis:	Analyses demonstrated achieved concentration and homogeneity were within protocol specification. No test article was detected in the vehicle control sample.
Clinical Chemistry:	Increased cholesterol, phosphate, and urea were recorded for animals in all groups administered Tetramethylthiuram monosulfide. A small dose-related decrease in chloride was recorded for animals administered Tetramethylthiuram monosulfide.

Histopathology:	On macroscopic examination, mottling of the liver was recorded for animals administered Tetramethylthiuram monosulfide. Distension of the stomach was recorded for occasional animals administered 140 or 280 mg/kg/day, and red areas on the mucosal surface were recorded for occasional animals administered Tetramethylthiuram monosulfide On microscopic examination, decreased hepatocyte glycogen was recorded for animals from all groups administered Tetramethylthiuram monosulfide, with a dose relationship observed. Centrilobular necrosis was recorded for occasional animals administered 140 or 280 mg/kg/day. Inflammation, primarily in the glandular stomach, was recorded for animals administered Tetramethylthiuram monosulfide. In the duodenum, single cell necrosis was recorded for animals from all groups administered Tetramethylthiuram monosulfide.
Bioanalysis/Exposure:	Terminal bleeds were not collected in error on Day 2. They were intended to be processed to plasma and held as a contingency for exposure. However, GI tract and liver (systemically exposed tissue) exposure was considered clear based on clinical chemistry, histopathology and dose route.
Assay validity:	The group mean vehicle control (tail intensity) data were within the laboratory’s historical vehicle control ranges for all three tissues. The positive control induced significant increases in tail intensity in the liver, stomach and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data. The assay was therefore accepted as valid.

There was no dose-related increases in %hedgehogs in liver, stomach, duodenum or gonad, thus demonstrating that treatment with Tetramethylthiuram monosulfide did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with Tetramethylthiuram monosulfide at 140 and 280 mg/kg/day exhibited group mean tail intensities (2.46% and 2.41%, respectively) in the liver that exceeded the 95% reference range of the laboratory's historical vehicle control data (0.04-1.80%). The tail intensity for all dose groups (70, 140 and 280 mg/kg/day) was statistically significantly (p<0.05) increased compared to the concurrent vehicle control. The linear trend test was also statistically significant (p<0.0001). It is acknowledged that in terms of group mean tail intensity the value for the high dose group (2.41% at 280 mg/kg/day) is equivalent to (marginally lower) the intermediate dose group (2.46% at 140 mg/kg/day). However, the highest individual animal tail intensity values are observed in the high dose group and there is a clear increase in %tail intensity in the liver in response to test article administration to the animals.

The individual animal tail intensity data showed that 4 out of 6 animals at 140 mg/kg/day and 3 out of 6 animals at 280 mg/kg/day exceeded the 95% reference range; although not all animals exceeded the 95% reference range, the tail intensity values for all animals in both 140 and 280 mg/kg/day dose groups did exceed all animal values in the concurrent vehicle control group (vehicle control: 0.15-0.69%; 140 mg/kg/day: 1.15-3.84%; 280 mg/kg/day: 1.25-4.14%). The evaluation criteria for a positive response have been met.

It must be noted that inflammatory responses (specifically centrilobular necrosis) were observed in the liver for occasional animals dosed at 140 mg/kg/day (R0202) and 280 mg/kg/day (minimal/slight in R0303, R0304 and R0305). These necrosis observations did not always correlate with the largest tail intensity animal values (e.g. R0203 and R0204 at 140 mg/kg/day; R0301 was noted as the second highest tail intensity value at 280 mg/kg/day, but no noted necrosis). Therefore, this microscopic histopathological observation confirm the test article does induce toxicological effects in the liver. However, toxicity and tail intensity responses were not directly linked.

Animals treated with Tetramethylthiuram monosulfide at 70, 140 and 280 mg/kg/day exhibited group mean and individual animal tail intensities in the stomach, duodenum and gonad that were similar to the concurrent vehicle control group and that fell within or marginally below the 95% reference range (stomach and duodenum) or observed range (gonad) of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. These data were considered clearly negative in the stomach, duodenum and gonad.

It is concluded that Tetramethylthiuram monosulfide met the criteria for a clear negative response and did not induce DNA strand breaks in the stomach, duodenum and gonad of male Sprague Dawley rats administered up to 280 mg/kg/day (the maximum tolerated dose determined in this study). In the same animals, Tetramethylthiuram monosulfide met the criteria for a positive induction of DNA strand breaks in the Liver.