5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione

Endpoint summary

Description of key information

Several relevant repeated-dose toxicity studies conducted via the oral route are available on the registered substance.

 

During an OECD 422, it was concluded that the NOAEL was 100 mg/kg/day based on:
Reduction in bodyweight gain at 1000 mg/kg/day in males considered adverse.
Microscopic changes observed for multiple organs at 300 and/or 1000 mg/kg/day (kidney, liver, thyroid) considered adverse, as it correlates with biochemical changes (lower alanine aminotransferase).

 

During an OECD 408, it was concluded that the NOAEL was 300 mg/kg/day based on:
Reduction in bodyweight gain at 1000 mg/kg/day in males considered adverse.
Higher thyroid gland weights, enlargement and increased incidences and severity of follicular cell hypertrophy/hyperplasia, including a possible related adenoma, and related changes in hormone levels (high TSH and low T3, T4).
Combination of findings in the kidneys at 1000 mg/kg/day.
Possible test material related diffuse hyperplasia in the urinary bladder and urethra of a single female.
Reduction in alanine aminotransferase was not considered as adverse in the absence of clear histopathological correlation.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 June 2023 - 24 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl: WI (Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent
species for nonclinical toxicity test by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 153 – 205 g (males) 116 – 165 g (females)
- Fasting period before study: no
- Housing: Polycarbonate cages containing sterilized wooden fibers as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: ad libitum, pellets of SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany.
- Water: ad libitum, municipal tap water.
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: It is considered that there aware no known contaminants in the feed, water or enrichment materials that could interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS (actual)
- Temperature (°C): 20 to 22
- Humidity (%): 45 to 74
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 June 2022 To: 23 September 2022

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were homogenized to visually acceptable levels, stored at room temperature and prepared within two hours prior to dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle chosen as it was previously used in the OECD 422 performed in the same species by oral route (gavage).
- Concentration in vehicle: 25, 75 or 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Specific gravity: 0.91

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis as indicated in table 1 ''Any other information on materials and methods incl. tables''. All samples to be analyzed were transferred (at room temperature) to the analytical laboratory at the Test Facility for same day analysis.

Concentration and homogeneity analyses were performed using a validated analytical procedure (Test Facility Study No. 20354298).

Acceptance criteria:
For concentration, mean sample concentration results within or equal to ± 10% for solutions and ± 15% for suspensions of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of = 10% for each group.

The stability of the material in peanut oil was not determined, since the available analytical method (US/Vis spectrophotometry) was not capable of indicating stability. Details of all attempts made during validation and development of the analytical method are extensively described in the analytical report (Test Facility Study No. 20354298). To limit the impact, all preparations were used within 2 hours after completion of the preparation of the formulation. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) was conducted on Wistar Han rats at doses of 30, 100, 300 and 1000 mg/kg bw/day of Bis DMTD via the oral route (gavage).
At 1000 mg/kg bw/day, one female was prematurely sacrificed on Day 40 due to adverse clinical signs, which appeared to be related with difficulties associated with parturition, even though relationship with treatment cannot be excluded. No mortality was reported on male animals. Ptyalism was noted in 8/12 female animals on three occasions or less. Piloerection was reported in four animals during gestation for up to three days, along with hunched posture in two animals. Lower body weight gain was apparent during gestation, correlating with a reduction in food consumption and reaching statistical significance, but not resulting in statistically significant reduction in body weight in females during gestation when compared to control animals. No relevant clinical signs nor effects on bodyweight and food consumption were reported in female animals receiving 300 mg/kg bw/day or less of the test substance.
Ptyalism was noted in 8/12 males on five occasions or less at 1000 mg/kg bw/day, and 4/12 males showed increased salivation on seven or eight occasions during the study. Ptyalism was also reported on some occasions in 8/12 males at 300 mg/kg bw/day and 5/12 males at 100 mg/kg bw/day. Noisy respiration was reported on one occasion in 1/12 males at 1000 mg/kg bw/day and 1/12 males at 300 mg/kg bw/day.
Piloerection and hunched posture were observed on Day 44 in 1/12 males at 1000 mg/kg bw/day. No relevant clinical signs were reported at 30 mg/kg bw/day. Statistically significant reduction in bodyweight gain appeared at 1000 mg/kg bw/day in male animals from Day 8, and this reduction was also occasionally significant in other treatment groups. This resulted in dose-related lower bodyweight in all treatment group at the end of treatment period (down to -9% vs control) but not reaching statistical significance. On the last week of treatment, males at 1000 mg/kg bw/day showed an overall reduction in bodyweight (-1,9 g between Day 36 to 43). Dose-related reduction in food consumption was reported in males at 300 and 1000 mg/kg bw/day (down to -27% vs control at the end of the treatment period).
For the main study, the highest dose should be chosen with the aim to induce some toxicity (clinical signs or a decrease in body weight), but not death or severe suffering. Considering the observations made on animals during the OECD 422 at 1000 mg/kg bw/day consisted of limited clinical signs, and a reduction in body weight gain (significant from Day 8 in male animals) without a statistically significant impact on body weight, the high dose selected for this study will be 1000 mg/kg bw/day.
The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity. 300 and 100 mg/kg bw/day are selected as the mid- and low-dose levels, respectively.

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: at least twice daily, except on days of receipt and necropsy where frequency was at least once daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily from start of administration onwards, up to and including the day prior to necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.

ARENA OBSERVATIONS: yes
- Time schedule: Once before the first administration of the test material and weekly during the Treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: weekly, starting from day 1.

WATER CONSUMPTION: Yes
- Time schedule for examinations: regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment period (all animals main study); week 13 of dosing period (group 1 and 4). The eyes were examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).

FUNCTIONAL TEST: Yes
- Time schedule for examinations: once during dosing period the first 5 animals per sex per group during Week 12-13. These tests were performed after clinical observations. The following evaluations were conducted: Posture/Body carriage, Convulsions, Stereotypy, Tremor, Palpebral closure/Ptosis, Ease of removal, Handling reactivity, Rearing, Arousal/Alertness, Gait/Mobility, Vocalizations, Respiration, Defecation, Urination, Appearance, Lacrimation, Salivation, Exophthalmus, Erected fur, Touch response/Tactile reflex, Startle response, Tail pinch response, Pupil response, Body temperature, Body tone, Air righting reflex, Grip strength (forelimb and hindlimb). No motor activity measurements were performed at the end of the treatment period. Since no relevant findings were made during the in-life phase (clinical observations and functional observations), it was considered that motor activity would not have been affected in this study.

ESTROUS STAGE DETERMINATION: Yes
- Time schedule for examinations: On the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all females, except from those that have to be euthanized in extremis or die spontaneously. Estrous stage was evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Sampled between 07:00 and 10:30 from the retro-orbital sinus on days of at the end of treatment, if possible, otherwise from the aorta.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes, (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters in table 2 under ''Any other information on materials and methods incl. tables'' were examined.

COAGULATION: Yes
- Time schedule for collection of blood: Sampled between 07:00 and 10:30 from the retro-orbital sinus on days of at the end of treatment, if possible, otherwise from the aorta.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes, (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters examined were: Prothrombin time (PT) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Sampled between 07:00 and 10:30 from the retro-orbital sinus on days of at the end of treatment, if possible, otherwise from the aorta.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes, (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters in table 3 under ''Any other information on materials and methods incl. tables'' were examined.

SERUM HORMONES (T3, T4, TSH): Yes
- Time schedule for collection of blood: Sampled between 07:00 and 10:30 from the retro-orbital sinus on days of at the end of treatment, if possible, otherwise from the aorta.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes, (overnight with a maximum of 24 hours)
- How many animals: all animals
- TSH was measured using the IMMULITE® 1000 analyser whilst measurement of T3 and T4 by using LC-MS according to the bioanalytical method validated in Test Facility Study No. 20213516.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (15-20 hours) at the end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters in table 4 under ''Any other information on materials and methods incl. tables'' were examined.

Sacrifice and pathology:

Study animals surviving until scheduled euthanasia had a terminal body weight recorded and were deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post-mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy. Animals euthanized before scheduled euthanasia were anesthetized using isoflurane and subsequently exsanguinated.

GROSS PATHOLOGY: Yes (all animals)
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

HISTOPATHOLOGY: Yes (all animals)
Tissues as detailed in table 5 under ''Any other information on materials and methods incl. tables'' were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin for histopathological evaluation.

ORGAN WEIGHTS: Yes (all animals)
The organs detailed in in table 5 under ''Any other information on materials and methods incl. tables'' were weighed at necropsy. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair was taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight) was calculated.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons was conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses were performed according to table 6 under ''Any other information on materials and methods incl. tables'' but excluded any group with less than 3 observations.

PARAMETRIC/NON-PARAMETRIC: Levene’s test was used to assess the homogeneity of group variances.The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

INCIDENCE: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was seen after dosing on a single occasion in one male at 100 mg/kg bw/day, on a few occasions in a single male and in three females at 300 mg/kg bw/day and in all males and females between Days 14 and 91 at 1000 mg/kg bw/day. Considering the nature of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
No findings were noted during the arena observations in this study.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered not to be test material-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test material.
One female at 1000 mg/kg bw/day was scheduled to be euthanized in extremis on Day 13, but died before anaesthesia in the necropsy room. On this day, shallow breathing with abnormal sounds, hunched and prostrate posture, erected fur, partly closed and dark eyes were noted. In addition, the animal was cold to touch. On days prior to death no clinical signs were observed and no effect on body weight or body weight gain was observed. At necropsy, clear watery content and yellow fluid was observed in the thoracic body cavity. Main microscopic findings were the presence of foreign material at the pleura and epicard and associated inflammations of organs in the thoracic cavity.
A male at 300 mg/kg bw/day was euthanized in extremis on Day 55. On the day of necropsy, deep breathing with abnormal breathing sounds, erected fur, partly closed eyes, and decreased activity was noted. In addition, this animal was cold to touch. On days prior to euthanasia no clinical signs were observed and no effect on body weight or body weight gain was observed. At necropsy abnormal clear watery content was observed in the thoracic body cavity and an accumulation of green gelatinous material was observed in the thoracic body cavity, including lung and diaphragm muscle. Related microscopic findings consisted of the presence of foreign material attached to and associated with acute inflammation of pleura of the lung, epicard of the heart, thymic capsule, and/or diaphragm. Furthermore, this animal showed test material-related findings in kidneys and adrenal gland, similar as in the scheduled euthanised females of the 300 mg/kg bw/day group.
Based on these findings, the premature death of these two animals was considered to be gavage procedure-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effect on body weight or body weight gain was observed in males at 100 and 300 mg/kg bw/day and in all females treated with the test material.
In males at 1000 mg/kg bw/day, a lower body weight gain was observed between Days 29-43 (13.3 and 12.2 grams body weight gain for Group 4 animals between Days 29-36 and 36-43, respectively, compared to 20.1 and 18.3 grams body weight gain for controls) and 50-57 (8.9 grams body weight gain for Group 4 males compared to 17.2 grams body weight gain for control animals), which resulted in an 11% lower body weight at the end of the treatment.
Any other changes in body weight (gain), irrespective of statistical significance, were considered to be unrelated to the test material since no trend was apparent regarding dose and duration of dosing.
Please see also tables 7, 8, 9 and 10 under ''Any other information on results incl. tables''.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related changes in food consumption were noted.
The slightly lower food consumption that was noted in all males dosed with the test material lacked a dose response and was therefore considered to be not test material-related.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmology findings in Week 13.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters were considered not to have been affected by the test material in males and females at 100 mg/kg bw/day.
Monocyte counts were higher in females at 1000 mg/kg bw/day (1.62x of control). A lower red blood cell distribution width was noted in males at 300 and 1000 mg/kg bw/day (0.94x for both). Furthermore, mean corpuscular hemoglobin concentration was lower in males at 1000 mg/kg bw/day (0.98x) and females at 300 and 1000 mg/kg bw/day (0.98x and 0.97x, respectively). Finally, a lower platelet count was noted in males at 1000 mg/kg bw/day (0.87x).
Hematocrit concentration was higher in males at 300 and 1000 mg/kg bw/day (1.07x and 1.06x, respectively, compared to controls), but in absence of a dose-response, considered to be not test material-related.
Individual higher neutrophil counts with concurrently reduced lymphocyte counts were noted among the dose groups without a dose-related distribution. This shift in type of white blood cells was considered to be a secondary nonspecific response to stress and not related to treatment with the test material.
Remaining differences in hematology parameters were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Coagulation parameters were considered not to have been affected by the test material in males up to 1000 mg/kg bw/day and in females at 100 and 300 mg/kg bw/day.
A longer prothrombin time was noted in females at 1000 mg/kg bw/day (1.10x of control).
Activated partial thromboplastin time was noted to be longer in females at 300 mg/kg bw/day. As this occurred without a clear dose-response and was mainly caused by a single animal with an abnormally high value, this change was regarded to be not test material-related.

Please see also table 11 under ''Any other information on results incl. tables''

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in clinical chemistry parameters are presented in table 12 under ''Any other information on results incl. tables''.
A lower alanine aminotransferase activity was noted in males and females at all dose levels, which was outside the historical control range (see Clinical Chemistry HCD under ''Any other information on results incl. tables''). Total protein concentrations were higher in males and females at 1000 mg/kg bw/day, and albumin concentration was higher in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, total bilirubin concentrations were lower in females, while urea concentration was higher. In addition, creatinine concentrations were higher in both males and females.
Glucose concentrations were lower in females at 100, 300 and 1000 mg/kg bw/day and in males at 1000 mg/kg bw/day. Furthermore, cholesterol and HDL cholesterol concentration were lower in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day and LDL cholesterol concentration was lower in females at 100, 300 and 1000 mg/kg bw/day and males at 1000 mg/kg bw/day. Triglyceride concentrations were higher in males at 1000 mg/kg bw/day, but were lower in females at 100, 300 and 1000 mg/kg bw/day.
Lastly, chloride concentration was higher in females at 1000 mg/kg bw/day.
Remaining differences in clinical chemistry parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in thyroid hormone parameters are presented in table 13 under ''Any other information on results incl. tables''
T3 values were statistically significant lower in males and females at 1000 mg/kg bw/day and T4 values were statistically significant lower in males at 100, 300 and 1000 mg/kg bw/day and females at 300 and 1000 mg/kg bw/day. TSH was statistically significant higher in males and females dosed at 300 and 1000 mg/kg bw/day. In addition, T4 values of males at 1000 mg/kg bw/day and females at 1000 mg/kg bw/day and TSH values of all males (including some control animals) and females at 300 and 1000 mg/kg bw/day were outside the historical control range.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Lower pH was noted in males and females at 100, 300 and 1000 mg/kg bw/day (absolute values; males: 6.20, 5.61 and 5.30, respectively; females: 6.30, 6.05 and 5.89, respectively, not statistically significant in females at 100 mg/kg bw/day) compared to the controls (absolute values; males: 6.70; females: 6.45).
In addition, the specific gravity was higher in males at 100, 300 and 1000 mg/kg bw/day (1.02, 1.03 and 1.02x of control, respectively; not statistically significant at 100 and 1000 mg/kg bw/day) and females at 1000 mg/kg bw/day (1.02x).
Furthermore, the color of the urine in males ranged from light yellow in 9/10 and dark yellow in 1/10 controls to light yellow in 4/10, dark yellow in 2/10 and brown in 4/10 at 1000 mg/kg bw/day. Also, the clarity changed from clear in 6/10 and clouded in 4/10 controls to clear in 2/10 and clouded in 8/10 males at 1000 mg/kg bw/day.
See also table 14 under ''Any other information on results incl. tables''

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations (activity, autonomic response, excitability as well as physiological, sensorimotor and neuromuscular observations) were considered to be unaffected by treatment with the test material.
Any observations noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test material-related findings were noted in the adrenal gland, kidneys, pituitary gland, thyroid gland and thymus weights (absolute and/or relative to body weights), as shown in table 15 under "Any other information on results incl. tables
Higher adrenal gland weights (relative to body weight) were observed in males at 300 and 1000 mg/kg bw/day.
Higher kidney weights (relative to body weight) were observed in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day (absolute and/or relative to body weight).
Higher pituitary gland weights (relative to body weight) were observed in males at 1000 mg/kg bw/day.
Lower thymus weights were observed starting in females at 300 mg/kg bw/day (absolute and relative to body weight) and in males (absolute) at 1000 mg/kg bw/day.
Higher thyroid gland weights were observed in males (relative to body weight) and females at 300 and 1000 mg/kg bw/day (absolute and relative to body weight, not statistically significant at 300 mg/kg bw/day).
The statistically significant higher liver weight relative to body weight in males at 100, 300 and 1000 mg/kg bw/day and higher testis weight relative to body weight at 1000 mg/kg bw/day were considered to be the result of a test material-related effect on final body weight. Any other differences, including those that reached statistical significance were considered to be not test material-related due to the direction of the change, lack of dose related pattern, lack of microscopic correlate and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test material-related macroscopic findings were present starting at 100 mg/kg bw/day in the kidneys and starting at 300 mg/kg bw/day in the thymus, adrenal and thyroid gland.
In the adrenal gland, pale discoloration was noted in 2/10 females at 300 mg/kg bw/day and in 1/10 males and 3/9 females at 1000 mg/kg bw/day. In the kidneys, pale discoloration was noted in 4/10 females at 100 mg/kg bw/day, 5/10 females at 300 mg/kg bw/day, and 2/10 males and 7/9 females at 1000 mg/kg bw/day. Enlargement of the kidneys was noted in 1/10 males at 1000 mg/kg bw/day. A small thymus was noted in 1/9 males at 300 mg/kg bw/day and in 4/10 males at 1000 mg/kg bw/day.
Enlargement of the thyroid gland was observed in 1/10 males of the control group, in 3/9 males and 1/10 females at 300 mg/kg bw/day and in 6/10 males and 6/9 females at 1000 mg/kg bw/day. The unilateral enlargement noted in a single male of the control group was considered to be a spontaneous finding.
The remainder of the recorded macroscopic findings of the scheduled sacrificed rats were within the range of background gross observations encountered in rats of this age and strain.
Please see also see also table 16 under ''Any other information on results incl. tables''

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test material-related microscopic findings after treatment with test material were noted in the adrenal gland, kidney, pituitary gland, stomach, sublingual gland, thyroid gland and urinary bladder, and are summarized in Table 17 under ''Any other information on results incl. tables''.
In the adrenal gland, vacuolation of the zona fasciculata and reticularis was recorded at increased incidence and/or severity in males starting at 100 mg/kg bw/day with minimal degrees and at 300 and 1000 mg/kg bw/day up to mild degree. Vacuolation of the zona reticularis area was noted in females starting at 300 mg/kg bw/day at a minimal degree.
In the kidneys, tubular pigment (yellow-brown) was present in both sexes starting at 100 mg/kg bw/day (up to moderate). Tubular degeneration/regeneration was present in both sexes starting in a single female and all males at 100 mg/kg bw/day (up to mild). An increased incidence and severity of interstitial mononuclear cell infiltration was noted in both sexes at 1000 mg/kg bw/day.
In the pituitary gland, hypertrophy of the pars distalis was noted in males at 300 mg/kg bw/day (minimal) and in males (up to mild) and females (minimal) at 1000 mg/kg bw/day.
In the stomach, basophilia of the glandular mucosa at the pyloric antrum was noted at a minimal degree in females starting at 300 mg/kg bw/day and in males at 1000 mg/kg bw/day. This was associated with an increased incidence and/or severity of mixed cell infiltration of the glandular mucosa at 1000 mg/kg bw/day (up to mild).
In the sublingual gland, atrophy was noted in both sexes at 1000 mg/kg bw/day (up to mild).
In the thyroid gland, follicular cell hypertrophy was noted at increased incidence and/or severity in males (up to moderate) and females (up to marked) at 1000 mg/kg bw/day. Furthermore, a follicular cell adenoma, a benign tumor, was noted in one male at 100 mg/kg bw/day) and in one female at 1000 mg/kg bw/day.
In the urinary bladder, hypertrophy/hyperplasia of the transitional epithelium was noted in a single male (minimal) and 3/10 females (up to mild) at 1000 mg/kg bw/day.
Findings of note were present in the mammary gland and vagina of females and are summarized in Table 18 under ''Any other information on results incl. tables''
Mucification of the vagina and lobuloalveolar hypertrophy/hyperplasia of the mammary gland were noted in 2/10 females at 100 mg/kg bw/day, 1/10 female at 300 mg/kg bw/day and 2/10 females at 1000 mg/kg bw/day. This combination of findings suggests increased progesterone or altered estrogen/progesterone ratios. This can occur as a part of the onset of reproductive senescence associated with aging. It is described in a study with 104 female Han Wistar rats, that at 24 weeks of age, 88% have an estrous cycle of regular pattern and duration (Mitchard and Klein, 2016). The females in the current study were just a bit younger (approximately 19-20 weeks of age at the end of the study) and the abnormal cycles did not show a clear dose response. Therefore, these findings were considered most likely not related to the test material.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

The estrous stage for female animals at the end of the Treatment Period can be found in Table 19 under ''Any other information on results incl. tables''. As only one smear was taken on the day of necropsy, these results are solely intended to assist in histological evaluation of oestrogen sensitive tissues. A higher number of females were in diestrus at 300 and 1000 mg/kg bw/day, but this distribution of females in different estrous stages can be related to chance, and is considered to be not test material-related.

Details on results:

Wistar Han rats were treated with the test item for at least 90 consecutive days by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The animals of the control group received the vehicle, peanut oil, alone.
In males at 1000 mg/kg bw/day a lower body weight gain was observed which resulted in an overall lower body weight of 11%. At the severity observed, this effect was considered to be adverse.
At hematology, higher monocytes, and lower red blood cell distribution width, platelets and mean corpuscular hemoglobin concentration were noted in males and/or females at 300 and/or 1000 mg/kg bw/day. In addition, a longer prothrombin time was noted in females at 1000 mg/kg bw/day. In absence of a histopathological correlation and at the magnitude of change, these findings were considered to be not adverse.
At clinical chemistry, lower alanine aminotransferase activity and total bilirubin concentrations and higher total protein, albumin, urea and creatinine concentrations were noted in males and/or females at 300 and/or 1000 mg/kg bw/day. In addition, lower glucose and cholesterol (including HDL and LDL) concentration and higher and lower triglycerides concentration, were noted in males and females at 100, 300 and/or 1000 mg/kg bw/day. In addition, higher chloride concentration were noted in females at 1000 mg/kg bw/day. In absence of a clear histopathological correlate, these findings were considered to be not adverse.
Effects on TSH, T3 and T4 in males and females at 1000 mg/kg bw/day do correlate to histopathology findings in the thyroid gland discussed below, and are considered to be adverse. The lower T4 in males at 100 and 300 mg/kg bw/day and females at 300 mg/kg bw/day and higher TSH in males and females at 300 mg/kg bw/day are considered to be not adverse, in absence of a histopathological correlate.
The changes observed in the pH of all animals dosed with the test material, specific gravity of males at 100, 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day and colour and clarity of the urine of males at 1000 mg/kg bw/day might be related to the observed changes in the kidney, urinary bladder and urinary tract discussed below. However, no direct correlation can be made, and the observed changes by itself are considered to be not adverse.
At histopathological assessment, test material-related microscopic changes were noted in the adrenal gland, pituitary gland, thyroid gland, kidneys, urinary bladder, stomach, sublingual gland and thymus.
In the adrenal gland, increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis of the adrenal gland was recorded in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day. In males, the vacuolation was mainly macrovesicular, noted up to mild degrees and most prominent in the distal zona fasciculata (close to the medulla) and zona reticularis. At 300 and 1000 mg/kg bw/day, this finding was considered to be related with higher adrenal gland weights (relative to body weight). In females, the vacuolation was mainly microvesicular in nature, noted at minimal degrees, and present in the zona reticularis area of the adrenal gland. There was no microscopic correlate for the pale discoloration of the adrenal gland.
In the pituitary gland, hypertrophy of the pars distalis was noted starting in males at 300 mg/kg bw/day and females at 1000 mg/kg bw/day (males up to mild, females minimal) and was regarded to correlate with higher pituitary gland weights in males at 1000 mg/kg bw/day. The hypertrophic cells stained chromophobic and/or basophilic. Based on the hematoxylin and eosin stained slides it is not possible to further identify the type of affected endocrine cell.
The findings of the adrenal gland and pituitary gland were considered non-adverse, based on the minimal to mild severities and in absence of degenerative, necrotic, or proliferative changes (Kerlin et al., 2016; Palazzi et al, 2016).
In the thyroid gland, follicular cell hypertrophy was noted at increased incidence and/or severity in males (up to moderate) and females (up to marked) at 1000 mg/kg bw/day. In the cases with moderate or marked severity, the lumen of the follicles was very small and there was some evidence of hyperplasia as well. At 1000 mg/kg bw/day, there were relationships between the follicular cell hypertrophy, macroscopic enlargement, higher organ weights and high TSH levels. Indirectly, there was a relationship with lower T3 and T4 levels.
In one female at 1000 mg/kg bw/day, a follicular cell adenoma (a benign tumor) was noted which was embedded in follicles with hypertrophic/hyperplastic epithelium (moderate degree) and showed a high TSH level. A relation with the test material could not be fully excluded.
A follicular cell adenoma was also noted in one male at 100 mg/kg bw/day but was present in a thyroid gland with minimal hypertrophy and was without signs of hyperplasia, and this animal showed only a minimal increase in TSH level. Therefore, a relation with the test material was regarded unlikely.
The thyroid gland findings at 1000 mg/kg bw/day (high organ weights, high TSH levels and low T3 and T4 levels, and follicular cell hypertrophy/hyperplasia up to marked degree (with a possible related adenoma) were considered adverse.
In the kidneys, yellow-brown tubular pigment was present in both sexes starting at 100 mg/kg bw/day. In general, at 1000 mg/kg bw/day this pigment consisted of irregularly shaped granular deposits and was located centrally in the cytoplasm of tubules located in the cortex and outer medulla. In the lower dose groups, the pigment was mostly present at the apical side of the epithelial cells or attached to the epithelium in the lumen and had a smaller size and round form.
Tubular degeneration/regeneration was present in both sexes starting in a single female and all males at 100 mg/kg bw/day (up to mild). This finding was characterized by a basophilic staining of the cytoplasm and nucleus of tubular the epithelium, tubules with degenerating epithelial cells, and rare mitotic figures. Especially at 1000 mg/kg bw/day, the degenerating/regenerating tubules showed pigment and/or karyomegaly (enlarged/abnormal nuclei). Additionally, at 1000 mg/kg bw/day, there was an increased incidence and severity of interstitial mononuclear cell infiltration associated with the areas with degenerating/ regenerating tubules in both sexes. These findings were related with higher kidney weights (starting at 100 mg/kg bw/day in males and at 300 mg/kg bw/day in females) and macroscopic enlargement (in males at 1000 mg/kg bw/day). There was no clear correlate for the macroscopic pale discoloration.
Only, the kidney findings at 1000 mg/kg bw/day were considered adverse, based on the somewhat higher degree of tubular degeneration/regeneration (all males and all but one female showed mild degrees with prominent karyomegaly) and associated increased incidence and severity of interstitial mononuclear cell infiltration.
In the urinary bladder, diffuse hypertrophy/hyperplasia of the transitional epithelium (minimal or moderate degree) was noted in a few animals at 1000 mg/kg bw/day. The minimal degree in a single male and 2/9 females at 1000 mg/kg bw/day was considered non-adverse. In the female with the highest degree of hyperplasia, there was also very prominent hyperplasia visible at the end of the urinary tract (in the urethra, non-protocollary organ which was present on the slide of the vagina). This amount of hyperplasia was regarded adverse and a relationship to treatment with the test material could not be excluded.
In the stomach, basophilia of the glandular mucosa at the pyloric antrum was noted starting in females at 300 mg/kg bw/day and in males at 1000 mg/kg bw/day. This was considered to be mainly caused by less differentiation, including less mucus. At 1000 mg/kg bw/day, this finding was associated with an increased incidence and/or severity of mixed cell infiltration of the glandular mucosa (up to mild). At the recorded severities, the combination of these findings was considered non-adverse.
In the sublingual salivary gland, atrophy was noted in both sexes at 1000 mg/kg bw/day (minimal and once mild). At the noted low incidence and severity and in absence of other findings in the sublingual gland, the atrophy was considered non-adverse.
In the thymus, a small thymus was noted in males starting in a single male at 300 mg/kg bw/day and correlated with lower thymus weights at 1000 mg/kg bw/day. In females, lower thymus weights were noted starting at 300 mg/kg bw/day. There was no microscopic correlate for the findings in the thymus. Therefore, these findings were considered non-adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

serum/plasma hormone analyses

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

kidney

urethra

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Dose Formulation Analyses

Accuracy

The concentrations measured in the formulations in Week 1 (low-, mid- and high-dose groups), Week 4 (mid-dose group), Week 7 (mid- and high-dose groups), Week 8 (low-dose group) and Week 12 (low-, mid- and high-dose groups) were within the target concentration range (i.e. mean sample concentration results within or equal to 85-115% of target concentration). The other samples (Week 4, mid-dose group and 4; Week 7, low-dose group and Week 8, mid- and high-dose groups) were outside the target concentration.

If the samples were to be corrected for the mean recoveries of the corresponding QC samples, the concentrations analyzed in all groups would be within the target concentration range, except for Group 3 samples in Weeks 4 and 7 (with mean recoveries of 84 and 121%, respectively). As the results on Group 3 are just outside the specifications and vary in direction, either too low or too high, and all other measurements are within specification, it can be assumed that on average the animals have received the target dose. Therefore, these values are indicative of the achieved concentration in the formulations.

Small responses were observed in the low-dose group formulation prepared for use in Week 1, Week 4, Week 7, Week 8 and Week 12. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

Homogeneity

The formulations of the low and high-dose groups were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Table 7. BW result summary for males

Males	Day(s)	1	8	15	22	29	36	43
0 mg/kg bw/day	Mean	188.5	227.3	265.8	293.1	312.7	332.8	351.1
100 mg/kg bw/day	Mean	183.1	219.1	253.3	274.0	293.5	311.1	325.7
300 mg/kg bw/day	Mean	187.2	225.3	259.4	284.3	305.0	322.4	337.2
1000 mg/kg bw/day	Mean	183.7	220.4	257.0	279.3	297.6	310.9	323.1
Males	Day(s)	50	57	64	71	78	85	91
0 mg/kg bw/day	Mean	360.5	377.7	385.2	396.0	406.0	408.5	411.1
100 mg/kg bw/day	Mean	334.0	346.5	354.4	364.0	371.1	374.6	380.5
300 mg/kg bw/day	Mean	347.0	359.3	367.2	377.0	384.4	386.7	393.4
1000 mg/kg bw/day	Mean	329.0	337.9	346.4	356.1	362.5	361.6	365.4

 

Table 8. BW result summary for females

Females	Day(s)	1	8	15	22	29	36	43
0 mg/kg bw/day	Mean	137.3	154.9	175.1	185.3	196.8	207.8	214.2
100 mg/kg bw/day	Mean	140.2	158.2	179.4	189.0	201.3	207.3	217.3
300 mg/kg bw/day	Mean	140.3	162.1	179.5	189.7	202.4	211.5	216.8
1000 mg/kg bw/day	Mean	141.8	161.6	182.0	194.7	205.4	214.2	218.2
Females	Day(s)	50	57	64	71	78	85	91
0 mg/kg bw/day	Mean	216.9	223.2	225.7	229.3	231.3	232.9	234.3
100 mg/kg bw/day	Mean	221.1	227.8	231.2	234.6	237.5	237.4	244.9
300 mg/kg bw/day	Mean	222.7	230.1	233.6	238.8	240.5	240.6	246.9
1000 mg/kg bw/day	Mean	224.3	226.4	232.8	235.4	241.7	241.2	246.1

 

Table 9. BW gain result summary for males

Males	Day(s)	1 → 8	8 → 15	15 → 22	22 → 29	29 → 36	36 → 43	43 → 50
0 mg/kg bw/day	Mean	38.8	38.5	27.3	19.6	20.1	18.3	9.4
100 mg/kg bw/day	Mean	36.0	34.2	20.7	19.5	17.6	14.6	8.3
300 mg/kg bw/day	Mean	38.1	34.1	24.9	20.7	17.4	14.8	9.8
1000 mg/kg bw/day	Mean	36.7	36.6	22.3	18.3	13.3*	12.2**	5.9
Males	Day(s)	50 → 57	57 → 64	64 → 71	71 → 78	78 → 85	85 → 91	1 → 91
0 mg/kg bw/day	Mean	17.2	7.5	10.8	10.0	2.5	2.6	222.6
100 mg/kg bw/day	Mean	12.5*	7.9	9.6	7.1	3.5	5.9	197.4
300 mg/kg bw/day	Mean	13.3	7.9	9.8	7.4	2.2	6.8*	207.0
1000 mg/kg bw/day	Mean	8.9**	8.5	9.7	6.4	-0.9	3.8	181.7**

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤0.001

 

Table 10. BW gain result summary for females

Females	Day(s)	1 → 8	8 → 15	15 → 22	22 → 29	29 → 36	36 → 43	43 → 50
0 mg/kg bw/day	Mean	17.6	20.2	10.2	11.5	11.0	6.4	2.7
100 mg/kg bw/day	Mean	18.0	21.2	9.6	12.3	6.0	10.0	3.8
300 mg/kg bw/day	Mean	21.8	17.4	10.2	12.7	9.1	5.3	5.9
1000 mg/kg bw/day	Mean	19.8	18.8	12.7	10.8	8.8	4.0	6.1
Females	Day(s)	50 → 57	57 → 64	64 → 71	71 → 78	78 → 85	85 → 91	1 → 91
0 mg/kg bw/day	Mean	6.3	2.5	3.6	2.0	1.6	1.4	97.0
100 mg/kg bw/day	Mean	6.7	3.4	3.4	2.9	-0.1	7.5	104.7
300 mg/kg bw/day	Mean	7.4	3.5	5.2	1.7	0.1	6.3	106.6
1000 mg/kg bw/day	Mean	2.1	6.3	2.7	6.2	-0.4	4.9	103.3

[G] - Anova & Dunnett

 

Table 11. Summary of selected Hematology and Coagulation Mean Values

Sex: Male	NEUT (10^9/L) [G1]	MONO (10^9/L) [G]	RDWG (%) [G]	HCT (L/L) [G]	MCH (pg) [G]	MCHC (g/L) [G]	PLT (10^9/L) [G]	PT (sec) [G]	APTT (sec) [G]
Males
0 mg/kg bw/day	0.907	0.107	12.02	0.4476	17.38	329.2	674.9	18.07	18.87
100 mg/kg bw/day	1.032	0.102	11.87	0.4702	17.15	325.8	650.6	17.25	18.99
300 mg/kg bw/day	1.002	0.080	11.29 **	0.4771 *	17.19	325.4	626.1	17.01	17.52
1000 mg/kg bw/day	1.226	0.107	11.29 **	0.4753 *	17.13	322.2 **	586.4 **	17.98	18.77
Females
0 mg/kg bw/day	0.542	0.059	10.85	0.4477	18.41	330.4	605.2	16.74	16.69
100 mg/kg bw/day	0.438	0.051	10.84	0.4515	18.10	325.2	689.9	16.44	16.51
300 mg/kg bw/day	0.577	0.053	10.47	0.4667	17.86	322.6 *	730.5	16.95	19.99 *
1000 mg/kg bw/day	0.980	0.096 *	10.38	0.4539	17.66	321.7 *	630.9	18.42 **	17.73

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Table 12. Test item-Related Clinical Chemistry Changes

Group	2		3		4
Dose (mg/kg bw/day)	100		300		1000
Sex (Number of Animals)	M (10)	F (10)	M (9)	F (10)	M (10)	F (9)
Alanine aminotransferase	0.38**	0.35**	0.43**	0.52**	0.48*	0.46**
Total protein	–	–	–	–	1.05**	1.06
Albumin	–	–	1.06**	–	1.09**	1.07*
Total bilirubin	–	–	–	–	–	0.64**
Urea	–	–	–	–	–	1.34*
Creatinine	–	–	–	–	1.20**	1.20
Glucose	–	0.86*	–	0.89	0.83*	0.73**
Cholesterol	–	–	0.84*	–	0.66**	0.69**
HDL cholesterol	–	–	0.83**	–	0.59**	0.64**
LDL cholesterol	–	0.68**	–	0.79	0.79*	0.77*
Triglycerides	–	0.68*	–	0.76	1.61**	0.77
Chloride	–	–	–	–	–	1.02**

M = Males F = Females
A dash (—) indicates absence of change when compared to control.  Numerical values indicate fold change of the treated group mean value relative to the control group mean value. *= P ≤ 0.05; **=P ≤ 0.01.

Historical control data for clinical chemistry for Wistar Han rats (period 2018-2022):

- Alanine Aminotransferase (U/L). Males: mean = 53.367; P5 – P95 = 34.48 – 80.51 (n=534). Females: mean = 51.103; P5 – P95 = 30.19– 8.76 (n =532)

- Cholesterol (mmol/L). Males: mean = 1.7883; P5 – P95 = 1.28 – 2.34 (n=534)

- HDL Cholesterol (mmol/L). Males: mean = 1.1814; P5 – P95 = 0.82 – 1.59 (n=509)

 

Table 13. Test item-Related Clinical Chemistry Changes

Groups	1	2	3	4	HCD
Dose (mg/kg bw/day)	0	100	300	1000	Mean (P5 - P95)
Males
T3 ng/mL	0.526	0.514 (0.98x)	0.491 (0.93x)	0.325 (0.62x) **	0.386 (0.21 – 0.6) n=521
T4 ng/mL	55.01	40.79 (0.74x) **	32.31 (0.59x) **	9.31 (0.17x) **	45.007 (31.47 – 62.57) n=529
TSH mU/L	0.5963	0.6752 (1.13x)	1.1421 (1.92x)	5.1776 (8.68x) **	0.167 (0.04 – 0.46) n=527
Females
T3 ng/mL	0.707	0.691 (0.98x)	0.744 (1.05x)	0.341 (0.48x) **	0.468 (0.28 – 0.7) n=528
T4 ng/mL	39.44	37.01 (0.94x)	28.50 (0.72x) **	11.85 (0.30x) **	28.092 (16.5 – 44.58) n=528
TSH mU/L	0.1961	0.1896 (0.97x)	0.7935 (4.05x)	10.432 (53.20x) **	0.089 (0.01 – 0.25) n=495

HCD = Historical Control Data (2018-2022), reported as mean values with the confidence interval P5 – P95 between brackets and the number of samples the historical data is based on represented as n.
Absolute values are reported. Numerical values between brackets indicate fold change of the treated group mean value relative to the control group mean value. *= P £ 0.05; **=P £ 0.01

 

Table 14. Summary of Urinalysis values

	VOLUME (mL) [G]	SPECIFIC GRAVITY [G]	URINE pH [G]
Males
0 mg/kg bw/day	10.55	1.0261	6.70
100 mg/kg bw/day	6.60	1.0443	6.20 *
300 mg/kg bw/day	5.22	1.0559 **	5.61 **
1000 mg/kg bw/day	10.80	1.0459	5.30 **
Females
0 mg/kg bw/day	11.60	1.0172	6.45
100 mg/kg bw/day	7.95	1.0249	6.30
300 mg/kg bw/day	5.90	1.0305	6.05 *
1000 mg/kg bw/day	11.39	1.0342 *	5.89 **

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

 

Table 15. Mean Percent Organ Weight Differences from Control Groups

	Males			Females
Dose level (mg/kg bw/day):	100	300	1000	100	300	1000
TERMINAL BODY WEIGHT	-8	-6	-13*	4	5	3
ADRENAL GLAND
Absolute	2	14	10	2	-2	-16
Relative to body weight	10	19*	26**	-3	-7	-18
KIDNEYS
Absolute	10	6	4	9	16**	11*
Relative to body weight	20**	12**	18**	5	11**	8
PITUITARY GLAND
Absolute	-7	-8	1	6	4	1
Relative to body weight	2	-4	16*	2	-1	-1
THYMUS
Absolute	-15	-10	-29*	-10	-20*	-29**
Relative to body weight	-7	-6	-19	-14	-24**	-31**
THYROID GLAND^$
Absolute	-2	23	20	5	21	82**
Relative to body weight	7	31	36*	0	15	78**

*: P≤0.05, **: P≤0.01; ^$ = including parathyroid weight.

 

Table 16. Summary Test Material-Related Macroscopic Findings – Scheduled Euthanasia Animals

	Males				Females
Dose level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Number of Animals	10	10	9	10	10	10	10	9
GLAND, ADRENAL
Submitted	10	10	9	10	10	10	10	9
No Visible Lesions	10	10	9	9	10	10	8	6
Discoloration, pale	0	0	0	1	0	0	2	3
GLAND, THYROID
Submitted	10	10	9	10	10	10	10	9
No Visible Lesions	9	9	6	3	10	10	9	3
Enlargement	1	0	3	6	0	0	1	6
Discoloration, dark; red	0	0	0	1	0	0	0	0
Focus, dark; brown	0	0	0	1	0	0	0	0
Discoloration, pale	0	1	0	0	0	0	0	0
KIDNEY
Submitted	10	10	9	10	10	10	10	9
No Visible Lesions	10	9	8	8	10	6	5	2
Dilatation; pelvis	0	1	1	0	0	0	0	0
Enlargement	0	0	0	1	0	0	0	0
Discoloration, pale	0	0	0	2	0	4	5	7
THYMUS
Submitted	10	10	9	10	10	10	10	9
No Visible Lesions	10	10	8	6	10	10	10	8
Focus, dark; red	0	0	0	1	0	0	0	1
Small	0	0	1	4	0	0	0	0

 

Table 17. Summary Test Material-Related Microscopic Findings – Scheduled Euthanasia Animals

	Males				Females
Dose level (mg/kg bw/day):	0	100	300	1000	0	100	300	1000
ADRENAL GLAND ^(a)	10	10	9	10	10	10	10	9
Vacuolation^*, zona fasciculata/zona reticularis
Minimal	2	8	5	6	0	0	5	4
Mild	0	0	4	4	0	0	0	0
KIDNEYS  ^(a)	10	10	9	10	10	10	10	9
Pigment, tubular
Minimal	0	8	6	3	0	5	0	3
Mild	0	2	2	7	0	5	10	6
Moderate	0	0	1	0	0	0	0	0
Degeneration/regeneration, tubular
Minimal	0	9	6	0	0	1	6	1
Mild	0	1	3	10	0	0	3	8
Infiltration, mononuclear cell, interstitial
Minimal	1	3	1	2	1	0	3	5
Mild	0	0	0	2	0	0	0	1
Moderate	0	0	0	0	0	0	0	1
PITUITARY GLAND  ^(a)	10	10	9	10	10	10	10	9
Hypertrophy, pars distalis
Minimal	0	0	3	6	0	0	0	5
Mild	0	0	0	1	0	0	0	0
STOMACH  ^(a)	10	10	9	10	10	10	10	9
Basophilia, glandular musosa, pyloric antrum
Minimal	0	0	0	9	0	0	3	4
Infiltration, mixed cell, mucosal, glandular
Minimal	0	1	4	7	0	0	2	6
Mild	0	0	0	1	0	0	0	0
SALIVARY GLAND, SUBLINGUAL  ^(a)	10	10	9	10	10	10	10	9
Atrophy
Minimal	0	0	0	3	0	0	0	3
Mild	0	0	0	1	0	0	0	0
THYROID GLAND  ^(a)	10	10	9	10	10	10	10	9
Follicular cell hypertrophy
Minimal	7	6	4	2	1	1	1	1
Mild	0	0	0	3	0	0	0	3
Moderate	0	0	0	3^(b)	0	0	0	4^(b)
Marked	0	0	0	0	0	0	0	1^(b)
Follicular cell Adenoma
Present	0	1	0	0	0	0	0	1
URINARY BLADDER  ^(a)	10	10	8	10	9	10	10	9
Hypertrophy/hyperplasia transitional epithelium
Minimal	0	0	0	1	0	0	0	2
Moderate	0	0	0	0	0	0	0	1

^a  =  Number of tissues examined from each group.

^b  =  Including minimal hyperplasia and decreased size of the follicular lumen.

* =  Vacuolation of the adrenal gland in males in zona fasciculata and reticularis and in females only in the zona reticularis area.

 

Table 18. Findings of note Females – Scheduled Euthanasia Animals

Dose level (mg/kg bw/day):	0	100	300	1000
MAMMARY GLAND ^(a)	10	10	10	9
Hypertrophy/hyperplasia, lobuloalveolar
Minimal	0	1	1	1
Mild	0	1	0	1
VAGINA ^(a)	10	10	10	9
Mucification
Minimal	0	2	1	1
Mild	0	0	0	1

^a  =  Number of tissues examined from each group.

 

Table 19. Distribution of estrous stages in females

Group	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000
Number of females	F (10)	F (10)	F (10)	F (9)
Estrus	2	2	2	-
Metestrus	-	-	1	-
Diestrus	6	6	7	8
Proestrus	2	2	-	1

Conclusions:

In conclusion, following the administration of the test material for at least 90 days to Wistar Han rats, adverse findings included lower body weight gain in males at 1000 mg/kg bw/day, and higher TSH levels and lower T3 and T4 levels in males and females at 1000 mg/kg bw/day. In addition, adverse morphologic alterations were present at 1000 mg/kg bw/day in the thyroid glands and kidneys of males and females and possible in the urinary bladder and urethra of a single female.
These adverse test material-related morphologic alterations consisted of:
• Higher thyroid gland weights, enlargement and increased incidences and severity of follicular cell hypertrophy/hyperplasia, including a possible related adenoma, and related changes in hormone levels (high TSH and low T3, T4).
• The combination of findings in the kidneys, consisting of the presence of large particles of yellow-brown pigment in tubules, tubular degeneration/regeneration at mild degree (except one) with prominent karyomegaly, associated increased incidence and severity of interstitial mononuclear cell infiltration and enlargement.
• Possible test material related diffuse hyperplasia in the urinary bladder and urethra of a single female.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for BIS DMTD of 300 mg/kg bw/day was established.

Executive summary:

The objective of this study was to determine the potential toxicity of BIS DMTD, when given orally by gavage for at least 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.
Chemical analyses of formulations were conducted on Day 1 and Weeks 4, 7, 8 and 12 to assess accuracy and homogeneity. It can be assumed that based on the formulation analyses on average the animals have received the target dose.
The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), macroscopic examination, stage of estrous, organ weights and microscopic examination.
Two animals, one at 300 mg/kg/day and one at 1000 mg/kg/day, were prematurely euthanized due to severe clinical signs. Based on microscopic pathology, both premature deaths were considered to be gavage procedure-related and not related to the treatment with the test material.
At 100 mg/kg/day, clinical pathology changes consisted of lower glucose levels and lower LDL cholesterol and triglyceride concentration in females. In addition, alanine aminotransferase activity was lower in males and females. Thyroid hormone changes consisted of lower T4 level in males. On urinalysis, changes consisted of lower pH and higher specific gravity in the urine of males.
At necropsy, pale discoloration of kidneys in females and higher kidney weight in males were noted. Microscopic changes were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) in males and in the kidney (tubular pigment and tubular degeneration/regeneration) in males and females which was considered to be related to the higher organ weights in males.
All observed findings at 100 mg/kg/day were considered to be non-adverse.
At 300 mg/kg/day, hematological changes consisted of lower red blood cell distribution width in males, and lower mean corpuscular hemoglobin concentration in females. Clinical chemistry changes consisted of lower alanine aminotransferase activity in males and females, higher albumin concentrations in males, lower glucose, LDL cholesterol and triglyceride concentration in females and lower cholesterol and HDL cholesterol concentrations in males.
Thyroid hormone changes consisted of higher TSH, and lower T4 level in males and females.
On urinalysis, changes consisted of lower pH in males and females and a higher specific gravity in males.
Macroscopic findings were noted in the kidneys and adrenal gland (pale discoloration) in females, in the thyroid gland (enlargement) in males and females, and in the thymus (small size) in a single male.
Higher organ weights were present in the kidneys and thyroid gland of males and females, and in the adrenal gland of males. Lower thymus weights were observed in females.
Microscopic findings were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) of males and females correlating with higher adrenal gland weight, pituitary gland (hypertrophy of the pars distalis) of males, kidneys (tubular pigment and tubular degeneration/regeneration) of males and females which related with the higher kidney weights in males and females and stomach (basophilia of the glandular mucosa of the pyloric antrum) of females.
All these parameters were considered to be non-adverse.
At 1000 mg/kg/day, an adverse lower body weight gain was noted in males. Hematological changes consisted of non-adverse higher monocytes count in females, lower red blood cell distribution width and platelet count in males and lower mean corpuscular hemoglobin concentration in males and females. On coagulation, non-adverse longer prothrombin time was noted in females. Clinical chemistry changes consisted of lower alanine aminotransferase activity, glucose and cholesterol (including HDL and LDL) concentrations in males and females and lower total bilirubin concentration in females. In addition, higher total protein, albumin and creatinine concentration in males and females, and higher urea and chloride concentration in females were noted. Furthermore, higher triglyceride levels in males, and lower triglyceride levels in females were noted. These clinical chemistry changes were considered to be non-adverse.
Observed thyroid hormone changes consisted of higher TSH, and lower T3 and T4 levels in males and females, which were considered to be adverse.
On urinalysis, changes consisted of a change in color and a lower pH and higher specific gravity in males and females. These urinalysis findings were considered to be non-adverse.
Non-adverse macroscopic findings at 1000 mg/kg/day, were noted in the kidneys (pale discoloration) in males and females and additionally enlargement in a single male, in the adrenal gland (pale discoloration) in females and in a single male, in the thyroid gland (enlargement) in males and females, and in the thymus (small size) of males.
Organ weight changes consisted of higher kidney and thyroid gland weight in males and females, and in the adrenal gland and pituitary gland of males. Furthermore, a lower thymus weight was observed in males and females. These organ weight changes were considered to be non-adverse.
Non-adverse microscopic findings at 1000 mg/kg/day were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) of males and females with correlating higher adrenal gland weight, pituitary gland (hypertrophy of the pars distalis) of males and females with correlating higher pituitary gland weights, stomach (basophilia of the glandular mucosa of the pyloric antrum) of males and females which was associated with an increased incidence and/or severity of mixed cell infiltration of the glandular mucosa in both sexes and the sublingual salivary gland (atrophy) in males and females.
An adverse microscopic finding was present in the thyroid gland in the form of increased incidence and severity of follicular cell hypertrophy in males and females, as well as the presence of hyperplasia in the animals with the highest severity where the follicular lumens decreased in size, and a possibly related follicular cell adenoma, a benign tumor, in a single female, for which a relation with the test material could not be fully excluded. In addition, relationships were present between the follicular cell hypertrophy, higher organ weights, high TSH and low T3 and T4 levels and these findings were considered adverse.
An adverse microscopic finding was present in the kidney in the form of yellow-brown tubular pigment and tubular degeneration/regeneration in males and females and were considered to be related to the higher kidney weights and enlargement in a single male. The pigment was more prominent intracellular present in the same tubules which also showed degeneration and karyomegaly and was associated with an increased incidence and severity of interstitial mononuclear cell infiltration.
Lastly, a microscopic finding was present in the urinary bladder consisting of diffuse hypertrophy/hyperplasia of the transitional epithelium in males and females at minimal degree (considered non-adverse) and in one female at moderate degree (adverse). This latter animal also showed marked hyperplasia of the urethra, which was considered adverse and possibly test material-related.
In conclusion, following the administration of the test material for at least 90 days to Wistar Han rats, adverse findings included lower body weight gain in males at 1000 mg/kg/day, and higher TSH levels and lower T3 and T4 levels in males and females at 1000 mg/kg/day. In addition, adverse morphologic alterations were present at 1000 mg/kg/day in the thyroid glands and kidneys of males and females and possible in the urinary bladder and urethra of a single female.
These adverse test material-related morphologic alterations consisted of:
• Higher thyroid gland weights, enlargement and increased incidences and severity of follicular cell hypertrophy/hyperplasia, including a possible related adenoma, and related changes in hormone levels (high TSH and low T3, T4).
• The combination of findings in the kidneys, consisting of the presence of large particles of yellow-brown pigment in tubules, tubular degeneration/regeneration at mild degree (except one) with prominent karyomegaly, associated increased incidence and severity of interstitial mononuclear cell infiltration and enlargement.
• Possible test material related diffuse hyperplasia in the urinary bladder and urethra of a single female.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for BIS DMTD of 300 mg/kg/day was established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is considered as reliable with a klimish score of 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Combined 28 -day repeated toxicity and reproduction screening study (Envigo, 2016) :

The test item was administered by gavage to four groups, each of twelve male and twelve female Wistar rats, for up to eight weeks, including a two week prepairing phase, pairing, gestation and early lactation (Days 1 through 4 post partum) for females, at dose levels of 30, 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

One female at 1000 mg/kg bw/day was killed prior to dosing on Day 40 due to adverse clinical signs including piloerection, lethargy, hunched posture, ptosis, decreased respiration rate and ataxia; piloerection had been observed on the previous day. Necropsy revealed enlarged adrenals, eleven dead foetuses in the uterus and one dead foetus in the vagina. While this death appears to be related to difficulties associated with parturition, an association with treatment cannot be discounted.

Treatment was associated with an increased post-dosing salivation at 100, 300 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day, one male showed piloerection and hunched posture on Day 44 and similar clinical signs were occasionally observed for four females (including the decedent) from Day 39. There were no treatment-related changes in the behavioural parameters, no changes in functional performance for either sex and no changes from sensory reactivity assessments of females with litters at Day 4 post partum.

At 1000 mg/kg bw/day, lower body weight gain and low food consumption were apparent for males. For females at this dosage lower body weight gain and lower food consumption were apparent during gestation and lactation but this was based on a small group size (6 and 3 females respectively). There was no clear effect of treatment on body weight gain and food consumption for either sex at 30, 100 or 300 mg/kg bw/day.

There was no effect of treatment on water consumption for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

There was no effect of treatment on hematology parameters for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

At all dosages, mean alanine aminotransferase levels were statistically significantly lower than control for both sexes. For males at 1000 mg/kg bw/day, this decrease was accompanied by a statistically significantly lower level of alkaline phosphatase. For males at 100 and 300 mg/kg bw/day and both sexes at 1000 mg/kg bw/day, lower mean total cholesterol was statistically significantly lower than control. For males at 1000 mg/kg bw/day, mean total protein and albumin levels were statistically significantly lower than control and were accompanied by a statistically significant increase in albumin/globulin ratio. For females at 1000 mg/kg bw/day, higher levels of inorganic phosphorus and creatinine and lower levels of potassium and billirubin attained statistical significance when compared with control.

Macroscopic necropsy examination did not indicate any obvious effect of treatment for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

For males at all dosages, absolute and body weight relative pituitary weights were statistically significantly lower than control. For males at 1000 mg/kg bw/day, absolute and body weight relative thymus weights were statistically significantly lower than control. For females at 300 and 1000 mg/kg bw/day, absolute and body weight relative liver and kidney weights were statistically significantly higher than control. Additionally for females at 1000 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative thyroid weights.

Changes were noted in the adrenal glands of males, kidneys, pituitary, thyroid glands and livers of both sexes when treated with 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione at 1000 mg/kg bw/day. Changes were also apparent in the spleens of females, lymph nodes, thymus and

salivary gland of both sexes at this dose, which are considered likely to be secondary. When 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was administered at 300 mg/kg bw/day changes were noted in the pituitary glands of males, thyroid glands and kidneys of both sexes.

Deposition of yellow/brown pigment in the tubular cells of the kidneys of animals treated with 30 and 100 mg/kg bw/day of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was considered unlikely to be of toxicological importance, within the confines of this study, as no associated pathology was present.

Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day. A No Observed Effect Level (NOEL) for toxicity was not established due to lower levels of alanine aminotransferase levels and deposition of yellow/ brown pigment in the tubular cells of kidneys in all animals treated with 5,5’-Dithiodi-1,3,4 -thiadiazole-2(3H)-thione, despite the absence of histopathological lesions at 30 and 100 mg/kg bw/day.

 

 

90-day repeated dose toxicity study via the oral route (CRL, 2023):
The objective of this study was to determine the potential toxicity of BIS DMTD, when given orally by gavage for at least 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.
Chemical analyses of formulations were conducted on Day 1 and Weeks 4, 7, 8 and 12 to assess accuracy and homogeneity. It can be assumed that based on the formulation analyses on average the animals have received the target dose.
The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), macroscopic examination, stage of estrous, organ weights and microscopic examination.
Two animals, one at 300 mg/kg/day and one at 1000 mg/kg/day, were prematurely euthanized due to severe clinical signs. Based on microscopic pathology, both premature deaths were considered to be gavage procedure-related and not related to the treatment with the test material.
At 100 mg/kg/day, clinical pathology changes consisted of lower glucose levels and lower LDL cholesterol and triglyceride concentration in females. In addition, alanine aminotransferase activity was lower in males and females. Thyroid hormone changes consisted of lower T4 level in males. On urinalysis, changes consisted of lower pH and higher specific gravity in the urine of males.
At necropsy, pale discoloration of kidneys in females and higher kidney weight in males were noted. Microscopic changes were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) in males and in the kidney (tubular pigment and tubular degeneration/regeneration) in males and females which was considered to be related to the higher organ weights in males.
All observed findings at 100 mg/kg/day were considered to be non-adverse.
At 300 mg/kg/day, hematological changes consisted of lower red blood cell distribution width in males, and lower mean corpuscular hemoglobin concentration in females. Clinical chemistry changes consisted of lower alanine aminotransferase activity in males and females, higher albumin concentrations in males, lower glucose, LDL cholesterol and triglyceride concentration in females and lower cholesterol and HDL cholesterol concentrations in males.
Thyroid hormone changes consisted of higher TSH, and lower T4 level in males and females.
On urinalysis, changes consisted of lower pH in males and females and a higher specific gravity in males.
Macroscopic findings were noted in the kidneys and adrenal gland (pale discoloration) in females, in the thyroid gland (enlargement) in males and females, and in the thymus (small size) in a single male.
Higher organ weights were present in the kidneys and thyroid gland of males and females, and in the adrenal gland of males. Lower thymus weights were observed in females.
Microscopic findings were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) of males and females correlating with higher adrenal gland weight, pituitary gland (hypertrophy of the pars distalis) of males, kidneys (tubular pigment and tubular degeneration/regeneration) of males and females which related with the higher kidney weights in males and females and stomach (basophilia of the glandular mucosa of the pyloric antrum) of females.
All these parameters were considered to be non-adverse.
At 1000 mg/kg/day, an adverse lower body weight gain was noted in males. Hematological changes consisted of non-adverse higher monocytes count in females, lower red blood cell distribution width and platelet count in males and lower mean corpuscular hemoglobin concentration in males and females. On coagulation, non-adverse longer prothrombin time was noted in females. Clinical chemistry changes consisted of lower alanine aminotransferase activity, glucose and cholesterol (including HDL and LDL) concentrations in males and females and lower total bilirubin concentration in females. In addition, higher total protein, albumin and creatinine concentration in males and females, and higher urea and chloride concentration in females were noted. Furthermore, higher triglyceride levels in males, and lower triglyceride levels in females were noted. These clinical chemistry changes were considered to be non-adverse.
Observed thyroid hormone changes consisted of higher TSH, and lower T3 and T4 levels in males and females, which were considered to be adverse.
On urinalysis, changes consisted of a change in color and a lower pH and higher specific gravity in males and females. These urinalysis findings were considered to be non-adverse.
Non-adverse macroscopic findings at 1000 mg/kg/day, were noted in the kidneys (pale discoloration) in males and females and additionally enlargement in a single male, in the adrenal gland (pale discoloration) in females and in a single male, in the thyroid gland (enlargement) in males and females, and in the thymus (small size) of males.
Organ weight changes consisted of higher kidney and thyroid gland weight in males and females, and in the adrenal gland and pituitary gland of males. Furthermore, a lower thymus weight was observed in males and females. These organ weight changes were considered to be non-adverse.
Non-adverse microscopic findings at 1000 mg/kg/day were present in the adrenal gland (increased incidence and/or severity of vacuolation of the zona fasciculata and/or reticularis) of males and females with correlating higher adrenal gland weight, pituitary gland (hypertrophy of the pars distalis) of males and females with correlating higher pituitary gland weights, stomach (basophilia of the glandular mucosa of the pyloric antrum) of males and females which was associated with an increased incidence and/or severity of mixed cell infiltration of the glandular mucosa in both sexes and the sublingual salivary gland (atrophy) in males and females.
An adverse microscopic finding was present in the thyroid gland in the form of increased incidence and severity of follicular cell hypertrophy in males and females, as well as the presence of hyperplasia in the animals with the highest severity where the follicular lumens decreased in size, and a possibly related follicular cell adenoma, a benign tumor, in a single female, for which a relation with the test material could not be fully excluded. In addition, relationships were present between the follicular cell hypertrophy, higher organ weights, high TSH and low T3 and T4 levels and these findings were considered adverse.
An adverse microscopic finding was present in the kidney in the form of yellow-brown tubular pigment and tubular degeneration/regeneration in males and females and were considered to be related to the higher kidney weights and enlargement in a single male. The pigment was more prominent intracellular present in the same tubules which also showed degeneration and karyomegaly and was associated with an increased incidence and severity of interstitial mononuclear cell infiltration.
Lastly, a microscopic finding was present in the urinary bladder consisting of diffuse hypertrophy/hyperplasia of the transitional epithelium in males and females at minimal degree (considered non-adverse) and in one female at moderate degree (adverse). This latter animal also showed marked hyperplasia of the urethra, which was considered adverse and possibly test material-related.
In conclusion, following the administration of the test material for at least 90 days to Wistar Han rats, adverse findings included lower body weight gain in males at 1000 mg/kg/day, and higher TSH levels and lower T3 and T4 levels in males and females at 1000 mg/kg/day. In addition, adverse morphologic alterations were present at 1000 mg/kg/day in the thyroid glands and kidneys of males and females and possible in the urinary bladder and urethra of a single female.
These adverse test material-related morphologic alterations consisted of:
• Higher thyroid gland weights, enlargement and increased incidences and severity of follicular cell hypertrophy/hyperplasia, including a possible related adenoma, and related changes in hormone levels (high TSH and low T3, T4).
• The combination of findings in the kidneys, consisting of the presence of large particles of yellow-brown pigment in tubules, tubular degeneration/regeneration at mild degree (except one) with prominent karyomegaly, associated increased incidence and severity of interstitial mononuclear cell infiltration and enlargement.
• Possible test material related diffuse hyperplasia in the urinary bladder and urethra of a single female.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for BIS DMTD of 300 mg/kg/day was established.

Justification for classification or non-classification

Based on the available, no classification for repeated toxicity is required for 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione according to the Regulation EC n°1272/2008.