4,4'-methylene bis(dibutyldithiocarbamate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2019 to 16 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2019 to 28 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Range-Finding Toxicity Study in the Rat, to inform dose selection in an OECD 408 and 414 study.

Version / remarks:

To bridge from a rat dietary OECD 422 study

Deviations:

no

Principles of method if other than guideline:

This non-GLP study was conducted in accordance with Good Laboratory Practice principles.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Test item: 4,4’-methylene bis(dibutyldithiocarbamate)
CAS number: 10254-57-6
Appearance: Amber-green liquid.
Storage conditions: Controlled ambient temperature (15 to 25°C), in the dark

Species:

rat

Strain:

other: Han-Wistar [RccHan™:WIST ]

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxicity in humans and the requirement for a rodent species by regulatory agencies. The Han Wistar [RccHan™:WIST ] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: 56 to 63 days
- Weight at study initiation:
Males: 184 to 219 g
Females: 133 to 168 g
- Fasting period before study: No data
- Housing: Polycarbonatebody cages, with a stainless steel mesh lid
- Diet (e.g. ad libitum): Non-restricted
- Water (e.g. ad libitum): Non-restricted
- Acclimation period: 14 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

There was limited access to the animal facility to minimise entry of external biological and chemical agents and to minimise the transfer of such agents between rooms.

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The required amount of test item for 200 mg/mL was weighed out into a suitable container. Approximately 50% of the final volume of vehicle was added to the test item and mixed with a magnetic stirrer until uniformly mixed. The solution was then made up to the required volume with vehicle. The formulation was transferred to a final container and mixed with a magnetic stirrer until homogenous.

A series of formulations at the required concentrations (66 and 20 mg/mL) were prepared by dilution of individual weighings of the test item.


Frequency of preparation: Weekly

Storage of formulation: Refrigerated (2 to 8°C) for up to eight days

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

330 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3 Males; 3 Females

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected following consultation between the Study Director and the Study Monitor for the Sponsor, using a dietary combined repeat dose toxicity study with reproduction/ developmental screening test in the rat (SafePharm Laboratories Study, 2006).

Observations and examinations performed and frequency:

Detailed observations were recorded daily at the following times in relation to dose administration:
- Pre-dose
- As each animal was returned to its home cage
- At the end of the dosing all groups
- One to two hours after completion of dosing
- As late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Three days before treatment commenced, on the day that treatment commenced (Day-1), twice weekly during the treatment period, and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and estimate of any spilled was recorded for the three days before treatment and twice weekly during the treatment period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily by visual observation.

Sacrifice and pathology:

TERMINATION
- Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in
the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Statistics:

No statistical analysis of the data was performed on this study.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Isolated clinical signs of chin rubbing were observed on one occasion in one male from all treated groups and one female receiving 330 mg/kg/day. Excessive salivation was observed in one male receiving 1000 mg/kg/day.

No other clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled sacrifices.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in body weight or body weight change that were considered to be treatment related.

Group mean body weight change was lower at the end of treatment in females receiving 1000 mg/kg/day when compared with controls. However, this was due to one animal, who only gained 13 g over the treatment period, whereas the remaining animals in the group gained a similar weight to controls. Group mean body weight change was also lower at the end of treatment in males receiving 100 or 330 mg/kg/day when compared with controls. However, due to the lack of a dose relationship, the large variation in all treated groups (not seen in controls), some of which were similar or higher than controls, these were considered not to be related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The daily visual assessment of water intake did not reveal any treatment related changes in water consumption.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences in any organ weight that could be positively attributed to treatment. The absolute and adjusted (relative to body weight) organ weights were within the historical control data range of this laboratory.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings observed in the necropsy at termination of the study.

Details on results:

Clinical signs of chin rubbing and salivation observed in some animals were considered to be caused by the administration method and palatability of the test item, whereby the animals were able to taste the formulation.

Differences in mean body weight change at the end of treatment were considered to be incidental due to the small group sizes and short duration of the study.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Critical effects observed:

not specified

Conclusions:

Based on the results of this study, the test material was well tolerated in Han Wistar rats at oral doses up to 1000 mg/kg/day.

Executive summary:

Daily administration of the test item by oral gavage to Han Wistar rats for 14 days was well tolerated at dose levels up to 1000 mg/kg/day with no deaths and no effects seen on clinical observations, body weight, food consumption, water consumption, organ weights or macropathology.

Clinical signs of chin rubbing and salivation observed in some animals were considered to be caused by the administration method and palatability of the test item, whereby the animals were able to taste the formulation.

Differences in mean body weight change at the end of treatment were considered to be incidental due to the small group sizes and short duration of the study.

Therefore, it was concluded that 1000 mg/kg/day would be a suitable high dose level in the subsequent longer term toxicity studies in rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylene bis(dibutyldithiocarbamate)

EC Number:

233-593-1

EC Name:

4,4'-methylene bis(dibutyldithiocarbamate)

Cas Number:

10254-57-6

Molecular formula:

C19H38N2S4

IUPAC Name:

N,N-dibutyl({[(dibutylcarbamothioyl)sulfanyl]methyl}sulfanyl)carbothioamide

Test material form:

liquid

Specific details on test material used for the study:

Test material: 4,4’-methylene bis(dibutyldithiocarbamate)
CAS number: 10254-57-6
Appearance: Amber-green liquid.
Storage conditions: Controlled ambient temperature (15 to 25°C), in the dark

Test animals

Species:

rat

Strain:

other: RccHan™:WIST

Details on species / strain selection:

The rat was chosen as the test species because it is accepted by regulatory agencies. The Han Wistar [RccHan™:WIST] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Females (if applicable) nulliparous and non-pregnant: Based upon the age of the animals at receipt (young and just approaching sexual maturity), and segregation of the sexes by the breeder, the females were nulliparous and non-pregnant, even though this is not stated specifically in the report. The husbandry and clinical observation data add support to this statement.
- Age at study initiation: 43 to 49 days
- Weight at study initiation:

Males: 135 to 183 g
Females: 117 to 152 g

- Housing: Polycarbonatebody cages, with a stainless steel mesh lid
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated

- Photoperiod (hrs dark / hrs light):
Artificial lighting, 12 hours light: 12 hours dark

There was limited access to the animal facility to minimise entry of external biological and chemical agents and to minimise the transfer of such agents between rooms.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered by gavage using a rubber catheter attached to a suitably graduated syringe.

Vehicle:

corn oil

Details on oral exposure:

The test material was administered to 10 animals/sex/group by oral gavage once daily at approximately the same time for 13 weeks. Doses of 100, 330 or 1000 mg/kg/day were given in a volume of 5 mL/kg body weight. A similarly constituted control group received the corn oil vehicle at the same volume dose as treated groups. Individual dose volumes were calculated from the most recently recorded body weight. Dose selection was based upon the results of a two week repeat dose oral gavage study in which 3 animals/sex/group were dosed with the test material in corn oil at 100, 330 and 1000 mg/kg/day with no toxicologically significant findings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulations prepared for administration in Weeks 1, 4 and 13 of treatment were analyzed for achieved concentration of the test material by HPLC using UV detection. The analytical procedure was validated for the test material in corn oil with respect to the specificity, LOD, LOQ, linearity, repeatability, calibration standard and stability, accuracy, and precision. The mean test material concentrations were within 7% of the nominal concentrations; the difference from mean remained within 3%, confirming precise analysis.

The homogeneity and stability were confirmed with respect to the level of concentration of the test item in corn oil formulations at nominal concentrations of 10 mg/mL and 220 mg/mL. Storage stability was confirmed at ambient temperature (15 to 25ºC) for up to 1 day and following refrigeration (2 to 8°C) for up to 15 days following fresh preparation.

Duration of treatment / exposure:

13 weeks (at least 90 days)

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 Males; 10 Females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

During the acclimatization period, observations of the animals were recorded at least once per day.

Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Any deviation from normal was recorded in respect of nature and severity, date and time of onset, duration, and progress of the observed condition.


Detailed observations were also recorded at the following times in relation to dose administration, at a minimum:

Detailed Physical Examination and Arena Observations:
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.


Sensory Reactivity and Grip Strength:
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment by an observer who was unaware of the treatment group.


Motor Activity:
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity, respectively.


Body Weight:
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced, weekly throughout the study, and before necropsy. More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.


Food Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.


Water Consumption:
Fluid intake was assessed by daily visual observation.


Ophthalmic Examination:
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope after the pupils were dilated.

All animals (including spares) were examined pretreatment; all animals of Groups 1 and 4 were examined during week 12.

The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.


Hematology, Peripheral Blood:
Blood samples were collected from all animals during week 13 after overnight withdrawal of food and prior to dosing at the following occasion:

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics:

Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

Additional blood samples (nominally 0.6 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:

Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.
Fibrinogen concentration (Fib) - using IL PT Fibrinogen reagent.


Blood Chemistry:
Blood samples were collected from all study animals during week 13 after overnight withdrawal of food and prior to dosing at the following occasion.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Bile acid (Bi Ac)
Urea
Blood urea nitrogen (BUN)
Creatinine (Creat)
Glucose (Gluc)
Cholesterol - total (Chol)
Cholesterol - high density level (HDL)
Cholesterol - low density level (LDL)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.


Thyroid Hormone Analysis:
Blood samples were collected from all study animals during week 13 after overnight withdrawal of food and prior to dosing for analysis of Triiodothyronine (T3), Thyroxine (T4), and Thyroid stimulating hormone (TSH).

To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.
Blood sample site Sublingual vein.
Anesthetic Isoflurane.
Anticoagulant None.
Blood volume 1.0 mL.
Tubes Greiner Minicollect - with clot activator.

Samples were kept at ambient temperature (15 to 25 deg. C) for a minimum of 30 minutes prior to centrifugation at 2000 g for 10 minutes at 4 deg. C. All available serum was transferred to an appropriately labelled polypropylene tube and was placed on dry ice following collection, prior to storage at -60 deg. C to -90deg. C. Samples were then dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance for analysis.


Vaginal Smears:
Wet smears were taken using pipette lavage for four days before scheduled termination and assessed to establish the stage of estrus.

Sacrifice and pathology:

Method of Kill:
Carbon dioxide asphyxiation with subsequent exsanguination.


Necropsy:
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

- Schedule Animals were killed following 13 weeks of treatment.
- Sequence To allow satisfactory inter-group comparison.

The following organs were weighed, if appropriate, and prepared for evaluation:

Adrenals
Aorta - thoracic
Bone marrow smear
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur with femorotibial joint (and bone marrow)
Harderian glands
Head
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands - submandibular
- parotid
- sublingual
Sciatic nerves
Seminal vesicles
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum and marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina

Statistics:

All statistical analyses were carried out separately for males and females. For all parameters, the analyses were carried out using the individual animal as the basic experimental unit.

The following data types were analyzed at each timepoint separately:

- Grip strength and motor activity
- Body weight, using gains over appropriate study periods
- Hematology
- Blood chemistry
- Organ weights, absolute and adjusted for terminal body weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs that were considered to be treatment related.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

All animals gained body weight throughout the treatment period. When compared with the control group, the mean body weight and body weight gains were statistically significantly lower at 89%, 81% and 86%, between weeks 1-13, in females receiving 100, 330, or 1000 mg/kg/day, respectively, of the test material, but were mild, lacked a dose- response relationship, occurred in the absence of food consumption changes, and were considered non-adverse. Body weight and body weight change were unaffected in males.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No significant effect was observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmoscopy findings were considered to be treatment related. On female in the 100 mg/kg/day group was observed with slight, superficial opacities pre-treatment. During week 12 of treatment faint, superficial opacities were observed in one control male, one control female, 4 high dose males and one high dose female.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were some statistically significant differences such as; hematocrit levels were high in males receiving 1000 mg/kg/day and low in females receiving 330 or 1000 mg/kg/day, haemoglobin levels were low in females receiving 330 or 1000 mg/kg/day, red blood cell count was low in females receiving 1000 mg/kg/day, red blood cell distribution width was low in all male treated groups, prothrombin time was slightly elongated in all male treated groups and in females receiving 1000 mg/kg/day, activated partial thromboplastin time was shorter in males receiving 100 mg/kg/day and males and females receiving 1000 mg/kg/day, when compared with controls, however, due to the small magnitude of change and the lack of any correlating histopathological changes these were considered not to be toxicologically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were some statistically significant differences such as; alkaline phosphatase was low in all male treated groups and in females receiving 330 or 1000 mg/kg/day, alanine aminotransferase was low in all female treated groups and in males receiving 330 or 1000 mg/kg/day, bile acid was low in all female treated groups, creatinine was high in females receiving 1000 mg/kg/day, glucose was low in males receiving 1000 mg/kg/day, potassium was low in females receiving 330 or 1000 mg/kg/day, cholesterol and low density lipoproteins were high in males receiving 1000 mg/kg/day and albumin/globulin ratio was low in all male treated groups, however, due to the small magnitude of change and the lack of any correlating histopathological changes these were considered not to be toxicologically significant. Serum total T3, T4 and TSH were measured on samples obtained from each animal during week 13 of treatment. There were no difference in thyroid hormone levels that were considered to be treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in any sensory reactivity observation that could be positively attributed to treatment with the test material. Fore grip strength was statistically significantly lower in 1000 mg/kg/day females, but was considered to be incidental to treatment due to 7 or 10 individual values (7/10) being within the control group range and also being within the historical control range. There were some isolated incidences of statistical significance in motor activity, however, due to lack of consistency and no dose response relationship these were considered incidental to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight adjusted liver weight was high in all treated groups when compared with controls, with statistical significance being achieved in males at 330 and 1000 mg/kg/day and all female treated groups. Absolute liver weight was also high in animals receiving 1000 mg/kg/day and males receiving 330 mg/kg/day when compared with controls.

Body weight adjusted thyroid and parathyroid weight was statistically significantly high in males receiving 330 or 1000 mg/kg/day when compared with controls. Absolute thyroid and parathyroid weight was also slightly high in males receiving 330 or 1000 mg/kg/day when compared with controls.

The organ weight changes were mild, lacked any correlation with histopathological findings, and therefore were considered non-adverse.

All other inter-group differences from control, including those attaining statistical significance in body weight adjusted pituitary, adrenal, and kidney weights in females, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after 13 weeks of treatment revealed no test material related lesions.

The incidence and distribution of all findings were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyaline droplet accumulation in the proximal convoluted tubules of the kidney was noted in males treated with 1000 mg/kg/day.

Hyaline droplet accumulation is frequently seen in male rats treated with xenobiotics and is usually indicative of α2u globulin nephropathy. This is generally considered a phenomenon specific to male rats and of no significance to humans (Alden, 1986). In the absence of any associated degenerative changes, and given the low severity of the finding, hyaline droplet accumulation as seen in this study is considered non-adverse.

All other histological changes were considered to be unrelated to treatment.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal smears were taken from females before termination for assessment of the estrus stage. With the exception of two females receiving 330 mg/kg/day, all females showed estrus at termination.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day.

Executive summary:

Daily administration of the test material by oral gavage to Han Wistar rats for 13 weeks resulted in low body weight gain over the treatment period in females receiving 330 or 1000 mg/kg/day. Changes in alkaline phosphatase, alanine aminotransferase, bile acid, creatinine, glucose, cholesterol, low density lipoproteins and albumin/globulin ratio were considered treatment related, and the slightly higher body weight adjusted liver, thyroid and parathyroid weights were considered to be treatment related, but none of these findings were considered toxicologically significant.

Accumulation of hyaline droplets in the proximal convoluted tubules of the kidney was observed in males treated with 1000 mg/kg/day, a phenomenon specific to male rats that is not relevant to humans, and therefore, is considered non-adverse.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day.