Registration Dossier

Administrative data

Description of key information

The NOAEL for general, systemic toxicity of the test substance was 10 mg/kg bw/d for male and female based on a sub-chronic repeated dose study according to OECD 408.

Key value for chemical safety assessment

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:
15 Dec 2016 to 17 mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 August 2001
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of the test material (as cited in study report): Zink bis(diethyldithiocarbamate)
- Batch No.: 60616004
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018
- Purity: ≥ 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature

OTHER SPECIFICS:
- Appearance: White powder
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. This rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The animals were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 157- 192 g (mean 177 g) for males and 124- 150 g (mean 135 g) for females.
- Housing: The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex. On the day of FOB testing and motor activity assessment, the animals were temporarily kept singly in macrolon cages. During urine collection, animals were kept singly in metabolism cages.
- Diet: Food was provided ad libitum from the arrival of the rats until the end of the study unless precluded by the collection of concentrated urine in week 13, or the collection of blood from overnight fasted rats prior to scheduled necropsy. From their arrival until the end of the study, the rats received a powdered, cereal-based rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England; batch 2619 and, from 2 February 2017, batch 2771). The food was provided to the rats as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. During the study, the food in the cans was replaced by fresh portions from the freezer weekly and filled up as needed.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study unless precluded by the collection of concentrated urine in week 13, or the collection of blood from overnight fasted rats prior to scheduled necropsy. Each cage was supplied with domestic mains tap-water. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Each batch of food is analysed by the supplier for nutrients and contaminants. The tap-water was suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables.

ENVIRONMENTAL CONDITIONS
From their arrival, the animals were housed under conventional, controlled conditions in one room. No other test system was housed in the same room during the study.
- Temperature (°C): 20 - 24
- Relative humidity (%): 45-65 (except during brief periods associated with room cleaning when the relative humidity reached maxima up to 78%). The relative humidity dropped below the lower limit on 1 December 2016 (min. 41%) and 17 January 2017 (min. 36%) during periods lasting an hour or less. This deviation from the study plan is considered not to have affected the validity of the study.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route was used because this is a possible route of human exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For each day of the study and for each dosing group the appropriate amount of test substance was weighed in a vial. The vials were stored at ambient temperature. Each day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension were stirred on a magnetic stirrer until visual homogeneity was obtained. During the dosing procedure, all suspensions were continuously stirred in order to maintain the homogeneity of the test substance in the vehicle. To maintain a constant dose level in terms of the animal’s body weight the dose volumes were adjusted weekly to for each individual rat.

VEHICLE
- Concentration in vehicle: 0-25 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
- Lot/batch no.: A1600985
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in the gavage liquid was conducted using ICP-MS. Samples were taken under continuous stirring (similar to the dosing procedure) in duplicate, because the suspensions in oil separated if not stirred. The following analyses were conducted:
- Stability: The presence of the test substance in the test dilutions was determined by analysis of the Zink content. Therefore it was not possible to determine the stability of the test substance under experimental conditions. Instead, fresh test dilutions were prepared daily.
- Homogeneity: The homogeneity (and content) of the test substance in the test dilutions was assessed in the batch prepared on 15 December 2016, by analyzing three samples (taken at top-, mid- and bottom- of the vial) of each test dilution in duplicate.
- Content: The content of the test substance in each test dilution was determined in five batches of test dilutions, viz. the batches prepared on 15 December 2016, and on 10 and 31
January, 14 February and 7 March 2017.
Duration of treatment / exposure:
13 consecutive weeks
Frequency of treatment:
Once daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Highest dose: First 34 days of the study
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Highest dose: Reduced high dose level from study day 35 onwards, mortality was observed at 250 mg/kg bw/day.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: These dose levels were selected based on the results of a 14 day dose range-finding study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to prevent loss of tissues by cannibalism or autolytic degeneration. All abnormalities, signs of ill health or reactions to treatment were recorded. Animals showing signs of severe debility or intoxication, particularly if death appeared imminent, were humanely killed.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical examinations (in an arena outside the home cage) were performed on all rats prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT
The body weight of each animal was recorded once during the acclimatization period (day -1), at initiation of treatment (day 0), and once per week thereafter. On day 5 and day 8, additional individual body weight measurements were conducted to monitor the health status of the rats. The animals were weighed prior to scheduled necropsy in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage by weighing the feeders. The consumption was measured over weekly periods of for all animals in the cage. The results were expressed in g per animal per day.

WATER CONSUMPTION
Water consumption was measured per cage, by daily weighing the drinking bottles, during 5-day periods in weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopy observations were made prior to the start of treatment (on day -8 and -7 for males and females, respectively) in all rats, and in the last week of the treatment period (on day 88 and 89 for males and females, respectively) in all rats of the control group, the middose group and the high-dose group. Because no treatment-related ocular changes were observed in the higher-dose groups, eye examination was not extended to the animals of the low-dose group at the end of the study. Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulfate.

HAEMATOLOGY
- Time schedule for collection of blood: At necropsy
- Anesthetic used for blood collection: Yes (iCO2/O2)
- Animals fasted: Yes
- How many animals: All surviving rats
- Parameters examined: hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells [Lymphocytes (Lympho), neutrophils (Neutro), eosinophils (Eosino), basophils (Baso) and monocytes (Mono)], prothrombin time and thrombocytes.
- Parameters calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes (water was freely available)
- How many animals: All surviving rats
- Parameters examined: alkaline phosphatase activity (ALP), bilirubin (total),aspartate aminotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin (calculated), potassium (K),urea, chloride (Cl), creatinine, inorganic phosphate (PO4) and (fasting) glucose.

URINALYSIS
- Time schedule for collection of urine: In week 13 (day 84-85 and 88-89 for males and females, respectively)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water for 24 hours and of food during the last 16 hours)
- Parameters examined: volume (To investigate the concentrating ability of the kidneys), occult blood, specific gravity*, ketones, appearance (color and clarity), protein, pH, bilirubin, glucose, urobilinogen, microscopy of the urinary sediment (Red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, worm eggs, sperm cells.)

NEUROBEHAVIOURAL EXAMINATION
Behavioral endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study (males in week 11, females in week 12).
Sacrifice and pathology:
GROSS NECROPSY
Early in week 14, after overnight fasting (water was freely available), the animals were killed on two successive working days (day 91 and 92 for males and females, respectively) in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anesthesia and then examined grossly for pathological changes. A thorough necropsy was also performed on animals that died intercurrently (if not precluded by cannibalism or autolysis) or that had to be killed because they were moribund.

ORGAN WEIGHTS
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated on the basis of the terminal body weight of the animals: adrenals, prostate, brain, seminal vesicles (with coagulating glands), epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroids, ovaries and uterus.

TISSUE PRESERVATION
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde: adrenals, oviducts (=fallopian tubes), aorta, pancreas, axillary lymph, nodes parathyroid, brain(1), parotid salivary glands, cecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles + coagulating glands, esophagus, skeletal muscle (thigh), exorbital lachrymal glands*, skin (flank), eyes, spinal cord(2), femur with joint*, spleen, GALT (gut associated lymphoid tissue including Peyer's patches), sternum with bone marrow, stomach(3) heart, sublingual salivary glands, ileum, submaxillary salivary glands, jejunum, testes, kidneys, thymus, liver, thyroid, lungs, trachea/bronchi, mammary gland (females), urinary bladder, mandibular (cervical) lymph nodes*, uterus (with cervix), mesenteric lymph nodes, vagina, nerve-peripheral (sciatic), all gross lesions and ovaries. The carcass containing any remaining tissues was also retained in formalin, but discarded after completion of the histopathological examination
* Preserved but not processed for histopathological examination, unless histopathological examination was considered necessary on the basis of gross observations.
1) Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
2) Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
3) Non glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.

HISTOPATHOLOGICAL EXAMINATION
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs examined (except for the brain, spinal cord and stomach) of all animals of the control group and the high-dose group and of the animals of the intermediate-dose groups that died intercurrently. Histopathological examination of thymus, spleen, pancreas and heart was extended to the rats of the low- and mid-dose groups. Gross lesions were examined in rats of all dose groups.
Statistics:
- Body weight data collected after initiation of treatment: AnCova & Dunnett’s Test with automatic data transformation. Day 0 body weight data are used as covariate unless removed during data preprocessing.
- Pretreatment body weight data, body weight change, clinical pathology (hematology, clinical chemistry, urinary volume and specific gravity) and organ weight data: Generalized Anova Test with automatic data transformation.
- Food/ water consumption: Dunnett’s multiple comparison test.
- Semi quantitative urinalysis results: Kruskal-Wallis & Dunnett Test” with “Rank as data transformation method.
- Functional observational battery: One-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: Total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Incidences of histopathological changes: Fisher’s exact probability test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs noted mainly in the high-dose group and occasionally also in the mid-dose group, included salivation, piloerection, soiled fur, soft/ mucoid/ yellowish feces, dyspnea, sniffing, grunting, weakness and hunched posture. In the low-dose group these signs were not observed or occurred only incidentally. Salivation was occasionally noted in all dose groups and was related to the dosing procedure (oral gavage).
Signs noted soon after dosing included: sliding with the ventral parts of the head and neck over the cage bottom, piloerection, muscle weakness, salivation and chewing movements. These findings were noted mainly in the high-dose group and to a lesser extent in the mid-dose group, and tended to diminishing in the course of the study. In the low-dose group post-dosing signs were generally not observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
- Four males and four females in the high-dose group were killed in moribund condition or were found dead. These deaths are ascribed to the treatment and also occurred after the high-dose level had been reduced from 250 to 125 mg/kg bw/day (from day 35).
- One male of the mid-dose group was killed on day 70 of the studies for a reason unrelated to treatment. This animal initially showed enlargement of the left eye, but later on the eye got damaged and became severely inflamed. This development justified killing the animal to avoid further suffering.
- One male of the low-dose group was found dead immediately after dosing on day 1. Because there was no other mortality in this group nor treatment-related mortality in the mid-dose group, the death of this single animal was not ascribed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean body weights were statistically significantly decreased in the mid- and high-dose groups in both sexes at most of the stages. The differences with the controls were relatively small (< c. 8%), except for the high-dose males (c. 16%).
- At most stages, mean body weight were statistically significantly decreased in females of the low-dose group. In this group, however, the differences with the controls were very slight (<4%) and the statistical significance was probably due to the higher body weights in this group at the start of the study (day 0) and the statistical test (“AnCova”) using day 0 body weight data as covariate. Therefore, the (very slight) statistically significant changes in females of the low-dose group are not considered relevant.
- In agreement with the above findings, the overall body weight changes (day 0-90) were statistically significantly decreased in males of the mid- and high-dose groups and in females of all dose groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the initial stage of the study, food intake tended to be lower in the mid- and high-dose groups. The differences with the controls were statistically significant in males of these groups in week 1 and in females of the mid-dose group in week 2. There were, however, no noticeable differences in overall food intake among the groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- The weekly overall water intake was clearly increased (21-67%) in the high-dose group in both sexes throughout the study; the differences with the controls were statistically significant at several stages.
- In the course of the study water intake also tended to increase in the mid-dose group (up to c. 25%).
- In the low-dose group the differences in water intake with the controls were below +10%, except for low-dose males at the end of the study (+19%).
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmoscopic examination of all surviving animals in the control dose, mid-dose and high-dose week 13 did not reveal any treatment-related changes. The few changes observed were already present at the start of the study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Packed cell volume was statistically significantly decreased in the mid- and high-dose groups in both sexes. In females of these groups, red blood cell count was statistically significantly decreased, and MCH and MCHC were increased.
- In males of the high-dose group, red blood cell count and hemoglobin concentration were statistically significantly decreased and reticulocyte count was slightly increased.
- Thrombocytes were statistically significantly increased in low-dose females, but this finding was not confirmed at the higher dose levels and is therefore considered a chance finding.
- The absolute neutrophils count and the percentage of neutrophils were statistically significantly increased and the percentage of lymphocytes was decreased in males of the high dose group.
- The lymphocyte/neutrophil ratio was also decreased in low-dose males. Because this finding was not reflected in significant changes in the absolute neutrophil count this change in the low-dose group was not considered relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in clinical chemistry variables:
- Total protein and albumin concentration were decreased in males of all dose groups and in females of the mid- and high-dose group. The values in these test groups were also low in comparison to historical control data. The albumin/globulin ratio was decreased in males of the high-dose group.
- ALAT and ASAT activities were decreased in the mid- and high-dose females (not statistically significantly for ASAT in high-dose females). ALAT activity was also decreased in mid-dose males.
- Creatinine was decreased in females of the high-dose group.
- Inorganic phosphate was increased in males of the high-dose group
- Sodium concentration was slightly increased in low- and mid-dose females. Because of the lack of a dose response relationship this is considered a chance finding.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- The renal concentration test showed a slight, though statistically significant decrease in specific gravity in females of all dose groups, associated with a slightly higher urinary volume (statistically significant in high-dose females only). These findings are probably related to the higher water intake in these groups and therefore not considered of toxicological significance.
- Semi-quantitative (dipstick) urinary measurements showed a statistically significant increase in the incidence of urinary ketones in females of the mid- and high-dose group. The incidence and severity was within the range of historical control data.
- The urinary pH was statistically significantly increased in females of all dose groups, but this finding may be considered of little, if any toxicological significance.- A decrease in urinary bilirubin in low-dose females was considered a chance finding.
- Microscopic examination of the urinary sediment did not reveal any noticeable differences among the groups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Weekly detailed clinical examinations confirmed the daily clinical observations described in 'Clinical signs'. In different animals of the mid- and high-dose groups, treatment-related effects were observed in various weeks of the study. These included sliding with the ventral parts of the head and neck over the bottom of the open field, dyspnea, grunting respiration, sniffing, piloerection, salivation, serous nasal discharge, low arousal, soft and/or mucoid feces, diarrhea, soiled perineum and soiled fur. Although treatment-related, these observations and the results of motor activity assessment, do not point to a neurotoxic effect of the test substance. Therefore it is concluded that the results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences (of surviving rats) with the controls were noted:
- The relative weight of the kidneys was (c. 18%) increased in the high-dose group in both sexes.
- The relative weight of the liver showed a dose-related increase in males of all dose groups and in females of the mid- and high-dose group. The difference with the controls was only small (<7%) in low-dose males, 11-19% in the mid-dose group and 29-40% in the highdose group. The absolute liver weights were increased in males of the mid- and high-dose group and in females of the high-dose group.
- The relative weight of the spleen was increased in the high-dose group in both sexes.
- The relative weight of the adrenals was increased in females of the mid- and high-dose group. The absolute weight of this organ was increased in high-dose females only.
- The relative weight of the thyroid was higher than in controls in males of the mid- and high-dose group, but in the high-dose group statistically significance was not obtained.
- The absolute weight of the thymus was decreased in the high-dose group (not statistically significant in females). Although the relative weight of this organ was not statistically significantly affected, this finding may correlate with the observed decreased cortical cellularity in the thymus in high-dose animals.

The following changes were statistically significant but were not considered to be of toxicological significance:
- The absolute weight of the heart was decreased in females of all dose groups and in males of the mid- and high-dose group. These findings were not reflected in significant changes in the relative weight of this organ and may be ascribed to growth retardation.
- The relative weight of the brain was increased in males of the high-dose group and the relative weight of the testes was increased in the mid- and high-dose group. These changes are ascribed to growth retardation. During growth retardation, rats tend to maintain their (absolute) weights of brain and testes, and there is a well-known inverse correlation between terminal body weight and the relative weight of these organs. Hence, the absolute weights of these organs were not affected or only slightly reduced (absolute brain weight in males of all dose groups and females of the high-dose group).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
ANIMALS KILLED TERMINALLY
- At scheduled necropsy at the end of the study, 3/6 of the surviving high-dose males showed red discoloured mesenteric lymph nodes, which was probably related to treatment.

ANIMALS FOUND DEAD/KILLED MORIBUND
- Five high-dose animals were found dead before the end of the study. In addition, three high-dose animals were killed in moribund condition before termination of the study. For the high-dose animals, respiratory distress was an important clinical sign. Some of them showed gasping which resulted in swallowing air. This was demonstrated at necropsy where in six animals the stomach was swollen/filled with air. The pressure on the diaphragm by the extended stomach aggravates the respiratory distress which eventually may lead to death.
- One mid-dose male was humanely killed because of severely inflammation of the eye. The cause of death for the low-dose animal that was found dead on day 1 could not be established.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
ANIMALS KILLED TERMINALLY
- Microscopic evaluation revealed treatment-related microscopic changes in the pancreas and the spleen.
- The microscopic changes in the pancreas comprised single cell necrosis in 6/6 surviving males and in 2/6 surviving females.
- The microscopic changes in the spleen were characterised by increased accumulation of brown pigment in 4/6 high-dose males and in 3/6 high-dose females and extramedullary erythropoiesis in 3/6 high-dose males.
- A decrease in the incidence of microhaemorrhages in the thymus in high-dose females was considered a chance finding and was not ascribed to treatment.

ANIMALS FOUND DEAD/KILLED MORIBUND
- The main histopathological findings in animals that died intercurrently were: myocardial degeneration in the heart (1/4 high-dose males and 2/4 high-dose females), single cell necrosis in the pancreas (1/2 high-dose females), increased accumulation of brown pigment in the spleen (4/7 high-dose animals), decreased cortical cellularity in the thymus (6/8 high-dose animals), one of which also showed necrosis. Decreased cellularity was also occasionally seen in the mesenteric and axillary lymph nodes.
- There were no other treatment-related histopathological changes. The remaining histopathological changes were about equally distributed between the different treatment group(s) and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
pancreas
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

ANALYTICAL VERIFICATION OF DOSES OR CONCENTRATIONS

- Linearity: The correlation coefficient was 1.000 during the 6 runs which were performed during this study and therefore the preset criterion (the correlation coefficient of the calibration curves should be ≥ 0.996) was met.

- Homogeneity: The RSD between the mean concentrations at three different locations was < 5% for all dose levels and/or p was ≥ 0.01. Therefore the test substance was considered to be homogeneously distributed in the gavage liquids.

- Content: The concentration of the test substance was close to intended (85-115%) for all gavage liquids at all dose levels, except for the mid-dose level of the gavage liquids prepared (-23%). This single deviation is considered acceptable. It is concluded that the animals received the intended levels of test substance at all dose levels.

Conclusions:
Based on the results, the no-observed-adverse-effect level (NOAEL) was determined to be 10 mg/kg bw/day.
Executive summary:

The repeated dose toxicity of the test substance was determined in a GLP-compliant 90-day study in accordance with OECD Guideline 408 (Triskelion B.V., 2017). The test substance was administered daily oral gavage as a suspension in corn oil to groups of 10 male and 10 female Wistar rats at dose levels of 0 (vehicle control), 10 (low-dose group), 50 (mid-dose group) and 250 mg/kg bw/day (high-dose group). Because of mortality observed in the high-dose group, the initial high-dose level in was reduced to 125 mg/kg bw/d from study day 35 and onwards. 

Four males and four females in the high-dose group were found dead or killed in moribund condition. These deaths were treatment-related and also occurred after the high-dose level had been reduced from 250 to 125 mg/kg bw/day. Clinical signs noted mainly in the high-dose group and to a lesser extent also in the mid-dose group, included salivation, chewing movements, sliding with the ventral parts of the head and neck over the cage bottom soon after dosing, piloerection, soiled fur, soft/ mucoid/ yellowish faeces, dyspnoea, sniffing, grunting, weakness and hunched posture. Results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Ophthalmoscopy examination of all surviving animals in week 13did not reveal any treatment-related changes. Mean body weights were decreased in the mid- and high-dose groups in both sexes.In the initial stage of the study, food intake tended to be lower in the mid- and high-dose groups, but there were no differences in overall food intake. Water intake was increased in the high-dose group in both sexes throughout the study. In the course of the study, water intake also tended to be elevated in the mid-dose group and in males of the low-dose group. Haematology was conducted in 10 rats/sex/group at necropsy. Packed cell volume was decreased in the mid- and high-dose groups in both sexes. In females of these groups, red blood cell count was decreased, and MCH and MCHC were increased. In males of the high-dose group red blood cell count and haemoglobin concentration were decreased and reticulocytes were slightly increased. There were no relevant changes in clotting variables. The absolute neutrophil count was increased in males of the high-dose group. Clinical chemistry, conducted in 10 rats/sex/group at necropsy, showed changes in total protein and albumin concentrations (mid- and high-dose), albumin/globulin ratio (high-dose), ALAT and ASAT activities (mid- and high-dose), creatinine concentration (high-dose) and inorganic phosphate concentration (high dose). Urinalysis conducted in 10 rats/sex/group in week 13 of the study, did not reveal any relevant changes in renal concentrating ability or in microscopy of the urinary sediment. Semiquantitative (dipstick) urinary measurements showed an increase in the incidence of urinary ketones in females of the mid- and high-dose group. Changes in organ to body weight ratios were noted in kidneys (high-dose), liver (mid- and high-dose), spleen (high-dose), adrenals (mid- and high-dose), thyroid (mid- and high-dose) and thymus (high-dose). Macroscopic examination at scheduled necropsy at the end of the study showed red discoloured mesenteric lymph nodes in surviving high-dose males. Histopathological findings in high-dose animals that died intercurrently were myocardial degeneration in the heart, single cell necrosis in the pancreas, increased accumulation of brown pigment in the spleen and decreased cortical cellularity in the thymus. Decreased cellularity was also occasionally seen in the mesenteric and axillary lymph nodes. Microscopic examination of surviving rats showed single cell necrosis in the pancreas of high-dose males and females and increased accumulation of brown pigment in the spleen of high-dose males and females and extramedullary erythropoiesis in high-dose males.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: hepatobiliary, immune system and gastrointestinal tract
Organ:
pancreas
liver
spleen

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

The repeated dose toxicity of the test substance was determined in a GLP-compliant 90-day study in accordance with OECD Guideline 408 (Triskelion B.V., 2017). The test substance was administered daily oral gavage as a suspension in corn oil to groups of 10 male and 10 female Wistar rats at dose levels of 0 (vehicle control), 10 (low-dose group), 50 (mid-dose group) and 250 mg/kg bw/day (high-dose group). Because of mortality observed in the high-dose group, the initial high-dose level in was reduced to 125 mg/kg bw/d from study day 35 and onwards. 

Four males and four females in the high-dose group were found dead or killed in moribund condition. These deaths were treatment-related and also occurred after the high-dose level had been reduced from 250 to 125 mg/kg bw/day. Clinical signs noted mainly in the high-dose group and to a lesser extent also in the mid-dose group, included salivation, chewing movements, sliding with the ventral parts of the head and neck over the cage bottom soon after dosing, piloerection, soiled fur, soft/ mucoid/ yellowish faeces, dyspnoea, sniffing, grunting, weakness and hunched posture. Results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Ophthalmoscopy examination of all surviving animals in week 13did not reveal any treatment-related changes. Mean body weights were decreased in the mid- and high-dose groups in both sexes.In the initial stage of the study, food intake tended to be lower in the mid- and high-dose groups, but there were no differences in overall food intake. Water intake was increased in the high-dose group in both sexes throughout the study. In the course of the study, water intake also tended to be elevated in the mid-dose group and in males of the low-dose group. Haematology was conducted in 10 rats/sex/group at necropsy. Packed cell volume was decreased in the mid- and high-dose groups in both sexes. In females of these groups, red blood cell count was decreased, and MCH and MCHC were increased. In males of the high-dose group red blood cell count and haemoglobin concentration were decreased and reticulocytes were slightly increased. There were no relevant changes in clotting variables. The absolute neutrophil count was increased in males of the high-dose group. Clinical chemistry, conducted in 10 rats/sex/group at necropsy, showed changes in total protein and albumin concentrations (mid- and high-dose), albumin/globulin ratio (high-dose), ALAT and ASAT activities (mid- and high-dose), creatinine concentration (high-dose) and inorganic phosphate concentration (high dose). Urinalysis conducted in 10 rats/sex/group in week 13 of the study, did not reveal any relevant changes in renal concentrating ability or in microscopy of the urinary sediment. Semiquantitative (dipstick) urinary measurements showed an increase in the incidence of urinary ketones in females of the mid- and high-dose group. Changes in organ to body weight ratios were noted in kidneys (high-dose), liver (mid- and high-dose), spleen (high-dose), adrenals (mid- and high-dose), thyroid (mid- and high-dose) and thymus (high-dose). Macroscopic examination at scheduled necropsy at the end of the study showed red discoloured mesenteric lymph nodes in surviving high-dose males. Histopathological findings in high-dose animals that died intercurrently were myocardial degeneration in the heart, single cell necrosis in the pancreas, increased accumulation of brown pigment in the spleen and decreased cortical cellularity in the thymus. Decreased cellularity was also occasionally seen in the mesenteric and axillary lymph nodes. Microscopic examination of surviving rats showed single cell necrosis in the pancreas of high-dose males and females and increased accumulation of brown pigment in the spleen of high-dose males and females and extramedullary erythropoiesis in high-dose males. Based on the results, the no-observed-adverse-effect level (NOAEL) was determined to be 10 mg/kg bw/day.

 

A supporting oral chronic study with rats was available for assessment (Blackwell Smith et al., 1953). Groups of 10 male and 10 female rats were administered diet containing the test substance at dose levels of 0, 500, 1000, 2500, 5000 and 10000 ppm for two years. Animals were weighed weekly. Haematological examinations were performed at the end of 11 and 22 months. Gross and microscopic pathologic examinations were performed on all animals, except of those whose autolysis was obviously too advanced. There was apparent increased mortality in the females at the 10000 ppm level and possibly at the 5000 ppm level, but no such effect appeared in the males at any dose level. A tendency toward diminished growth appeared in both sexes at 10000 ppm level. Findings in haematological studies were within normal limits. Kidney pathology (congestion, nephritis or nephrosis) was seen in 6 of the 19 animals of 10000 group. No kidney changes were seen in the animals of 500 ppm group. One rat from 1000 ppm group, one from 2500 group and one from 5000 ppm groups showed nephritis. No other differences between the control and experimental groups were observed. In addition, hyperplasia of thyroid gland was observed in test animals at all dose levels. The degree of hyperplasia was graded from 0 to 5+. No hyperplasia was observed in 9 out of 17 control animals, while 2 had hyperplasia of 1+ degree and 6 had hyperplasia of 2+ degree. In 500 ppm dose group, hyperplasia in degrees 1+ and above (including one animal with 4+ hyperplasia) was observed in 15 out of 16 animals. At dose levels 1000 ppm and above all animals (17 in 2500 ppm group, 14 in 2500 and 5000 ppm groups and 18 in 10000 ppm groups) showed hyperplasia of degree 2+ and above. The microscopic studies revealed malignancies in both treated and control animals, however, there was no apparent evidence of any relationship between the treatment with the test substance and the incidence of neoplastic lesions. Based on the results of this study, no NOAEL for systemic toxicity could be established. The LOAEL for systemic effects was set at 500 ppm zinc bis(diethyldithiocarbamate) in diet, based on increased incidence of thyroid gland hyperplasia at all dose levels. Using the default parameters as reported by Paulussen et al., 1998, TNO report V98.390, this value was calculated to correspond to average dose of 36 mg/kg bw/day.

Justification for classification or non-classification

There are conclusive and sufficient data available for classification of the test substance with regard to the effects following repeated dosing. Based on the data available, the test article is classified as STOT Rep. Exp. 2 H373: 'May cause damage to the liver and spleen prolonged or repeated exposure' in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.