Ziram

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 24 Aug 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted in 1998

Deviations:

yes

Remarks:

Not all requested organs were weighed. The highest dose level produced one fatality and therefore exceeded the MTD (maximum tolerable dose)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ziram

EC Number:

205-288-3

EC Name:

Ziram

Cas Number:

137-30-4

Molecular formula:

C6H12N2S4Zn

IUPAC Name:

ziram

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Consort Limited, Herefordshire, UK
- Age at study initiation: approximately 21 - 24 weeks (males) and 20 - 21 weeks (females)
- Weight at study initiation: 6.2 - 10 kg (males) and 6.8 - 9.4 kg (females)
- Housing: 2 dogs of the same sex were housed in kennels.
- Diet: Standard ground dry diet (Diet A: Special Diets Services) 400 g/animal/day
- Water: Tap water, ad libitum
- Acclimation period: at least 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): not provided
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Fresh treated diet was prepared weekly, usually one day prior to the beginning of the dose week. The required amount of test compound was weighed and ground diet added until the desired concentration of pre-mix was achieved. This was blended in a mixer for a minimum of 2 min. Diets for each group were prepared by direct dilution of the pre-mix. Homogeneity of all diets was achieved by mixing for at least 7 min in a double cone blender. Formulated diets were stored at -20 °C, protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the commencement of the study, the proposed formulations were checked by chemical analysis (spectrophotometry at 435 nm after carbon disulphide extraction) to confirm that the method was acceptable and that the stability of the formulation was satisfactory. Samples of the dietary formulations prepared in Weeks 1 and 13 were also analysed to check the accuracy of preparation. In addition, samples of the 100 ppm formulation prepared in Week 1 were analysed following storage at room temperature for 8 h.
The mean concentrations of the test substance in dose formulations were within 7% of the nominal concentrations. It was also confirmed that the test substance was homogeneously blended in the diet. The stability data indicate that the test substance was stable at 5000 ppm in the diet for up to 14 days storage at ambient temperature. For the 100 ppm diet stability was not affected for an 8 h storage at ambient temperature but after storage periods of 24 h and 14 days, and therefore, a storage at -20 °C was recommended for diet formulations, fed on a daily basis and consumed within 8 h.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously via diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

corresponding to 4.07 mg/kg bw/day for males and 4.31 mg/kg bw/day for females

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

corresponding to 12.15 mg/kg bw/day for males and 13.04 mg/kg bw/day for females

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 41.81 mg/kg bw/day for males and 41.07 mg/kg bw/day for females

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Regularly throught the working day.
- Cage side observations checked: All signs of ill health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to feeding, once a week throughout the pre-dosing and dosing periods

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per dog per week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: From the food consumption data obtained, the quantity of the test substance ingested weekly by each group was calculated. The approximate dose equivalents expressed as mg/kg bw/day were calculated at weekly intervals from the mean body weight data.
The quantity of food left by individual animals was recorded daily throughout the experimental period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before dosing and during Week 13 by means of a Keeler indirect ophthalmoscope
- Dose groups that were examined: all animals
Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing and during Weeks 6 and 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: erythrocyte count (RBC), haemoglobin, haematocrit, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total leukocyte count, differential leukocyte count, platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), cell morphology and packed cell volume (PCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing and during Weeks 6 and 13; collection period of 16 h, water was removed from the kennels about 5 h prior to the start of collection
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: total protein, albumin, sodium, potassium, glucose, urea (BUN), alanine aminotransferase (GPT), aspartate aminotransferase (GOT), alkaline phosphatase (AP), creatinine, calcium, phosphorus, chloride, total cholesterol, total bilirubin, gamma-glutamyltransferase (GT), creatine phosphokinase (CPK) and ornithine carbamoyl transferase (OCT)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: before dosing and during Weeks 6 and 13
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, overnight
- Parameters checked: volume, specific gravity, glucose, pH, protein, ketones, haem pigments, bile pigments, urobilinogen, total reducing substances, epithelial cells (E), polymorphonuclear leukocytes (P), mononuclear leukocytes (M), erythrocytes (R), organisms (O), renal tubule casts (C), other abnormal constituents (A)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Prior to terminal autopsy, bone marrow was obtained from each animal by sternal puncture and a smear prepared for subsequent staining and examination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of 13 weeks dosing each animal was killed by exsanguination under pentobarbitone anaesthesia. All animals were fasted overnight prior to post mortem examination. Since the post mortem procedures took 3 days to complete, the dosing of individual treated animals continued until the day prior to their sacrifice.
At autopsy the macroscopic appearance of the tissues was noted and the weights of the following organs recorded (paired organs being weighed separately): adrenals, brain, heart, kidneys, liver, lungs, pancreas, pituitary, spleen, testes or ovaries, thymus, thyroids (and parathyroids) and uterus or prostate.

HISTOPATHOLOGY: Yes
The tissues listed below were examined for all animals by light microscopy: Adrenals, aorta (arch and abdominal), femur and articular surface, sternum, brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebellum), epididymides, oesophagus, gall bladder, heart, kidneys, caecum, colon, rectum, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), ovaries, pancreas, pituitary, prostate, mammary gland, sciatic nerve, duodenum, jejunum, ileum, spleen, skeletal muscle, skin, salivary gland (submandibular), spinal cord (cervical, thoracic and lumbar), stomach, testes, thymus, thyroids/parathyroids, trachea, tongue, eyes, urinary bladder, uterus, vagina and all gross lesions.

The fixative routinely used was 10% buffered formalin. The eyes were preserved in Davidson's fixative whilst additional pieces of liver and kidney were placed in formol calcium. Tissues were embedded in 56 °C M.P. paraffin wax and sections were cut at 4 µm and stained with haematoxylin and eosin. Cryostate sections of liver previously fixed in formol calcium were cut at 12 µm and stained with Oil Red O. Liver sections were stained using PAS for glycogen.

Statistics:

All analyses were carried out both together and separately for male and female.
Data relating to food consumption were analysed as totals over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
- For pre-dose data, analyses of variance were followed by Student’s ‘t’ test. For data from the dosing period, analyses of variance were followed by Williams’ test for a dose-related response. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Convulsive episodes were noted for the dog of the high dose group (1000 ppm) being sacrificed for humane reasons in Week 5 as discussed under "mortality". In addition, a female dog of the high dose group (1000 ppm) was noted to be trembling on one occasion in Week 8.
There were no other clinical signs attributable to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male dosed at 1000 ppm was sacrificed during week 5, because of a seizure without apparent significant recovery. Convulsive periods had previously been observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reductions in body weight were noted for one female dog (1000 ppm) from Week 4 to 6, with subsequent recovery from Week 7 and for one male dog (1000 ppm) from Week 2 until its death in Week 5, and were considered to be related to the treatment. Body weight loss and inappetence were also noted for a male dog of the high dose group (1000 ppm) during Week 3 and 4, however, as this was associated with a retropharyngeal abscess it was considered to be unrelated to treatment. With resolution of the abscess from Week 5, appetite restoration and body weight gain were noted for this animal. It was concluded that treatment with the test substance produced a slight reduction in body weight gain for the 1 male and 1 female dog at 1000 ppm.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From weeks 3 to 5, a gradually reduced food intake was recorded for one female dog of the high dose group (1000 ppm) with some subsequent recovery from Week 6. Inappetence was also noted for one male dog (1000 ppm) from Week 2 until its death in Week 5. These changes were considered to be associated with treatment. In addition, inappetence and associated body weight loss were noted for a male dog of the high dose group (1000 ppm) during weeks 3 and 4 and was associated with a retropharyngeal abscess, as previously discussed, and was considered to be unrelated to treatment. It is concluded that treatment with the test substance was associated with a slightly reduced food intake for 1 male and 1 female animal receiving 1000 ppm.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no changes observed during Week 13 that were considered to be related to the treatment with the test substance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At Weeks 6 and 13, group mean red cell indices (PCV, Hb, RBC) were slightly reduced in comparison to concurrent control values, for males and females receiving 1000 ppm. However, these differences were not progressive and did not always attain statistical significance. In addition, during Week 6, slightly elevated circulating reticulocyte counts, with associated slight polychromasia, hypochromasia and/or moderate anisocytosis, were noted for one male and one female animal at 1000 ppm. Slightly elevated circulating reticulocyte count was also noted for one female dog (1000 ppm) during Week 13. Group mean APTT values for males receiving 300 ppm during Week 13 and 1000 ppm during Weeks 6 and 13 were slightly but statistically significantly higher than corresponding control values. A similar change was apparent for females at 1000 ppm during Weeks 6 and 13, although these did not attain statistical significance.
There were no other changes considered to be attributable to treatment. As the EDTA sample for haematological investigations was clotted, no haematology results were obtained for the animal sacrificed at Week 5. However, values for PT and APTT were slightly lower than pre-dose values.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 6, high bilirubin, AP, GPT, GOT, OCT and cholesterol values were recorded for one female dog (1000 ppm), with the enzyme levels remaining clearly elevated, though to a lesser extent, on repeat investigation in Week 7. These values, however, were essentially unremarkable at Week 13, with the exception of AP which was still slightly elevated. Mean AP values were increased among females receiving 1000 ppm at Weeks 6 and 13 in comparison with values for concurrent controls, attaining statistical significance in Week 13. In addition, there was an apparent dose-related increase in cholesterol for males and females at Weeks 6 and/or 13, attaining statistical significance at 300 and 1000 ppm. Group mean albumin concentrations were slightly, but statistically significantly lower than corresponding control values for animals at 1000 ppm during Week 6 and for animals from all treated groups during Week 13. This was associated with a corresponding increase in globulin concentrations, such that total protein levels were unaltered. There were no other changes considered to be attributable to treatment.
The male dog sacrificed at Week 5 showed slightly higher serum GPT, sodium and chloride levels and markedly higher serum AP, GOT, CPK and cholesterol
values than the corresponding pre-dose values.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

A high urinary SG, protein and traces of total reducing substances were noted in the urine of the male dog killed for humane reasons in Week 5. There were no other effects on urinalysis parameters that were considered to be related to the treatment with the test substance.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean liver weights for males and females at 1000 ppm were statistically significantly higher than control values, with individual liver weights for 3 males and 1 female at this dose exceeding the normal upper limit of 4% of body weight, and this was considered to be related to treatment. A slightly high liver weight for one male dog at 300 ppm, equaling the normal upper limit of 4% body weight, was also considered to be related to treatment. The individual liver weight for one female dog at 100 ppm also exceeded the normal upper limit of 4% of body weight. In isolation, this finding is considered to be of uncertain relationship to treatment.
In addition, a statistically significantly lower group mean heart weight for males at 1000 ppm and lower group mean lung weight for females at this dose was also noted in comparison to controls. However, as differences were slight, not dose-related and confined to 1 sex only and as all individual values were within normal limits for dogs of this age, these findings are considered to be incidental and unrelated to the treatment with the test substance.
In the male animal of the high dose group (1000 ppm) killed for humane reasons in Week 5, liver weight was increased, which at 4.33% of body weight exceeded the normal upper limit of 4.0%. Spleen and prostate weights were also considered to be lower than expected in this animal.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, multiple depressed pale areas were noted on the liver of one female dog receiving 1000 ppm, which correlated with histopathological findings.
In the male dog sacrificed at Week 5 (1000 ppm group) pallor of the endocardium, extending into the myocardium of the left ventricle and an area of red endocardial discolouration on the left atrioventricular valve of the heart were noted. Further pallor of the liver, with accentuation of the lobular markings, and a friable cut surface, was seen grossly. All these macroscopic findings were accompanied by histopathological changes.

There were no other findings considered to be of toxicological importance, in particular no treatment-related changes were seen in the hearts of any dogs killed at termination.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight relative increase in myeloid cells in the smear from a male dog of the control group. For all other animals sacrificed after 13 weeks of treatment, all smears were normal in cellularity, distribution or cell morphology.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings were noted in liver and lungs as follows:
Liver:
Corresponding to the macroscopic observations in the liver, one female dog receiving 1000 ppm showed focal necrosis, together with loss of cells and dilated sinusoids. This finding correlates with the high individual AP, GPT, GOT and OCT values previously noted for this dog. Focal necrosis was also seen in one male dog treated with 300 ppm. Minimal amounts of pigment were seen in the Kupffer cells of one male and one female animal receiving 300 ppm and for two males and one female at 1000 ppm. These changes were not seen in control dogs or dogs receiving 100 ppm.

Lungs:
Minimal foci of eosinophilic homogeneous material were seen in the alveoli of the lungs of one male and one female animals receiving 100 ppm, one male animal receiving 300 ppm and one male animal receiving 1000 ppm. In the male dog receiving 1000 ppm, this change was associated with acute inflammatory cell infiltration. The toxicological significance of these findings, which were not related to dose, is not clear.

There were no other findings considered to be of toxicological importance.

In the male dog sacrificed in Week 5, focal myocardial necrosis with acute inflammatory cell infiltration and haemorrhage was seen histologically. Further, centrilobular hepatocyte degeneration was reported histologically. This was possibly secondary to the heart changes seen. These changes were considered likely to be related to convulsions seen clinically rather than a direct effect of the test compound. Areas of polymorphonuclear leucocyte infiltration into the alveoli were also noted for this dog.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 4.07 mg/kg bw/day for males and 4.31 mg/kg bw/day for females

2

Key result

Dose descriptor:

LOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, clincial signs, mortality, changes in body weight, food consumption, clinical biochemistry, and gross pathology were observed at 1000 ppm.

Remarks on result:

other: corresponding to 12.15 mg/kg bw/day for males and 13.04 mg/kg bw/day for females

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The present study was conducted to assess the sub-chronic toxicity of the test substance on dogs when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 409 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 4 male and 4 female dogs at dose levels of 100, 300 and 1000 ppm corresponding to approximately 4.07, 12.15 and 41.81 mg/kg bw/day for males and 4.31, 13.04 and 41.07 mg/kg bw/day for females. Under the conditions of the test, the test substance caused clinical signs, reduced body weights and food consumption and changes in haematology and clinical biochemistry parameters, organs weights, gross pathology and histopathology at 300 and/or 1000 ppm. Therefore, the NOAEL was set at 100 ppm for males and females (corresponding to 4.07 and 4.31 mg/kg bw/day respectively).