Thiophanate-methyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 20, 1990 - August 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 20, 1990 - August 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

survival rate in high-dose male group was less than required, excessive mortality was partly due to accidental deaths but was also clearly substance related indicating that MTD was exceeded; survival rate in other three groups corresponded to guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Atsugi, Kanagawa
- Age at study initiation: 6 weeks
- Weight at study initiation: mean 121g (males); mean 99 g (females)
- Housing: housed individually in a stainless steel mesh cage
- Diet: ad libitum, basal diet (Diet M®, purchased from Oriental Yeast Co., Ltd., Tokyo, Japan
- Water: ad libitum, tap water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 40-80
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
The test-item was ground and incorporated with the basal diet (M diet, purchased from Oriental Yeast Co., Ltd., Tokyo) to make separate premixes for each dietary concentration. Each premix was mixed with the basal diet by using a blender (SS-161, Kanto Kongoki, Tokyo). The diets were prepared monthly and stored at about -20 °C until used. Diet samples were taken from all dose levels, and homogeneity and concentration analysis were performed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dietary mean concentration values ranged from 94.1 to 110.9% of the nominal concentration.

Duration of treatment / exposure:

52 weeks (chronic study)
104 weeks (carcinogenicity study)

Frequency of treatment:

continously

Dose / conc.:

75 ppm

Dose / conc.:

200 ppm

Dose / conc.:

1 200 ppm

Dose / conc.:

6 000 ppm

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, morbidity and overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through week 14, then at week 16 and once every four weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- recorded weekly through Week 14, then at Week IB and once every four weeks thereafter
Once each week, food consumption was determined for a 24-hour period for each rat. The following formulas were used to calculate food consumption values (g/kg body weight/day), food efficiency values (%) and compound consumption values (mg/kg/day). Food consumption value=[Food consumed(g/day)]/[Body weight(kg)]

FOOD EFFICIENCY:
- Food efficiency value =[Body weight gain(g/day)]/[Food consumed (g/day)] x 100

WATER CONSUMPTION AND COMPOUND INTAKE: At Months 18 and 24, water consumption was measured, because a increased urinary volume was noted in males of the 6000 ppm group at Month 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
- performed on all animals prior to the study initiation and at Months 12 and 24 of the study. At Months 6 and 18, all animals from the control and the highest dose (6000 ppm) groups were also examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At Months 3, 6, 12, 18 and 24 of the study, blood was taken from the retro-orbital sinus
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals:
10 rats/sex/group
- Parameters checked: Erythrocyte count (RBC), Leukocyte count (WBC),Hemoglobin (Hb), Platelet count (Pla), Hematocrit (PCV), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration) were calculated. Differential leukocyte count was conducted on the control and the highest dose groups. The slides from the other groups were only examined at Months 12, 18 and 24

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At Months 6, 12, 18 and 24 of the study
- Animals fasted: Yes
- How many animals:10 rats/sex/group
- Parameters checked: Alkaline phosphatase (ALP), Aspartate aminotransferase (GOT), Alanine aminotransferase (GPT), Lactic dehydrogenase (LDH), Creatinine phosphokinase (CPK), Cholinesterase (Ch-E), Glucose (Glu), Total bilirubin (T-Bil), Total cholesterol (T-Cho), Creatinine (Crea), A/G ratio, Total protein (T-Pro), Albumin (Alb), Urea nitrogen (BUN), Inorganic phosphate (P), Calcium, Potassium and Sodium, Chloride, Thyroxine (T-4) and Triiodothyronine (T-3), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine:
At Months 6, 12, 18 and 24 of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: occult blood, protein, ketone, glucose, bilirubin and urobilinogen, specific gravity, pH, volume, appearance, sediment, protein

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

A complete necropsy was performed on all rats dying spontaneously, sacrificed in extremis or sacrificed at the interim and final necropsies. After administration for 52 weeks, 10 rats/sex/group were fasted for about 16 hours and weighted, anesthetized with sodium pentobarbital and sacrificed by exsanguination. The number of high dose (6000 ppm) males for interim sacrifice was reduced to 5 rats, because 8 rats of this group died at Weeks 11 or 12. After Week 104, all survivals were fasted for about 16 hours and weighted, killed by inhalation with chloroform, and examined for macroscopic lesions. The rats sacrificed in extremis were not fasted prior to sacrifice, and were also killed by chloroform inhalation.

GROSS PATHOLOGY: Yes
The following tissues from each animal were preserved in 10% neutral buffered formalin.
Accessory genital organs: Uterus, Vagina, Oviduct and Clitoral gland Prostate, Seminal vesicle, Coagulating gland, Preputial gland), Adrenal, Aorta, Bone and marrow (Sternum and Femur), Brain (Medulla/Pons, Cerebrum and Cerebellum), Optic nerve, Eye and Harderian gland, Gastro intestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum) Gonad (Ovary and Testis, Epididymis) Heart, Joint (knee), Kidney, Liver, Lung, Lymph node (mesenteric, mandibular), Mammary gland, Musculature (thigh), Pancreas, Pituitary, Salivary glands (Mandibular, sublingual, parotid), Sciatic nerve, Skin, Spinal cord (3 levels), Spleen, Thymic region, Thyroid and Parathyroid, Trachea, Urinary bladder (inflated with formalin), Unusual lesions, Regional lymph node (when applicable)
The following organs from each animal were weighed: Brain, Liver, Adrenals,Thyroid/Parathyroids, Heart, Lung, Kidneys, Pituitary,Testes, Spleen, Ovaries

HISTOPATHOLOGY: Yes
The organs and tissues, which were listed were fixed in buffered 10% formalin, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The following histopathology was performed:
(a) :All organs in all animals sacrificed at interim necropsy.
(b) :All organs in all animals in the control and 6000 ppm groups, and all males in the 1200 ppm group.
(c) :All organs of all animals that died or were killed during the study.
(d) :Lung, liver, kidney, thyroid, parathyroid, pituitary, adrenal and gross lesions of all remaining animals.
A five-step grading system (from 0 to 4) was used to define lesions. Tumors were classified as benign, malignant or metastatic.

Other examinations:

MECHANISTIC STUDY
The study also contained mechanistic investigations. The mechanistic investigations were performed on Fischer 344 rats or ICR mice (both Charles River, Japan) with the substance TPM (same purity and batch as in the main study, dose level 6000 ppm) and with the reference substances phenobarbital (500 ppm) and propylthiouracil (1000 ppm). The test item and phenobarbital were given by diet, propylthiouracil was given in distilled water.
TPM or the reference substances were given to male and female rats (6 weeks old) for 2 or 8 days (males) and 8 days (females). Measurement of T3, T4 and TSH in serum, and determination of drug intake, liver and thyroid weights and total cholesterol in serum were performed. Measurement of thyroid weight was performed after a recovery period of 8 days. Additional administration of TPM to male rats (8 weeks old) was performed for 8 days with daily T4 supplementation (30 μg/kg bw subcutaneously injected). Isolation of microsome fraction of each liver were performed, including measurements of enzyme induction of the following enzymes: cytochrome P450, cytochrome b5, NADPH-cytochrome c reductase, UDP-glucuronosyltransferase) and microsomal protein. Isolation of microsome fraction of porcine thyroids was performed, purchased from a butchery. Measurement of peroxidase activity in the presence of TPM (10E-3 M to 10E-4 M) or propylthiouracil (10E-4 M to 10E-6M) was performed.

Materials: For feeding study, TPM and phenobarbital were incorporated in basal food (M diet, ORIENTAL YEAST Inc., Tokyo) at 6000 ppm and 500 ppm, respectively. Propylthiouracil (PTU, Tokyo kasei Co, Ltd., Purity :over 99%) was dissolved in distilled water at 1000 ppm.
Animals and housing: F 344 rats and ICR mice (Charles River Breeding Laboratories, Atugi, Kanagawa, Japan) were used. Each group consisted of 5 animals, except notified. The environmental conditions of the animals were maintained temperature at 21-24°C, humidity at 40-80%, air exchange at 10 times an hour and 12-hr light-dark cycle.
Sampling: All rats and mice were not restricted food before sampling of blood and necropsy. Blood was taken under pentobarbital sodium anesthesia, and serum was used for analysis. Porcine thyroids, purchased from a butchery, were stored at -80"C until preparation of microsome.
Statistical analysis: All data are presented as mean value ± SD and the Student's t-test and Mann-Whitney U-test were used to detect significance (*:p<0.05, **:p<0.01, ***:p<0.001) between control and dosed groups.

Statistics:

Mortality, clinical observation, ophthalmoscopic examination and macroscopic observation data were analyzed by the chi-square test. When the number of animals were below 5/group, Fisher's exact test (two-tailed) was used to access significance between control and dose groups. Comparisons of all data collected at each interval on body weights, food consumption values, clinical laboratory values and organ weights (absolute and relative to body weights) were performed using Bartlett's test for homogeneity of variances. If variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test or Scheffe's test was used to determine which means were significantly different from control. If a non-parametric procedure for testing equality of means were needed, the Kruskal-Wallis test were used, and if differences were indicated, Dunnett-type test or Scheffe-type test was used to determine which means were significantly different from the control (Gad and Weil, 1982).
The graded data from urinalysis were analyzed by the Mann-Whitney test. The microscopic observation data were analyzed by the Mann-Whitney test (non-neoplastic lesions) or by the chi-square test (neoplastic lesions). When the number of animals were below 5/group, Fisher's exact test (two-tailed) was used to access significance between control and dose groups.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males, the incidence of pale discoloration of skin and mucous membranes was significantly higher in the 6000 ppm group than control from Week 77 to study termination. Tissue masses in the subcutaneous tissue, including the lip, were found more frequently at 1200 ppm from Weeks 95 to 101. Tissue masses on the skin were also slightly increased in the 1200 ppm group at Weeks 91 and 92, and in the 6000 ppm group at Week 91. In females, no increases in pale skin coloration or masses were noted.
In females, the incidence of alopecia at Week 52 was 2/60, 0/60, 4/60, 5/60 and 7/59 at the doses of 0, 75, 200, 1200 and 6000 ppm, respectively. Statistical significance was attained in the 6000 ppm group from Weeks 79 to 104. No increase in alopecia was seen in treated males.

Mortality:

mortality observed, treatment-related

Description (incidence):

Dose-related and accidental deaths occurred in males of the 6000 ppm group. Eight males of this group were killed in extremis or found dead at Weeks 11 or 12. The cause of death was fracture of the nasal bone and subsequent dyspnea (rhinorrhagia). All cages and feeders for males had been changed to a larger type to accommodate the increased size of growing males. The new feeder was equipped with a stainless plate through which some holes had been punched. Because the deaths occurred after changing the feeders, it is considered that the fractures were related to the feeder plate. The plates were removed after 3 days and no accidental deaths were noted thereafter. After Week 80, the mortality of the high-dose males increased significantly, reaching 96% at Week 104. No dose related effect on mortality was noted in females (for more information please see table 1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose-related and statistically significant growth depression and/or weight loss were noted in both sexes of the 1200 and 6000 ppm groups. Although rats typically lose weight, as a group, near the end of a 2-year study, the 6000 ppm males began losing weight by Week 52 in this study, as shown in Table 2. At termination, the mean body weights of the 1200 and 6000 ppm males were 84 and 73 % of the controls, respectively. The net gains throughout the study were 256.7 g (79 %) and 204.5 g (63 %) in the 1200 and 6000 ppm males, while that of the control males was 324.8 g. In females, body weight depressions were observed at 1200 and 6000 ppm, and were statistical significant after Week 2 in the 6000 ppm group and between Weeks 20 and 52 in the 1200 ppm group. At termination, the mean body weights of the females were 91 and 78% in the 1200 and 6000 ppm groups compared to controls. The net gains throughout the study were 199.4 g (88 %) and 156.5 g (69 %) in the 1200 and 6000 ppm females, while that of the control females was 227.8 g. Based on the > 10 % body weight depressions in the 1200 ppm group, it was determined that 1200 ppm was the Maximum Tolerated Dose (MTD). The body weight changes in the 75 and 200 ppm groups were comparable to those of the control groups (both sexes).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

As shown in Table 3, at Week 1, food consumption (g/animal/day and g/kg body weight/day) was decreased in males of 75 and 6000 ppm groups (p<0.01) and in females of all dosed groups (p<0.01). At Week 2, high dose females ate less food than control.
The males and females from 6000 ppm group ate slightly less food than controls at Week 78 and thereafter, with statistical significance occasionally attained. For much of this 2-year study, however, the food consumed per animal per day was similar to sex-matched controls, regardless of dose level. Due to the lower body weights of the 1200 and 6000 ppm rats, food consumption values (g/kg body weight/day) of these rats were significantly higher than controls at various time points (see table 3).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency of the 6000 ppm males and females were slightly lower than controls at several test times during the study.

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All rats placed on study had normal ophthalmoscopy.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related anaemia (decreases of red blood cell count, haemoglobin, haematocrit, MCV, MCH and MCHC) was noted in both sexes at 6000 ppm at months 3, 6, 12 and 18. In the 1200 ppm groups, occasionally statistically significant changes in haemoglobin, MCV and MCH were seen. As these changes, however, occurred only at one or two time points and were seen sometimes in males and sometimes in females, they are not considered to be related to treatment. Increased platelet counts and white blood cell counts were frequently seen in males of the 6000 ppm group. Differential leukocyte counts remained normal at all test times for all groups but the 6000 ppm males at month 24. For this time point no correct evaluation was possible since only one animal could be investigated.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry revealed several statistically significant and dose related changes. Increases in total cholesterol and total protein, and a decrease in A/G ratio were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and/or 18. Only at month 24, treated males exhibited increased BUN (75, 200 and 1200 ppm groups) and creatinine levels (1200 ppm group). Decreased levels of chloride, potassium and decreased activities of LDH, ALAT, ASAT and CPK were seen predominantly in females of the 1200 and 6000 ppm groups at the 6- and 12-month examinations. In high dose males chloride, ALAT and ASAT were found to be decreased after 12 months. Serum cholinesterase activity was increased in males at the highest dose level at the time intervals 6 and 12 months. No changes were seen after 18 and 24 months. The serum cholinesterase activity in females was decreased at the two highest dose levels at the time intervals 6 and 12 month. No changes were seen after 18 and 24 months. In males of the 6000 ppm group, thyroxine (T4) and triiodothyronine (T3) values were numerically lower than those of the control at month 6, 12 and 18 (not evaluable at month 24). Thyroid stimulating hormone (TSH) values of this group were higher than those of the respective centrals. Similar, but weaker effects on the thyroid hormones and TSH were also found in males of the 1200 ppm and females of the 6000 ppm groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A dose-related and statistically significant increase of urinary protein (semi-quantitative analysis) was noted in both sexes of the 6000 ppm group at various test times. By using a qualitative method (nephelometry), protein was also increased in males of the 1200 ppm dose and the 200 ppm dose (the latter only noted at month 24 and lacking a histological correlate). In males of the 6000 ppm group ketone bodies, urinary volume and water consumption were increased, and pH and specific gravity were decreased significantly at various test times.

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):

Dose-related and statistically significant increases in the liver, kidney and thyroid weights and their body weight ratios were noted in both sexes of the 1200 and 6000 ppm groups at both interim and final sacrifice. Other changes in organ weight were noted in high dose males and females and in 1200 ppm males, but these changes are most likely related to the decrease in body weight and thus not considered as a primary toxicological effect of the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At interim sacrifice, treatment related changes were found in the liver (brownish-black colour) and kidneys (granular surface, brownish-black colour) of the 6000 ppm males and females. At final sacrifice, males of the 1200 ppm group exhibited granular kidneys. In the 6000 ppm females, kidneys were brownish-black and the thyroids were enlarged. Animals which died during the study also had treatment related changes. At 6000 ppm, swelling of the thyroid (both sexes) and granular kidney (male) were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examinations were performed on all rats which died on study or were killed at interim and final sacrifices. An independent peer review of relevant microscopic findings was conducted. Dose-related changes were found in the thyroid, liver, kidney and adrenals.
Interim:
Thyroid: A treatment-related change, follicular hyperplasia and hypertrophy, was found in both sexes of the 6000 ppm group. Morphologically, the follicles were small and irregular, composed of cuboidal to columnar cells, and deficient in colloid (grade 2). At 1200 ppm, the lesion consisted of slightly irregular follicles, cuboidal to columnar epithelial cells, and had weakly stained colloid (grade 1). No follicular hyperplasia was noted at 200 and 75 ppm in either sex. Focal hyperplasia of follicular cells was found in 2 females from 6000 ppm group. This change was also considered to be related to compound administration.
Liver: The only lesions in the liver which were interpreted to have been related to the test compound were hepatocellular hypertrophy and deposition of lipofuscin pigment. The hypertrophy was centrilobular (grade 1), centrilobular and mid zonal (grade 2) or all lobules (grade 3). The amount of lipofuscin pigment increased related to the severity of hypertrophy. These changes were found in both sexes of the 1200 and 6000 ppm groups. Other liver lesions, such as foci of cellular alteration were seen microscopically, but were not considered to be related to treatment. Foci, when seen, were classified according to Eustis et al., (1990) into vacuolated, eosinophilic, basophilic, clear or mixed. The number of foci per 2 sections (2 different lobes) for each rat was counted and graded (grade 1: <5 foci, grade 2: 5 - 10 foci).
Adrenal cortex: Cytoplasmic lipid in adrenal cortical cells is a normal finding. In this study, a slightly increased amount of lipid was seen in 1200 ppm interim females and in both sexes at 6000 ppm.
Kidney. Nephropathy, which is an age-related progressive change in the rat, appeared to be accentuated in 6000 ppm males. Morphologically, there were numerous protein casts in the corticomedullary junction with interstitial fibrosis, regenerative tubules, thickened tubular basement membrane and glomerular lesions (crescents, thickened basement membranes). When 25 to 50 % of the cortex was affected, these lesions were graded 3 (moderate). Mild change, which was noted in females from 6000 ppm and males from the control and other dosed groups, consisted of several to many protein casts and less than 25% of superficial cortex area affected. A minimal lesion (grade 1) was found mostly in females. Here, there were only a few or no casts. However, slight glomerular lesions, such as crescents or thick basement membranes were present.
Although pale skin color had been seen clinically, and the clinical pathology data indicated mild anemia, the bone marrow was free of detectable treatment-related lesions.
Numerous other lesions were noted microscopically, but all were unrelated to dose, and were considered to be spontaneous changes.
Final sacrifice:
Microscopic changes interpreted to have a relationship to treatment were noted in the skin (females), thyroid, liver and kidney.
Skin: A dose-related lesion, atrophy of hair follicles, was seen in female rats which had bilateral alopecia noted clinically. In females of the 6000 ppm group, the incidence of this lesion was significantly increased. No significant change was noted in males.
Thyroid: A dose-related and statistically significant increase in diffuse thyroid follicular cell hyperplasia and hypertrophy was noted at Month 24 in both sexes of the 1200 and 6000 ppm groups. The incidence of focal (adenomatous) hyperplasia of follicular cells was slightly, but not statistically significantly, increased in females of the 1200 and 6000 ppm groups. In males, no dose-related increase of focal hyperplasia was detected.
Liver: As seen in 12 month interim rats, the livers of 1200 and 6000 ppm rats (both sexes) had compound-related hepatocellular hypertrophy with deposition of lipofuscin pigment. The incidences of altered foci (vacuolated, clear, eosinophilic, basophilic and mixed) were not changed among groups, except for basophilic foci, which were decreased in females from 6000 ppm group. The incidences of multiple focal hyperplasia, which were usually seen in rats with mononuclear cell leukemia, were not changed among groups.
Kidney: The severity of nephropathy was increased in both sexes of 1200 and 6000 ppm groups. Statistical significance was attained only in females. A small amount of lipofuscin pigment was found in the tubular epithelium, especially in the proximal tubules of males and females from the 6000 ppm group.
Adrenal cortex: Increased adrenal cortical lipid, seen microscopically in dose-related fashion at Month 12, had no relation to dose at Month 24. Focal hyperplasia in the adrenal cortex was a common finding in all groups, with no statistical significance in the incidences among treated rats.
The spleen and bone marrow remained free of detectable dose-related alterations, even in the presence of mild anemia which had been noted in hematological tests. Numerous other miscellaneous non-neoplastic lesions were observed and are recorded in the microscopic data. None of these lesions were distributed in a manner which suggested a relationship to treatment, and are interpreted to be spontaneous age or disease-related changes.
Animals found dead or sacrificed in extremis:
Eight rats died or were sacrificed in extremis in Week 11 or 12. The clinical observation indicated fracture of the nasal bone, and this finding was confirmed by histopathological examination (fracture of nasal bone accompanied by bleeding into nasal turbinate and meatus). Four of the eight rats had eosinophilic crystals in the lungs, probably of hemoglobin. All eight rats had centrilobular necrosis of the liver, indicating acute hypovolemic shock. These were clearly differentiated from drug-induced lesions (hepatocellular hypertrophy). Treatment-related changes were found in the liver, kidney and thyroid.
Liver: Hepatocellular hypertrophy with lipofuscin deposition was found frequently in males and females of the 6000 ppm group. Males of the 6000 ppm group frequently also had microgranulomas and focal fatty degeneration (Greaves 1984), interpreted to be degenerative/inflammatory changes related to age and debilitation, rather than due to treatment.
Kidney: The severity (grade 4: >50 % of kidney affected) of nephropathy was extreme in the males of the 6000 ppm group, and was sometimes accompanied by hypertrophy of the parathyroid, with resorption of bone and metastatic calcification in the aorta (medial calcification :Mc), lung (calcium deposition :Ca), heart (Ca) and stomach (Ca).The end-stage renal lesion was interpreted to be the cause of death for many males of the 6000 ppm group. In females, nephropathy was not as severe as in males, even at the level of 6000 ppm.
Thyroid: Diffuse hypertrophy and hyperplasia of follicular cells were noted in females of the 1200 ppm and in both sexes of the 6000 ppm groups. Although not statistically different, focal hyperplasia of follicular cells was frequently found in males of the 6000 ppm group.
Other non-neoplastic changes noted in unscheduled-death rats were incidental to treatment.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim: Only one tumor was found at the interim sacrifice which could be linked to a treatment-related effect. This was a thyroid follicular cell adenoma, seen in a male from the 1200 ppm group.
Final Sacrifice: Tumors were found in various organs. However, there was neither statistically significant nor dose-related increases of any tumor incidences at final sacrifice. Only 3 tumors were seen with possible relationship to treatment. These were thyroid follicular cell adenomas in one 1200 ppm male and one 6000 ppm female, and a thyroid follicular cell adenocarcinoma in a 6000 ppm male.
Animals found dead or sacrificed in extremis: No tumors were found in unscheduled-death rats which could be related to treatment, with the exception of thyroid follicular cell (FC) neoplasms.

Description (incidence and severity):

MECHANISTIC STUDY:
Thiophanate-methyl caused decreases in T4 and T3 1evels and increases in TSH level and thyroid weights on days 2 and/or 8. The liver weight and amount of total cholesterol in serum were also increased. There was a close correlation between TSH level and thyroid weight. The T4 supplementation counteracted the hypertrophy of the thyroid and the TSH response, indicating that thiophanate-methyl caused the hypertrophy by negative feedback mechanism. The T4 supplementation had no influence on the increased liver weight and total cholesterol level. The effect of thiophanate-methyl on the thyroid was reversible after a recovery period without administration of the test substance.Thiophanate-methyl inhibited the thyroid peroxidase (30 fold weaker than propylthiouracil). Thiophanate-methyl caused hypertrophy of the liver, accompanied by a proliferation of liver cells which was observable in rats only after 2 days but in mice after 2 and 8 days.
Thiophanate-methyl induced cytochrome P 450 and related drug metabolising enzymes. One of them, the UDP-glucuronosyltransferase plays an important role in the clearance of T4 in the liver. UDP-glucuronosyltransferase was raised by 335% (20.6 +/-3.9 in control and 69.2 +/- 19.5 nmol/min/ng microsomal protein in the 6000 ppm dose group). Phenobarbital which also induced liver cell proliferation and the drug metabolising enzymes revealed only a very slight increase of TSH without thyroid hypertrophy.
Thus, the inhibition of hormone synthesis in the thyroid due to an inhibition of peroxidase could be the main cause of the T4 depression.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Table 1: Mortality (%)

	Male			Female
Dose (ppm)	wk 52	wk 80	wk 104	wk 52	wk 80	wk 104
0	0/60 (0)	2/50( 4)	13/50(26)	0/60 (0)	3/50 (6)	13/50 (26)
75	0/60 (0)	2/50( 4)	15/50(30)	0/60 (0)	1/50 (2)	12/50 (24)
200	0/60 (0)	8/50(16)	24/50(48)a	0/60 (0)	1/50 (2)	8/50 (16)
1200	0/60 (0)	3/50( 6)	21/50(42)	0/60 (0)	0/50 (0)	12/50 (24)
6000	8/60(13) a	18/55(33)c	53/55(96)c	1/60 (2)	3/60 (6)	11/50 (22)

Significantly different from control, a: p<0.05, c: p<0.001 (Chi-square test)

Table 2: Mean body weights (g) and ratios to control (%)

	Male				Female
Dose (ppm)	Week 13	Week 42	Week 80	Week 104	Week 13	Week 52	Week 80	Week 104
0	353.8 (100)	471.9 (100)	454.5 (100)	445.6 (100)	193.6 (100)	258.1 (100)	306.7 (100)	327.9 (100)
75	353.2 (100)	473.7 (100)	480.6 ( 99)	432.4 ( 97)	194.5 (100)	263.8 (102)	312.3 (102)	327.5 (100)
200	354.7 (100)	476.4 (101)	481.5 ( 99)	438.9 ( 98)	194.1 (100)	258.8 (100)	305.7 (100)	316.6 ( 97)
1250	350.6 ( 99)	469.2 ( 99)	467.1 ( S5)	376.3 ( 84)c	190.3 ( 98)	248.4 (96)b	291.4 ( 95)	299.6 ( 91)
6000	353.6 (100)	451.4 ( 96)b	402.3 ( 83)c	323.1 ( 73)d	182.5 ( 94)b	227.4 (88)c	251.4 ( 82)b	256.4 (78)c

Significantly different from control. b:p<0.01. c:p<0.001 (Chi2 test)

d: Statistical analysis was not performed due to the small number of surviving animals

Table 3: Mean food consumption

	Food consumption				Compound consumption
	g/animal/day		mg/kg body weight/day		mg/kg body weight/day
Dose (ppm)	Male	Female	Male	Female	Male	Female
0	18.8	12.9	43.9	50.6	0.0	0.0
75	18.9	12.8	44.3	50.0	3.3	3.8
200	18.9	12.8	44.1	50.9	8.8	10.2
1200	18.9	12.8	45.3	52.9	54.4	63.5
6000	18.4	12.4	46.8	55.8	280.6	334.7

Table 4: Thyroid changes

	Incidence of thyroid follicular cell (FC) changes
Males	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Diffuse FC hyperplasia hypertrophy	0/60 (0)	0/58 (0)	0/60 (0)	23/60 (38)**	58/60 (97)***
Focal FC hyperplasia	3/60 (5)	2/58(3)	2/60 (3)	3/60 (5)	15/60 (25)**
FC adenoma	1/60 (2)	0/58 (0)	0/60 (0)	4/60 (7)	12/60 (20)**
FC adenocarcinoma	0/60 (0)	0/60 (0)	0/60 (0)	0/60 (0)	3/60 (5)
Females	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Diffuse FC hyperplasia hypertrophy	1/60 (2)	1/59 (2)	0/60 (0)	28/60 (47)***	59/60 (98)***
Focal FC hyperplasia	0/60 (0)	1/59 (2)	0/60 (0)	4/60 (7)	8/60 (13)*
FC adenoma	0/60 (0)	0/59 (0)	0/60 (0)	1/60 (2)	2/60 (3)
FC adenocarcinoma	0/60 (0)	0/59 (0)	0/60 (0)	0/60 (0)	0/60 (0)
*p<0.05, **p<0.01, ***p<0.00

 Table 5: Hypertrophy and lipofuscin observed at liver histopathology

	Incidence of liver hypertrophy and lipofuscin
	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Males, 12 months	0/10	0/10	0/10	0/10	5 (moderate) /5**
Males, 24 months	0/37	0/33	0/26	16/27***	1/2a
Females, 12 months	0/10	0/10	0/10	10 (minimal) 10**	10 (mild) /10**
Females. 24 months	0/37	0/37	0/42	25 (minimal) 38***	36 (mild) /39***

*p<0.05, "p -0.01. ***p<0.001

a Statistical analyses were not performed in the high dose group due to low number of animals as a result of mortalities

Table 6: Kidney changes

	Incidence of thyroid follicular cell (FC) changes
Males	0 ppm	75ppm	200 ppm	1200 ppm	6000 ppm
Nephropathy, 12 months	10 (mild)/10	10 (mild)/10	10 (mild)/10	10 (mild)/10	5 (moderate). 5**
Nephropathy, 24 months
Lipofuscin pigmentation, 12 months	0/10	0/10	0/10	0.10	4 (minimal)/5*
Lipofuscin pigmentation, 24 months
Females	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Nephropathy. 12 months
Nephropathy, 24 months
Lipofuscin pigmentation, 12 months
Lipofuscin pigmentation, 24 months

*p<0.05, **p<0.01, ***p< 0.001

Table 7: Terminal organ weights   Organ weights, grams (% change) Dose level (ppm) 0 75 200 1200 6000a Males Liver s.d 12.841 2.521 13.454 2.080 13.811 1.996 16.021 (25)*** 2.984 19.141 (49) 2.299 Thyroid s.d 0.033 0.013 0.034 0.007 0.071 0.188 0.041 (24) *** 0.007 0.320b 0.368 Kidney (right) s.d 1.623 0.160 1.696 0.194 1.752 0.293 1.943(20)*** 0.345 1.901 0.243 Kidney (left) s.d 1.611 0.160 1.655 0.196 1.770 0.265 1.922 (20)*** 0.332 1.852 0.238 Females Liver s.d 7.685 0.860 8.038 1.411 8.271 1.192 9.548 (24) *** 1.652 10.807 (41) *** 1.472 Thyroid s.d 0.022 0.004 0.027 0.014 0.033 0.038 0.028 (27)** 0.006 0.039 (77)*** 0.006 Kidney (right) s.d 1.061 0.092 1.059 0.099 1.093 0.113 1.148 (8)* 0.121 1.216(15)*** 0.127 Kidney (left) s.d 1.062 0.093 1.057 0.090 1.092 0.104 1.128 0.099 1.208(14)*** 0.140

* and ** and *** significantly different compared to control at 0.05, 0.01 and 0.001 level, respectively.

Only organs where significant changes occurred are included in the table and that were considered treatment related.

a Statistical analyses were not performed in the high dose group due to low number of animals (2) as a result of mortalities.

.s.d: standard deviation

b: one value was 0.580, the other was 0.059.

Conclusions:

In males and females rats at dose levels of 1200 or 6000 ppm treatment-related effects were seen which included body weight depression, anemia, accelerated nephropathy, adrenal cortical lipidosis, hepatocellular hypertrophy with an associated increase in serum cholesterol and total protein, decreased T3/T4, increased TSH, and thyroid follicular hyperplasia. A treatment-related increase in organ weights of liver, thyroid and kidneys also occurred. Based upon body weight depression and mortality, the MTD was determined to be 1200 ppm for both sexes. At 6000 ppm, approximately 5 times the MTD, an increase in thyroid follicular cell adenomas was observed in the males. These adenomas were considered to be a secondary effect, related to the treatment-related changes in hormonal homeostasis of the pituitary-thyroid axis. The NOAEL was 200 ppm (8.8 and 10.2 mg/kg bw/day in male and female rats, respectively).

Executive summary:

A study according EPA OPP 83-5 (GLP) and similar to OECD TG 453 was conducted. 60 Fischer 344 rats/sex/dose group were treated via the diet with the test item at dietary concentration of 0, 75, 200, 1200 and 6000 ppm for up to 24 months. The dietary concentrations were equivalent to 0, 3.3, 8.8, 54.4 and 280.6 mg/kg bw/day for males and 0, 3.8, 10.2, 63.5 and 334.7 mg/kg bw/day for females. Mortality, clinical signs, the presence of masses (palpation), body weight, food consumption, ophthalmological signs, endpoints of clinical laboratory investigations (hematology, clinical chemistry including thyroid hormone analysis and determination of Cholinesterase activity, urinalysis), organ weights, gross pathology and histopathology were assessed. After administration for 12 months, 10 rats/sex/dose group were sacrificed, except for males of the 6000 ppm group of which only 5 animals were used due to several premature decedents.

Due to technical reasons, 8 high-dose males were killed in extremis at weeks 11 and 12. These animals showed a fracture of the nasal bone and subsequent dyspnoea (rhinorrhagia). After week 80, the mortality in the 6000 ppm group increased extremely and only two males survived the course of study. The main causes of death noted in males of 6000 ppm group were nephropathy (22 rats), thyroid follicular cell tumours (10 rats) and leukemia (6 rats). The severe nephropathy was associated with hyperplasia of the parathyroid, demineralization of the bone and metastatic calcification in various organs. At this dose level, the MTD was exceeded.

No clinical signs attributable to the test item were noted in any dose groups during the first 52 weeks. After Week 52, dose related clinical signs included pale appearance of skin and mucous membrane (6000 ppm males), alopecia (6000 ppm females), tissue masses on the skin and in the subcutis (1200 and 6000 ppm males).

Dose-related and statistically significant depressions of body weight and weight gain were noted in both males and females of the 1200 and 6000 ppm groups. At termination, the mean body weights of the 1200 and 6000 ppm males were 84 and 73 % of the controls. The mean body weights of the 1200 and 6000 ppm females were 91 and 78 % of the controls, respectively. Thus, > 10% body weight depression was noted in both males and females at 1200 ppm, indicating that this dose was the MTD. The net gains throughout the study were 79 and 63% in males and 88 and 69% in females from the 1200 and 6000 ppm groups, respectively.

Dose-related decreases in food consumption values (g/animal/day) were noted in both sexes at 6000 ppm starting at Week 76. Food consumption per body weight values (g/kg/day) was occasionally higher in both sexes of the 1200 ppm and 6000 ppm groups than those of controls as a result of the lower body weights of these rats.

No dose-related abnormalities were observed in the ophthalmoscopic examinations at months 6, 12, 18 and 24.

Treatment-related anemia (decreases of red blood cell count, hemoglobin, hematocrit, MCV, MCH and MCHC) was noted in both sexes at 6,000 ppm at months 3, 6, 12 and 18. In the 1,200 ppm groups, occasionally statistically significant changes in hemoglobin, MCV and MCH were seen. As these changes, however, occurred only at one or two time points and were seen sometimes in males and sometimes in females, they are not considered to be related to treatment. Increased platelet counts and white blood cell counts were frequently seen in males of the 6,000 ppm group. Differential leukocyte counts remained normal at all test times for all groups but the 6000 ppm males at month 24. For this time point no correct evaluation was possible since only one animal could be investigated.

Blood chemistry revealed several statistically significant and dose related changes. Increases in total cholesterol and total protein, and a decrease in A/G ratio were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and/or 18. Only at month 24, treated males exhibited increased BUN (75, 200 and 1200 ppm groups) and creatinine levels (1200 ppm group). Decreased levels of chloride, potassium and decreased activities of LDH, ALAT, ASAT and CPK were seen predominantly in females of the 1200 and 6000 ppm groups at the 6- and 12-month examinations. In high dose males chloride, ALAT and ASAT were found to be decreased after 12 months. Serum cholinesterase activity was increased in males at the highest dose level at the time intervals 6 and 12 months. No changes were seen after 18 and 24 months. The serum cholinesterase activity in females was decreased at the two highest dose levels at the time intervals 6 and 12 month. No changes were seen after 18 and 24 months. In males of the 6000 ppm group, thyroxine (T4) and triiodothyronine (T3) values were numerically lower than those of the control at month 6, 12 and 18 (not evaluable at month 24). Thyroid stimulating hormone (TSH) values of this group were higher than those of the respective centrals. Similar, but weaker effects on the thyroid hormones and TSH were also found in males of the 1200 ppm and females of the 6000 ppm groups.

A dose-related and statistically significant increase of urinary protein (semi-quantitative analysis) was noted in both sexes of the 6000 ppm group at various test times. By using a qualitative method (nephelometry), protein was also increased in males of the 1200 ppm dose and the 200 ppm dose (the latter only noted at month 24 and lacking a histological correlate). In males of the 6000 ppm group ketone bodies, urinary volume and water consumption were increased, and pH and specific gravity were decreased significantly at various test times.

Dose-related and statistically significant increases in the liver, kidney and thyroid weights and their body weight ratios were noted in both sexes of the 1200 and 6000 ppm groups at both interim and final sacrifice. Other changes in organ weight were noted in high dose males and females and in 1200 ppm males, but these changes are most likely related to the decrease in body weight and thus not considered as a primary toxicological effect of the test substance.

At interim sacrifice, treatment related changes were found in the liver (brownish-black colour) and kidneys (granular surface, brownish-black colour) of the 6000 ppm males and females.

At final sacrifice, males of the 1200 ppm group exhibited granular kidneys. In the 6000 ppm females, kidneys were brownish-black and the thyroids were enlarged.

Animals which died during the study also had treatment related changes. At 6,000 ppm, swelling of the thyroid (both sexes) and granular kidney (male) were noted.

Microscopic examinations were performed on all rats which died on study or were killed at interim and final sacrifices. An independent peer review of relevant microscopic findings was conducted. Dose-related changes were found in the thyroid, liver, kidney and adrenals.

Thyroid: Follicular cell hyperplasia and hypertrophy were noted in both sexes from the 1200 and/or 6000 ppm groups at months 12 and 24. In addition, an increase of focal follicular cell hyperplasia was observed in the 6000 ppm males and females. In males, the incidence of thyroid follicular cell adenoma was increased at 1200 ppm and above, but reached statistical significance only at 6000 ppm.

Liver: Centrilobular hepatocellular hypertrophy and occurrence of lipofuscin were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and 24. These changes were also seen in majority of 6000 ppm males and females which died on study.

Adrenals: Lipidosis of the adrenal cortex was noted in females of the 1200 ppm group and in both sexes at 6000 ppm at month 12. However, no treatment related incidence of this finding was seen in males and females which died on study or were terminally sacrificed.

Kidney: The severity of nephropathy was increased in both sexes of the 6000 ppm group compared to controls at month 12 and in both sexes of the 1200 and 6000 ppm groups at month 24. The lesions were more severe in males than in females and grew worse with increasing dose and time. For many animals affected in that way, the renal failure was considered to be the main lesion/cause of death. Microscopic evidence of renal failure in these rats was accompanied by hyperplasia of the parathyroid, demineralization of the bone and metastatic calcification in various organs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPS Guideline 83-5

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

survival rate in high-dose male group was less than required, excessive mortality was partly due to accidental deaths but was also clearly substance related indicating that MTD was exceeded; survival rate in other three groups corresponded to guideline

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiophanate-methyl

EC Number:

245-740-7

EC Name:

Thiophanate-methyl

Cas Number:

23564-05-8

Molecular formula:

C12H14N4O4S2

IUPAC Name:

methyl N-{[2-({[(methoxycarbonyl)amino]methanethioyl}amino)phenyl]carbamothioyl}carbamate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Atsugi, Kanagawa
- Age at study initiation: 6 weeks
- Weight at study initiation: mean 121g (males); mean 99 g (females)
- Housing: housed individually in a stainless steel mesh cage
- Diet: ad libitum, basal diet (Diet M®, purchased from Oriental Yeast Co., Ltd., Tokyo, Japan
- Water: ad libitum, tap water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 40-80
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test-item was ground and incorporated with the basal diet (M diet, purchased from Oriental Yeast Co., Ltd., Tokyo) to make separate premixes for each dietary concentration. Each premix was mixed with the basal diet by using a blender (SS-161, Kanto Kongoki, Tokyo). The diets were prepared monthly and stored at about -20 °C until used. Diet samples were taken from all dose levels, and homogeneity and concentration analysis were performed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dietary mean concentration values ranged from 94.1 to 110.9% of the nominal concentration.

Duration of treatment / exposure:

52 weeks (chronic study)
104 weeks (carcinogenicity study)

Frequency of treatment:

continously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 ppm

Doses / concentrations 2

Dose / conc.:

200 ppm

Doses / concentrations 3

Dose / conc.:

1 200 ppm

Doses / concentrations 4

Dose / conc.:

6 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale:
The sub-chronic study in Fischer rats (Nishibe and Takaori, 1990), which had been conducted primarily as a range-finding study, revealed apparent toxicity at 2200 ppm and higher doses (4200, 6200 and 8200 ppm). Anemia, hepatocellular hypertrophy accompanied by lipofuscin deposition, increased severity of nephropathy accompanied by hyaline droplets in the glomeruli, and thyroid follicular hypertrophy and hyperplasia were noted at 2200 ppm and higher doses. No body weight depression was noted at 8200 ppm, the highest dose employed in the sub-chronic toxicity study. It was estimated that 6000 ppm could be the maximum tolerated dose (MTD) for a study of 2 years duration.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, morbidity and overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through week 14, then at week 16 and once every four weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- recorded weekly through Week 14, then at Week IB and once every four weeks thereafter
Once each week, food consumption was determined for a 24-hour period for each rat. The following formulas were used to calculate food consumption values (g/kg body weight/day), food efficiency values (%) and compound consumption values (mg/kg/day). Food consumption value=[Food consumed(g/day)]/[Body weight(kg)]

FOOD EFFICIENCY:
- Food efficiency value =[Body weight gain(g/day)]/[Food consumed (g/day)] x 100

WATER CONSUMPTION AND COMPOUND INTAKE: At Months 18 and 24, water consumption was measured, because a increased urinary volume was noted in males of the 6000 ppm group at Month 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
- performed on all animals prior to the study initiation and at Months 12 and 24 of the study. At Months 6 and 18, all animals from the control and the highest dose (6000 ppm) groups were also examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At Months 3, 6, 12, 18 and 24 of the study, blood was taken from the retro-orbital sinus
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 10 rats/sex/group
- Parameters checked: Erythrocyte count (RBC), Leukocyte count (WBC),Hemoglobin (Hb), Platelet count (Pla), Hematocrit (PCV), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration) were calculated. Differential leukocyte count was conducted on the control and the highest dose groups. The slides from the other groups were only examined at Months 12, 18 and 24

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At Months 6, 12, 18 and 24 of the study
- Animals fasted: Yes
- How many animals: 10 rats/sex/group
- Parameters checked: Alkaline phosphatase (ALP), Aspartate aminotransferase (GOT), Alanine aminotransferase (GPT), Lactic dehydrogenase (LDH), Creatinine phosphokinase (CPK), Cholinesterase (Ch-E), Glucose (Glu), Total bilirubin (T-Bil), Total cholesterol (T-Cho), Creatinine (Crea), A/G ratio, Total protein (T-Pro), Albumin (Alb), Urea nitrogen (BUN), Inorganic phosphate (P), Calcium, Potassium and Sodium, Chloride, Thyroxine (T-4) and Triiodothyronine (T-3), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: At Months 6, 12, 18 and 24 of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: occult blood, protein, ketone, glucose, bilirubin and urobilinogen, specific gravity, pH, volume, appearance, sediment, protein

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

A complete necropsy was performed on all rats dying spontaneously, sacrificed in extremis or sacrificed at the interim and final necropsies. After administration for 52 weeks, 10 rats/sex/group were fasted for about 16 hours and weighted, anesthetized with sodium pentobarbital and sacrificed by exsanguination. The number of high dose (6000 ppm) males for interim sacrifice was reduced to 5 rats, because 8 rats of this group died at Weeks 11 or 12. After Week 104, all survivals were fasted for about 16 hours and weighted, killed by inhalation with chloroform, and examined for macroscopic lesions. The rats sacrificed in extremis were not fasted prior to sacrifice, and were also killed by chloroform inhalation.

GROSS PATHOLOGY: Yes
The following tissues from each animal were preserved in 10% neutral buffered formalin.
Accessory genital organs: Uterus, Vagina, Oviduct and Clitoral gland Prostate, Seminal vesicle, Coagulating gland, Preputial gland), Adrenal, Aorta, Bone and marrow (Sternum and Femur), Brain (Medulla/Pons, Cerebrum and Cerebellum), Optic nerve, Eye and Harderian gland, Gastro intestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum) Gonad (Ovary and Testis, Epididymis) Heart, Joint (knee), Kidney, Liver, Lung, Lymph node (mesenteric, mandibular), Mammary gland, Musculature (thigh), Pancreas, Pituitary, Salivary glands (Mandibular, sublingual, parotid), Sciatic nerve, Skin, Spinal cord (3 levels), Spleen, Thymic region, Thyroid and Parathyroid, Trachea, Urinary bladder (inflated with formalin), Unusual lesions, Regional lymph node (when applicable)
The following organs from each animal were weighed: Brain, Liver, Adrenals,Thyroid/Parathyroids, Heart, Lung, Kidneys, Pituitary,Testes, Spleen, Ovaries

HISTOPATHOLOGY: Yes
The organs and tissues, which were listed were fixed in buffered 10% formalin, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The following histopathology was performed:
(a) :All organs in all animals sacrificed at interim necropsy.
(b) :All organs in all animals in the control and 6000 ppm groups, and all males in the 1200 ppm group.
(c) :All organs of all animals that died or were killed during the study.
(d) :Lung, liver, kidney, thyroid, parathyroid, pituitary, adrenal and gross lesions of all remaining animals.
A five-step grading system (from 0 to 4) was used to define lesions. Tumors were classified as benign, malignant or metastatic.

Other examinations:

MECHANISTIC STUDY
The study also contained mechanistic investigations. The mechanistic investigations were performed on Fischer 344 rats or ICR mice (both Charles River, Japan) with the substance TPM (same purity and batch as in the main study, dose level 6000 ppm) and with the reference substances phenobarbital (500 ppm) and propylthiouracil (1000 ppm). The test item and phenobarbital were given by diet, propylthiouracil was given in distilled water.
TPM or the reference substances were given to male and female rats (6 weeks old) for 2 or 8 days (males) and 8 days (females). Measurement of T3, T4 and TSH in serum, and determination of drug intake, liver and thyroid weights and total cholesterol in serum were performed. Measurement of thyroid weight was performed after a recovery period of 8 days. Additional administration of TPM to male rats (8 weeks old) was performed for 8 days with daily T4 supplementation (30 μg/kg bw subcutaneously injected). Isolation of microsome fraction of each liver were performed, including measurements of enzyme induction of the following enzymes: cytochrome P450, cytochrome b5, NADPH-cytochrome c reductase, UDP-glucuronosyltransferase) and microsomal protein. Isolation of microsome fraction of porcine thyroids was performed, purchased from a butchery. Measurement of peroxidase activity in the presence of TPM (10E-3 M to 10E-4 M) or propylthiouracil (10E-4 M to 10E-6M) was performed.

Materials: For feeding study, TPM and phenobarbital were incorporated in basal food (M diet, ORIENTAL YEAST Inc., Tokyo) at 6000 ppm and 500 ppm, respectively. Propylthiouracil (PTU, Tokyo kasei Co, Ltd., Purity :over 99%) was dissolved in distilled water at 1000 ppm.
Animals and housing: F 344 rats and ICR mice (Charles River Breeding Laboratories, Atugi, Kanagawa, Japan) were used. Each group consisted of 5 animals, except notified. The environmental conditions of the animals were maintained temperature at 21-24°C, humidity at 40-80%, air exchange at 10 times an hour and 12-hr light-dark cycle.
Sampling: All rats and mice were not restricted food before sampling of blood and necropsy. Blood was taken under pentobarbital sodium anesthesia, and serum was used for analysis. Porcine thyroids, purchased from a butchery, were stored at -80"C until preparation of microsome.
Statistical analysis: All data are presented as mean value ± SD and the Student's t-test and Mann-Whitney U-test were used to detect significance (*:p<0.05, **:p<0.01, ***:p<0.001) between control and dosed groups.

Statistics:

Mortality, clinical observation, ophthalmoscopic examination and macroscopic observation data were analyzed by the chi-square test. When the number of animals were below 5/group, Fisher's exact test (two-tailed) was used to access significance between control and dose groups. Comparisons of all data collected at each interval on body weights, food consumption values, clinical laboratory values and organ weights (absolute and relative to body weights) were performed using Bartlett's test for homogeneity of variances. If variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test or Scheffe's test was used to determine which means were significantly different from control. If a non-parametric procedure for testing equality of means were needed, the Kruskal-Wallis test were used, and if differences were indicated, Dunnett-type test or Scheffe-type test was used to determine which means were significantly different from the control (Gad and Weil, 1982).
The graded data from urinalysis were analyzed by the Mann-Whitney test. The microscopic observation data were analyzed by the Mann-Whitney test (non-neoplastic lesions) or by the chi-square test (neoplastic lesions). When the number of animals were below 5/group, Fisher's exact test (two-tailed) was used to access significance between control and dose groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males, the incidence of pale discoloration of skin and mucous membranes was significantly higher in the 6000 ppm group than control from Week 77 to study termination. Tissue masses in the subcutaneous tissue, including the lip, were found more frequently at 1200 ppm from Weeks 95 to 101. Tissue masses on the skin were also slightly increased in the 1200 ppm group at Weeks 91 and 92, and in the 6000 ppm group at Week 91. In females, no increases in pale skin coloration or masses were noted.
In females, the incidence of alopecia at Week 52 was 2/60, 0/60, 4/60, 5/60 and 7/59 at the doses of 0, 75, 200, 1200 and 6000 ppm, respectively. Statistical significance was attained in the 6000 ppm group from Weeks 79 to 104. No increase in alopecia was seen in treated males.

Mortality:

mortality observed, treatment-related

Description (incidence):

Dose-related and accidental deaths occurred in males of the 6000 ppm group. Eight males of this group were killed in extremis or found dead at Weeks 11 or 12. The cause of death was fracture of the nasal bone and subsequent dyspnea (rhinorrhagia). All cages and feeders for males had been changed to a larger type to accommodate the increased size of growing males. The new feeder was equipped with a stainless plate through which some holes had been punched. Because the deaths occurred after changing the feeders, it is considered that the fractures were related to the feeder plate. The plates were removed after 3 days and no accidental deaths were noted thereafter. After Week 80, the mortality of the high-dose males increased significantly, reaching 96% at Week 104. No dose related effect on mortality was noted in females (for more information please see table 1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose-related and statistically significant growth depression and/or weight loss were noted in both sexes of the 1200 and 6000 ppm groups. Although rats typically lose weight, as a group, near the end of a 2-year study, the 6000 ppm males began losing weight by Week 52 in this study, as shown in Table 2. At termination, the mean body weights of the 1200 and 6000 ppm males were 84 and 73 % of the controls, respectively. The net gains throughout the study were 256.7 g (79 %) and 204.5 g (63 %) in the 1200 and 6000 ppm males, while that of the control males was 324.8 g. In females, body weight depressions were observed at 1200 and 6000 ppm, and were statistical significant after Week 2 in the 6000 ppm group and between Weeks 20 and 52 in the 1200 ppm group. At termination, the mean body weights of the females were 91 and 78% in the 1200 and 6000 ppm groups compared to controls. The net gains throughout the study were 199.4 g (88 %) and 156.5 g (69 %) in the 1200 and 6000 ppm females, while that of the control females was 227.8 g. Based on the > 10 % body weight depressions in the 1200 ppm group, it was determined that 1200 ppm was the Maximum Tolerated Dose (MTD). The body weight changes in the 75 and 200 ppm groups were comparable to those of the control groups (both sexes).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

As shown in Table 3, at Week 1, food consumption (g/animal/day and g/kg body weight/day) was decreased in males of 75 and 6000 ppm groups (p<0.01) and in females of all dosed groups (p<0.01). At Week 2, high dose females ate less food than control.
The males and females from 6000 ppm group ate slightly less food than controls at Week 78 and thereafter, with statistical significance occasionally attained. For much of this 2-year study, however, the food consumed per animal per day was similar to sex-matched controls, regardless of dose level. Due to the lower body weights of the 1200 and 6000 ppm rats, food consumption values (g/kg body weight/day) of these rats were significantly higher than controls at various time points (see table 3).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency of the 6000 ppm males and females were slightly lower than controls at several test times during the study.

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All rats placed on study had normal ophthalmoscopy.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related anaemia (decreases of red blood cell count, haemoglobin, haematocrit, MCV, MCH and MCHC) was noted in both sexes at 6000 ppm at months 3, 6, 12 and 18. In the 1200 ppm groups, occasionally statistically significant changes in haemoglobin, MCV and MCH were seen. As these changes, however, occurred only at one or two time points and were seen sometimes in males and sometimes in females, they are not considered to be related to treatment. Increased platelet counts and white blood cell counts were frequently seen in males of the 6000 ppm group. Differential leukocyte counts remained normal at all test times for all groups but the 6000 ppm males at month 24. For this time point no correct evaluation was possible since only one animal could be investigated.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry revealed several statistically significant and dose related changes. Increases in total cholesterol and total protein, and a decrease in A/G ratio were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and/or 18. Only at month 24, treated males exhibited increased BUN (75, 200 and 1200 ppm groups) and creatinine levels (1200 ppm group). Decreased levels of chloride, potassium and decreased activities of LDH, ALAT, ASAT and CPK were seen predominantly in females of the 1200 and 6000 ppm groups at the 6- and 12-month examinations. In high dose males chloride, ALAT and ASAT were found to be decreased after 12 months. Serum cholinesterase activity was increased in males at the highest dose level at the time intervals 6 and 12 months. No changes were seen after 18 and 24 months. The serum cholinesterase activity in females was decreased at the two highest dose levels at the time intervals 6 and 12 month. No changes were seen after 18 and 24 months. In males of the 6000 ppm group, thyroxine (T4) and triiodothyronine (T3) values were numerically lower than those of the control at month 6, 12 and 18 (not evaluable at month 24). Thyroid stimulating hormone (TSH) values of this group were higher than those of the respective centrals. Similar, but weaker effects on the thyroid hormones and TSH were also found in males of the 1200 ppm and females of the 6000 ppm groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A dose-related and statistically significant increase of urinary protein (semi-quantitative analysis) was noted in both sexes of the 6000 ppm group at various test times. By using a qualitative method (nephelometry), protein was also increased in males of the 1200 ppm dose and the 200 ppm dose (the latter only noted at month 24 and lacking a histological correlate). In males of the 6000 ppm group ketone bodies, urinary volume and water consumption were increased, and pH and specific gravity were decreased significantly at various test times.

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):

Dose-related and statistically significant increases in the liver, kidney and thyroid weights and their body weight ratios were noted in both sexes of the 1200 and 6000 ppm groups at both interim and final sacrifice. Other changes in organ weight were noted in high dose males and females and in 1200 ppm males, but these changes are most likely related to the decrease in body weight and thus not considered as a primary toxicological effect of the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At interim sacrifice, treatment related changes were found in the liver (brownish-black colour) and kidneys (granular surface, brownish-black colour) of the 6000 ppm males and females. At final sacrifice, males of the 1200 ppm group exhibited granular kidneys. In the 6000 ppm females, kidneys were brownish-black and the thyroids were enlarged. Animals which died during the study also had treatment related changes. At 6000 ppm, swelling of the thyroid (both sexes) and granular kidney (male) were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examinations were performed on all rats which died on study or were killed at interim and final sacrifices. An independent peer review of relevant microscopic findings was conducted. Dose-related changes were found in the thyroid, liver, kidney and adrenals.
Interim:
Thyroid: A treatment-related change, follicular hyperplasia and hypertrophy, was found in both sexes of the 6000 ppm group. Morphologically, the follicles were small and irregular, composed of cuboidal to columnar cells, and deficient in colloid (grade 2). At 1200 ppm, the lesion consisted of slightly irregular follicles, cuboidal to columnar epithelial cells, and had weakly stained colloid (grade 1). No follicular hyperplasia was noted at 200 and 75 ppm in either sex. Focal hyperplasia of follicular cells was found in 2 females from 6000 ppm group. This change was also considered to be related to compound administration.
Liver: The only lesions in the liver which were interpreted to have been related to the test compound were hepatocellular hypertrophy and deposition of lipofuscin pigment. The hypertrophy was centrilobular (grade 1), centrilobular and mid zonal (grade 2) or all lobules (grade 3). The amount of lipofuscin pigment increased related to the severity of hypertrophy. These changes were found in both sexes of the 1200 and 6000 ppm groups. Other liver lesions, such as foci of cellular alteration were seen microscopically, but were not considered to be related to treatment. Foci, when seen, were classified according to Eustis et al., (1990) into vacuolated, eosinophilic, basophilic, clear or mixed. The number of foci per 2 sections (2 different lobes) for each rat was counted and graded (grade 1: <5 foci, grade 2: 5 - 10 foci).
Adrenal cortex: Cytoplasmic lipid in adrenal cortical cells is a normal finding. In this study, a slightly increased amount of lipid was seen in 1200 ppm interim females and in both sexes at 6000 ppm.
Kidney. Nephropathy, which is an age-related progressive change in the rat, appeared to be accentuated in 6000 ppm males. Morphologically, there were numerous protein casts in the corticomedullary junction with interstitial fibrosis, regenerative tubules, thickened tubular basement membrane and glomerular lesions (crescents, thickened basement membranes). When 25 to 50 % of the cortex was affected, these lesions were graded 3 (moderate). Mild change, which was noted in females from 6000 ppm and males from the control and other dosed groups, consisted of several to many protein casts and less than 25% of superficial cortex area affected. A minimal lesion (grade 1) was found mostly in females. Here, there were only a few or no casts. However, slight glomerular lesions, such as crescents or thick basement membranes were present.
Although pale skin color had been seen clinically, and the clinical pathology data indicated mild anemia, the bone marrow was free of detectable treatment-related lesions.
Numerous other lesions were noted microscopically, but all were unrelated to dose, and were considered to be spontaneous changes.
Final sacrifice:
Microscopic changes interpreted to have a relationship to treatment were noted in the skin (females), thyroid, liver and kidney.
Skin: A dose-related lesion, atrophy of hair follicles, was seen in female rats which had bilateral alopecia noted clinically. In females of the 6000 ppm group, the incidence of this lesion was significantly increased. No significant change was noted in males.
Thyroid: A dose-related and statistically significant increase in diffuse thyroid follicular cell hyperplasia and hypertrophy was noted at Month 24 in both sexes of the 1200 and 6000 ppm groups. The incidence of focal (adenomatous) hyperplasia of follicular cells was slightly, but not statistically significantly, increased in females of the 1200 and 6000 ppm groups. In males, no dose-related increase of focal hyperplasia was detected.
Liver: As seen in 12 month interim rats, the livers of 1200 and 6000 ppm rats (both sexes) had compound-related hepatocellular hypertrophy with deposition of lipofuscin pigment. The incidences of altered foci (vacuolated, clear, eosinophilic, basophilic and mixed) were not changed among groups, except for basophilic foci, which were decreased in females from 6000 ppm group. The incidences of multiple focal hyperplasia, which were usually seen in rats with mononuclear cell leukemia, were not changed among groups.
Kidney: The severity of nephropathy was increased in both sexes of 1200 and 6000 ppm groups. Statistical significance was attained only in females. A small amount of lipofuscin pigment was found in the tubular epithelium, especially in the proximal tubules of males and females from the 6000 ppm group.
Adrenal cortex: Increased adrenal cortical lipid, seen microscopically in dose-related fashion at Month 12, had no relation to dose at Month 24. Focal hyperplasia in the adrenal cortex was a common finding in all groups, with no statistical significance in the incidences among treated rats.
The spleen and bone marrow remained free of detectable dose-related alterations, even in the presence of mild anemia which had been noted in hematological tests. Numerous other miscellaneous non-neoplastic lesions were observed and are recorded in the microscopic data. None of these lesions were distributed in a manner which suggested a relationship to treatment, and are interpreted to be spontaneous age or disease-related changes.
Animals found dead or sacrificed in extremis:
Eight rats died or were sacrificed in extremis in Week 11 or 12. The clinical observation indicated fracture of the nasal bone, and this finding was confirmed by histopathological examination (fracture of nasal bone accompanied by bleeding into nasal turbinate and meatus). Four of the eight rats had eosinophilic crystals in the lungs, probably of hemoglobin. All eight rats had centrilobular necrosis of the liver, indicating acute hypovolemic shock. These were clearly differentiated from drug-induced lesions (hepatocellular hypertrophy). Treatment-related changes were found in the liver, kidney and thyroid.
Liver: Hepatocellular hypertrophy with lipofuscin deposition was found frequently in males and females of the 6000 ppm group. Males of the 6000 ppm group frequently also had microgranulomas and focal fatty degeneration (Greaves 1984), interpreted to be degenerative/inflammatory changes related to age and debilitation, rather than due to treatment.
Kidney: The severity (grade 4: >50 % of kidney affected) of nephropathy was extreme in the males of the 6000 ppm group, and was sometimes accompanied by hypertrophy of the parathyroid, with resorption of bone and metastatic calcification in the aorta (medial calcification :Mc), lung (calcium deposition :Ca), heart (Ca) and stomach (Ca).The end-stage renal lesion was interpreted to be the cause of death for many males of the 6000 ppm group. In females, nephropathy was not as severe as in males, even at the level of 6000 ppm.
Thyroid: Diffuse hypertrophy and hyperplasia of follicular cells were noted in females of the 1200 ppm and in both sexes of the 6000 ppm groups. Although not statistically different, focal hyperplasia of follicular cells was frequently found in males of the 6000 ppm group.
Other non-neoplastic changes noted in unscheduled-death rats were incidental to treatment.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim: Only one tumor was found at the interim sacrifice which could be linked to a treatment-related effect. This was a thyroid follicular cell adenoma, seen in a male from the 1200 ppm group.
Final Sacrifice: Tumors were found in various organs. However, there was neither statistically significant nor dose-related increases of any tumor incidences at final sacrifice. Only 3 tumors were seen with possible relationship to treatment. These were thyroid follicular cell adenomas in one 1200 ppm male and one 6000 ppm female, and a thyroid follicular cell adenocarcinoma in a 6000 ppm male.
Animals found dead or sacrificed in extremis: No tumors were found in unscheduled-death rats which could be related to treatment, with the exception of thyroid follicular cell (FC) neoplasms.

Description (incidence and severity):

MECHANISTIC STUDY:
Thiophanate-methyl caused decreases in T4 and T3 1evels and increases in TSH level and thyroid weights on days 2 and/or 8. The liver weight and amount of total cholesterol in serum were also increased. There was a close correlation between TSH level and thyroid weight. The T4 supplementation counteracted the hypertrophy of the thyroid and the TSH response, indicating that thiophanate-methyl caused the hypertrophy by negative feedback mechanism. The T4 supplementation had no influence on the increased liver weight and total cholesterol level. The effect of thiophanate-methyl on the thyroid was reversible after a recovery period without administration of the test substance.Thiophanate-methyl inhibited the thyroid peroxidase (30 fold weaker than propylthiouracil). Thiophanate-methyl caused hypertrophy of the liver, accompanied by a proliferation of liver cells which was observable in rats only after 2 days but in mice after 2 and 8 days.
Thiophanate-methyl induced cytochrome P 450 and related drug metabolising enzymes. One of them, the UDP-glucuronosyltransferase plays an important role in the clearance of T4 in the liver. UDP-glucuronosyltransferase was raised by 335% (20.6 +/-3.9 in control and 69.2 +/- 19.5 nmol/min/ng microsomal protein in the 6000 ppm dose group). Phenobarbital which also induced liver cell proliferation and the drug metabolising enzymes revealed only a very slight increase of TSH without thyroid hypertrophy.
Thus, the inhibition of hormone synthesis in the thyroid due to an inhibition of peroxidase could be the main cause of the T4 depression.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Mortality (%)

	Male			Female
Dose (ppm)	wk 52	wk 80	wk 104	wk 52	wk 80	wk 104
0	0/60 (0)	2/50( 4)	13/50(26)	0/60 (0)	3/50 (6)	13/50 (26)
75	0/60 (0)	2/50( 4)	15/50(30)	0/60 (0)	1/50 (2)	12/50 (24)
200	0/60 (0)	8/50(16)	24/50(48)a	0/60 (0)	1/50 (2)	8/50 (16)
1200	0/60 (0)	3/50( 6)	21/50(42)	0/60 (0)	0/50 (0)	12/50 (24)
6000	8/60(13) a	18/55(33)c	53/55(96)c	1/60 (2)	3/60 (6)	11/50 (22)

Significantly different from control, a: p<0.05, c: p<0.001 (Chi-square test)

Table 2: Mean body weights (g) and ratios to control (%)

	Male				Female
Dose (ppm)	Week 13	Week 42	Week 80	Week 104	Week 13	Week 52	Week 80	Week 104
0	353.8 (100)	471.9 (100)	454.5 (100)	445.6 (100)	193.6 (100)	258.1 (100)	306.7 (100)	327.9 (100)
75	353.2 (100)	473.7 (100)	480.6 ( 99)	432.4 ( 97)	194.5 (100)	263.8 (102)	312.3 (102)	327.5 (100)
200	354.7 (100)	476.4 (101)	481.5 ( 99)	438.9 ( 98)	194.1 (100)	258.8 (100)	305.7 (100)	316.6 ( 97)
1250	350.6 ( 99)	469.2 ( 99)	467.1 ( S5)	376.3 ( 84)c	190.3 ( 98)	248.4 (96)b	291.4 ( 95)	299.6 ( 91)
6000	353.6 (100)	451.4 ( 96)b	402.3 ( 83)c	323.1 ( 73)d	182.5 ( 94)b	227.4 (88)c	251.4 ( 82)b	256.4 (78)c

Significantly different from control. b:p<0.01. c:p<0.001 (Chi2 test)

d: Statistical analysis was not performed due to the small number of surviving animals

Table 3: Mean food consumption

	Food consumption				Compound consumption
	g/animal/day		mg/kg body weight/day		mg/kg body weight/day
Dose (ppm)	Male	Female	Male	Female	Male	Female
0	18.8	12.9	43.9	50.6	0.0	0.0
75	18.9	12.8	44.3	50.0	3.3	3.8
200	18.9	12.8	44.1	50.9	8.8	10.2
1200	18.9	12.8	45.3	52.9	54.4	63.5
6000	18.4	12.4	46.8	55.8	280.6	334.7

Table 4: Thyroid changes

	Incidence of thyroid follicular cell (FC) changes
Males	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Diffuse FC hyperplasia hypertrophy	0/60 (0)	0/58 (0)	0/60 (0)	23/60 (38)**	58/60 (97)***
Focal FC hyperplasia	3/60 (5)	2/58(3)	2/60 (3)	3/60 (5)	15/60 (25)**
FC adenoma	1/60 (2)	0/58 (0)	0/60 (0)	4/60 (7)	12/60 (20)**
FC adenocarcinoma	0/60 (0)	0/60 (0)	0/60 (0)	0/60 (0)	3/60 (5)
Females	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Diffuse FC hyperplasia hypertrophy	1/60 (2)	1/59 (2)	0/60 (0)	28/60 (47)***	59/60 (98)***
Focal FC hyperplasia	0/60 (0)	1/59 (2)	0/60 (0)	4/60 (7)	8/60 (13)*
FC adenoma	0/60 (0)	0/59 (0)	0/60 (0)	1/60 (2)	2/60 (3)
FC adenocarcinoma	0/60 (0)	0/59 (0)	0/60 (0)	0/60 (0)	0/60 (0)
*p<0.05, **p<0.01, ***p<0.00

 

Table 5: Hypertrophy and lipofuscin observed at liver histopathology

	Incidence of liver hypertrophy and lipofuscin
	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Males, 12 months	0/10	0/10	0/10	0/10	5 (moderate) /5**
Males, 24 months	0/37	0/33	0/26	16/27***	1/2a
Females, 12 months	0/10	0/10	0/10	10 (minimal) 10**	10 (mild) /10**
Females. 24 months	0/37	0/37	0/42	25 (minimal) 38***	36 (mild) /39***

*p<0.05, "p -0.01. ***p<0.001

a Statistical analyses were not performed in the high dose group due to low number of animals as a result of mortalities

 

Table 6: Kidney changes

	Incidence of thyroid follicular cell (FC) changes
Males	0 ppm	75ppm	200 ppm	1200 ppm	6000 ppm
Nephropathy, 12 months	10 (mild)/10	10 (mild)/10	10 (mild)/10	10 (mild)/10	5 (moderate). 5**
Nephropathy, 24 months
Lipofuscin pigmentation, 12 months	0/10	0/10	0/10	0.10	4 (minimal)/5*
Lipofuscin pigmentation, 24 months
Females	0 ppm	75 ppm	200 ppm	1200 ppm	6000 ppm
Nephropathy. 12 months
Nephropathy, 24 months
Lipofuscin pigmentation, 12 months
Lipofuscin pigmentation, 24 months

*p<0.05, **p<0.01, ***p< 0.001

Table 7: Terminal organ weights   Organ weights, grams (% change) Dose level (ppm) 0 75 200 1200 6000a Males Liver s.d 12.841 2.521 13.454 2.080 13.811 1.996 16.021 (25)*** 2.984 19.141 (49) 2.299 Thyroid s.d 0.033 0.013 0.034 0.007 0.071 0.188 0.041 (24) *** 0.007 0.320b 0.368 Kidney (right) s.d 1.623 0.160 1.696 0.194 1.752 0.293 1.943(20)*** 0.345 1.901 0.243 Kidney (left) s.d 1.611 0.160 1.655 0.196 1.770 0.265 1.922 (20)*** 0.332 1.852 0.238 Females Liver s.d 7.685 0.860 8.038 1.411 8.271 1.192 9.548 (24) *** 1.652 10.807 (41) *** 1.472 Thyroid s.d 0.022 0.004 0.027 0.014 0.033 0.038 0.028 (27)** 0.006 0.039 (77)*** 0.006 Kidney (right) s.d 1.061 0.092 1.059 0.099 1.093 0.113 1.148 (8)* 0.121 1.216(15)*** 0.127 Kidney (left) s.d 1.062 0.093 1.057 0.090 1.092 0.104 1.128 0.099 1.208(14)*** 0.140

* and ** and *** significantly different compared to control at 0.05, 0.01 and 0.001 level, respectively.

Only organs where significant changes occurred are included in the table and that were considered treatment related.

a Statistical analyses were not performed in the high dose group due to low number of animals (2) as a result of mortalities.

.s.d: standard deviation

b: one value was 0.580, the other was 0.059.

Applicant's summary and conclusion

Conclusions:

In males and females rats at dose levels of 1200 or 6000 ppm treatment-related effects were seen which included body weight depression, anemia, accelerated nephropathy, adrenal cortical lipidosis, hepatocellular hypertrophy with an associated increase in serum cholesterol and total protein, decreased T3/T4, increased TSH, and thyroid follicular hyperplasia. A treatment-related increase in organ weights of liver, thyroid and kidneys also occurred. Based upon body weight depression and mortality, the MTD was determined to be 1200 ppm for both sexes. At 6000 ppm, approximately 5 times the MTD, an increase in thyroid follicular cell adenomas was observed in the males. These adenomas were considered to be a secondary effect, related to the treatment-related changes in hormonal homeostasis of the pituitary-thyroid axis. The NOAEL was 200 ppm (8.8 and 10.2 mg/kg bw/day in male and female rats, respectively).

Executive summary:

A study according EPA OPP 83-5 (GLP) and similar to OECD TG 453 was conducted. Fischer 344 rats/sex/dose group were treated via the diet with the test item at dietary concentration of 0, 75, 200, 1200 and 6000 ppm for up to 24 months. The dietary concentrations were equivalent to 0, 3.3, 8.8, 54.4 and 280.6 mg/kg bw/day for males and 0, 3.8, 10.2, 63.5 and 334.7 mg/kg bw/day for females. Mortality, clinical signs, the presence of masses (palpation), body weight, food consumption, ophthalmological signs, endpoints of clinical laboratory investigations (hematology, clinical chemistry including thyroid hormone analysis and determination of Cholinesterase activity, urinalysis), organ weights, gross pathology and histopathology were assessed. After administration for 12 months, 10 rats/sex/dose group were sacrificed, except for males of the 6000 ppm group of which only 5 animals were used due to several premature decedents.

Due to technical reasons, 8 high-dose males were killed in extremis at weeks 11 and 12. These animals showed a fracture of the nasal bone and subsequent dyspnoea (rhinorrhagia). After week 80, the mortality in the 6000 ppm group increased extremely and only two males survived the course of study. The main causes of death noted in males of 6000 ppm group were nephropathy (22 rats), thyroid follicular cell tumours (10 rats) and leukemia (6 rats). The severe nephropathy was associated with hyperplasia of the parathyroid, demineralization of the bone and metastatic calcification in various organs. At this dose level, the MTD was exceeded.

No clinical signs attributable to the test item were noted in any dose groups during the first 52 weeks. After Week 52, dose related clinical signs included pale appearance of skin and mucous membrane (6000 ppm males), alopecia (6000 ppm females), tissue masses on the skin and in the subcutis (1200 and 6000 ppm males).

Dose-related and statistically significant depressions of body weight and weight gain were noted in both males and females of the 1200 and 6000 ppm groups. At termination, the mean body weights of the 1200 and 6000 ppm males were 84 and 73 % of the controls. The mean body weights of the 1200 and 6000 ppm females were 91 and 78 % of the controls, respectively. Thus, > 10% body weight depression was noted in both males and females at 1200 ppm, indicating that this dose was the MTD. The net gains throughout the study were 79 and 63% in males and 88 and 69% in females from the 1200 and 6000 ppm groups, respectively.

Dose-related decreases in food consumption values (g/animal/day) were noted in both sexes at 6000 ppm starting at Week 76. Food consumption per body weight values (g/kg/day) was occasionally higher in both sexes of the 1200 ppm and 6000 ppm groups than those of controls as a result of the lower body weights of these rats.

No dose-related abnormalities were observed in the ophthalmoscopic examinations at months 6, 12, 18 and 24.

Treatment-related anemia (decreases of red blood cell count, hemoglobin, hematocrit, MCV, MCH and MCHC) was noted in both sexes at 6,000 ppm at months 3, 6, 12 and 18. In the 1,200 ppm groups, occasionally statistically significant changes in hemoglobin, MCV and MCH were seen. As these changes, however, occurred only at one or two time points and were seen sometimes in males and sometimes in females, they are not considered to be related to treatment. Increased platelet counts and white blood cell counts were frequently seen in males of the 6,000 ppm group. Differential leukocyte counts remained normal at all test times for all groups but the 6000 ppm males at month 24. For this time point no correct evaluation was possible since only one animal could be investigated.

Blood chemistry revealed several statistically significant and dose related changes. Increases in total cholesterol and total protein, and a decrease in A/G ratio were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and/or 18. Only at month 24, treated males exhibited increased BUN (75, 200 and 1200 ppm groups) and creatinine levels (1200 ppm group). Decreased levels of chloride, potassium and decreased activities of LDH, ALAT, ASAT and CPK were seen predominantly in females of the 1200 and 6000 ppm groups at the 6- and 12-month examinations. In high dose males chloride, ALAT and ASAT were found to be decreased after 12 months. Serum cholinesterase activity was increased in males at the highest dose level at the time intervals 6 and 12 months. No changes were seen after 18 and 24 months. The serum cholinesterase activity in females was decreased at the two highest dose levels at the time intervals 6 and 12 month. No changes were seen after 18 and 24 months. In males of the 6000 ppm group, thyroxine (T4) and triiodothyronine (T3) values were numerically lower than those of the control at month 6, 12 and 18 (not evaluable at month 24). Thyroid stimulating hormone (TSH) values of this group were higher than those of the respective centrals. Similar, but weaker effects on the thyroid hormones and TSH were also found in males of the 1200 ppm and females of the 6000 ppm groups.

A dose-related and statistically significant increase of urinary protein (semi-quantitative analysis) was noted in both sexes of the 6000 ppm group at various test times. By using a qualitative method (nephelometry), protein was also increased in males of the 1200 ppm dose and the 200 ppm dose (the latter only noted at month 24 and lacking a histological correlate). In males of the 6000 ppm group ketone bodies, urinary volume and water consumption were increased, and pH and specific gravity were decreased significantly at various test times.

Dose-related and statistically significant increases in the liver, kidney and thyroid weights and their body weight ratios were noted in both sexes of the 1200 and 6000 ppm groups at both interim and final sacrifice. Other changes in organ weight were noted in high dose males and females and in 1200 ppm males, but these changes are most likely related to the decrease in body weight and thus not considered as a primary toxicological effect of the test substance.

At interim sacrifice, treatment related changes were found in the liver (brownish-black colour) and kidneys (granular surface, brownish-black colour) of the 6000 ppm males and females.

At final sacrifice, males of the 1200 ppm group exhibited granular kidneys. In the 6000 ppm females, kidneys were brownish-black and the thyroids were enlarged.

Animals which died during the study also had treatment related changes. At 6,000 ppm, swelling of the thyroid (both sexes) and granular kidney (male) were noted.

Microscopic examinations were performed on all rats which died on study or were killed at interim and final sacrifices. An independent peer review of relevant microscopic findings was conducted. Dose-related changes were found in the thyroid, liver, kidney and adrenals.

Thyroid: Follicular cell hyperplasia and hypertrophy were noted in both sexes from the 1200 and/or 6000 ppm groups at months 12 and 24. In addition, an increase of focal follicular cell hyperplasia was observed in the 6000 ppm males and females. In males, the incidence of thyroid follicular cell adenoma was increased at 1200 ppm and above, but reached statistical significance only at 6000 ppm.

Liver: Centrilobular hepatocellular hypertrophy and occurrence of lipofuscin were noted in both sexes of the 1200 and/or 6000 ppm groups at months 12 and 24. These changes were also seen in majority of 6000 ppm males and females which died on study.

Adrenals: Lipidosis of the adrenal cortex was noted in females of the 1200 ppm group and in both sexes at 6000 ppm at month 12. However, no treatment related incidence of this finding was seen in males and females which died on study or were terminally sacrificed.

Kidney: The severity of nephropathy was increased in both sexes of the 6000 ppm group compared to controls at month 12 and in both sexes of the 1200 and 6000 ppm groups at month 24. The lesions were more severe in males than in females and grew worse with increasing dose and time. For many animals affected in that way, the renal failure was considered to be the main lesion/cause of death. Microscopic evidence of renal failure in these rats was accompanied by hyperplasia of the parathyroid, demineralization of the bone and metastatic calcification in various organs.