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REACH

Thiophanate-methyl

EC number: 245-740-7 | CAS number: 23564-05-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Repeated dose oral:

A study according EPA OPP 83-5 (GLP) and similar to OECD TG 453 is available. 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).

Repeated dose dermal:

A study according to EPA 82-2 and similar to OECD TG 410 was conducted. New-Zealand White rabbits were treated with the substance at doses of 100, 300 and 1000 mg/kg bw/day. The NOAEL is considered to be 1000 mg/kg bw/day under the conditions of this study.

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPS Guideline 83-5

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:

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

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

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:

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

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.

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:

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. 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.

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.

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.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 8.8 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: similar to OECD TG 453

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: inhalation
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: inhalation
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 8, 1990 - November 15, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

Analytical data on the stability of the test substance were not reported

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The animal model, the New Zealand White rabbit, is recognized as appropriate for dermal toxicity studies and is a widely used strain for which significant historical control data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, PA.
- Age at study initiation: 15 weeks
- Weight at study initiation: 2.1 to 2.5 kilograms for the males and 2.1 to 2.3 kilograms for the females
- Fasting period before study: no
- Housing: all animals were housed individually in clean, wire-mesh cages
- Diet: ad libitum, Purina# Certified Rabbit Chow #5322
- Water: ad libitum, tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Contaminants present in animal feed or water were not expected to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67 ± 4
- Humidity (%): 58 to 78
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: The test material was applied to the shaved intact dorsal skin of each test animal.
- % coverage:
The test material covered approximately 2-3% of the total body surface on the animals in the 100 mg/kg/day group, approximately 5-6% of the total body surface on animals in the 300 mg/kg/day group and approximately 8-10% of the total body surface on animals in the 1000 mg/kg/day group.
- Type of wrap: Doses were applied under gauze binders and secured with nonirritating tape.
- Time intervals for shavings or clipplings: The animals were shaved twice weekly during the study to facilitate dosing and dermal observation.

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of a six-hour exposure period, the dressings were removed and the test sites were washed with disposable paper towels moistened with deionized water.

TEST MATERIAL
- Amount(s) applied: Individual animal doses for the treated groups were moistened with deionized water to form a paste immediately prior to application. Immediately prior to application the individual doses were moistened with approximately 0.2, 0.5 and 1.5 milliliters of deionized water, respectively for the 100, 300 and 1000 mg/kg/day doses.
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied: The control material, deionized water, was administered at a dose volume of 1.0 mL/kg/day.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

Duration of treatment / exposure:

three weeks

Frequency of treatment:

six hours per day, five days per week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: for mortality and overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION: Yes
- Time schedule for examinations: Each application site was examined for erythema, edema and other dermal findings once daily (immediately prior to dosing on days of dose administration). The sites were graded approximately 18 hours after removal of the wrappings. Erythema and edema were evaluated based on a four-step grading system of very slight, slight, moderate and severe. Other dermal findings, if present, were noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured weekly beginning one week prior to dose administration.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day prior to study termination,
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count, Differential WBC Count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day prior to study termination,
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Glucose, Blood Urea Nitrogen, Blood Creatinine, Total Protein, Albumin, Albumin/Globulin Ratio (A/G Ratio), Sodium, Potassium, Chloride, Calcium, Phosphorus, Aspartate Aminotransferase (Aspartat Transfer), Alanine Aminotransferase (Alanine Transfer), Serum Alkaline Phosphatase (Alkaline Phoshatase), Total Bilirubin (Total Bili), Total Cholesterol, Globulin, Gamma Glutamyltransferase (Glutamyl Transfer)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy, the following tissues and organs were collected and preserved in 10% neutral buffered formalin: Kidneys (2), Liver (sections of two lobes), Skin (treated and untreated), Gross lesions

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled sacrifice: brain, kidneys, liver, ovaries, testes
Paired organs were weighed together. Organ to final body weight ratios were calculated.

HISTOPATHOLOGY: Yes
Tissues listed were examined from animals in the control and high dose groups. In addition, treated skin, untreated skin, and gross lesions were examined from animals in the low and mid dose group.

Statistics:

All analyses were conducted using two-tailed tests for significance levels of 5% and 1% comparing the treatment groups to the control group by sex. All means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a DEC (Digital Equipment Corporation) computer with appropriate programming. Analysis of body weights, body weight changes, food consumption, clinical pathology parameters and absolute and relative organ weight values were analyzed by a one-way analysis of variance, following by Dunnett´s test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical findings were limited. Decreased defecation was noted in two 1000 mg/kg/day group males and one 1000 mg/kg/day group female on the third day of dose administration and one other study day for each animal. The two 1000 mg/kg/day group males also had single occurrences of decreased urination noted. Head tilt was observed in one 100 mg/kg/day group male on the last 15 days of the study. This finding was not related to the test material.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

No dermal irritation was present in the control group. Signs of dermal irritation were limited in each treated group to sporadic occurrences of desquamation and/or very slight erythema, generally observed during the second week of test material administration. No dose-related patterns were apparent. No signs of edema were present in any dose group.
At a dose level of 100 mg/kg/day, two males had findings of very slight erythema on one or three days. Desquamation was noted for one 100 mg/kg/day male on one occasion. Two 100 mg/kg/day females had desquamation observed on one or three days. A single incidence of very slight erythema was noted for one 300 mg/kg/day male. Desquamation was present on a few days for three males and all females in the 300 mg/kg/day group. At 1000 mg/kg/day, a single occurrence of very slight erythema was noted for one male. All 1000 mg/kg/day group males had desquamation present on one to four occasions. Very slight erythema was noted on the last three study days for a single 1000 mg/kg/day female. Desquamation was noted on two females in the 1000 mg/kg/day group on a few days.
Males displayed no dermal effects after day 14. Except for 1000 mg/kg/day female no. 10986, females displayed no dermal effects after day 10.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related effects on mean body weights or mean body weight gains. Mean body weights were slightly decreased in the 1000 mg/kg/day group males at weeks 1, 2 and 3 and mean body weight gains in these males appeared to be reduced beginning weeks 0 to 1. It should be noted that the comprehensive (weeks 0-3) mean body weight gain value was also reduced, however, this value was influenced by one male (no. 10957) with consistently lower body weight gains. When the comprehensive (weeks 0-3) body weight gain for male no. 10957 was excluded from the mean for the 1000 mg/kg/day male group, no remarkable differences were observed between the control group and the 1000 mg/kg/day male group. Therefore, the decreases in body weights and body weight gains were considered to be the result of biological variability (especially notable in small sample sizes). There were no numerical trends in mean body weights or body weight gains in the treated female groups when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption (g/animal/day and g/kg bw/day) was significantly decreased at 1000 mg/kg bw/day throughout the study (20-30% compared to control). This was considered to be a possible treatment-related effect. It was however not of sufficient magnitude to produce a significant decrease in weight gain.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related effects were seen in the hematology parameters at any dose level. All mean hematology values in the treated groups were comparable to the corresponding control group values. An evaluation of the mean absolute and relative numbers of leukocytes revealed statistically significant decreases (p<0.05 or p<0.01) in mean percentages and absolute numbers of monocytes in males dosed at levels of 100 and 1000 mg/kg/day. These differences were minimal, no dose response was apparent and therefore, the decreases were not considered to be toxicologically significant. No statistically significant differences in the numbers or types of leukocytes were observed for the 100 and 1000 mg/kg/day females or the 300 mg/kg/day males and females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed in the serum chemistry parameters. Some differences occurred between the treated groups and the control group when the values were compared, however, no trends were apparent and the differences were not statistically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect was apparent in the organ weight values at any dose level. The mean brain weight was increased (statistically significant at p<0.05) in the 300 mg/kg/day males when compared to the control group. All other mean organ weight values and organ weight relative to final body weight values in the 300 mg/kg/day male group were comparable to the control group. All mean organ weight values and organ weight relative to final body weight values were comparable to the control group for the 100 and 1000 mg/kg/day groups and the 300 mg/kg/day group females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related lesions were observed at necropsy. Accessory spleens were present in the control and all treated groups. One control group male and one 1000 mg/kg/day female had white areas on the liver. Ovarian cysts were present in one 1000 mg/kg/day female. Reddened renal cortico-medullary junctions were observed in one 1000 mg/kg/day female. One 300 mg/kg/day group male had white foci on the liver. Another 300 mg/kg/day group male had a depressed area on the kidney. One 100 mg/kg/day male had pitted kidneys. White foci on the kidneys were noted for a single control group female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Hyperkeratosis of the treated skin was observed for one female in each of the 100, 300 and 1000 mg/kg bw/day groups. The 300 mg/kg bw/day female also had nonsuppurative subdermal inflammation. No internal histopathological changes related to treatment were observed at dose levels of 100, 300 and 1000 mg/kg/day. Kidney changes (infiltrate lymphocyte, tubular necrosis, interstitial fibrosis, cortical infarct) and liver changes (infiltrate lymphocyte, fatty change, hyperemia, suppurative inflammation, hepatocellular necrosis) were generally seen at a higher incidence in the control group when compared to the high dose group. Several histopathologic changes were observed in other tissues and organs; these were also considered to be normal background changes.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Key result

Critical effects observed:

Conclusions:

The NOAEL is considered to be 1000 mg/kg bw/day under the conditions of this study.

Executive summary:

A study according to EPA 82-2 and similar to OECD TG 410 was conducted. New-Zealand White rabbits were treated with the substance at doses of 100, 300 and 1000 mg/kg bw/day. The test material was moistened with deionized water and applied five days per week for three weeks to the shaved intact dorsal skin of each rabbit for a total of 15 applications per animal. The test material was applied under gauze binders and secured with non-irritating tape for a period of six hours per day. Each group consisted of five males and five females. A concurrent control group received deionized water (1.0 mL/kg bw) on a comparable regimen. All animals wore plastic Elizabethan cellars during the daily exposure period. The animals were observed for signs of overt toxicology, dermal irritation and effects on body weight, food consumption, clinical pathology parameters and organ weights. Complete necropsy examinations were performed on all animals. A microscopic examination was conducted on selected guideline tissues.

Analytical data on the stability of the test substance were not reported. No compound related effects were observed. No compound related effects were observed on body weight. Mean food consumption (g/animal/day and g/kg bw/day) was significantly decreased at 1000 mg/kg bw/day throughout the study (20-30% compared to control). This was considered to be a possible treatment-related effect. It was however not of sufficient magnitude to produce a significant decrease in weight gain. No compound related effects were observed. No compound related effects were observed on organ weights or during the gross pathology. Dermal irritation was limited to sporadic occurrence of very slight erythema and desquamation in all treated groups, generally during the second week of test material administration. Edema was not present in any treated group. Microscopic tissue changes were limited to the treated skin. Hyperkeratosis of the treated skin was observed in one female in each of the treated groups. Non-suppurative subdermal inflammation was also present for the 300 mg/kg bw/day female that had hyperkeratosis. Although there was an effect on food consumption that seems to be dose-related, it gave no significant effect on body weights or other investigated parameters and was therefore not adverse for the duration of the study. Therefore, the NOAEL(systemic and local) is considered to be 1000 mg/kg bw/day under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rabbit
Quality of whole database:: similar to OECD TG 410

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 8, 1990 - November 15, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

Analytical data on the stability of the test substance were not reported

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The animal model, the New Zealand White rabbit, is recognized as appropriate for dermal toxicity studies and is a widely used strain for which significant historical control data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Analytical verification of doses or concentrations:

Duration of treatment / exposure:

three weeks

Frequency of treatment:

six hours per day, five days per week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Key result

Critical effects observed:

Conclusions:

The NOAEL is considered to be 1000 mg/kg bw/day under the conditions of this study.

Executive summary:

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Additional information

Repeated dose oral

Key study- 24 month rats

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.

Dose-related decreases in food consumption values 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 6000 ppm at months 3, 6, 12 and 18. In the 1200 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. 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.

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.

Discussion

The adrenal glands of interim-sacrifice males (6000 ppm) and females (1200 and 6000 ppm) exhibited a very slight increase in cytoplasmic lipid in the zona fasciculata cells of the cortex. In untreated rats, these cells normally have a mild degree of cytoplasmic lipid, as they are a site of endogenous steroid production. It is unknown why the higher dosages of test item would have caused an increase in endogenous steroid production (manifested by the observed adrenal "lipidosis"). It is possible that some substance-induced change in metabolism of lipid occurred, as cholesterol levels were also increased by the higher doses. It is also possible that these high(er)-dose animals were simply under more "stress", since they were being dosed at or above the MTD. Animals respond to "stress" by increasing their production of endogenous steroids. In either event, this slight "lipidosis" appears to have had little biological or toxicological significance, and was not readily apparent morphologically by the study conclusion. The inability to detect subtle differences in adrenal cortical lipid in very aged or dying rats is not surprising, since such animals are certainly being challenged by additional stresses as they approach their termination.

Administration of test item also caused a consistent mild anemia in rats of both sexes at 6000 ppm. This anemia, microcytic in character, manifested itself as decreased hemoglobin and decreased RBC indices. Clinically, the anemia correlated with the observed pale discoloration of skin (ear pinna) and mucous membrane. Platelet and WBC counts were often elevated slightly in the anemic rats. These latter findings are not surprising, as the body generally responds in a nonspecific manner to a mild anemia (that is, both erythroid and granulocytic cell production may increase together). Thus, if no toxic effect was induced on the platelets or WBC series, these cells would tend to equilibrate at a higher than normal level in the face of an anemic effect (Schalm et. al., 1975). The significance of the anemia in this study appears to be minimal, since evidence of decreased hematopoiesis was not detectable in the spleen or bone marrow of affected rats.

Kidneys were significantly affected substance administration at dosages of 1200 and 6000 ppm in both sexes. Alterations detected by clinical pathology included increased blood urea nitrogen (BUN) and serum creatinine (CR), increased excretion of protein into the urine, and decreased serum albumin. The increased BUN and CR indicate loss of proper renal function/clearance of proteins. The increased urinary excretion of protein (presumably albumin, leading to the decrease in albumin noted above) indicate significant renal damage has occurred.

These in-life determinations were confirmed at necropsy, where the mean absolute kidney weights of 1200 and 6000 ppm males and females were found to be increased, and color and/or cortical surfaces were macroscopically different from the control kidneys. Histologically, the rats exposed to the two highest doses had more severe nephropathy than lower-dose or control rats. Kidney lesions were dose, time, and sex dependent, in that lesions were more severe in males than females, and grew worse with increasing dose and time.

Although some degree of nephropathy was noted for almost all rats on study, this is a common "spontaneous" and progressive change in aging Fischer 344 rats (Montgomery et. al., 1990). Accentuation of this lesion in rats fed 1200 and 6000 ppm was clear. The nephropathy was so severe in high-dose males that renal failure was considered to be the main lesion/cause of death for many of these animals. Microscopic evidence of renal failure in these rats included parathyroid hyperplasia, cortical bone resorption, and disseminated calcification of somatic tissues. The reason why substance administration may have accelerated the spontaneous "nephropathy" is unknown. It is possible that test item-induced metabolic alterations, mediated either through liver or pituitary/thyroid changes (discussed below), contributed to exacerbating this condition. It is suspected that the high-protein diet routinely fed to rats plays a role in spontaneous, chronic rat nephropathy (Montgomery et. al., 1990). Possibly, substance-induced hepatic hypertrophy caused a change in the composition/ratio of hepatic proteins released to the serum, and these proteins adversely affected the "normal" development of the nephropathy.

The rat liver appears to be directly affected by test item administration. In 1200 and 6000 ppm rats of both sexes, a constellation of effects were observed which relate to substance induced hepatocellular hypertrophy. At necropsy, increased liver weights were observed, as were some dark discolorations of the livers in rats from the two highest doses. Microscopically, classical centrilobular hepatocellular hypertrophy was observed, with extension of affected areas of the lobule in more severely affected animals.

Hepatocellular hypertrophy is the morphologic expression of a subcellular increase in liver microsomal enzymes. Such "enzyme induction" has been classically associated with an increased concentration of cytochrome P-450. It is known that such enzyme induction may also increase the activity of other hepatic enzymes, including those which are involved with the production of cholesterol, thus the increased cholesterol levels seen in this study are not unexpected (Hill, R.N. et. al., 1989). The increased total serum protein (TP) seen in females, and the increased serum globulins seen in both males and females (with decreased albumin and normal or elevated TP, serum globulins must be increased), may also be directly related to increased enzyme activity due to "enzyme induction".

A mechanistic investigation to provide supplementary data regarding the enzyme-inducing potential of the substance was conducted outside the scope of this study. That investigation clearly showed that the substance produces dramatic induction of hepatic microsomal enzymes. Briefly, the studies show a doubling of P-450 and cytochrome b5 activity and a tripling of UDP-glucuronosyI transferase (UDP-GT) activity after 8 days of exposure to 6000 ppm. Total cholesterol was also significantly increased after 8 days.

The role of T4 was investigated by measuring thyroid and liver weights, TSH levels and total cholesterol in the presence of T4, test item, and test item plus T4 supplementation. Similar to the experiment described above, substance feeding for 8 days at 6000 ppm resulted in statistically significant (p<0.001) increased liver and thyroid weights compared to control. T4 alone did not affect the organ weights. However, T4 in the presence of 6000 ppm test substance resulted in normal thyroid weight and increased liver weight. Thus the effects of test item in the thyroid were ameliorated by the supplement of T4. In addition, T4 alone depressed TSH levels slightly, test item increased TSH nearly 5 fold yet T4 plus test item resulted in normal levels of TSH compared to controls.

Thyroid follicular cell carcinogenesis in laboratory animals and humans and related interactions and responses to a variety of chemicals have been reviewed in detail by Hill, et. al. (1989). In this comprehensive review of published data including NTP carcinogenic bioassays (about 300 chemicals of which approximately 21 resulted in thyroid neoplasms), the relationship between the pituitary and thyroid glands and the circulating hormones (T4, T3 and TSH) levels are discussed and establishes a hormonal basis for thyroid follicular cell carcinogenesis for non-genotoxic chemicals. The results of this chronic study indicate that microscopic findings in the thyroid at high dietary concentrations of test item also have a hormonal basis. Simplistically, a decrease in circulating T4 through a feedback mechanism triggers TSH release from the pituitary which stimulates the thyroid to synthesize and release T4. Prolonged decreases in T4 result in continuous TSH stimulation of the thyroid resulting in thyroid cell changes ranging from hypertrophy to hyperplasia and potentially to neoplasia. Decreases in circulating T4 can result from decreased synthesis of T4 or from elimination through enhanced metabolism of T4 as a result of induction of hepatic drug metabolizing enzymes (P-450 and the endoplasmic reticulum). For the substance, the latter mechanism (enhanced metabolism of T4) appears to be involved. As discussed above, chronic ingestion of test item results in decreased T4, increased TSH, increased liver weights and hepatic centrilobular hypertrophy. Induction of specific metabolizing enzymes by the substance have been demonstrated in the mechanistic studies. Substance exposure at the MTD and in excess of the MTD led to an increase in thyroid follicular cell adenomas in male rats. In the absence of genotoxicity and confirming clinical/biochemistry and pathology, these adenomas are considered to be secondary changes related to alterations of hormonal homeostasis of the pituitary-thyroid axis.

In conclusion, 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).

Supporting Study- 90 day (rats)

A sub-chronic (13 weeks) feeding study of thiophanate-methyl was performed using Fischer 344 rats. The dose levels were 0, 200, 2200, 4200, 6200 and 8200 ppm and each group consisted of 10 males and 10 females.

No dose-related clinical sign of toxicity was observed in dosed rats. One male from 200 ppm group died at study week 11. Fracture of nasal bone was noted.

No dose-related changes were noted in body weights and food efficiencies. Slight decreases of food consumption were noted in both sexes of 4200 ppm and/or higher dose groups during study weeks 1-2.

No abnormalities were observed in the ophthalmoscopic examinations.

In hematological tests, dose-related anemia (decreases of hemoglobin, hematocrit, MCV, MCH and MCHC) was noted in both sexes of 2200 ppm and/or higher dose groups. Total and differential leukocyte counts remained normal.

Blood chemistry revealed dose-related increase in total cholesterol, total protein and albumin in both sexes of 2200 ppm and/or higher dose groups. Triiodothyronine was increased in females of the 8200 ppm group. No dose-related change was noted in thyroxine.

Dose-related and statistically significant increases of urinary protein and ketone bodies were noted in males of 6200 ppm and/or 8200 ppm groups.

Dose-related increases of liver, kidney and thyroid weights, and decreases of thymus weights were noted in both sexes of the 2200 ppm and/or higher dose groups.

Thyroid weights were increased in both sexes treated with dose levels 2200 ppm and above. At 200 ppm there was a 31% increase compared to controls that was statistically significant. Liver weights were increased in females starting at 2200 ppm and males starting at 4200 ppm. Kidney weights were increased in males at 2200 ppm and in females at 8200 ppm. Thymus weight was decreased in females starting from 4200 ppm and in males from 6200 ppm. Testes weights were increased in males treated with dose levels 4200 ppm and above.

Dose related histopathological changes, accompanied by weight changes of the affected organs, were observed in the thyroid, liver, adrenal and kidney. Follicular hyperplasia and hypertrophy were noted in the thyroid in both sexes at doses of 2200 ppm and above. Despite the increased thyroid weight in females at 200 ppm, no lesion was noted in this dose group. Hepatocellular swelling and increased lipofuscin pigment were noted in both sexes of the 2200 ppm and higher dose groups. Fatty degeneration of adrenal cortex was noted in females of the 4200 ppm and in both sexes of the two higher dose groups. The severity of glomerulonephrosis, which was characterised by tubular lesions and hyaline droplets in the glomerular epithelium, was increased in males of the 2200 ppm and higher dose groups and in females of the highest dose group.

Based on the results, it is concluded that the effects of the treatment with thiophanate-methyl are anemia, follicular hyperplasia and hypertrophy of the thyroid, hepatocellular swelling accompanying lipofuscin and glomerulonephrosis. The NOAEL for males was 200 ppm (15.7 mg/kg bw/day), based on haematological effects, increased total cholesterol, increased kidney and thyroid weights as well as histopathological findings in these organs at 2200 ppm (corresponding to approximately 173 mg/kg bw/day). Thyroid weights were increased in both sexes at higher doses in a dose-related manner. Starting from 2200 ppm there were also histopathological findings in the thyroid (hypertrophy and hyperplasia). The increase in thyroid weight seen at 200 ppm is therefore considered to be an early stage of the effects seen at higher doses and is considered adverse. The LOAEL was thus 200 ppm in females (13.9 mg/kg bw/day). A NOAEL for females cannot be established as no lower dose was tested.

Supporting study – 1-year dog

In a further study the test item was administered to Beagle dogs for one year. Four animals per sex and dose group received the test substance orally via gelatine capsules daily at dose levels of 0, 8, 40 and 200 mg/kg bw/day (groups I, II, III, IV). The animals were observed for mortality and clinical signs of toxicity at least twice daily. Detailed physical examinations took place once before the start of the study, then weekly during treatment and at terminal sacrifice. Body weight and food consumption were determined weekly. Ophthalmoscopic examinations took place once before the start of the study and then weekly during treatment and at terminal sacrifice. Clinical chemistry, haematology and urinalysis were carried out before commencement of treatment and after approximately 3, 6 and 12 months of administration on all animals. The clinical chemical investigations also included the determination of the thyroid hormones T3 and T4 as well as the thyroid stimulating hormone (TSH). All animals were subjected to a complete gross pathological evaluation. Organ weights were determined and histopathology was carried out on the full set of organs required in the guideline.

The stability of the test substance was guaranteed over the entire administration period. All animals survived throughout the study. Tremors were seen in all high dose animals and one mid-dose animal shortly after dosing on one or more occasions during the initial three weeks of study but were not observed subsequently. No other signs of clinical toxicity were noted that could be associated with treatment. No compound-related ocular abnormalities were revealed. Most animals in group IV exhibited a slight body weight loss during the first week of treatment and decreased body weight gain thereafter. At the end of the study, absolute body weight was reduced by approximately 20% and body weight gain by 45% in males and 64% in females relative to controls. Slightly decreased food consumption values were also evident during the early months of the study.

Haematological effects in animals of group IV consisted of slight decreases, relative to control values, in total erythrocyte counts (-13%), haemoglobin (-14%) and haematocrit (-14%) values for males (but not females) at 12 months (the effects were seen, but did not attain statistical significance, also at month 3 and 6 in males). Several biochemistry alterations which appeared to be related to administration were evident in groups III and IV after 6 and/or 12 months. Changes consisted of elevated serum cholesterol levels in group III and IV males and in group IV females, elevated serum alkaline phosphatase levels and slightly decreased serum alanine aminotransferase levels in group IV males and females. The latter observation is generally not considered indicative of toxicity but appears to represent an effect of the test material. Decreases in the albumin/globulin ratio resulting from decreased and/or increased globulin levels were seen in group III and IV males at month 6 and 12. The slight decrease in BUN values was not considered indicative of toxicity. Alterations in serum electrolytes included decreased calcium, potassium, and phosphorous levels in group IV males and decreased calcium levels in group III males (month 6 and 12). Effects on thyroid function were limited to statistically significant decreases in T4 levels for group III and IV males at month 6 and for group IV males at month 12. No effects on T3 or TSH levels were evident. Female thyroid function parameters (T3, T4 and TSH) were not affected at any dose.

Post-mortem abnormalities included increased absolute and relative liver weights in group IV animals. Thyroid weights were increased as follows results in groups II, III, IV compared to controls): males, absolute weights: 11%, 34%, 41%, relative (to body) weights: 4%, 42%, 72%; females, absolute weights: 29%, 45%*, 39%, relative (to body) weights: 10%, 54%**, 75%** (* p <0.05, ** p <0.01). Microscopic alterations attributed to test material administration were limited to the follicular epithelium of the thyroid gland with minimal to moderate hypertrophy in males (0/0, 0/0, 0/0, 4/4) and females (0/0, 0/0, 2/4, 3/4) and slight hyperplasia (group IV, one male and one female).

A LOAEL for females of 8 mg/kg bw/day is derived due to increased thyroid weights. Based on this LOAEL, no NOAEL for females can be established as no lower dose was tested. NOAEL for males was 8 mg/kg bw/day.

General Conclusion

The study according OECD TG 453 performed in rats was used as key study because of the chronic exposure duration compared to the 1-year dog study.

The Committee for Risk Assessment (RAC) concluded in their Opinion on proposed harmonized classification and labeling of the substance (15th March 2019) the following on repeated dose toxicity:

“Whereas thyroid is clearly a target organ in test item toxicity, it is noted that in none of the studies effects indicative of significant or severe toxicity were observed at dose levels within the guidance values for classification. At dose levels at or below the guidance value there were either no effects (most studies), or effects on thyroid weight without histopathological findings (90-d rat, 1-yr dog). Only in two studies some histopathological findings were observed. In the 90-d dog study it concerned minimal hypertrophy in 2/8 dogs, in the 2-gen rat study hypertrophy and hyperplasia (no severity score reported) [...] Overall these effects were observed at dose levels below the guidance value, possibly indicating an adaptive response, and are not sufficient to warrant classification.

In conclusion, the mortality observed in rabbits in a developmental toxicity study [see section 7.8.2] and the effects on the thyroid (increased weight, hypertrophy and hyperplasia) in rats and dogs observed upon short- to long-term exposure were considered by the RAC not sufficient to support the classification as STOT RE 2.”

Repeated dose dermal

Key study

Analytical data on the stability of the test substance were not reported. No compound related effects were observed. No compound related effects were observed on body weight. Mean food consumption (g/animal/day and g/kg bw/day) was significantly decreased at 1000 mg/kg bw/day throughout the study (20-30% compared to control). This was considered to be a possible treatment-related effect. It was however not of sufficient magnitude to produce a significant decrease in weight gain. No compound related effects were observed. No compound related effects were observed on organ weights or during the gross pathology. Dermal irritation was limited to sporadic occurrence of very slight erythema and desquamation in all treated groups, generally during the second week of test material administration. Edema was not present in any treated group. Microscopic tissue changes were limited to the treated skin. Hyperkeratosis of the treated skin was observed in one female in each of the treated groups. Nonsuppurative subdermal inflammation was also present for the 300 mg/kg bw/day female that had hyperkeratosis.Although there was an effect on food consumption that seems to be dose-related, it gave no significant effect on body weights or other investigated parameters and was therefore not adverse for the duration of the study. Therefore, the NOAEL(systemic and local) is considered to be 1000 mg/kg bw/day under the conditions of this study.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the seventeenth time in Regulation (EU) 2021/849.

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