FUNDAMENTAL

AND

APPLIED

16,667-686

TOXICOLOGY

(199 1)

Chronic Toxicity Studies with Thiram in Wistar Rats and Beagle Dogs K. MAITA, Toxicology

Division,

The Institute

S. TSUDA,

of Environmental

Received

May

AND Y. SHIRASU

Toxicology,

21, 1990; accepted

Suzuki-cho

November

2-772,

Kodaira-shi,

Tokyo

187, Japan

28, 1990

Chronic Toxicity Studies with Thiram in Wistar Rats and Beagle Dogs. MAITA, K., TSUDA, S., AND SHIRASU, Y. (1991). Fundam. Appl. Toxicol. 16, 667-686. Groups of 64 male and 64 female Wistar rats were given thiram at constant dietary doses of 0, 3, 30, and 300 ppm (0, 0.1, 1.2, and 11.6 mg/kg/day for males and 0,O. 1, 1.4, and 13.8 mg/kg/day for females) for 104 weeks. Eight males and eight females in each group were killed after Weeks 13, 26, and 52. For the dog study, four male and four female beagle dogs were allotted to each group and treated with the compound at 0,0.4,4, and 40 mg/kg/day for 104 weeks. The dogs in the 40 mg/kg/day group had severetoxic signs, including nausea or vomiting, salivation, and occasional clonic convulsion, and all were subjected to unscheduled necropsy before Day 203 of treatment. The dogs also had ophthalmological changes such as fundal hemorrhage, miosis, and desquamation of the retina which were consistent with the retinal lesions shown by histopathology. The rats of the high-dose group had retarded growth with a slightly decreased food intake. Anemia was evident in highdose female rats and in middle- and high-dose dogs. Liver failure in male and female dogs and kidney damage in female dogs were detected in middle- and high-dose groups by blood biochemistry and/or histopathology. Regressive changes of the sciatic nerve accompanied by atrophy of the calf muscle were seen in female rats of the high-dose group but not in male rats. In high-dose rats, progression of myocardial lesions of the heart and chronic nephrosis of the kidney were depressed in males and females, respectively. Female rats of the middle- and high-dose groups had decreased occurrences of mammary fibroadenoma and decreased development of skin masses. 0 1991 Society of Toxicology.

Thiram, bis(dimethylthiocarbamoy1) disulfide, is a dithiocarbamate compound which has been widely used as a bacteriocide or fungicide in agriculture as well as a vulcanizing agent in the rubber industry. In Japan, a total of 600 tons of thiram was produced and used in combination with other agrochemicals in 1989. With regard to the toxicity of thiram, skin lesions including hand eczema or dermatitis have long been recognized among workers in the rubber industry and hospital surgical staff who use rubber gloves. An allergic response to the thiram that is secreted by the rubber is suspected as the cause (Van Ketel et al., 1984; Rudzki et al., 1976). In broiler chickens, tibial dyschondroplasia is induced by the addition of thiram to feed at a concentration of as little as 30 ppm for 20 days. De667

pletion of trace elements is proposed as one of the causative factors of this lesion (Edwards, 1987). Toxicities of dithiocarbamate compounds have been extensively investigated in a series of rodent studies by Lee and co-workers since the middle seventies. They found that thiram could produce adverse effects on the reproductive activity of CD rats when the chemical was given at 132 mg/kg for 13 weeks to males and at 30 mg/kg or more for 2 weeks to females (Short et al., 1976). When CD rats were treated during organogenesis or the periand postnatal periods, toxic changes were observed in the fetuses or offspring only at levels at which dams or adults experienced significant depression of body weight and food consumption. A cytotoxic effect was also reported in Chinese hamster ovary cells in culture 0272-059019

1 $3.00

Copyright 0 199 I by the society of Toxicology. All rights of reproduction in my form reserved.

668

MAITA,

TSUDA, AND SHIRASU

(Hodgson and Lee, 1977). A 13-week subacute studies with the compound in Wistar rats and treatment of male CD rats produced testicular beagle dogs. changes at 132 n&kg/day and mild elevations of blood biochemical parameters indicating MATERIALS AND METHODS renal or hepatic dysfunction at 58 and 132 mg/kg/day (Lee et al., 1978). During the 80 Test muferid Thiram, bis(dimethyhhiocarbamoy1) diweeks of treatment, female CD rats given 66.9 sulfide, with a purity of 98.7%, was obtained from Ouchimg/kg/day had a neurologic syndrome of the Shinko Chemical Industrial Co., Ltd. (Chuo-ku, Tokyo). hind feet and tail beginning at 5 months of In the rat study, the compound was incorporated into treatment (Lee and Peters, 1976). This neu- powdered chow M (Oriental Yeast Co., Ltd., Itabashi-ku, Tokyo) at concentrations of 0, 3, 30. and 300 ppm and rotoxicity was confirmed by histological and administered to rats for a period of 104 weeks. Since the neurofunctional examinations. Because compound was stable in test diets for 1 week, test diets thiram is a pesticide for edible plants, its mu- were prepared twice a week during the treatment. The concentrations of the compound in test diets were not tagenicity or carcinogenicity could be another concern for human health. Its mutagenic po- adjusted to the growth curve of the animals during the treatment. The dose levels were set on the basis of the tential has been shown by the Ames test and results of a 4-week preliminary study in which groups of in mammalian somatic cells (Hedenstedt et five male and five female rats were given test diets of 0, al., 1979; Zdzienicka et al., 1979; Paschin and 10,100,1000, and 2500 ppm. Males in the 100 ppm group Bakhitova, 1985). Moreover, Zdzienicka et al. and males and females in the 1000 and 2500 ppm groups showed moderate to marked unpalatability of the test diets (1982) also reported sperm-head abnormalities which was reflected by retarded growth. In the dog study, in (CFW X C57BL)Fi mice. Although these the compound was administered in gelatin capsules at results strongly suggest a possible carcinogenic doses of 0, 0.4, 4, and 40 mg/kg/day, 7 days a week, for potential of the compound, no increased tu- 104 weeks. The control animals received gelatin capsules mor incidences were reported in a mouse study only. The highest dose was set on the basis of results of a (IARC, 1976) in which groups of 18 male and preliminary study in which the animals receiving the compound at more than 40 mg/kg/day had frequent vom18 female (C57BL/6 X C3H/Anf)F, mice and iting. 18 male and 18 female (C57BL/6 X AKR)F, Animals. Male and female Wistar rats (JckWistar) were mice were administered the compound for up purchased at 4 weeks of age from Japan CLEA Co., Ltd. (Meguroku, Tokyo) and housed four per cage in stainlessto 78 weeks of age. Recently, a dose-dependent steel cages with wire-mesh floors, 3 10 W X 440 D X 230 reduction in the incidence of mononuclear cell H mm. The cages were placed in a barrier system animal leukemia was reported in F344 rats by Hase- room maintained under the following conditions: temgawa et al. (1988), who gave the compound perature, 23 t- 1“C, humidity, 55 f 10%; ventilation, 12 to the rats in their feed at a concentration of times per hour; and lighting from 5:OO AM to 7:OO PM 0.1 or 0.05% for 104 weeks. Although previous After acclimatization to the test environment for a week, groups of 64 males and 64 females were randomly selected reports have clearly demonstrated the toxicity and allotted to each dose group. They were allowed free of the compound, most of the studies were accessto the test diets and tap water. The rats were uniquely done using doses higher than the maximal tol- identified by ear punch and fur staining with saturated erated dose (MTD), where the physiological picric acid solution. After Weeks 13, 26, and 52, eight state of the animals was compromised, result- males and eight females in each group were randomly seing in 10% or more depression of body weight. lected and killed for the laboratory examinations. Eighteen beagle dogs of both sexeswere purchased from In estimating human risk from environmental CSK Experimental Animal Inc. (Suwa, Nagano) at 4 toxicants such as pesticides, it is important to months of age. Refore being shipped they received vacevaluate chronic effects of the compound in cinations against distemper and infectious canine hepatitis. long-term rodent studies covering most of the Parasites in the feces and microfilaria in the blood were life span using maximal tolerated doses or less. also checked. Dogs were housed individually in cages,700 W X 250 D X $00 H mm, which were placed in an animal Information from a chronic study in a non- room that was maintained under the same conditions as rodent animal should be also useful. The pres- that of rats except that temperature was regulated at 24 ent paper reports the results of 2-year chronic + 2°C. The dogs were supplied 200 g of biscuit diet (Fun-

TOXICITY

OF THIRAM

abashi Farm Co., Ltd., Funabashi, Chiba) daily and allowed free access to tap water in a jar. After 32 days, 16 males and 16 females were selected and divided into four groups of four males and four females each through a randomization procedure. Examinations. For rats, measurements of body weight, food intake, and water intake were taken weekly up to Week 26, biweekly up to Week 52, and every 4 weeks thereafter. Urinalysis, hematology, and blood biochemistry were performed for all rats scheduled to be killed. Urinalysis included pH, protein, glucose, ketones, and occult blood. Hematological parameters included hematocrit, hemoglobin, erythrocyte count, leukocyte count, and differential leukocyte count. Biochemical examinations of plasma included total protein, alkaline phosphatase, glucose, blood urea nitrogen, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, total cholesterol, and calcium. Ophthalmological examinations with a halogen ophthalmoscope (BX-13, Neitz Instruments Co., Ltd., Tokyo) were also conducted for all rats scheduled to be killed after Weeks 26 and 52 and for 10 rats of both sexes randomly selected from each dose group at the terminal kill. The areas examined included eyeball, eyelid, cornea, anterior chamber, pupil, iris, lens, vitreous body, and fundus. Organ weight analyses of brain, pituitary, thyroids, heart, thymus, liver, kidneys, spleen, adrenals, testes, ovaries, and calf muscle (M. triceps surae, unilateral) were performed for all rats scheduled to be killed. In addition to these organs, sciatic nerve, bone with marrow (sternum and femur), lymph nodes (cervical, mesenteric), salivary glands, stomach, pancreas, duodenum, jejunum, ileum, cecum, colon, rectum, lung, urinary bladder, epididymides, prostate, seminal vesicle, coagulating gland, uterus, eyes and contiguous glands, skin (lumbodorsal region), mammary gland (female only), and all gross lesions were sampled from all rats scheduled to be killed, preserved in neutral buffered 10% formalin, and processed for histopathology. The organs and tissues described above were also sampled from rats found dead or killed in extremis. In the dog study, the body weight of each animal was measured weekly during the treatment. The portion of the diet not eaten was weighed in the early morning every day. At Weeks 0, 4, 13, 26, 39, 52, 78, and 104, water consumption, ophthalmology, urinalysis, hematology, and blood biochemistry evaluations were done for all surviving animals. Ophthalmological examinations performed with a Kowa RC-II portable fundus camera (Kowa Co., Ltd., Tokyo) included the same parameters as those in the rat study. Urinalysis, hematology, and blood biochemistry parameters were also identical to those in the rat study. Blood was obtained from the Saphenus vein. In organ weight analysis, the lung was weighed but the calf muscle was not included. In addition to the tissues examined in rat study, the nose, tongue, esophagus, trachea, aorta, gallbladder, urethra, and spinal cord (cervical, thoracic, and sacral) were processed for histopathology. Statistical ana/yses. In the rat study, mortality was evaluated by a life-table analysis. Body weight, food intake,

IN RATS AND DOGS

669

water intake, hematology, blood biochemistry, and organ weight were analyzed by the Student t test. The MannWhitney U test was used for urinalysis data. Ophthalmology and pathological findings were assessed by the Fisher exact probability test. A 5% level of probability was used as the criterion of significance. No statistical analyses were performed in the dog study.

RESULTS In Vivo Observations and Mortality Rat. Clinical signs indicative of toxicity were not observed in the rats of either sex during the treatment. The mortality of females in the 30 and 300 ppm groups was slightly higher than that of the control group during the last 8 weeks of the treatment (Fig. 1). These higher mortality rates were apparently correlated to incidental higher occurrences of pituitary tumors during this period, although overall incidences of the tumors in these females were comparable to that of the control group (Tables 5 and 6). In the 300 ppm group, the body

-

0 PPM 10 PPM 30 PPM

-_ . . -_+ 16

32

46 WEEKS

16

32

48 WEEKS

64

64

300 60

60

PPM 96

96

FIG. 1. Survival curves of rats receiving thiram in feed for 104 weeks.

670

MAITA,

TSUDA

AND

weights of both sexes were remarkably depressed to rates of 10% or more of the controls during the early weeks of the treatment and remained at significantly lower levels during almost all of the treatment (Fig. 2). Food intake of the 300 ppm group was 30% or more less than that of the controls for both sexes at the first week of treatment (Table 1). Although the animals began to recover their appetite, average daily food intakes in the 300 ppm group were 8.4 and 11 .O% lower than those of the controls for males and females, respectively. Chemical intake per body weight was significantly decreased during the first 13 weeks of treatment due to the rapid weight gain of the animals (Table l), and the average values in the 3,30, and 300 ppm groups during the entire treatment period were 0.1, 1.2, and 11.6 mg/kg/day for males and 0.1, 1.4, and 13.8 mg/kg/day for females. No treatment-related changes in water intake (Table 1) and ophthalmology were observed. Dog. Males and females in the 40 mg/kg/ day group showed marked toxic signs beginning with frequent nausea or vomiting which occurred as early as the first week of treatment. Excessive salivation after dosing, soft stools,

SHIRASU

and anorexia became evident a few weeks later. Neurological disturbance was first observed at Week 8 in a female that showed fits of clonic convulsion beginning just after dosing and continuing for 10 min. Although the frequency, duration, or severity of the clonic convulsion varied in each animal and in each occurrence of a fit, this sign became common in both males and females after Week 10. The general condition of these animals became worse with prolonged dosing and all males and females were found dead or killed in extremis by Day 152 or Day 203, respectively. Body weights of these animals were slightly or moderately depressed (Figs. 3 and 4) concomitant with a slightly lowered food consumption. In ophthalmology fundal hemorrhage was detected as early as the first week of treatment. Miosis and desquamation of the retina were observed frequently thereafter. There were no changes in water consumption attributable to the treatment. In the 4 mg/kg/day group, nausea or vomiting and salivation were common findings in both sexes. One female showed clonic convulsion from Week 37. She sometimes revealed counterclockwise rolling shortly after

(MALE)

3 PPM 30 PPM 300 PPM

---

0

6

16

24

32

40

46

56

64

72

60

66

WEEKS

FIG. 2. Growth

curves of rats receiving

thiram

in feed for 104 weeks.

96

104

TOXICITY

OF THIRAM

IN RATS AND DOGS

671

TABLE 1 DAILY

FOOD,

CHEMICAL,

AND WATER INTAKE OF WISTAR RAT TREATED

WITH THIRAM

IN FEED FOR 104 WEEKS

Week of treatment Dose group

(wm)

1

4

13

26

52

16

104

Male Food intake (g) 0 3 30 300 Chemical intake @x/kg MY 4 3 30 300 Water intake (g) 0 3 30 300

19.7 19.3 18.9 13.3

23.5 21.3 21.7 19.9

20.4 18.8 18.5 17.8

20.1 18.5 18.9 18.2

19.9 19.5 19.1 18.9

19.5 20.0 19.3 17.7

19.6 19.6 17.7 18.1

0.3 3.0 22.4

0.2 2.1 21.3

0.1 1.4 14.0

0.1 1.1 11.6

0.1 1.0 10.6

0.1 9.5

0.1 1.0 9.5

36.2 35.4 35.8 28.3

35.0 36.9 35.8 35.5

32.3 32.4 32.2 32.4

21.9 29.5 31.7 26.7

27.4 29.3 28.7 25.9

27.6 29.4 28.2 26.2

35.2 36.6 34.2 30.1

1.0

Female Food intake (g) 0 3 30 300 Chemical intake (mg/kg body ~0 3 30 300 Water intake (g) 0 3 30 300

14.5 15.1 14.5 10.0

14.8 15.7 15.6 12.8

13.9 14.6 14.2 12.6

14.0 14.2 14.2 12.1

14.8 16.1 15.3 13.9

16.2 18.6 16.3 14.4

15.8 16.1 14.7 13.1

0.3 2.9 21.3

0.2 2.3 20.8

0.2 1.6 15.8

0.1 1.5 13.5

0.1 1.3 13.5

0.1 1.2 12.3

0.1 1.0 10.4

26.6 30.4 28.0 23.8

33.5 34.7 30.4 30.5

26.8 32.1 29.6 28.5

24.9 27.5 27.1 28.3

22.6 26.7 27.5 23.1

25.0 30.4 30.0 23.0

31.4 35.2 30.0 22.5

convulsion. No toxic changes of in vivo parameters attributable to the treatment were observed in the animals receiving the chemical at 0.4 m&kg/day. Clinical Examinations Rat. There were no changes in urinalysis attributable to the treatment. Upon hemato-

logic examination, females of the 300 ppm group revealed decreases or decreasing tendencies in hematocrit, hemoglobin, and erythrocyte count at interim kills after Weeks 13, 26, and 52, although the values of these parameters were comparable to those of the controls at terminal kill (Table 2). Hematologic examination of males in this group revealed no significant changes except for a slight decrease in erythrocyte count at interim kill

672

MAITA.

TSUDA, AND SHIRASU

MALE

0

6

16

24

32

40

46 56 WEEKS

64

72

60

88

96

104

FIG. 3. Growth curves of male dogs receiving thiram by gelatin capsule for 104 weeks.

after Week 13 (Table 2). Blood biochemical examinations revealed incidental increases in GPT for both sexes in all treated groups at terminal kill, although the values were comparable to those of the controls in the examinations at three interim kills during the treatment (Table 2). Incidental increases in GOT were observed in the 30 ppm group females and in the 300 ppm group males only at terminal kill. There were no abnormal changes in other parameters, including ALP and BUN for all treated groups of both sexes during the treatment.

Dog. There were no changes in urinalysis attributable to the treatment. Hematological examinations revealed that the anemia parameters including hematocrit, hemoglobin, and erythrocyte count were concomitantly depressed in the 40 mg/kg/day group of both sexes (Table 3). These parameters were also Iowered in the 4 mg/kg/day group of both sexes at Week 4. Anemic conditions persisted in the animals of the middle dose group thereafter and females showed lower hemoglobin and erythrocyte counts at terminal kill, while in males these values were almost comparable

FEMALE

0

8

16

24

32

40

40

56

64

72

00

86

96

104

WEEKS

FIG. 4. Growth curves of female

dogs

receiting t&ram by gelatin capsule for 104 weeks.

Week of treatment:

examined:

(ppm):

44.7 15.9 8.0

15.6 55 34 71 18

12.7 55 35 75 18

8

3

93 21

44.4 15.9 8.1

8

0

76 20

27

47 21.4

44 20.0

24

45.7 16.7 10.2

3 8

45.7 16.5 10.0

0 8

30 8

8

30

12.8 60 27 75 17

41.0 15.1* 7.1*

8

300

165 20

29

48 18.8

45.0 15.9 9.5*

300 8

11.0 70 56 85 17

42.9 15.3 7.4

8

0

95 15

34

51 13.9

44.5 16.2 8.3

0 8

t test).

11.2 63 51 84 16

42.6 15.3 7.4

8

3

78 15

38

54 17.7

45.8 16.6 8.3

3 8

26

26

BKXHEMISTRY

(Student’s

AND BLWD

at a 5% probability

16.4 53 37 75 17

45.8 16.5 8.1

13

79 22

29

51 19.5

46.3 16.6 10.0

13

IN HEMATOLOGY

a Alkaline phosphatase. b Glutamic oxaloacetic transaminase. c Glutamic pyruvic transaminase. d Blood urea nitrogen. * Significantly different from the control

Hematology Hematocrit (a) Hemoglobin (g/dl) Erythrocyte(X106/mm3) Blood biochemistry ALP” (K-A unit) GOTb(Karmen unit) GPT’ (Karmen unit) Total cholesterol (mg/dl) BUNd (mg/dl)

No. of animals

Dose group

Week of treatment:

(mg/dl)

Total cholesterol BUNd (mg/dl)

Female

unit)

GPT’(Kannen

Hematology Hematocrit (%) Hemoglobin (g/d]) Erythrocyte (X 106/mm3) Blood biochemistry GOTb ALP’ (K-A (Karmenunit) unit)

Dose group (ppm): No. of animals examined:

Male

CHANGES

49 13.5

45.2 16.5 8.4

30 8

9.7 69 54 89 16

41.8 15.0 7.3

8

30

99 17

35

2

12.7 45 27 87 17

40.2* 14.2* 6.6*

8

300

98 17

30

49 17.5

45.0 16.5 8.3

300 8

IN WISTAR

TABLE

12.2 93 59 110 21

41.6 14.2 10.7

8

0

137 18

112

110 17.2

44.4 14.7 11.3

0 8

3 8

13.4 87 77 92 18

42.8 14.6 10.8

8

3

132 19

66

88 19.6

52

52

10.9 89 60 103 17

42.8 14.6 10.8

8

30

121 17

76

94 16.3

44.8 14.7 11.2

30 8

WITH THIRAM

44.4 14.5 11.4

RATS TREATED

17.5 117 63 103 20

39.9 13.9 9.3*

8

300

131 18

71

90 13.4

44.4 14.9 11.1

300 8

12.6 88 40 129 18

39.0 14.0 6.5

27

0

177 20

33

61 15.9

43.5 15.4 7.5

0 26

FOR 104 WEEKS

13.1 94 54: 130 17

42.3 15.3 7.1

28

3

157 20

43*

70 16.7

43.1 15.0 7.6

3 26

104

104

9.8 113* 55* 135 18

39.5 14.2 6.6

20

30

201 20

41*

69 15.6

44.6 15.7 7.6

30 24

10.8 99 52* 108 18

40.0 15.0 6.6

20

300

159 19

46*

16* 17.9

43.9 15.4 7.5

300 28

5

E CA

2

F z

2

%

Et x 0” 3

of treatment:

43.5 15.0 5.8

6.9 14 22 200 11

7.2 11 17 193 11

7.4 13 17 181 I1

45.8 15.9 6.2

7.7 12 21 217 10

7.4 11 19 181 11

8.7 11 17 195 9

45.3 15.6 6.0

43.8 14.9 5.9

4 4

41.0 14.1 5.5

0.4 4

43.8 15.0 6.0

0 4

0

8.0 13 20 200 10

43.4 14.8 5.8

7.1 12 18 197 10

40.8 14.4 5.6

40 4

IN HEMATOL~CY

due to machine trouble. ’ Alkaline phosphatase. b Glutamic oxaloacetic transaminase. ’ Glutamic pyruvic transaminase. d Blood urea nitrogen. e Two males or three females were examined.

Note. ND: Not determined

Hematology Hematocrit (‘%) Hemoglobin (g/dl) Erythrocyte (X 106/mm3) Blood biochemistry ALP” (K-A unit) GOT b (Karmen unit) GPT’ (Karmen unit) Total cholesterol (mg/dl) BUNd (mg/dl)

Hematology Hematocrit (%) Hemoglobin (g/dl) Erythrocyte (X 106/mm3) Blood biochemistry ALP” (K-A unit) GOT ’ (Karmen unit) GPT’ (Karmen unit) Total cholesterol (mg/dl) BUNd (mg/dl)

Dose group (ppm): No. ofanimals examined:

Week

CHANGES

6.5 18 15 195 11

42.9 15.4 6.9

7.1 23 23 195 11

44.6 15.9 7.0

0 4

6.1 24 22 203 11

42.1 15.5 6.5

7.2 21 18 213 12

39.0 13.7 6.1

0.4 4

AND BLOOD 4

7.3 17 14 243 15

39.5 14.2 6.0

7.5 15 14 237 12

37.1 13.7 5.7

4 4

7.5 24 53 246 12

17.6 7.5

11.1 16 21 ND 13

46.3

10.4 4.1

243 10

6.8 24 44

44.9 16.9 7.5

0 4

6.6 27 41 262 10

17.6 7.5

45.9

245 13

7.8 23 37

40.8 15.5 6.8

0.4 4

13

8.4 22 29 295 12

15.4 6.9

43.3

298 11

8.1 19 46

39.6 14.0 5.1

4 4

28.0 30 63 359 11

10.8 4.7

33.7

376 13

37.7 50 114

32.5 10.6 4.7

40 213’

0 4

7.8 18 19 245 15

17.1 9.9

48.3

197 14

3.7 20 27

6.1 19 26 245 14

16.9 9.5

49.5

226 16

7.0 21 23

45.8 16.1 9.6

0.4 4

52

8.9 20 45 300 15

16.5 9.1

48.3

268 13

8.8 18 43

45.1 15.3 8.6

4 4

FOR 104 WEEKS

49.8 16.9 10.0

IN DCGS TREATED WITH THIRAM

3

31.0

Female

320 12

13.3 18 27

31.0 10.2 4.0

Male

40 4

BIOCHEMISTRY

TABLE

-

-

-

-

-

-

40 -

5.9 19 23 198 13

20.1 8.6

51.8

164 12

3.2 25 44

49.1 18.5 8.1

0 4

7.1 21 29 230 10

18.6 8.0

49.6

180 14

5.5 22 27

47.0 18.1 7.6

0.4 4

104

8.9 21 46 311 13

17.0 7.1

45.1

267 11

8.1 21 39

48.6 19.0 8.1

4 4

40 -

-

-

-

-

-

-

$ 2

!z

s .s

;1

5 -fJ

TOXICITY

OF

THIRAM

to those of the controls. In the 0.4 mg/kg/day group of both sexes, there were no changes in the anemia parameters. Although males showed slightly decreased hematocrit, hemoglobin, and erythrocyte counts at Week 4, the values were comparable to those determined before commencement of treatment. In blood biochemistry, the levels of ALP and total cholesterol were already increased in males of the 40 mg/kg/day group at Week 4 (Table 3). These animals also showed higher values of GOT and GPT at the terminal stage of the course of toxicity. These parameters were also elevated in females of this group. In the 4 mg/ kg/day group, the total cholesterol level in males and females was higher during the treatment, whereas males showed no significant differences from controls in this parameter. Elevations of GPT were observed in males and females at Week 52 and in females at terminal kill. Pathology Rat. In organ weight analyses, a significant change in the calf muscle (M. triceps surae) was observed (Table 4). In males, the calf muscle seemed to develop in proportion to growth of the body during the first year of life and the relative weights to body weights were almost the same among the three interim kills, Weeks 13, 26, and 52, whereas the absolute weights increased with age during this period. Both absolute and relative weights of the tissue decreased nearly 40% during the next year, although body weight was almost stable during this period. In females, however, the absolute weights of the tissues were almost consistent in the first half of the treatment period. Relative weight showed a tendency to decrease with prolonged treatment. The values decreased in the latter half of the treatment, showing 7 and 12% decreases for absolute and relative weight, respectively. In the 300 ppm group, both absolute and relative weights of the calf muscle were significantly decreased in both males and females at scheduled kills after

IN

RATS

AND

DOGS

675

Weeks 52 and 104, although they were comparable to the controls at interim kills after Week 13 or Week 26. Kidney weight of males and females showed a tendency to decrease in the latter half of the treatment (Table 4). Females in this group exhibited an increased thyroid weight at terminal kill. The thyroid weights of females in the 30 and 3 ppm groups appeared to be increased at terminal kill, corresponding to the greater body weight of these animals. No treatment-related changes in the weights of the liver, testes, or ovaries were observed. At necropsy, the lowered weights of the calf muscles appeared to correspond to an increased incidence of atrophy of the calf muscles in females of 300 ppm group, although males did not show an increased occurrence of this change (Table 5). A striking change in gross lesions observed at terminal kill was that only 1 of 20 females in the 300 ppm group had skin mass versus 16 of 28 females in the control group (Table 5). The overall incidence of skin mass was significantly decreased in females of the 300 ppm group, 7 of 64, compared with the control group, 19 of 64. In females of 30 ppm group, occurrence of skin mass was also considerably depressed, 7120 versus 16/28 in the control group, at terminal kill, although there was no statistically significant difference between the groups. In females these skin masses arose largely on the ventral or dorsal surface of the body, with considerably higher occurrences at the axillary, lower abdominal, or upper thigh region. They appeared to be solid nodular or multilobular and sometimes had cysts of various sizes containing brownish-white, proteinous, or milky mateIidS.

In histology, atrophy of the calf muscle observed at necropsy was revealed to be atrophy or degenerative changes of muscle fibers of the tissue (Table 5). The changes were frequently observed in females found dead or killed in extremis in the last quarter of the treatment as well as in females at terminal kill. The muscle fibers were atrophic, losing their acidophilic tincture and were replaced partially by colla-

Week

of treatment:

WEIGHTS

Week

2.80 0.64

3.55 0.81

2.70 0.6 I

(g) (%)

(g) (%)

(g) (%)

17.0 0.006

9.30 3.50

1.81 0.68

72.3 0.27

1.93 0.73

(mg) (W)

(g) (%)

(g) (%)

(g) (W)

(g) (%)

1.84 0.68

70.6 0.26

1.82 0.61

9.51 3.51

14.8 0.006

3 8

2.81 0.64

3.51 0.82

2.76 0.63

16.1 3.66

17.6* 0.004*

3 8

13

13

30 8

1.93 0.73

75.4 0.29

1.75 0.66

8.94 3.38

15.1 0.006

30 8

2.7 I 0.62

3.44 0.79

2.85 0.65

16.8 3.84

1.82 0.77

67.8 0.29

1.62* 0.69

8.33 3.52

15.5 0.007

300 8

2.38 0.60

3.40 0.86

2.63 0.66

15.2 3.81

18.6* 0.005

300 8

KIDNEYS,

20.9 0.005

LIVER,

1.79 0.64

71.3 0.26

1.82 0.65

8.93 3.18

15.5 0.006

8

0

2.93 0.59

3.60 0.72

3.12 0.63

16.7 3.34

22. I 0.004

0 8

TESTES,

1.98 0.66

84.4 0.28

2.00 0.67

9.41 3.15

0.006

18.0

3 8

2.99 0.59

3.45 0.69

3.24 0.64

17.1 3.38

22.8 0.005

3 8

OVARIES,

26

26 30 8

CALF

1.88 0.65

77.5 0.27

I .92 0.67

9.00 3.1 I

18.5 0.006

30 8

2.96 0.58

3.61 0.73

3.24 0.64

17.7 3.49

22.9 0.005

AND

’ Ratios of organ weight to body weight are expressed as percentage (76). * Significantly different from the control at a 5% probability (Student’s t test).

Thyroids Absolute Relative Liver Absolute Relative Kidneys Absolute Relative Ovaries Absolute Relative Calf muscle Absolute Relative

0 8

16.2 3.61

(g) (o/o)

of treatment:

20.9 0.005

0 8

OF THE THYROIDS,

(mg) (%)

Dose group (ppm): No. of animals examined:

Female

Thyroids Absolute Relative” Liver Absolute Relative Kidneys Absolute Relative Testes Absolute Relative Calf muscle Absolute Relative

Dose group (ppm): No. of animals examined:

Male

ORGAN

1.73 0.65

72.6 0.27

1.71 0.64

8.96 3.24

18.5 0.007

300 8

2.95 0.60

3.78 0.78

2.93 0.60

17.7 3.59

23.3 0.005

300 8

MUSCLE

TABLE 4

1.93 0.62

77.4 0.25

2.2 I 0.71

9.97 3.20

18.8 0.006

8

0

3.25 0.59

3.67 0.67

3.82 0.69

19.3 3.50

21.9 0.004

0 8

OF WISTAR

2.05 0.59

83.7 0.24

2.31 0.67

10.66 3.05

20. I 0.006

3 8

3.05 0.55

3.64 0.66

3.63 0.66

18.3 3.31

23.5 0.004

3 8

RATS

52

52

1.98 0.58

65.4 0.18

2.23 0.65

10.46 3.04

22.5* 0.007

30 8

3.25 0.58

3.80 0.69

3.72 0.66

18.2 3.23’

27.8 0.005

30 8

TREATED

I .66* 0.54;

60.8 0.20

2.03 0.65

9.35 3.03

19.7 0.006

300 8

2.46’ 0.47’

3.72 0.71

2.96* 0.56*

17.5 3.32

21.5 0.004

300 8

WITH

1.60 0.40

72. I 0.18

2.92 0.72

15.1 3.70

22.6 0.006

0 28

1.94 0.34

3.48 0.59

4.2 1 0.74

19.6 3.37

37.2 0.006

0 26

THIRAM

1.77 0.41

64.3 0.15

3.30 0.76

14.9 3.37

30.4* 0.007

3 29

1.82 0.32

3.63 0.63

4.44 0.76

19.7 3.34

36.7 0.006

3 26

IN FEED

104

104

1.73 0.41

86.0 0.20

3.08 0.7 1

15.4 3.51

30.6* 0.007

30 20

2.09 0.37

3.59 0.65

4.39 0.79

20.4 3.68

41.0 0.007

30 24

300 28

I .23* 0.338

66.8 0.18

2.46* 0.67

12.6* 3.39

29.9* 0.008’

300 20

1.47* 0.27*

3.64 0.66

3.66’ 0.66

18.5 3.32

44.0 0.008

FOR 104 WEEKS

5

5

17

12

7

27

3

0

1

7

14

0 0

2

18

17

12

25

1

0 2

9

16

0 0

15

15

0 0

19

5

3 0

4

26

1

0 0

9

15 0

3

0 2

1 0 6

1

20

10

13

14

1

2

21

5*

23

10

8

6

1

4

64

8

0

26

300

3

64

7

30

3

64

7

0 63

0 0

7

0

0 1

2

20

5

12

17

4

6

26

3

0 0

0 0

6

4

1 7

1 0

2

18

2 0

2

17

5*

14

11 7

20

3

8

28

300

8

0

3

24

30

Week 1O46

’ Incidence in all animals examined. * Incidence in the animals subjected to Week 104 terminal kill. * Significantly different from the control at a 5% probability (Fisher’s exact test).

Gross lesion Calf muscle Atrophy Skin Mass Microscopic lesion Calf muscle: Atrophy/degeneration Sciatic nerve Atrophy/degeneration Heart Myocardial atrophy/fibrosis Kidney Chronic nephrosis Pancreas Acinar atrophy/pancreatitis Thyroid Follicular cell hyperplasia C-cell hyperplasia Testis Seminiferous tubular atrophy Pituitary Adenoma/adenocarcinoma Mammary gland Hypersecretion/hyperpplasia Fibroadenoma

Dose group (ppm): No. of animals examined:

Overall”

Male

0

3 16

21

1 0

0

18

0

9

6

19

I

64

3

5 13

21

0 1

0

15

6*

11

8

15

I

64

4 9

25

0 2

1

16

3

8

9

14

2

64

30

Overall

7 3*

21

0 0

0

6*

4

15

19:

7*

11*

64

300

0

15 4 9

0 15

1 4’

0 1

1 0

15

0

0 0

1

7

0 0

2*

10 14

12*

11*

1*

9*

20

300

4*

9

7

2

20

30

3

9

7

10

I

29

3

Week 104

0

8

6

16

1

28

Female

MAJOR GROSSAND MICROSCOPICLESIONS IN WISTAR RATS TREATED WITH THIRAM IN FEED FOR 104 WEEKS

TABLE

678

MAITA,

TSUDA,

gen fibers. The lesions were limited in the calf muscle but not in the muscles of other parts of the body, including the thigh. A variety of degenerative processes including atrophy, vacuolation, fibrosis, and mineralization of the nerve fibers were found in the sciatic nerve in the aged animals of both treated and control groups. In the last quarter of the treatment period, the lesions were observed more frequently in females of the high-dose group than in the control group. In most cases the muscular lesions of the calf muscle were found concomitant with the alterations in the sciatic nerve. There were no abnormalities in the histology of the osseous and cartilaginous tissues from the sternum and femur. In males, the high-dose group showed a significantly depressed occurrence of myocardial atrophy/fibrosis, which is a common senile change of male rats of this strain (Table 5). In females, the incidence of this lesion was incidentally increased in the 3 or 300 ppm groups as shown in Table 5. Occurrence of chronic nephrosis, which is also a typical senile lesion of the rat, was considerably depressed in females of the 300 ppm group (Table 5), but no similar tendency was observed in males. There were no remarkable changes in the pancreas, thyroid, and testis. During the last 8 weeks of the treatment period, pituitary adenoma occurred more frequently in females of the 30 and 300 ppm groups (four of five and seven of eight, dead animals bore the tumor, respectively) than in those of the 3 ppm and control groups (one of three in both groups), resulting in slightly higher mortalities in middle- and high-dose females during this period (Fig. 1). However, since the occurrence of this tumor in these two female groups at terminal kill was less than that in the 3 ppm and control groups, overall incidences of the tumor were comparable in all groups (Table 5). The decreased occurrences of skin masses in females of the middle- and high-dose groups were comparable to the decreased incidences of mammary fibroadenoma (Tables 5 and 6). Although this negative trend in incidence of

AND

SHIRASU

this tumor was not confirmed statistically for overall incidence of females in the 30 ppm group, the incidence (9/64) was lower than that of the control ( 16/64). The incidence of mammary tumors was also considerably depressed in females of the 3 ppm group at terminal kill, 9/29 versus 15/28 in the control group, although no statistical significance was seen. The incidences of other types of tumors were comparable to those of the controls in both sexes (Table 6). Dog. In organ weight analyses, high values were recorded for both absolute and relative weights of the liver in males of the 4 mg/kg/ day group, whereas those of females were comparable to the controls (Table 7). The weights of other organs in the treated groups of both sexes were comparable to those in the controls. Although no particular gross lesions were observed in the animals at both scheduled and unscheduled necropsy, a variety of lesions attributed to the treatment were observed at histopatholic examination (Table 8). Males and females in the 40 mg/kg/day groups showed fatty degeneration, atrophy, and focal necrosis of hepatocytes at centrilobular and midlobular regions in the liver. Edema of the retina was prominent in animals of this dose group, and one male revealed a partial loss of the retina accompanied by fibrous thickening of the tapetum nigrum. One female had diffuse necrosis of cardiac muscle with hemorrhage in the heart and another female had a pyloric ulcer in the stomach (not shown; Table 8). Hemorrhagic foci were also evident in other parts of the body, including liver. There were no histological lesions in the central and peripheral nervous system comparable to the behavioral disturbance observed in the dogs of the high-dose group. In the 4 mg/kg/day group, animals of both sexes showed atrophy of hepatocytes and granuloma with brown pigment deposits positive to hemosiderin staining (Prussian blue method) in the liver. Two females in the 4 and 40 mg/kg/day groups had swelling and vacuolation of proximal tubular epithelium in the kidney (Table 8).

TOXICITY

OF THIRAM

679

IN RATS AND DOGS

TABLE 6- 1 HISTOLCKXCAL TYPES AND NUMBER OF ANIMALS WITH TUMORS IN WISTAR RATS TREATED WITH THIRAM IN FEED FOR 104 WEEKS Males Dose level (ppm): No. of rats examined: Site and type of tumors

0 63

3 64

Females 30 64

300 64

No.

Hematopoietic system Multiple organs Leukemia Lymphangioma Reticulum cell sarcoma Spleen Fibrosarcoma Respiratory system Lung Adenoma Adenocarcinoma Squamous cell carcinoma Digestive system Duodenum Papillary adenoma Liver Adenoma Cholangioma Pancreas Exocrine grandular cell adenoma Islet cell adenoma Urinary system Kidney Adenoma Clear cell adenoma Liposarcoma Reproductive system Testis Interstitial cell tumor Sertoli cell tumor Ovary Granulosa cell tumor Uterus Endometrial polyp Leiomyosarcoma

0 64

3 64

30 64

300 64

of rats with tumors

1 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 1 0

2 0 0

0 0 1

0

0

0

0

0

0

1

0

0 0 0

0 1 0

2 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 I

0

0

0

0

0

0

1

0

0 0

0 0

0 0

0 0

I 1

0 0

0 0

0 1

0 3

3 5

0 0

0 0

0 0

0 0

0 0

0 0

0 1 0

0 0 0

0 0 0

1 0 0

1 0 0

0 0 0

0 0 I

0 1 0

2 1

1 0

0 0

3 0 0

0

1

0

0 0

0 0

0 0

1 2

DISCUSSION In the assessment of human risk from a compound based on the results of chronic studies, it is preferable that the doses used inelude both a toxic level showing mild but pos-

itive toxic signs and a no-observable-effect level (NOEL). In the rodent study, the former can be equivalent to the MTD in a carcinogenicity study, where the animals have an approximately 10% reduction in body weight compared to the controls. In the present rat

MAITA.

680

TSUDA, AND SHIRASU TABLE 6-2

HISTOL~CICAL

TYPES AND

NUMBER WITH

OF ANIMALS

THIFCAM

WITH

IN FEED

TUMORS

IN WISTAR

RATS

TREATED

FOR 104 WEEKS

Males Dose level (ppm): No. of rats examined: Site and type of tumors

0 63

Females

3 64

30 64

300 64

No. Nervous

3 64

30 64

300 64

tumors

system

Cerebrum Meningioma Cerebellum Meningioma Endocrine system Pituitary Anterior adenoma Anterior adenocarcinoma Thyroid gland Adenoma C-cell adenoma Adrenal gland Cortical adenoma Pheochromocytoma Integumentary system Skin Trichoepithelioma Fibroma Lipoma Hemangioma Osteosarcoma Giant cell sarcoma Unclassified sarcoma Mammary gland Adenoma Fibroadenoma Adenocarcinoma Thoracic cavity Squamous cell carcinoma Reticulum cell sarcoma Abdominal

of rats with

0 64

1

0

0

0

0

0

1

0

0

0

1

0

0

0

0

0

16 0

14 0

19 0

15 0

20 1

20 1

23 2

21 0

0 0

0 0

2 0

2

0 0

2

1 0

0 1

0 1

2 0

0

0

2

1 0

0

1

0 1

0 0

0 2 0 0 0 0 0

2 3 0 0 0 1 0

0

0 3 0 0 0 0 0

0 1

0 1 0 1 1 0 0

0 0 0

0 0 0

0 0 0

1 0

1

0 0

0 0

I 0 0

0 1 I

0

2 0 0 1 0 I

1

0

1

0

0 0 0

1 16 2

0 0 0

1 0 0 0

0

0

1

1

13 1

9 0

0 3* 0

0 0

0

0 0

0 0

0 0 1

0

0 0 0

I 0 0

0

cavity

Lipoma Liposarcoma Mesothelioma

0 0 0

0 0

* Significantly different from the control at 5% probability.

study, body weights in the 300 ppm group were depressed at rates of more than 10% compared to those of the controls a few weeks after initiation of treatment, although the weight gain soon returned to normal causing reduced

weights in 10% of the controls during the remainder of the treatment period. It is reasonable to say that the high dose in the present study was close to the MTD. In the dog study, the animals in the high-dose group 40 mg/kgJ

TOXICITY

OF THIRAM

681

IN RATS AND DOGS

TABLE 7 ORGAN

WEIGHTS

OF THYROIDS,

LIVER, KIDNEYS, TESTES, AND OVARIES OF DOGS TREATED WITH THIRAM FOR 104 WEEKS

Thyroids Dosegroup“ h&/day)

No. of animals examined

Absolute k4

Liver

Relativeb @)

Absolute k)

Kidneys Relative (S)

Absolute (9)

Testes/ovaries

Relative (%I

Absolute cd

Relative @)

Male 0 0.4 4.0

4 4 4

0.60 0.59 0.58

0.006

0.005 0.005

282.9 311.0 394.6

4 4 4

0.73 0.68 0.56

0.007 0.006 0.005

352.3 312.0 331.6

2.6 1 2.70 3.57

51.4 47.9 54.3

0.48 0.42 0.50

3.38 2.85 3.15

44.0 49.0 43.0

0.42 0.45 0.42

12.5 12.5 14.4

0.012

0.011 0.013

Female 0 0.4 4.0

0.63 0.74 0.74

0.006 0.006 0.007

a Animals in the 40 mg/kg/day group died during the treatment period and their organ weights were not measured. * Ratios of organ weight to body weight are expressed as percentage (9’0).

TABLE 8 MAJOR

MICRO~CZOPIC

LESIONS IN DOGS TREATED

WITH THIRAM

104 WEEKS

FOR

Female

Male Dose group (mg/kg/day): No. of animals examined: Heart Diffuse necrosis/hemorrhage Edematous thickening of endocardium Liver Hepatocellular degeneration Hepatocellular swelling/necrosis Hemorrhage/inflammation Pigmentation in Kupffer cell Granuloma with pigmentation Microgranuloma Kidney Tubular swelling/vacuolation at pars recta Thyroid Follicular cell hyperplasia C-cell hyperplasia Interstitial lymphocytes infiltration Eye Edema of retina Desquamation of retina Fibrous thickening of tapetum nigrum

0 4

0.4 4

4.0 4

40 4

0 4

0.4 4

4.0 4

40 4 1 1

2

2 2 2 1

1

2 2

3 1

1

2

1 2

1 1 1

1 3

1

1 1

2 1 1

2

1 3

682

MAITA,

TSUDA,

day, died but those in middle-dose group, 4 mg/kg/day, showed clear toxic changes in both clinical and laboratory examinations. The low doses in either the rat or the dog study induced no treatment effect and may be considered NOELs. Neurological disturbances including frequent nausea, salivation, and clonic convulsion observed in the dogs receiving doses of 4 or 40 mg/kg/day were the most conspicuous symptoms in the present study, although there were no histological abnormalities in the central or peripheral nervous system. Female rats given the compound in feed at a concentration of 300 ppm (13.8 mg/kg/day as average) for 104 weeks revealed an increased incidence of atrophy and degeneration of the sciatic nerve along with atrophic changes of the calf muscle, although these changes induced no neurological clinical signs in the animals. Lee et al. (1978) treated CD rats with the compound in feed for 80 weeks at average doses of 5, 20, and 52 mg/kg/day for males and 6, 26, and 67 mg/kg/day for females and observed ataxia or paralysis of the hind legs in females but not in males. The signs were first observed at Week 20 of treatment and a total of 8 of 24 females developed ataxic syndromes. Two rats also had curvature of the thoracic spine and atrophy of both hind legs. Lee and Peters (1976) conducted detailed histopathological examinations of the ataxic female rats, which received the test diet at an average dose of 65.8 mg/kg/ day for 36 weeks, and found degeneration of the sciatic nerve, severe atrophy and degeneration of muscle fibers of the gastrocnemius muscle, and degenerative changes in the ventral horn of the lumbar spinal cord. They suggested that the primary lesion was at the peripheral axon. The neurological symptoms of the dogs in the present study appeared to have been caused by damage to the autonomic nervous system, suggesting that the neurological target site was different for beagle dogs and CD rats. Moreover, in the dog study, there was no sex difference in response to the treatment, whereas CD rats showed a clear predisposition toward females. The results indicate

AND

SHIRASU

that species and sex differences in metabolism of the compound might play an important role in the induction of neurological disturbances in the animals and further studies are needed to elucidate the mechanism of this toxicity. In the present rat study, histological examinations revealed an increased incidence of atrophy and degeneration of the sciatic nerve along with regressive changes in the fibers of the calf muscle. Since the major part of the calf muscle is the gastrocnemius muscle, the lesions observed in the present study are comparable to those reported by Lee and Peters (1976) and Lee et al. ( 1978). Radiculoneuropathy, one of the senile changes in aged rats, is composed of segmental demyelination of the spinal nerve root and sciatic nerve accompanied by atrophy of the calf muscle (Berg et al., 1962; Van Steenis and Kroes, 197 1; Gilmore, 1972; Mitsumori et al., 1981). The lesions can be seen more frequently in males than in females, probably because of the males’ greater body weights. It was difficult to distinguish the histological features of the lesions observed in the high-dose group from those of radiculoneuropathy in the control group. Lee et al. (1978) reported no histological changes of the sciatic nerve in the rats that received a dietary dose of the compound 26 mg/kg/day or less for 80 weeks. These results suggest that the increased incidence of the lesion in the high-dose group might have been incidental and not a reflection of toxicity. However, the incidence of the lesion at terminal kill in the high-dose group, 60%, was more than double that of the control group, 29%. Atrophy and degeneration of the calf muscle are considered to be changes secondary to the sciatic nerve lesion. While the muscular lesions occurred 2.6 times more frequently in the high-dose group than in the controls at terminal kill, the overall incidence, including the dead animals during the study, was three times greater in the former group than in the latter group. In general, histological changes in the sciatic nerve are easily complicated by autolysis but those of the muscle are preserved longer after death. Therefore, it is likely that the rats with the muscular changes

TOXICITY

OF THIRAM

had the sciatic nerve lesions as well and that the overall occurrence of the sciatic nerve lesion was increased in the high-dose group. This may indicate that the elevated incidence of sciatic nerve lesions in females of the highdose group was attributable to the treatment. One female dog that received a dose of 4 mg/kg/day showed clonic convulsion accompanied by occasional fits of counterclockwise rolling. It was 37 weeks before she manifested the first neurological sign. Female rats in the present study as well as those in previous reports (Lee and Peters, 1976; Lee et al., 1978) also required long-term treatments before producing the peripheral nerve lesions. These findings suggest that this toxicity is of a chronic nature. Future studies should include a timecourse chemical analysis of the nervous tissue to investigate whether there is accumulation of the compound or its metabolites in the tissue. In the dog study, ocular lesions, which were readily seen in the ophthalmological examinations during the first week of treatment, were confirmed as retinal changes in the histopathological examination. Several thiocarbamate compounds, including diphenylthiocarbazone (Budinger, 1961; Delahunt et al., 1962) and sodium diethyldithiocarbamate (DuBois et al., 1961) have been reported to cause retinal changes in animals with a tapeturn, such as dog and rabbits, but not in species without this membrane, such as rats and monkeys. The tapetum has a high content of Zn2+. A possible mechanism of retinal toxicity with thiocarbamate compounds may be the active chelation of Zn*+ by the compounds, resulting in regressive changes of the retina. Another hypothesis for the mechanism of retinal damage concerns depression of catabolic activity in the tissue. DuBois et al. (196 1) conducted a biochemical analysis on beagle dogs following treatment with sodium diethyldithiocarbamate and reported that oxygen consumption in the liver and kidney was depressed in addition to a suppression of the utilization of pyruvate and a lowered synthesis of oxalate in the tissue. They suggested that

IN RATS AND DOGS

683

similar biochemical failure would have taken place in retina following treatment with the chemical. Since thiram has also a thiocarbamoyl moiety, it is plausible that the retinal damage by thiram treatment might be induced through a similar mechanism. Anemia was seen in both Wistar rats and beagle dogs in the present study. Although male and female dogs dosed at 40 mg/kg/day showed anemia to the same degree of severity, in the 4 mg/kg/day group the anemic condition was more prominent in females than in males. In the rat study only females in the 300 ppm group (11.6 mg/kg/day) had anemia. These results may suggest that females are more predisposed to anemia induced by the treatment than are males. There have been few descriptions of anemia in previous studies with thiram or other dithiocarbamate compounds. Among the toxicity studies with thiocarbamate-related compounds, mild anemia was reported in female CD rats receiving Noxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide in feed at a concentration of 600 ppm for 6 or 12 months (Hinderer et al., 1986). Although Lee et al. (1978) treated female CD rats with thiram in feed at an average dose of 67 mgfkgfday for 80 weeks and Hasegawa et al. ( 1988) gave female F344 rats the compound at an average dietary dose of 42.3 mg/kg/day for 104 weeks, they observed no anemia in those animals. There may be a strain difference as well as a species difference in hematological response to the treatment. An effect on liver function by the compound was reported by Lee et al., 1978, who found elevations of SGOT and SGPT in male CD rats following a dietary treatment as high as 132 mg/kg/day for 13 weeks. In the present study, the dogs of both sexes in the 40 mg/kg/ day group had increases in plasma ALP, GOT, GPT, and total cholesterol accompanied by marked histological changes in the liver, indicating that the cause of death of these animals might have been liver injury. In the 4 mg/kg/day group, the dogs of both sexes also had high values for plasma GPT and total cholesterol. Moreover, males had increases of

684

MAITA,

TSUDA.

absolute and relative liver weights, and both males and females had granulomas with brown pigment deposition in the liver. These changes might indicate that necrotic changes of hepatocytes with a slight hemorrhage were induced by the treatment. All these findings would suggest that the liver is a target organ of the compound for dogs. Dogs seemed to be more vulnerable than CD rats to the compound. A contradictory result was obtained for the renal lesions of female rats and dogs in the present study. The high-dose female rats had a reduced incidence of chronic nephrosis, whereas mid- and high-dose female dogs had degeneration of proximal tubular cells at pars recta. Lee et al. ( 1978) reported a sparing effect of the compound on renal lesions in both male and female CD rats. The mechanism of metabolic activation of the compound might differ in rats and dogs. Although the incidence of chronic nephrosis in high-dose male rats was similar to that in the controls, the occurrence of myocardiac lesions was considerably depressed in the treated rats. Atrophy and fibrosis of the heart are senile lesions commonly seen in various strains of rats, including F344, Sprague-Dawley, and Wistar (Imai et al.. 1978a). Males are more predisposed to the lesion than females, suggesting the role of androgenic hormones in the onset of this pathological condition (Imai et al., 1978b). Longterm treatment with testosterone propionate or atenolol, a ,&androgenic blocking agent, can aggravate the severity of the lesion (Yoshimura et al., 1988). On the other hand, Yoshimura et al. (1989) reported a depressed occurrence of the lesion in Sprague-Dawley rats that received captopril, an inhibitor of angiotensin I converting enzyme, at doses of 30 or 100 mg/ kg/day for 52 weeks, suggesting that a longacting positive inotropic effect of angiotensin II might play a role in the onset of the lesion. It is interesting that the occurrence of two major aging diseases in rats, chronic nephrosis and myocardiac lesion, was significantly depressed following treatment with the compound. Speculation concerning an antioxi-

AND

SHIRASU

dative effect of the compound as a possible mechanism of the phenomenon could be raised. Bartoli et al. (1983) reported an antioxidant effect of diethyldithiocarbamate (DDTC), a reduction product of disulfiram. Since thiram has a chemical structure very close to that of disulfiram with only the diethyl radical replaced with a dimethyl radical, it is likely that thiram or its metabolites has an antioxidant effect. A slight increase in thyroid weight was observed in the treated female rats, but no histological evidence was obtained to support the weight change. There were no increased incidences of any types of tumors in the present study. However, the incidence of mammary fibroadenoma was significantly depressed in females of the 30 and 300 ppm groups. They also had a lower occurrence of skin masses, most of which were considered to arise from the mammary gland. It is generally recognized that tumor incidences are lower when animals have an intensively retarded growth. Although the body weights of females in the 300 ppm group were significantly decreased during most all of the course of the treatment, the degree of depression was only 10% at most, and the body weights of females in the 30 ppm group were comparable to those of the controls. It is more plausible that the occurrence of mammary tumors in the present study was depressed directly by thiram treatment. Disulfiram and its reduction product, DDTC, have been shown to have an anticancer potential which might be attributable to their antioxidative property (Wattenberg, 1974, 1980; Perchellet et al., 1987). As mentioned above, given the structural similarity between thiram and disulfuam, it is reasonable to suggest that tumorigenesis can be also reduced by the treatment of thiram. Recently, Hasegawa et al. ( 1988) conducted a 2-year oncogenicity study with thiram in F344 rats and reported a dose-dependent reduction of spontaneous leukemia (so called Fischer leukemia) in both sexes, but not of mammary fibroadenoma. Fischer leukemia and fibroadenoma are major tumors for F344 rats of both

TOXICITY

OF

THIRAM

sexes and female Wistar rats, respectively. Fischer leukemia rarely occurs in Wistar rats. It seems that thiram treatment may suppress the occurrence of major tumors in those two rat strains. However, although mammary fibroadenoma is also a major tumor for female F344 rats and pituitary anterior adenoma occurs more dominantly in female Wistar rats than does mammary fibroadenoma, no suppressive responses were demonstrated in the incidence of these tumors. Leukemia is a tumor originating from the hematopoietic tissue and mammary fibroadenoma arises from the epithelial tissue. These two tissues originate from different germ layers and must have different physiological behaviors. Further experiments are necessary to elucidate why thiram treatment does not depress the occurrence of tumors other than leukemia for F344 rats and fibroadenoma for female Wistar rats, and why the treatment suppresses the tumorigenesis of the tissues from different germ layers when the rat strain used in the experiment is different. REFERENCES BARTOLI, G. M., MULLER, A., CADENAS, E., AND SIES, H. ( 1983). Antioxidant effect of diethyldithiocarbamate on microsomal lipid peroxidation assessedby low-level chemiluminescence and alkane production. FEBS Left. 164,37

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BERG, B. N., WORF, A., AND SIMMS, H. S. (1962). Degenerative lesions of spinal roots and peripheral nerves in aging rats. Gerontologia 6, 72-80. BUDINGER,J. M. (196 I). Diphenylthiocarbazone blindness in dogs. Arch. Pathol. 71, 304-310. DELAHUNT, C. S., STEBBINS,R. B., ANDERSON, J., AND BAILEY, J. (1962). The cause.of blindness in dogs given hydroxypyridinethione. Toxicol. Appl. Pharmacol. 4, 286-291.

DuBoq K. P., RAYMUND, A. B., AND HIETBRINK, B. E. (1961). Inhibitory action of dithiocarbamates on enzymes of animal tissues. Toxicol. Appl. Pharmacol. 3, 236-255.

EDWARDS, H. M., JR. (1987). Effects of thiuram, disulfiram, and a trace element mixture on the incidence of tibia1 dyschondroplasia in chickens. J. Nutr. 117, 964969.

GILMORE. S. A. (1972). Spinal nerve root degeneration in aging laboratory rats. A light microscopic study. Anat. Rec. 174, 251-258.

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HASEGAWA, R., TAKAHASHI, M., FURUKAWA, F., ToYODA, K., SATO, H., JANG, J. J., AND HAYASHI, Y. ( 1988). Carcinogenicity study of tetramethylthiuram disulfide (thiram) in F344 rats. Toxicology 51, 155165.

HEDENSTEDT, A., RANNUG, U., RAMEL, C., AND WACHTMEISTER, C. A. (1979). Mutagenicity and metabolism studies on 12 thiuram and dithiocarbamate compounds used as accelerators in the Swedish rubber industry. Mutat. Res. 68, 3 13-325. HINDERER, R. K., LANKAS, G. R., KNEZEVICH, A. L., AND AULETTA, C. S. (1986). The effects of long-term dietary administration of the rubber accelerator, N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide, to rats. Toxicol. Appl. Pharmacol. 82, 52 I-53 I. HODGSON, J. R., AND LEE, C. C. (1977). Cytotoxicity studies on dithiocarbamate fungicides. Toxicol. Appl. Pharmacol. 40, 19-22. IMAI, K., HAYASHI. Y., AND KONO, M. (1978a). Testosterone-induced focal myocarditis in rats. I. Morphological and biochemical studies with special reference to pathogenetical mechanism and species differences. J. Toxicol. Sci. 3, 2 15-228. IMAI, K., YOSHIMURA, S., AND IGUCHI, H. (1978b). Testosterone-induced focal myocarditis in rats. II. Morphological and biochemical observations of the adrenal in relation to the pathogenetical mechanism of cardiac lesions. J. Toxicol. Sci. 3, 229-242. International Agency for Research on Cancer, IARC (1976). IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. Vol. 12. Thiram, pp. 225-236. Lyon. LEE, C. C., AND PETERS, P. J. (1976). Neurotoxicity and behavioral effects of thiram in rats. Environ. Health Perspect.

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LEE, C. C., RUSSELL,J. Q., AND MINOR, J. L. (1978). Oral toxicity of ferric dimethyldithiocarbamate (ferbam) and tetramethylthiuram disulfide (thiram) in rodents. J. Toxicol.

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MITSUMORI, K., MAITA, K., AND SHIRASU,Y. ( 198 1). An ultrastructural study of spinal nerve roots and dorsal root ganglia in aging rats with spontaneous radiculoneuropathy. Vet. Pathol. 18, 7 14-726. PASCHIN, Y. V., AND BAKHITOVA, L. M. (1985). Mutagenic effectsof thiram in mammalian somatic cells. Food Chem.

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PERCHELLET, J. P., ABNEY, N. L., THOMAS, R. M., PERCHELLET, E. M., AND MAATTA, E. A. (1987). Inhibition of multistage tumor promotion in mouse skin by diethyldithiocarbamate. Cancer Res. 47,63026309.

RUDZKI, E., OSTASZEWSKI, K., GRZYWA, Z., AND KoZLOWSKA, A. (1976). Sensitivity to some rubber additives. Contact Dermatitis 2, 24-27. SHORT, R. D., RUSSEL, J. Q., MINOR, J. L., AND LEE, C. C. (1976). Developmental toxicity of ferric dimethyldithiocarbamate and bis(dimethylthiocarbamoy1) di-

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(1984). The problem of the sensitization to dithiocarbamates in thiuram-allergic patients. Dermatologica 169, 70-75. VAN STEENIS,G., AND KROES, R. (197 1). Changes in the nervous systemand musculature of old rats. Vet. Pathol. 8, 320-332. WATTENBERG, L. W. (1974). Inhibition of carcinogenic and toxic effects of polycyclic hydrocarbons by several sulfur-containing compounds. J. Natl. Cancer Inst. 52, 1583-1587. WA~TENBERG. L. W. (1980). Inhibition of chemical carcinogenesis by antioxidants. In Carcinogenesis, Vol. 5, Modifiers ofChemical Carcinogenesis (T. J. Slaga, Ed.), pp. 85-98. Raven Press, New York.

YOSHIMURA, S., AND HASHIMOTO. K. (1988). Role of endogenous Padrenetgic mechanism in the pathogenesis of spontaneous myocaridal fibrosis in rats. Tohoku J. Med. 155,327-333. YOSHIMURA,

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T. (1979). Mutagenic activity of thiram in Ames tester strains of Salmonella typhimurium. Mutat. Res. 68, 9-13. ZDZIENICKA, M., HRYNIEWICZ, M., AND PIE~~KOWSKA, M. (1982). Thiram-induced sperm-head abnormalities in mice. Mutat. Res. 102, 2 16-264. CZYK,