Toxicity of toxaphene in the rat and beagle dog

FUNDAMENTALANDAPPLIEDTOXICOLOGY

7,406-418(1986)

Toxicity of Toxaphene

in the Rat and Beagle Dog

IH CHU,* DAVIDC.VILLENEUVE,*CHONG-WLJ SUN,~VIC SECOURS,* BRYAN~ROCTER,$ ELSIE ARNOLD,~ DAVID CLEGG,~ LINCOLN REYNOLDS,~~ AND V. E. VALLI# *Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario, $Bio-Research Laboratories, Montreal, Quebec, §Toxicotogical Evaluation Division, Bureau of Chemical Safety, Food Directorate, Ottawa, Ontario, “Ontario Research Foundation, Mississauga. Ontario, and *Biopath Analysts Limited, Guelph, Ontario, Canada; and TBureau of Environmental Protection, Ministry of Urban and Rural Construction and Environmental Protection, Beijing, China

Toxicity of Toxaphene in the Rat and Beagle Dog. CHU, I., VILLENEUVE, D. C., Sm, C., L., ANDVALLI,V. E.(l986). Fundam. Appt. Toxicol. 7,406-4 18. Residues of the insecticidal mixture, toxaphene, have been found in Great Lakes fish. The purpose of the present study was to assessthe subchronic toxicity oftoxaphene in the rat and beagle dog. In the rat study, groups of 10 male and 10 female animals were fed diets containing 0, 4, 20, 100, or 500 ppm of the test compound for 13 weeks. No clinical signs of toxicity or spontaneous deaths were observed. Toxaphene treatment up to 500 ppm had no effectson weight gain or food consumption. The liver/body weight ratio and hepatic microsomal enzyme activities (phenobarbital type) were increased in both sexesfed 500 ppm of the test compound. Toxaphene at the highest dose also caused kidney enlargement in male but not in female rats. Dose-dependent histological changes were seen in the kidney, thyroid, and liver. Changes in the liver and thyroid were considered to be adaptative but the injury in the proximal tubules of the kidney was focally severe.Groups of six male and six female beagle dogs were fed toxaphene in gelatin capsules at 0,0.2, 2.0, and 5.0 mg/kg body wt/day for 13 weeks. Food consumption and growth rate were not affected. All animals survived the entire treatment period. No clinical signs of toxicity were observed. The liver/body weight ratio and serum alkaline phosphatase were increased in dogs of both sexes fed 5.0 mg/kg. Mild to moderate dosedependent histological changes were observed in the liver and thyroid. Toxaphene was accumulated in a dose-dependent manner in the fat and liver of dogs and rats. Based on the biochemical, histological, and residue data, it was concluded that the no-adverse-effect levels of the pesticide were 4.0 ppm (0.35 mg/kg) for the rat and 0.2 mg/kg for the dog. o 1986 k&tyofToti~logy. SECOURS,V.,F%WER,B.,ARNOLD,E.,CLECG,D.,REYNOLDS,

Toxaphene is a technical mixture of more than 177 different polychlorinated camphenes (Casida et al., 1977); it was produced as a pesticide from 1947 to 1983 and was used primarily on cotton, soybeans, peanuts, and cattle (Korte et al., 1979). Toxaphene was used extensively in the south and southeast United States where most cotton farming is located (Carey et al., 1978, 1979). Recently, this pesticide has been found in Great Lakes fish at levels ranging from 0.02 to 3.9 ppm (Schmitt et al., 1981, 1983). Toxaphene has not been used in Canada. For the Great Lakes, long-range air transport probably represents the major source of toxaphene con0272-0590186 $3.00 Coptire 986bytbeSocietyofToxiwlogy. AIlrightsofreproductioninanyformreserved.

406

tamination since drainage basin and river runoff to the Great Lakes contribute negligible amounts of this pesticide (Rice and Evans, 1984). Many toxicity studies including acute, chronic, carcinogenicity, mutagenicity, and reproduction have been reported from various laboratories, and a recent World Health Organization document has summarized these and other studies (WHO, 1984). An examination of existing toxicity data indicated that most of the studies were not adequate by present standards due to small group size, inadequate dosing levels or procedures, or insufficient parameters examined. It was concluded that further animal

TOXAPHENE

studies were needed for the regulatory agencies to assessthe potential for adverse effects in humans associated with the ingestion of toxaphene-contaminated fish. To this end, subchronic studies (13 weeks) of toxaphene in two animal species, beagle dogs and Sprague-Dawley rats, were carried out. METHODS Toxaphene (polychiorinated camphenes) was provided by FBC Chemicals (Scarborough, Ontario) as a 90% w/w solution in xylene. This solution was vacuum distilled, with more than 8% w/w of the xylene being removed. Other chemicals and solvents were of reagent grade and were procured commercially. Rat experiment. Weanling Sprague-Dawley rats, purchased from Charles River Laboratories (Wilmington, Mass.), were acclimatized to the laboratory conditions for 1 week before the study (temperature: 2 1 ? 2°C; relative humidity: 40-60%). These animals were randomly divided into 10 per sex per group and were fed diets (Master Fox, Ralston-Purina) containing 0, 4.0, 20, 100, or 500 ppm toxaphene for 13 weeks. Corn oil (Mazola, 4% w/w of diet) was used as a vehicle to dissolve toxaphene and to facilitate its incorporation into the diets. All animals were housed individually in stainless steel mesh cages with free accessto food and water. Clinical observations were made daily; body weight gain and food consumption were measured weekly. At the termination of the toxaphene exposure all animals were lightly anesthetized with ether and exsanguinated via the abdominal aorta. All animals were examined grossly at the time of necropsy. The brain, heart, liver, spleen, and kidneys were excised and weighed. Blood samples collected at necropsy were analyzed for the following parameters: hemoglobin, packed cell volume, erythrocyte count (Baker 7000), total and differential leukocyte counts, platelet count, and prothrombin time. Serum biochemical profiles were determined in a Technicon microanalyzer (Model 12/60) and included sodium, potassium, inorganic phosphate, total bilirubin, alkaline phosphatase, asparate aminotransferase, total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase. Hepatic aniline hydroxylase (AH; Fouts, I963), aminopyrine demethylase (APDM; Cochin and Axelrod, 1959), and ethoxyresorufin deethylase (ER, Burke and Mayer, 1974) activities were determined based on the literature methods adapted to automated instruments. At necropsy, gross examination was performed. A section of femoral bone marrow was aspirated, spread on the slide from which thin films were made, and stained with MayGriinwald-Giemsa stain for cytological evaluation. The following tissues were taken and fixed in 10% buffered formalin (pH 7.4) for routine histological examination: eye, optic nerve, spinal cord, skin, tongue, brain, pitu-

TOXICITY

407

itary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, jejunum, ileum, pancreas, colon, cecum, kidneys, spleen, bone marrow, mesenteric and mediastinal lymph nodes, skeletal muscle, ovaries, uterus, vagina or testes, prostate, epididymis, sciatic nerve, urinary bladder, salivary gland, mammary gland, and heart. Potential fatty change in the liver was determined in frozen sections as previously described (Villeneuve et al., 1979). Sections of liver and perirenal fat were excised and kept at -70°C pending residue analysis using a gas chromatographic method as described previously (Villeneuve et al., 1979). Quantitation of toxaphene was made by integrating peak areas of total peaks. The GC operating parameters are described as follows: A Hewlett-Packard 5700 gas chromatograph equipped with a 63Ni electroncapture detector and a 1.83 m X 4 mm i.d. packed column was used (3% SE-30, 6% OV-215 on Chromosorb W HP, 80/ 100 mesh). The column and detector temperatures were 225°C (isothermal) and 35o”C, respectively. The carrier gas was 5% methane in argon at a tIow rate of 60 ml/min. Quantitation was made by use of a Spectra Physics Computer, Model 4000, to calculate the total peak areas. Selected samples were also analyzed by gas chromatograph using a DB- 170 1 capillary column (J. W. Scientific, 30 m X 0.3 mm i.d. X 0.25 pm) to confirm the chemical identity of residues in tissue samples. The operating conditions were as follows: column temperature, starting from 90 to 28o’C at 4C/min; carrier gas, helium at 30 cm/set; splitless mode; injector temperature: 250°C; makeup gas, 5% methane in argon at 60 ml/min. Dog experiment. Beagle dogs, 7-8 months of age, were purchased from White Eagle Laboratories (Doylestown, Pa.), and were acclimatized to laboratory conditions for 2 weeks before the study (temperature, 2 1 + 2°C; relative humidity, 40-60%). The dogs were divided into four groups of six animals per sex. Toxaphene was administered via gelatin capsules at 0.2 (low), 2.0 (intermediate), and 5.0 (high)’ mg/kg body wt/day around 8:00 AM. Gelatin capsules were prepared weekly using corn oil as a solvent in an amount of 50 mg/kg/day, and the control groups received corn oil only. Each animal was offered 400 g/day of standard dog food (Purina Chow No. 5007) during a 1-hr feeding period in the afternoon. Fresh water

‘ Dose adjustment was required for the high-dose groups. The dogs were initially treated with toxaphene at 10 m&kg body wt/day for 2 days, but convulsions, with salivation and vomiting, were observed in animals of both sexes. From the third day onward the highest dose was 5.0 mg/kg body v&/day. From the 5th week of study the high-dose groups inadvertently received 2.5 mg/kg body wt/day for 4 weeks, but the dose was adjusted back to 5.0 mg&g from the 9th week to the 13th week.

408

CHU ET AL.

was provided ad libitum. A 12-hr alternating light and dark cycle was maintained. Observations for clinical signs were made twice daily white food consumption was determined daily. Body weights were monitored weekly. Serum biochemistry, hematology and urinalysis parameters were measured 2 weeks before commencement, and at Weeks 5, 10, and 13 oftreatment. Blood samples were collected from the jugular vein of nonfasted animals while urine samples were obtained over a 16-hr period from animals not deprived of water or food. At necropsy, the animals were sedated with an iv dose of sodium pentobarbital and exsanguinated via the axillary artery. Serum biochemistry, hematology, hepatic mixed function oxidase activity, histology, and tissue residue analysis were carried out in a manner simliar to that described for the rat experiment. Urine samples collected before and during the study were analyzed for glucose, creatinine, urea nitrogen, protein, and albumin using methods similar to those described for serum. Data were treated with a one-way analysis of variance. When a significant difference was noted among the groups, the data were further analyzed by Duncan’s multiple range test in order to determine which groups were significantly different (Nie et al., 1977).

RESULTS Rat Study Clinical observation. No clinical signs of toxicity were observed. All animals survived until the termination of the study. Growth rate andfood consumption. Weight gain and food consumption of toxaphenetreated rats were not significantly different from those of control rats. Based on the food consumption data the amount of toxaphene ingested by the rats ranged from 0.35 to 45.9 mg/kg body wt/day for the males, and 0.50 to 63 mg/kg body wt/day for the females (Table 1). Gross pathology. Five male rats had fatty liver; of these one was found in the control group and the remainder in the 4(2), 20(l), 1OO(1) ppm groups. Three males and one female had kidney enlargement (2 males in the 500 ppm group; 1 male and 1 female in the 4.0 ppm group). Organ weight. There was a significant increase in the liver weight of rats of both sexes fed 500 ppm toxaphene. Slight increases in liver weight were also observed in lower-dose

groups but they were not statistically significant (Table 2). The relative kidney weights of male rats fed 500 ppm toxaphene diet were higher than the control (control: 0.30 f 0.03; treated: 0.37 + 0.04). Biochemical changes. The serum biochemical parameters measured in the present study were not affected by treatment. Hepatic aniline hydroxylase and aminopyrine demethylase activities in both sexes were increased by 500 ppm toxaphene (Table 2). Hematology. Hematological data revealed no effect of toxaphene. Histopathology. The liver, thyroid, and kidney were the target organs of toxaphene treatment. Mild and dose-dependent adaptive changes occurred in the liver (Figs. 1 and 2). Architectural changes were minimal to moderate and consisted of an accentuation of zonation seen at low magnification. Nuclear changes were characterized by mild to moderate anisokaryosis occurring in all dose groups (and in 2 control males), with some vesiculation and focal necrosis occurring only at doses of 20 ppm or more. In the hepatocellular cytoplasm a mild increase in homogeneity was noted in all groups except control females. In control males and in both sexes at 4 ppm this observation was made only in part of the perivenous area. In the 20 to 100 ppm groups, the homogeneity extended increasingly to all of the perivenous and to part of the midzone area. Only the observations in the 20 ppm and greater groups were considered to be biologically significant. In the highest-dose group, some animals had increased perivenous and midzone cytoplasmic volume accompanied by peripheralized basophilia. Minimal changes were observed in the biliary ducts. The prevalence of lesions is shown in Table 3. Dose-dependent renal injuries were seen in the proximal tubules. In males these consisted of large eosinophilic inclusions which protruded into the tubular lumen in the 20 ppm group (Figs. 3 and 4). In the two highestdose groups these inclusions were smaller and more refractive in appearance and more prevalent, occupying 50% of the tubular area.

TOXAPHENE

409

TOXICITY

TABLE

1

BODY WEIGHTS AND FOOD CONSUMPTION OF RATS FED TOXAPHENE Treatment (ppm toxaphene)

Initial weight (9)

Weight gain (g)

Food consumption WWday)

FOR 13 WEEKS’

Approximate amount of chemical ingested h/b/day)

Male Control 4.0 20 100 500

9Ok 11 91+ 8 94+ 8 97+ 9 92+ 9

397 430 433 460 431

+ 32 f 37 f 54 + 46 k37

22 f 23 f 23 f 24 k 24 f

1.8 1.6 2.3 2.2 2.3

0 0.35 1.8 8.6 45.9

15 + 15 + 16 f 16 + 15 f

1.0 1.2 0.9 1.3 1.7

0 0.50 2.6 12.6 63

Female Control 4.0 20 100 500

92k 8 86~ 8 94* 10 92+ 9 93 f 12

155 153 155 163 144

+ 2 + -t f

19 21 17 22 24

LIValues represent means + SD obtained from 10 rats.

These cytoplasmic changes were accompanied by anisokaryosis, and in severe cases, by focal tubular necrosis and casts. In females pyknosis was observed but tubular injury was

less severe. In the 4 and 20 ppm groups injury was minimal and focal and was not considered to be biologically significant even though no tubular injury was observed in the control

TABLE 2 LIVER WEIGHTS AND HEPATIC MIXED FUNCTION OXIDASE ACTIVITIES OF RATS FED TOXAPHENE FOR 13 WEEKS a Treatment (ppm in the diet)

Liver/body weight ratio (76 of body wt)

Aniline hydroxylase (nmol/mg protein/hr)

Aminopyrine demethylase (nmol/mg protein/hr)

Male Control 4.0 20 100 500

3.8 3.7 3.9 3.9 4.5

f 0.27 f 0.29 + 0.29 f 0.18 f 0.58’

6.7 k 1.1 6.6 +- 2.2 8.5 + 1.7 7.7 + 2.6 13.3 + 3.2”

28 + 4.2 3Ok 7.1 33 1?I.5.3 31 f 7.1 55 f 10.2b

10.2 + 8.7 + 10 + ll.O+ 17.6 +

25 + 25 + 27 + 29+ 46 k

Female Control 4.0 20 100 500

3.4 f 0.32 3.6 iz 0.3 1 3.7 + 0.22 3.7 f 0.36 4.5 k 2ob

’ Results denote means + SD from 9 or 10 rats. b Significantly different from controls (p G 0.05).

2.2 2.4 3.7 1.6 3.9b

1.9 1.7 2.4 1.1 5.9b

410

CHU ET AL.

FIG. 1. Liver from rat fed control diet for 90 days. Functional lobule with portal (left) and perivenous area (right). Hepatocytes and nuclei are of uniform size with a slight increase in perivenous cytoplasmic hlomogeneity. H & E, X275.

FIG. 2. Liver from rat fed diet containing 500 ppm toxaphene for 13 weeks. There is a moderate increase in cytoplasmic volume with reduced density in the perivenous and midzone areas. H & E, X275.

TOXAPHENE

TOXICITY

411

TABLE 3 PREVALENCEOFHISTOLOGICCHANGESINRATSFEDTOXAPHENEFOR~

3

WEEK.?/

Toxaphene (ppm in the diet) Male Tissuesb Liver Accented zonation Anisokaryosis Nuclear necrosis Increased cytoplasmic volume Peripheralized basophilia Vesiculation of biliary nuclei Increased cytoplasmic homogeneity Kidney Glomerular adhesions Tubular injury-secondary Tubular injury-primary Tubular necrosis Anisokaryosis Pyknosis Cast formation Interstitial sclerosis Thyroid Reduced follicular size Follicular collapse Increased epithelial height Cytoplasmic vacuolation Reduced colloid density Papillary proliferation

0 (control)

2/o

Female

4

20

100

500

l/O

311 711

510

614 317 9/o

4/O

9/O

510 2/o

4

20

100

500

4/O 6/O

5/O 9/O 3/O

7/O 911 3/O

317 317 710

4/O

215

6/O

5t2

410

7/O

811

511

8/O

610

($1

712

612

712

l/O

210

311

6/O

711

O/1

212

ai2

w

411

7/O

119 911 416

112

0 (control)

510

212

412

812

7/O

5/O

9/O

218

612

10/o

9/O

9/o

l/O

210

2/O WO 4/O

110 9/O 911 110

W’

10/o

8/O 612

8/O 911 810

l/O

l/O

l/O

513 3/O

7~2 2/O

5/5

212 110

3/O

4/O

($0

712

4/O

2/o

312’

511

711

5/l 712

611’

611

712’

l/8’

210

512

713

317

O/SC

O/9’

613

10/o

911

812

111

211 212

5/4=

512

812

8/O

811

317

6/l 111

7/3 5/O

l/8’ 4/O

O/SC

9/O

9/O

10/o

317 l/O

317 411

612

l/O

l/O

8/l

a Values denote number of animals with minimal-mild changes/number of animals with moderate-severe changes. b Number examined was 10 unless indicated otherwise. ’ Number examined = 9.

females. Glomerular changes were confined to adhesionsof the visceral and parietal layers of Bowman’s capsule and were observed in control as well as treated groups. Interstitial changes consisted of areas of sclerosis with varied amounts of lymphoid reaction and were not clearly indicative of a treatment effect at 4 ppm.

Morphological changes in the thyroid were mild to moderate in nature, and consisted of an angular collapse of follicles, increased epithelial height with multifocal papillary proliferation, and reduced colloid density. The interstitium was normal. Mild changes were observed in both the control and the 4 ppm males and were considered to be similar in

412

CHU ET AL.

FIG3. Renal proximal tubules from rat fed the control diet. Cytoplasmic volume and density are uniform. Nuclei have regular size and placement with compact chromatin. H & E, X400.

FIG.4. Renal proximal and distal (center) tubules from rat fed 500 ppm toxaphene diet for 13 weeks. The cytoplasm of proximal tubules has increased volume and basilar density due to closely packed hyaline inclusions. Nuclei are enlarged and vesicular with irregular central displacement and multifocal pyknosis. H &E, x400.

TOXAPHENE

413

TOXICITY

TABLE 4 TOXAPHENE RESIDUESIN LIVER AND FAT OF DOGS AND RATS’ Female

Male Treatment

Dog b-&g/day) Control 0.2 2.0 5.0

Rat (ppm in diet) Control 4 20 100 500

Liver

Fat

NDb 0.91 + 0.85 5.1 LO.47 9.8 + 1.6

ND 5.8 f 1.4 61 + 9.7 92 +40

0.17 0.70 3.6 7.1

kO.12 + 0.4 1 + 1.8 k0.96

2.9 2.3 47 105

0.05 0.06 1.7 2.3 7.4

0.84 0.47 1.8 4.5 57

1.2 1.3 1.5 2.7 10.6

kO.51 kO.14 kO.13 + 0.29 k4.9

1.0% 0.18 1.8 + 0.46 12 + 4.9 63 -c97 103 +28

f 0.02 f 0.02 kO.52 20.23 + 3.9

+ 0.21 f 0.07 +- 0.39 + 2.7 +14

Liver

Fat + 0.88 f 1.3 + 8.2 k26

u Results represent means + SD ppm, obtained from 5 rats or 6 dogs per group. b ND: Nondetectable < 0.03 ppm.

these groups and not related to treatment. No similar effects were observed in control females but the changes noted in 4,20, and 100 ppm females did not differ from those in control and low-dose males and, therefore, were not considered to be treatment related. Thus, only the moderate changes observed at 20 ppm or higher in males and at 500 ppm in females were considered to be clearly treatment related. The histological changes in the target organs of the males were generally more severe than in those of the females. Residue analysis. A dose-dependent accumulation of toxaphene residues was found in the fat and liver of rats (Table 4). Higher concentrations of toxaphene were retained in the adipose tissues. In addition, levels of toxaphene in female rats appeared to be higher than those of males. Dog Study Due to adjustments in the high-dose regimen, caution should be taken in interpreting the data pertaining to these groups of animals. The animals received 5.0 mg/kg/day for the first 4 weeks, 2.5 mg/kg by error dur-

ing the next 4 weeks, and back to 5.0 mg/kg until the termination of the study. However, since the purpose of the study was to establish a no-observable-effect level, it was decided to continue with the study to completion even though it was realized that the high-dose data would have only limited significance. Clinical observation. Brief convulsions, salivation, and vomiting were observed in one male and two females fed the test compound at 10 mg/kg body wt for 2 days. However, these signs were no longer evident when the dose was adjusted to 5 mg/kg body wt on the third day. No other signs of toxicity were noted. Growth rate and food consumption. No treatment-related changes in the growth rate were observed. Low-dose males and intermediate-dose females showed smaller weight gains than did the controls, but there was no indication of a dose-response relationship. During the first week of treatment some of the high-dose males and females consumed little or no diet when these animals were fed 10 mg toxaphene/kg body wt. This effect was no longer evident when the dose was adjusted to 5 mg/kg body wt on the third day of study. For the remainder of the study, although in-

414

CHU ET AL. TABLE 5 LIVER WEIGHTS AND SERUM ALKALINE PHOSPHATASEOF Doris FED TOXAPHENE FOR 13 WEEKS Alkaline phosphatase (mIU/ml)”

Treatment Ow&G-W

Liver/body weight ratio (% body wt)

2 Weeks

5 Weeks

10 Weeks

13 Weeks

Male 0 (control) 0.2 2.0 5.0

3.1 t 0.54 3.6 zk 0.46 3.6 zk 0.21 4.4 t 0.456

90 + 38 89 IL 45 (5) 87 f 26 (5) 91 ~36

74 + 22 (5) 83?47 100 f 44 (5) 147 +- 59b

93+ 130-t 132_+ 160 t

48 69 67 102

70+26 73 f 49 (5) 94 + 47 168k66b

99 * 135 + 127 + 205 f

117+ 161? 208 + 276 f

27(4) 93 107 162

14 f 153 f 145 + 185 f

Female 0 (control) 0.2 2.0 5.0

3.0 -c 0.6 3.4 +- 0.4 3.6 k 0.31’ 3.9 k 0.46’

93 I!I 21 99*35 85 * 25 67+22

30 80 81 76’

22 80 (5) 95 (5) 68 (5)(,

a Serum alkaline phosphatase levels were analyzed at 2 weeks before dosing and at 5, 10, and 13 weeks after dosing. Unless otherwise indicated in uarentheses. results reuresent means + SD from groups of 6 animals. b Significantly different from controls at p G 0.05 level.

dividual food intake varied, there was no indication of a treatment-related effect on any of the groups. Gross pathological changes. Common necropsy findings were red and dark patches on the ileocolic mucosa and lungs, and pale areas on the spleen and stomach. These occurred in control as well as treated groups. There were no changes which appeared to be treatment related. Organ weights. Significant increases in the liver/body weight ratios were observed in males receiving 5 .O mg and in females receiving 2.0 and 5.0 mg toxaphene (Table 5). Biochemical changes. The only serum biochemical parameter affected by toxaphene treatment was alkaline phosphatase in both male and female dogs (Table 5). There was a dose-dependent increase in this serum enzyme in both sexes of all three treated groups; however, statistically significant increases were found only in the high-dose group. Urinalysis. None of the urinary parameters examined were affected by toxaphene treatment. Hematology. No overt hematological changes were observed. Female dogs receiv-

ing 5.0 mg toxaphene/kg showed slight reduction in red cells (control: 7.0 + 0.50; treated: 6.5 f 0.28 X 106/& hemoglobin (control: 17.1 + 1.1; treated: 15.9 & 0.50 g/100 ml), and packed cell volume (control: 46 & 28; treated: 42.5 t- 1.1%) at Week 13. Hemoglobin content was also reduced at Week 10. Histopathology. The liver and, to a lesser extent, the kidney and thyroid were the organs affected by toxaphene treatment. However, changes in these organs were considered mild in nature even in the high-dose groups. Changes in the liver consisted of increased cytoplasmic eosinophilia of the periportal hepatocytes with increased density and granular appearance in the midzone and perivenous areas. Large eosinophilic inclusions were found in the high-dose males while cytoplasmic vacuolation was found in the mid- and high-dose groups (Figs. 5 and 6). Changes in the hepatocellular nuclei were minimal. These changes were considered to be dose dependent. The prevalence of histological changes is presented in Table 6. Changes in the thyroid included a mild increase in epithelial height and reduced colloid density of

TOXAPHENE

TOXICITY

FIG,5. Liver from dog of the control group. There is uniformity of nuclear and cellular size and density in the portal (left) and perivenous area (right). H & E, X275.

FIG.6. Liver from dog dosed with 5.0 mg/kg/day toxaphene. There is increased cytoplasmic density in the portal area (left) with fine fatty vacuoles and an irregular increase in perivenous hepatocellular volu~me (right). H & E, X275.

416

CHU ET AL. TABLE 6 PREVALENCE

OF HISTOLOGIC

CHANGES

IN Dam

ADMINISTERED

TOXAPHENE”

Treatment (mg toxaphene/kg body wt/day) Female

Male Tissue Liver Accented zonation Anisokaryosis with vesiculation Periportal eosinophilia Cytoplasmic vacuolation Peripheralized basophilia Biliary lymphoid reaction Interstitial lymphoid reaction Increased cytoplasmic density Eosinophilic inclusions Kidney Enlarged glomernli Glomerular adhesions Tubular anisokaryosis/ pyknosis Tubular cytoplasmic granularity/basophilia Thyroid Reduced follicle size/follicular collapse Increased epithelial height Reduced colloid density Proliferation of interstitial cells

Ob

0.2

2.0

5.0

Ob

0.2

2.0

5.0

216

216

116

316

116

216

316

416

116

116

316

416 216

6l6 216

216

116

416

116 616

116

116 116

216

116 216

116

316

316

216 616 216

116 216

216 216 116

416

516

l/6

316

216

416

116

316

116

216

116

216

116

116

116 116 116

116

116 316 116

316

316

116

416 416 5lf.3

216 216

516 416 516

316 216 216

216

’ Values denote number of animals showing changes/number of animals examined. b Control group.

thyroid follicles observed in the mid- and high-dose males. Although similar morphological changes were seen in females at all dose levels, the incidence was not dose related. Particularly in the low dose females, the changes were very mild in nature and were not considered to be biologically significant. Very mild changes were found in the kidneys of dogs treated with toxaphene. Glomerular adhesions in both sexes and tubular anisokaryosis/pyknosis in females occurred more frequently in treated groups than in controls but the incidence was not dose related and did not appear to be of biological significance. Tubular cytoplasmic granularity and basophilia were seen in one mid-dose male and

one high-dose female and may have been related to treatment. Residue analysis. A dose-dependent accumulation of toxaphene residue was found in the fat and liver of dogs. Concentrations of toxaphene in the fat were generally one order of magnitude higher than those found in the liver (Table 4). DISCUSSION Although toxaphene has been extensively studied the previously reported studies did not provide sufficient information required for a full health hazard assessment. Some of

TOXAPHENE

the deficiencies of previous studies include small group size of test animals and insufficient numbers of parameters examined. The present study was, therefore, carried out to provide information required for risk assessment. In the present study administration of toxaphene to dogs for 13 weeks produced toxicological effects: hepatomegaly and elevated serum alkaline phosphatase at 5.0 mgfkg; hepatocellular cytoplasmic vacuolation at 2 and 5.0 mg/kg, and eosinophilic inclusions at 5.0 mg/kg in males only; kidney tubular cytoplasmic changes at 2 and 5.0 mg/kg; and mild thyroid changes in the 2 and 5.0 mg/kg dosed males. Elevation of serum alkaline phosphatase was an interesting observation because this functional change correlated well with hepatomegaly and the microscopic changes observed in this organ. Our results agreed with those of Treon et al. (1952) who reported that dogs fed 40 and 200 ppm toxaphene diet (equivalent to 0.6-1.47 mg/kg body wt and 3.1-6.5 mg/kg body wt, respectively) showed hepatomegaly and degenerative changes in the kidney and liver. However, Treon et al. failed to observe mild treatment-related changes in the thyroid. Similarly, Lackey (1949) noted that dogs fed the pesticide at a dose of 4 mg/kg body wt had degenerative changes in the kidney and liver. In the present study, lack of changes such as urinary glucose, creatinine, and protein was not surprising as morphological changes in the kidney were mild even in the high-dose group. Toxicological changes which occurred in rats were generally similar to those in dogs but involved focally severe kidney changes at the highest dose level. Toxaphene treatment resulted in hepatomegaly, hepatic microsomal enzyme induction, and histological changes in the kidney, liver, and thyroid. This pesticide is an enzyme inducer of the phenobarbital type as it increases the hepatic aniline hydroxylase and aminopyrine demethylase but not ethoxyresorufin deethylase activities (Burke and Mayer, 1974). The results of the present rat study are consistent with those of

417

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Kinoshita et al. ( 1966) who reported the microsomal enzyme induction in rats fed toxaphene at 5 mg/kg and higher for 12 weeks. In a similar study, Peakall (1976) reported increases in liver weight and microsomal enzyme activities of rats administered 2.4 mg/ kg body wt/day for 1,3, and 6 months. Histological changes in the target organs of rats for the most part were mild to moderate. Only the kidneys of males fed 500 ppm toxaphene diet exhibited tubular changes which were considered to be severe and nonadaptive. Metabolism studies revealed that toxaphene underwent considerable metabolic degradation in rats (Pollack and Hillstrand, 1982; Pollack and Kilgore, 1976). Following a single oral dose of [i4C]toxaphene to rats, a total of 62% of the administered radioactivity was excreted. These metabolism results are supported by our residue data. Accumulation of toxaphene in the liver and fat of rats is lower than that which occurs with other organochlorine pesticides such as hexachlorobenzene. Analysis of the adipose tissues of rats fed 500 ppm hexachlorobenzene diet for 1 month showed that the levels of this compound ranged from 5500 to 83 17 ppm compared to 57 to 103 ppm toxaphene observed in the present 13-week study (Chu et al., 1983). Based on the biochemical, histological, and residue data it would appear that the no-observable-adverse-effect level of toxaphene is 0.2 mg/kg for the dogs and 4.0 ppm (0.35 mg/ kg) for the rats. ACKNOWLEDGMENTS The authors thank Mr. A. Viau, Mr. J. Kelly, Mr. A. Yagminas, Mrs. N. Beament, and Mrs. B. Reed for technical assistance; Mrs. M. Beaudette for data handling; and Mrs. J. Ireland for typing the manuscript.

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