Document Rpopbk94wJbggvZy3kkpwXv0X

IIINOAMINIM AND M'l'l 111) IO\KOI(XD 17. 376-3X9(19911 90-Day Toxicity Study of Dichloroacetate in Dogs1 J. L. ClCMANEC,* L. W. CONDIE,* G. R. OLSON.t AND SlIIN-Ru WaNOJ tTo\icoloi;v and Mierolnoloin Division, 11cullIt I,Ik'd s Research l.ithoiiilorv, I'tncninaii. Ohio J330S. il'inliiiloi'v Issociaics. Inc . 6217 Centre Park Dine. I) esicheslei. Ohio 43099. unil ^Computer Sciences Corporation. Cincinnati. Ohio 432M Received September 23. 1990. accepted April IS. 1991 90-Day Toxicity Study of Dichloroacetate in Dogs. Cicmanec. J. L, Condii.. L W,, Oi,.son, G. R.. and WaNG. S R (1991). Fnndam Appl Toxicol. 17, 376-389, Male and Icmalc lU'emle beagle dogs were dosed daily for 90 days with dichloroacetate (OCA) The compound was ad ministered orally via gelatin capsules at doses of 0, 12.5. 39.5. and 72 mg/hg/day. Each dose group consisted of live males and five females. The dogs were observed clinically and blood samples were taken at 15-day intervals for hematologic and serum chemistry values. Decreased total erythrocyte count and hemoglobin levels were observed in mid- and high-dose dogs beginning at Day 30. Serum concentrations of LDH were elevated at Days 30 and 45 in females and at Day 75 in males treated with DCA at 72 mg/kg/day. One female of the high-dose group died at Day 50 and two high-dose males died at Days 51 and 74. Hindlimb partial paralysis was observed in many high-dose dogs V acuolization of myelinated white tracts of cerebrum, cerebellum, and/or spinal cord was observed in many high-dose dogs as well as some mid- and low-dose subjects. Degeneration of testicular germinal epithelium and syncytial giant cell formation was noted in males of all dose groups. Hepatic vacuolar change and chronic hepatitis appeared only in DCAtreated dogs. In addition, suppurative bronchopneumonia and chronic pancreatitis were noted in many high-dose and some middose subjects A "no-adverse-effect level" was not determined in this Study. ic 1991 Soctew ofToxicolog> Chlorinated acids, alcohols, ketones, and al dehydes are formed when natural water which contains humic and fulvic acids is treated with chlorine. Current research activity in many laboratories is concerned with defining which of these compounds are significant risks to human health as the result of long-term ex posure in drinking water, Dichloroacetic acid, (DCA), is one of three principal nonvolatile compounds formed when natural water is chlorinated and it has been recognized that 1 This document has been reviewed in accordance with U S- Environmental Protection Agency policy and ap proved for publication. Approval does not signify that the contents necessarily reflect the views or policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. the nonvolatile compounds contain three to five times the amount of chlorine in the vol atile compounds (Coleman el at., 1986). In one study (Johnson et at., 1982), it was found that upon chlorination of water from a North Carolina lake, DCA was formed in concentra tions varying from 14 to 34 ppm. In GC/MS analysis of drinking water samples, DCA was found in concentrations ranging from 10 to 500 ppb (Coleman el at.. 1986). Interest in DCA also arises from many of the clinical side effects that were noted in ther apeutic applications. The diisopropylammonium salt of DCA has been used in the treat ment of diabetes, hypercholesterolemia, and hyperlacticemia in humans (Moore cl at.. 1979; Ribesc/tf/., 1979;Stacpoolee//., 1978). This compound directly reduces serum lactate, glucose, triglycerides, and cholesterol in all 0272-0590/91 S3.00 Copvnghi 0 |99l by ibe Society of 1 oxicologv All nghis of reproduction m uny form revrved 37h TOXICTIY SIUDY Of DICIILOROACK 1 ATE IN [XX'.S 377 animal species tested. These effects appear to be related to its capacity to activate pyruvate dehydrogenase. The toxicity of DCA has been demonstrated in many animal species and humans (Katz el al.. 1981; Evans, 1982; Evans and Stacpoole, 1982; Stacpoole el al.. 1978). In subchronic studies, dogs appear to be more sensitive to DCA than mice or rats. Whereas mice and rats show clinical signs and carcinogenesis at dosages greater than 1000 mg/kg in repeated doses, dogs demonstrate severe toxic effects at dosages below 100 mg/kg (Katz et al., 1981). Toxicity for many organ systems including lungs, liver, eyes, gall bladder, and pancreas has been demonstrated with DCA but most notable is the toxic effect upon the central ner vous system and testes in dogs (Katz et al., 1981). The purpose of this study was to extend the findings of Katz to further elucidate the lesions of the central nervous system and to establish a "no-adverse-effect level" for DCA in dogs. METHODS Test Article Dichloroacetic acid was purchased from Sigma Chem ical Company (St. Louis, MO) as reagent grade. Due lo its low pH and corrosive properties it was neutralized with NaOH lo a final pH of 7.4. The stock solution was pipetted into gelatin capsules for dosing. Animals and Maintenance Twenty 4-month old, male beagle dogs (weight range 8.6-13.6 kg) and twenty 4-month old. female beagle dogs (weight range 6.1-9,4 kg) were purchased from Laboratory Research Enterprises of Kalamazoo. Michigan, and ac climated to the laboratory environment for I month prior to the start of the study. Four-month-old dogs were selected because they would still be growing but would be past their most pronounced period of weight gain and this age group is most likely to be free of lntercurrcnt disease. The dogs were housed individually in stainless-steel cages and were given distilled drinking water and Purina Hi-pro dog chow ad libitum The animal room was held at a temper ature of 25 2C and 40-60% relative humidity. The lighting cycle was 12 hr light/12 hr dark controlled by an automatic timer. The dogs were allowed lo mm and ex ercise freely for 30 min. 5 days per week. Siudv Design The study design incorporated three ticaimem groups 12.5, 39.5, and 72 mg/kg. DCA was administered orally in gelatin capsules daily 7 days a week. The control group received gelatin capsules containing distilled watci only Five male and five female dogs were assigned to each ex perimental group. Oral dosing was performed daily. 7 days per week, be tween 9:00and 10:00 am. fora total of90 days. The IX'A was prepared each day just prior to dosing and was ad ministered at least I hr before the dogs were led Following dosing the dogs were observed for 10 min to ensure that the capsule had been swallowed. Doses of EXTA throughout each week were determined on the basis of individual body weights which were measured the preceding Fnday. All serum chemistry determinations were performed on a Baker Encore Serum Analyzer, Baker Instrument Com pany (Allentown, PA). Blood was collected from each dog on Days 0, 15, 30, 45, 60. 75, and 90. Alanine amino transferase (ALT) and aspartate aminotransferase (AST) analyses were carried out by the NADH oxidation method, lactic dehydrogenase (LDH) was determined by the NAD reduction method: creatinine was measured using alkaline picrate colorimetry: blood urea nitrogen (BUN) was mea sured with an enzymatic method using urease and gluta mate dehydrogenase; total bilirubin was determined by diazo colorimetry: and calcium was measured using aliza rin sulfonate. Hematologic values were established on a Coulter Counter Model 5. Modifications were made in the aperture current so that counts ofcanine erythrocytes, leukocytes, and platelets could be determined accurately. The determinations made included total erythrocyte count, hematocrit, hemoglobin, and leukocyte count Mean cor puscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration values were then calculated. Leukocyte differentials were determined manually. At the end of the 90-day dosing period, the dogs were euthanized with an overdose of sodium pentobarbital given intravenously. Food had been withheld for 24 hr prior to euthanasia. At necropsy all major organs were weighed, gross lesions were described, and tissues were placed in 10% neutral buffered formalin. Following fixation the tis sues were trimmed, embedded in paraffin, sectioned at 5 iim. and stained with hematoxylin and eosin 1 he tissues were then examined microscopically. Statistical Analysis of Data Statistical analysis of final body and organ weights and organ weights per IOC) g body wt was performed as a onefactor (dose) analysis of variance (ANOVA) with Tukey's multiple comparison procedure; this analysis tested for an overall dose-related effect The assumption of the homo geneity of variance necessary for the validity of ANOVA procedures was upheld by Levene's test. ANOVA procc- 378 CICMANl-X' I I AI B u) e o7 to 17 27 34 41 66 76 B3 40 Days I Control fcWS Low fe..-W.d Medium tSSSS High Fig. I Changes in female body weights for the various DCA groups dures with contrast comparisons were used in the pairwise analyses- A linear trend analysis was also done for each response measure using ANOVA. The pathology lesion data were analyzed by the exact test for trend. This generalization of the Fisher-lrwin test was used to test for a positive linear dose-related trend in the number of animals w ith particular lesions. A one-tailed Fisher exact test was used in the pairwise comparisons of each dose group with its appropriate control Given the sample sizes per treatment group, it is noted that the power of the tests to detect a dose-related effect is low relative to the trend test. The distribution of hematology and serum enzyme data contained many extreme data points. For this reason no analysis of raw data values was performed Instead the numbers of animals outside the normal range were ex amined, The exact lest for trend and the one-tailed Fishei exact test were also used in the analysis Within the data tables significant differences are pre sented on a gradient scale: */> - 0 03-0,05, **/i = 0 010.03. ***/; < 0.0! RESULTS Clinical Signs Dyspnea was the most significant clinical sign observed. It was noted around Day 45 in TOXICITY STUD'S OF DICHLOROACF.TaTE IN DOC.S 379 TABLE I Hematology Vai its for Fi-.mai f Dogs Treated with Dichloroacetk' acid for 90 Days" Dose group Da\ (mg/kg) Erythrocytes < HP/mm') Hemoglobin (g%> Leukocytes (10'/mm') 0 0 7.06 0.40* 17.2 + 2.4 9.5 1.9 12.5 6 43 0.39 16.3 + 0.5 10.5 + 1.2 39.5 6.79 0.54 16.7 + 1.4 9.8 1.5 72 6 48 0.45 15.4 l.l 11.2 3.0 15 0 7.51 0.69 17.2 1.7 7.8 1.2 12.5 6.89 0.44 16.2 + 0.9 10.0 2.1 39.5 6.85 0.75 16.0 1.7 9.8 + 1.8 72 6.35 i 0 22 14.0 + 0.4 14.9 + 7.1 30 0 7.27 - 0 70 16.6 + 1.9 7.1 + 1.2 12.5 6.48 0.28 15.0 + 0.7 8.3 + 2.5 39.5 6.24 - 0 52 13.8 + 1.1 8.9 + 3.2 72 5.23 = 0.36** 11 4 1.2 11.8 + 4.4 45 0 7.08 + 0 50 16.4 1.1 9.0 3.8 12.5 6.30 0.41 14 6 + 0.8 9.3 + 2.2 39.5 5.84 0.95 12.7 1.7 9.6 + 3.7 72 4.43 + 0 79*** 9.7 + 1.9*** 14.1 + 6.5 60 0 7.00 0 62 16.1 + 2.0 8.1 + 1.3 12.5 6.55 i 0 55 14.9 1.1 9.8 + 3.1 39.5 5.61 0.71 12.4 + 1.4 11.2 + 3.7 72 4.88 0.76*** 10.6+ 1.8** 14.0 + 4.1 75 0 7.18 + 0.33 16.6 + 1.0 8.9 + 1.3 12.5 6,64 0.47 15.2 + 1.1 8.0 + 1.5 39.5 6 47 + 0,46 14.0+ 1.0 8.6 + 0.9 72 5.53 + 0.66 11.8 1.6 14.1 + 5.0 90 0 7.10 + 0.28 16.6 1.0 8.7 + 2.3 12.5 6.57 = 0.48 15.3 + 1.0 10.7 + 4.7 39.5 ofl 13.2 1.9 7.7 + 1.5 72 5.33 0 43* 11.4 + 0.9*** 12.2 + 6.6* " Normal values: total erythrocyte count. 5.5-8.5 x lO6: total leukocyte count, 6-17 X 10': hemoglobin, 12-18 g% ' Values represent means SD. Significant results are presented on a gradient scale. * p = 0.05-0.05. ** p = 0 01-0.03. *** P < 0 01. 2 males and 2 females in the middose groups and in 8 of 10 dogs in the high-dose groups. Forced expiratory effort was the primary char acteristic of dyspnea noted in the affected dogs. In general, once dyspnea appeared it became noticeably worse within 5-7 days and general depression and reduced activity were noted. When the affected dogs did move there was an obvious increase in respiratory rate, and efforts at forced expiration were so pronounced that abdominal heaving became apparent im mediately. In some cases coughing would ac company difficult breathing. All dogs in the high-dose groups exhibited severe dyspnea by the end of the study. Bilateral conjunctivitis accompanied by a slight clear ocular discharge was a commonly observed sign. This change was observed in 24 380 C1CMANEC ET AL. ]ABLE2 Hi matoi oc> Vains cor Mali; DogsTreated with Dighioroaci iaii tor 90 Days" Dose group Day (mg/kg) Erythrocytes (Itf'/mm') Hemoglobin <g%) leukocytes (10'/mm') 0 0 6.40 0.51 * 14.6 + 1.15 10,9 i *) 12 1 6.82 0.51 15 2 1.11 10.5 + 1.89 19 5 6.69 0 66 15 1 1 05 10.2 + 1.58 12 6.26 0.47 14.1 0.71 9.4 105 15 0 6 78 0.89 15.9 2.27 ll.4 2.4(1 12 5 6.66 0.31 15.4 0.41 1 1,1 1 1.01 19 5 5.98 0.75 72 5.90 0.11 1.1.1 1.41 11.5 0.78 11,7 4.16 11.6 1.12 10 0 6.85 0.67 15.6 1.58 9.2 1.01 12.3 6.41 0.21 14,1 0 71 96 1.54 39.5 5.92 0 47 12.7 + 1.16 10.9 + 5.82 7"* 5.44 0.10" 11.7 0.74*" 8,5 1 19 A5 0 7.09 0.71 16.1 1.76 8 1 2.11 12.5 6.95 0.29 15.0 0 81 12.5 + 2.62 39 5 5.72 + 0.27 11.9 0.71 10.0 2.65 72 4.86 0.41"* 10.4 0 .87"* 15.7 + 12.19 60 0 6.81 0.82 15.5 + 2.02 ll.ll 1 16 12.5 6.05 0 24 11.0 0.90 86 + 1.08 19.5 5,19 + 0 47 10.6 1.20 9 1 2.61 72 4 73 0.15"* 9.9 + 0.62"* 14,6 + 4.91 75 0 6.72 0 87 15.1 i 1.90 12.2 l 1.41 12,5 6.06 + 0,15 12.9 + 0 71 10.2 + 2.11 39.5 5.51 0.62 10.9 1.29 1 1.1 + 5.97 72 4.50 0.72*** 9 ) 1.15*" 18.1 6.04* 90 0 6.72 0.72 16.0 + 2.09 11.2 2.24 12.5 6.20 0 54 11.4 1.21 11.8 + 2.17 19.5 5.68 0 61 11.4 + 1.30 10.9 1.72 71 5.1 1 0.41"* 9.0 + 0.85*** 17.8 16.08 *1 Normal values1 total erythrocyte count, 5.5-8.5 x 10*:1total leukoevte count. 6-17 x 10';; hemoelohin, 12-ISg'T h Values represent means a: SD. Significant results are presented on a gradient scale. * P = (1,0.1-0 0? ** /> = 0.01-0.03 *" p < 0 01, of 30 treated dogs during the initial month of the studs and \sas occasionally noted in a few control dogs. The conjunctivitis progressed to a pronounced degree of swelling, with the ocular discharge becoming more purulent in 8 of 10 high-dose animals. The discharge re mained clear throughout the study in affected low- and middose dogs. Slight hilteral posterior paresis was observed beginning about Day 50 in one female and two males in the high-dose group. Among the affected dogs the paralysis was only partial and was not evident each time that the dog at tempted to move. This sign was difficult to clearly verify because the dogs were reluctant to move and when they did the> would drag the hind limbs intermittent!) rather than take coordinated steps. Once obsened. the paralysis persisted but did not progress until near the termination of the studs. TOXICITY STUDY OF DICHL.OROACETATE IN DOGS .181 TABLE 3 Serum Chemistry Values for Female Dogs Treated with Dichloroacftic Arm tor 90 Days" Dose group Day (mg/kg) AST ALT LDH 00 29.4 6 07* 19.2 + 3 70 87.2 32.36 12.5 32.0 18.40 20.0 2.92 137.6 96.24 39.5 28.2 8.53 26.2 6.57 74.6 + 29.65 72 32.0 4.74 21.8 4 15 93.4 16.59 15 0 12.5 39.5 72 28.4 20.6 19.8 21.2 7.20 5.03 4.15 5.67 24.4 15.8 25.2 18.0 5.41 2.59 4.72 4.18 57.6 77.0 55.0 59.0 40.30 47.49 39.56 18.10 30 0 24.4 + .3.65 20.6 6.50 66.0 29.18 12.5 33.0 + 7.35 20.8 5.63 47.6 26.43 39.5 27.0 + 7.18 36.0 + 24.64 109.0 24.12 72 26.2 11.19 13.0 6.67 217.0 112.36*** 45 0 26.2 + 5.36 35.6 16.27 62.8 + 13.42 12.5 603.8 + 186.27 385.4 250 83 218.4 69.26 39.5 92.2 + 119.21 52.0 50.40 339.0 196.43 72 34.0 13.78 22,2 8.87 360.0 + 158.14*** 31.2 + 8.04 52.0 + 17.86 60 0 28.8 5.07 198.8 + 36.62 104,4 + 75.65 12.5 175.8 97.24 42.0 + 44.96 257."' + 150.30 39.5 44.2 28.10 23.0 + 9.76 111.5 + 74.29 72 28.5 10.66 30.8 7.50 30.6 10.16 75 0 28.8 2.86 38.8 8 44 178.0 118.44 12.5 34.2 7.26 24.8 10.92 158.8 + 118.44 39.5 31.6 9.56 72 22.8 4.86 22.0 15.12 65.5 23.33 90 0 25.2 5.63 22.2 6.26 52.4 16.64 12.5 25.4 7.33 21.0 3.16 80.8 21.06 39.5 20.2 5.67 17.8 6.72 74.0 30.80 72 24.8 4.79 20.8 6.34 100.8 + 32.60 0 Normal values: AST. 10-80 IU; ALT, 10-65 IU; LDH, 0-110 IU. * Significant results are presented on a gradient scale. * p = 0.03-0.05. ** p = 0.01-0.03. *** p < 0.01. Diarrhea was observed sporadically in dogs in the mid- and high-dose groups. Feces of af fected dogs were very watery and had a very fetid odor. Once diarrhea was noted it became progressively worse. The quantity of fluid lost each day in severely affected dogs was esti mated to be 750 ml. In some animals fluid therapy was needed to avoid severe dehydra tion. One high-dose female died at Day 50 and two high-dose males died on Days 51 and 74. Based upon clinical observation of animals and microscopic examination of postmortem tissues, it was concluded that these deaths re sulted from pneumonia and dehydration. High-dose males exhibited a weight loss of 16%. Middose males and high-dose females showed a 9% loss and middose females showed 382 CIC'MANLC I T Al tabu: 4 Serum Cm mimr'i v m ui:s for Mai.i DogsTri aiid wim Diem okoac'i nc Acid for 90 Days Dose group Da> (mg/kg) AST ALT LDH 00 12.5 39 5 72 29.4 1 31.8 30.8 26.8 3.96'' 10.92 5.63 4.87 24 4 i 57.0 22.4 25,4 5 91 74 98 5 13 6 31 79 4 105.8 83 8 69,4 45.51 50.87 37.37 34.08 15 0 25.6 4.67 37.0 + 14.87 51 8 31.17 12 5 111.2 209.54 105.8 182.97 43.2 33.72 39.5 25.0 10.30 17 4 + 4.04 6 17 37 72 26.8 11.53 20.8 5,40 39.0 17.1 1 30 0 12.5 39.5 72 26.6 57.8 30.4 26.2 4.72 71.12 7.80 9.20 30.6 12' 105.6 185,27 20.8 5 45 16.2 2.77 49.4 4 75.8 86 6 79.8 21.31 33.30 34 79 64 25 45 0 33.6 7.09 35.8 - 5.36 41.8 21.18 12.5 881.6 685.52 571.0 514.66 121.6 67.30 39.5 934.6 884.22 317 8 + 293.06 200.2 187.94 72 114.4 130.40 48.0 29.61 83.6 90.85 60 0 32.6 16.65 41.6 18 66 42.4 15.61 12.5 204.6 158.16 245.6 162.29 57.2 18.21 39 5 308.8 211.12 193.2 + 134.99 110.2 34.02 72 101.0 62.34* 54.0 36.60* 76.6 49.07 75 0 12.5 39.5 72 27.0 53.6 38.0 34.0 7.38 53.85 11.90 5.29 38.6 + 83.4 4 36.8 32.0 6.99 67.80 16.56 7.53 48.8 41.2 119.2 170.8 14.13 17.08 61.97 89,75*** 90 0 12.5 39 5 72 35.2 42.6 27.4 33.3 20.44 23.48 3.51 17.39 37 4 + 4.28 76.4 101 91 21.4 + 9.29 25.3 1.53 74.2 42.19 82.4 76.44 143.6 88.18 727.0 105.42*** " Normal values: AST. 10-80 IU; ALT. 10-65 IU; LDH. 0-110 IU. h Significant results arc presented on a gradient scale. * p = 0.03-0.05. ** p = 0,01-0 03 *** p <0.01. an 11% loss in body weight during the 90-day study. Changes in body weights for the various dose groups are depicted in Figs. 1 and 2. Re duction in food and water consumption was noted in dogs of all treatment groups but not the control groups. However, large individual variation precludes a conclusive statement re garding these changes. Anorexia did not ap pear to be dose related in that great variation was also observed in some low-dose subjects. Hematology All hematologic results are summarized in Tables 1 and 2. Reduced total erythrocyte counts and hemoglobin levels were noted in both high-dose males and females on Day 30 of the study. At Days 45. 60, 75, and 90, he moglobin values were also below normal for middose males. By trend analyses, decreases in total erythrocyte counts and hemoglobin TOXICITY STUDY OF DICHLOROACETATE IN DOGS 383 TABLE 5 Summary of Organ Weigh i s of Female Dogs Treated with Dichloroacetic Acid for *KI Das s Liver Kidney Ovanes Control Low Medium High g g/IOOg 274.6 35.91" 366.8*** 13.42 336.2*** 19.98 384.8*** 20.30 3.28 0.21 4.69*** 0.498 4 72*** 0.358 5.52*** 0.731 Heart g g/IOOg 44.98 3.83 46.66 2.82 48.56 6.35 62.50*** 6.70 0.54 0.022 0.60 0.084 0.68** 0.087 0.90*** 0.132 Lungs g g/IOOg 0.95 0.32 0.73 0.14 0.72 0.16 0.83 0.05 0.012 0.0048 0.009 0.0025 0.010 0.0028 0.012 0.0014 Brain Conlrol Low Medium High g 82.0 7.98 81.1 7.18 80.6 18.26 79.5 8.27 g/100 g 0.98 0.064 1.04 0.147 1.12 0.149 1.14 0.139 g 74.7 14.55 70.2 7.80 69.2 3.54 93.8* 19.28 g/IOOg 0.89 0.095 0.89 0.113 0.98 0.107 1.34*** 0.250 g 75.8 5.81 77.8 5.35 72.3 3.68 76.6 0.86 g/100 g 0.91 0.110 0.99 0.104 1.02 0.116 1.10** 0.082 " Significant results are presented on a gradient scale. * /> = 0.03-0.05. ** p = 0.01-0.03. *** p < 0.01. levels were found to be statistically significant at Days 30, 45, 60, and 90 for both sexes and at Day 75 for males. One of the male dogs with a markedly elevated total leukocyte count of 33,000/mm3 on Day 45 died at Day 51. Clinical Chemistry The clinical chemistry results are presented in Tables 3 and 4. Among the parameters tested only ALT, AST, and LDH showed sig nificant changes and therefore only these val ues are presented. Trend analysis revealed sig nificant changes in the females for LDH at Days 30 and 45. By trend analysis, there was a significant change for AST and ALT at Day 60 for male dogs. At days 75 and 90 there was significant change for LDH in the males. Pathology The following tissues were examined grossly and microscopically at necropsy: cerebrum, cerebellum, medulla, salivary gland, pancreas, axillary' lymph node, pituitary, adrenals, thy mus, mesenteric lymph node, thyroid, para thyroid, trachea, esophagus, heart, colon, je junum. aorta, stomach, duodenum ileum, spleen, urinary bladder, lungs, sciatic nerve, spinal cord, kidneys, liver, ovanes/testes, uterus/prostate gland, skin, mammary gland, eyes, sternum/bone marrow, femur, and gall bladder. Many organs in the high-dose group showed distinct gross changes at necropsy. The lungs were mottled and showed moderate red dis coloration. The kidneys were pale and were discolored yellow-brown. White frothy mate- 384 CICMANLC t l AL TABLK 6 Summary oi Oruan Weitarrsor Mai.l Doc.s I ri atw> wnu Dichiokoaci nr At in iok 90 im-, s Liver Kidney Iestes e g/lOOg g E/100 g g g/lOOe Control Low Medium High 373.8 69.41" 539.2*** 95.95 596.6*** 57.52 417.7 115.52 3.01 0.121 4.69* 0 436 5.60*** 0.849 5.26** 2.579 Heart 63.6 11.64 71.6 9.72 74.5 12.37 61.5 12.25 0.52 0.089 0.63 0,091 0.70** 0.130 0.73** 0.057 Lungs 20.1 5.06 23.4 5.68 14.9 3.96 13.2 2.54 0 17 0 046 0.21 0.049 0 14 0,045 0 16 0.034 Brain Control Low Medium High g 104.0 6.86 108 4 20 33 91.6 11.78 76 3** 8.50 g/l00g 0.86 0.138 0.94 0.117 0.86 0.135 0.91 0 108 6 106.4 6.02 97.0 17.31 107.2 29.49 103.3 3.21 g/100g 0.88 0.157 0.85 0.138 1.00 0.288 1.25* 0.242 g 82.8 8.58 81.4 5.55 79.0 2.74 76.7 4 04 g/IOOg 0,70 0,214 0 72 0.065 0,74 0 062 0.92* 0 157 `'Significant results are presented on a gradient scale, * /) = 0.03-0,05 ** p = 0.01-0.03. *** p < 0.01, rial was present in the trachea and the liver showed mild yellow discoloration. The hepatic lesions were considered primary lesions and the changes in the kidneys and lungs were considered secondary'. Although changes in the cerebrum and cerebellum were only observed microscopically, they were considered primary lesions. Changes in the pancreas and gall blad der were also primary. At necropsy, the weights of liver, kidney, testes/ovaries, heart lung, and brain were de termined and summary data for each study group are presented in Tables 5 and 6. The liver weights (expressed as percentages of body weight) of both males and females were sig nificantly higher than those of the controls at all doses of DCA. Percentage kidney weights in middose and high-dose females and males were significantly increased. The lung weights of high-dose animals of both sexes (expressed as a percentage of body weight) were signifi cantly higher than those of controls. No sig nificant weight changes were found in testes or ovaries. High-dose males and females showed increased relative brain weights. Microscopie Findings Within the brain, vacuolization of white myelinated tracts was observed in all DCAtreated dose groups: in all cases the severity was mild (see Fig. 3). In some dogs this was present in both cerebrum and cerebellum, while in others it was noted only in cerebrum or cerebellum (Table 7). In addition, vacuolar change was observed in the medulla and spinal cord of some males and meningoencephalitis TOXIC ITY STUDY Of DICHLOROACIH A IT- IN DOCiS 38? Fig. 3. Photomicrograph of the cerebrum ol a high-dose dog. The dear areas represent vacuolization within the white myelinated tract. X400. was present in one-high dose female; again, the severity was mild. Hepatic, lung, pancreatic, and testicular pathologic changes are summarized in Table 8. Chronic hepatitis was present in 2 high-dose males and one middose and 2 high-dose fe males. Hemosiderosis was prevalent in midand high-dose animals of both sexes. Although TABLE 7 Summary of Histopatholocucal Chasc.es Foi 'no is the Central Nervous System or Dogs Treated with Dichloroacfhc Acid for 90 Days" Vacuolization of white mvelmated tracts-- cerebrum and cerebellum Females Males Vacuolization of white mvelinated tracts-- cerebrum on!> Females Males Vacuolization of white myelinated tracts-- medulla only Females Mcningoencephalitis Females Vacuolization of white myelinated tracts-- cerebellum onlv Males Vacuolar change-- medulla and spinal cord Males Low (l/? 1/5 0/5 7/5 0/5 0/5 0/5 0/5 Medium 0/5 V5 1/5 0/5 0/5 0/5 1/5 s/5 High 0/5 1/5 1/5 7/5 1/5 1/5 0/5 t/s " All CNS lesions were of mild severity 386 Clt'MANl.C 1:1 \L TABLE 8 Summary or Histopatholooical Changes Sf.en in mi- Liver. Lungs, Pancreas. and Ti siivoi Dixin Treated with Dichi.oroacetic acid for 10 Days Dose group Control Low Medium High Hepatic lesions Vacuolar change Chrome inflammation Hemosiderosis Lung lesions Suppurative bronchopneumonia Chronic pneumonia Granulomatous pneumonia Edema Pancreatic lesions Chronic inflammation Acinar degeneration Testicular lesions Degeneration 2/5(1. I)"-* 2/5(1, 1) 0/5 0/5 0/5 0/5 0/5 0/5 1/5(1) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5(1. 1, 2. 2) 5/5(1. 1, 1. 1. 2 0/5 0/5 1/5(1) 0/5 0/5 0/5 1/5(1) 1/5(1) 0/5 0/5 1/5(1) 0/5 1/5(1) 0/5 0/5 0/5 4/5(1. 2. 2. 3) 3/5(1. 2, 2) 2/5(1. 1) 0/5 1/5(1) 1/5(2) 3/5(1. 1. 1) 2/5(1.21 2/5(1, 1) 2/5(1, 1) 2/5(1. 1) 2/5(1, 1) 3/5(1, 1. 2) 2/5 (3. .3) 1/5(3) 0/5 3/5(1. 1. 1) 0/5 0/5 2/5(1. 1) 0/5 2/5 (2. 3) 4/5(1, 2. .3, 3) 1/5(3) 0/5 0/5 1/5(2) 3/5 (1.2, 2) 0/5 2/5(1. 1) 1/5(2) 2/5(1. 1) 1/5 (2) 2/5 (1.2) 2/5 (1.2) 2/5(1. 2) 3/5(1. 2, 2) 5/5 (2. 3. 3, 3) 5/5 (3. 3. .3, 3, 3 " Top row of each group shows incidence in males and second row shows incidence in females * Numbers in parentheses indiate degree of seventy: I. mild. 2, moderate, 2, severe. hepatic vacuolization was observed in most DCA-treated dogs, it was also present in some control animals. Mucosal epithelial vacuoli zation and hyperplasia of the gallbladder were prominent in all DCA-treated dogs (see Fig. 4). Pneumonia and bronchopneumonia were observed in nearly all DCA-treated dogs, being more severe in mid- and high-dose animals. Marked suppuration was evident in the highdose animals. Pancreatic acinar degeneration associated with chronic inflammation was noted in many DCA-treated dogs in the mid- and high-dose groups (see Fig. 5). Testicular changes featur ing syncytial giant cell formation and degen eration of germinal epithelium were present in nearly all DCA-treated males. The mid- and high-dose groups had an increased severity oi lesions. The testicular lesions are thought to occur as a primary effect of DCA. The testes of affected males did not show lesions upon gross necropsy. Prostatic glandular atrophy characterized b\ a significant reduction of glandular aveoli was noted in the mid- and high-dose groups Thymic atrophy was observed in most highdose males and was characterized by a marked depletion of lymphoid tissue. DISCUSSION In contrast to reports that DCA produces minimal acute toxicity whether given intra- TOXICITV STUDY OF DICHLOROACETATE IN DOGS 387 Fig. 4. Photomicrograph of the gall bladder of a high-dose dog. (A, Piling up of epithelial cells; B. Mild vacuolar change within the epithelium X400, venously or orally (Davis, 1986; Evans, 1982; Stacpoole el ai, 1979), the results ofthis study showed marked changes in clinical signs, he matology, and pathology in a 90-day subchronic study. Three of eight high-dose (72 mg/kg) dogs died before completion of the dosing period and significant pathological changes were observed in the cerebrum, cer ebellum, testicles, lungs, pancreas, and liver of most high-dose dogs and some of the mid- and low-dose subjects. A primary objective of this study was to es tablish a no-adverse-effect level, but due to the vacuolar changes present in the white matter of the cerebrum in two males of the low-dose group and chronic active pneumonia in three of five males also in this group, we were unable to establish a no-adverse-effect level with the dose levels chosen. Among the various clinical laboratory tests performed during the study, the serum chem istry changes for ALT, AST, and LDH were most difficult to interpret. In contrast to other DCA studies performed in dogs, we have ob served marked elevations of these enzymes. It is perplexing that marked elevations (ALT and AST) were observed for individual low- and middose dogs but not in the high-dose groups. It is possible that marked elevations occurred in high-dose animals but for only short periods and they did not occur when blood samples were taken. The hepatic changes that were ob served upon postmortem examination of the liver indicated that these changes most likely did occur during the study for all dose groups. We did not observe the lenticular opacities previously described (Katz et a/., 1981), but conjunctival inflammation was present early in the study in all dose groups. The ocular changes were among the clinical signs most frequently observed and they became more severe later in the study. Due to the wide range 388 C'lCMANIC 1 1 Al Fig 5. Photomicrograph of the pancreas of a high-doso dog (A. Mild acinar necrosis x400). of tissues affected, the toxicity of DCA might involve metabolic processes rather than direct tissue alteration. Most toxic chemicals do not affect such a wide spectrum of tissues with such a broad range of functions as nervous tissue, lung, liver, and testes. In dogs, the relatively widespread organ toxicity may relate to the long biological half-life (20.8 hr) for this com pound. The known metabolic effect of acti vating pyruvate dehydrogenase (Crabb el al. 1981) and the drug's effects on lipid and amino acid metabolism may explain the widespread effects on many separate organ systems. Stacpoole <7 al (1984) have reasoned that since DCA stimulates at least two thiamine-depen dent enzymes. in long-term studies it max in duce a thiamine deficiency which may explain why lesions are observed in a variety of tissues. In summary, this study confirmed most of the acute findings of Katz el al (1981). More extensive hepatic serum chemistry alternations were noted, more extensive hematologic changes were observed, and microscopic pan creatic lesions were detected- Vacuolar changes in the white myelinated tracts of cerebellum and spinal cord were observed in middose and even low-dose group animals. Safety concerns regarding use of DCA for therapeutic purposes have curtailed its use in clinical settings (Stacpoole et al. 1979). 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