FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19, 307-3
14 (1992)
One-Year Dietary Toxicity Study with Methidathion JANE C. F. CHANG,*” *Environmental
Health Center, Agricultural and tAnimal Health Division,
JAMES A. WALBERG,*
AND WILLIAM
in Beagle Dogs
R. C,uN?BELLt
Division, CIBA-GEIGY Corporation, 400 Farmington Avenue, Farmington, CIBA-GEIGY Corporation, P.O. Box 18300, Greensboro, North Carolina
Connecticut 27419
06032;
Received December 12, 199 1; accepted April 9, 1992
the U.S. population.
The current acceptable daily intake
One-Year Dietary Toxicity Study with Methidathion in Beagle (ADI), or the reference dose, was estimated to be 0.001 mg/ Dogs. CHANG, J. C. F., WALBERG, J. A., AND CAMPBELL, kg/day by the EPA. The AD1 was established on the basis W. R. (1992). Fundam. Appl. Toxicol. 19,307-3 14.
The purposeof this study wasto determinethe chronic toxicity of methidathion, an organophosphate insecticide, in dogs. Groups of beagledogs,four/sex/dose, were fed methidathion at constant dietary concentrations of 0, 0.5, 2, 4, 40, or 140 ppm for 1 year. The equivalent daily dosageswere approximately 0, 0.02, 0.07, 0.15, 1.4, and 4.7 mg/kg. There were no deaths or adverseclinical signsassociatedwith the treatment. Weekly body weights and weight gains were not affected. Mean daily food consumption wasreducedin male dogsgiven the 140-ppmdiet. Major treatment-related effects were cholestasis,chronic inflammation in the liver, and cholinesterase(ChE) inhibition. The liver effectswereindicated by grossand microscopicpathologic findings aswell asmoderate increasesin serumbile acids and enzyme activities (alanine aminotransferase,aspartateaminotransferase,sorbitol dehydrogenase, and alkaline phosphatase) in all dogsreceiving >40 ppm. RBC ChE wasinhibited in males at 340 ppm and in femalesat 140ppm. Brain ChE wasinhibited in both sexesat 140 ppm; the magnitude of inhibition relative to control wasslightly greater with the cerebellarfraction than with the cerebral fraction. Serum ChE was not affected at any doselevel. In conclusion, liver was the target organ in beagle dogsgiven a40 ppm (equivalentto 1.4 mg/kg/day) methidathion in diet for I year. The no-observable-effectlevel was4 ppm (0.15 mg/kg/day) for both liver cholestasisand ChE inhibition. o 1992
of results of a 2-year chronic toxicity study in dogs and a safety factor of 100 (EPA, 1988, 1989). In the previous study (Johnston, 1967), beagle dogs were provided for 2 years with methidathion technical in diet at a concentration of 0, 4, 16, or 64 ppm, 6 days/week (200 g of food daily except on Saturdays when 400 g was fed and on Sundays when no test diets were provided). All dogs survived and remained in good condition throughout the study. Major findings at the high dose, 64 ppm, included marked elevations of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities, retention of sodium sulfobromophthalein, and increased pigment in liver and nephrons. A slight inhibition of RBC ChE was also seen at 64 ppm whereas no inhibitory effects on plasma or brain ChE were noted. The no-observable-effect level was judged to be 4 ppm or 0.08 mg/kg (FAO/WHO, 1973; Hayes, 1982). The purpose of the present study was to determine the toxicity of methidathion in beagle dogs after continuous exposure in diet for 1 year. The daily dosage of methidathion (mg/kg) received by dogs given the highest dietary concentration, 140 ppm, was approximately four times higher than that used in the previous study. This study was conducted
to satisfy regulatory requirements by the California Department of Food and Agriculture to update the data base. The EPA FIFRA guidelines on nonrodent chronic toxicity studies (Pesticide Assessment Guidelines, Part 158, Section 83-l) Methidathion (GS-13005,S-2,3-dihydro-5-methoxy-2oxo- 1,3,4-thiadiazol-3-yl-methyl O,O-dimethyl phospho- 1984) and EPA-FIFRA GLP Standards (40 CFR, Part 160) rodithioate) is an organophosphorus insecticide. The chem- were followed. Our results demonstrate that the major in beagle dogs are hepaical is marketed by the CIBA-GEIGY Corp. under the trade chronic toxicities of methidathion but not inname of Supracide 2E Insecticide-Miticide for use on certain tobiliary dysfunction and RBC ChE inhibition citrus fruits, almonds, pecans, walnuts, prunes, safflowers, hibition of serum or brain ChE. Society of Toxicology.
grain sorghum, alfalfa, artichoke, tobacco, nursery stock, and severalother crops (CIBA-GEIGY Corp., 1991; EPA, 1989).
Based on residue tolerances established for methidathion in food and feed crops, the average anticipated residue contribution (ARC) was established at 0.000935 mg/kg/day for
METHODS Test substance. Technical methidathion (GS-1300&S-2,3-dihydro5-methoxy-2-oxo-l,3,4-thiadiazol-3-yl-methyl-O,O-dimethyl phosphorodithioate) was supplied by the CIBA-GEIGY Corp., Agricultural Division (Greensboro, NC). The test material, a solid with a purity of 96%. was stored at 4°C.
’ To whom correspondence should be addressed. 307
0272-0590/92 $5.00 Copyright 0 1992 by the Society of Toxicology. All rights of reproduction in any form reserved.
308
CHANG,
WALBERG,
Animals. Laboratory-reared beagle dogs were obtained from Marshall Farms (North Rose, NY) and maintained in compliance with the U.S. Animal Welfare Act (1985). The dogs were housed individually in stainless steel cageswith approximately 8 ft’ of floor space. The animal room was provided with at least I5 air changes per hour with the temperature maintained at 19-24°C and relative humidity at 40-60%. Fluorescent lighting was provided on a 12-hr light/dark cycle. During the quarantine/acclimation period, dogs were provided with approximately 350 g/day of Purina Certified Canine Diet No. 5007 kibble during the first few days: thereafter, Purina Certified Canine Diet No. 5007 ground meal was provided. The feeding periods were limited to 4 hr daily (midday). Local water was provided ad libitmn by an automatic watering system and in water bowls during feeding hours. After a quarantine period of 28 days, dogs were assigned to the study groups such that all groups had similar mean body weight at study start. Littermates were not assigned to the same group. All dogs were approximately 5 months old at the study start. Test diets. Methidathion technical was used as received without adjustment for purity. The bulk technical material, upon receipt, was melted at xSO”C, aliquoted into small jars. and stored at 4°C until used. To prepare the test diet, the aliquoted technical material was melted, mixed with corn oil, and incorporated into a portion of Purina Certified Canine Diet No. 5007 ground meal. The resulting mixture was further diluted with ground meal in a Patterson-Kelly twin-shell blender to achieve the desired dietary concentrations. The control diet consisted of ground meal blended with corn oil in amounts equivalent to those used for the 140-ppm blends (0.01% final concentration). The test diets were prepared approximately biweekly. All test diets were stored at 4°C until scooped into food bowls. Methidathion was determined to be stable in bulk dietary mixtures (0.5 and 4 ppm) at 4°C for 6 weeks and in food bowls at 25°C for 5 days. The concentration and homogeneity of methidathion in test diet mixtures met the specifications for most blends analyzed. The grand means of the analytical concentrations of the test diets were 0.48, 1.88. 3.67. 37.7. and 133.4 ppm for the nominal concentration of 0.5, 2. 4. 40, and 140 ppm, respectively. The test diets (350 g/dog) were provided for approximately 4 hr daily. Afterward. unconsumed feed was removed. Observations and measurements. The dogs were observed at least twice daily (AM and PM; before and after feeding) for general appearance, behavior, signs of toxicity. and mortality. General physical examinations were performed weekly. In addition, detailed physical examinations, which included measurements of heart rate, respiration. and temperature as well as examination of sensory/motor systems,were performed by the clinical veterinarian at pretest and once every 3 months during the study. Complete ophthalmologic examinations were performed at pretest and before study termination. Body weight was measured weekly. Daily food consumption by the individual animal was measured twice per week for the first I3 weeks and twice monthly thereafter. Complete hematology, clinical chemistry, and urinalysis were performed at pretest and at 3.6, and I2 months. Fecal analyses were performed at pretest and at study termination. Blood samples for hematology and clinical chemistry were obtained from the jugular veins of dogs fasted for at least I8 hr prior to sample collection. Urine samples were collected overnight using a collection tray. Hematology parameters (hematocrit. hemoglobin, platelet count. white and red blood cell counts) were determined by an Ortho ELT-8/ds counter (Ortho Diagnostic System, Inc., Westwood, MA). Differential leukocyte counts were performed manually. Prothrombin time and activated partial thromboplastin time were determined by using Coag-A-Mate (Akzo, Oklahoma City, OK). Clinical chemistry tests (serum ChE, bile acids, aspartate aminotransferase (AST), ALT, ALP. y-glutamyltransferase (GGT). sorbitol dehydrogenase (SDH). creatine phosphokinase, blood urea nitrogen. cholesterol. triglycerides, total bilirubin, total protein, albumin. globulin, creatinine, glucose. calcium, inorganic phosphate, sodium, potassium. and chloride) were performed on an IL Monarch 2000 Chemistry System (Instrumentation Laboratory, Lexington, MA). Test reagents used included the IL Test Reagents (all parameters except bile acids, SDH, and ChE), the
AND CAMPBELL Sigma Diagnostics Reagents (bile acids, SDH) and the Boehringer-Mannheim Diagnostics Reagents (serum and brain ChE). Urinalyses (pH, protein, glucose, ketone. bilirubin, blood, urobilinogen, specific gravity) were performed on an Ames Clini-Tek 10 Urine Chemistry Analyzer (Miles Diagnostics Division. Elkart, IN). RBC ChE was analyzed by the Baker Centrifichem 500 Analyzer (ScronoBaker Instruments Corp., Pleasantsville, NY) based on the Ellman’s method (Ellman rf al.. 196 1). Blood samples were collected in EDTA anti-coagulated tubes. RBC hemolysates were prepared by adding 1 part of saline-washed, packed RBC to 19 parts of 1% Triton X- 100 in saline. The sulthydryl groups in the RBC hemolysate were removed by preincubation with dithiobisnitrobenzoic acid (final concentration. 3 X 10e4 M) at room temperature for 15 min. Subsequently. the substrate, acetylthiocholine (final concentration, 5 X 10m4M in 0. I M phosphate buffer, pH 8.0) was added and the reaction was allowed to proceed at 37°C for 30 sec. The formation of the colored endproduct, thionitrobenzoic acid, was measured at 405 nm. Both serum and brain ChE were determined by using the IL Monarch 2000 Chemistry Systemand the Boehringer-Mannheim Diagnostic Reagents. which are also based on the Ellman’s method. Serum samples were used as obtained. Brain tissuesobtained from animals at necropsies were immediately placed in ice-cold 0. I M phosphate-buffered saline, pH 8.0 (l/IO. w/v). and homogenized with a Brinkman Polytron. The homogenized fractions were next centrifuged at 4°C at 7000g for IO min and the supernatant fractions were aliquoted for analysis. Brain ChE from two homogenized fractions were determined. One homogenized fraction was prepared from the vermis of the cerebellum whereas the other was from the right cerebral hemisphere. The assaytemperature was 30°C for both serum and brain ChE. To determine the sensitivity of serum and RBC ChE to methidathion irr vitro. serum or whole blood from one control male and one control female dog was separately incubated with methidathion (dissolved in methanol: final concentration, 2,20,200.2000, or 20,000 FM) at 37°C for 2 hr. Thereafter, plasma was separated from the RBC and ChE activity in either RBC, plasma, or serum was analyzed. Serum or whole blood samples incubated with or without methanol served as controls. The dogs were terminated by iv phentobarbital injection followed by exsanguination, Complete necropsy examinations were performed and complete tissue collections were made. Organ weights were taken for adrenals. brain. heart, kidneys, liver, ovaries, pituitary, testes, thyroid, and spleen. Tissues were trimmed, embedded in paraffin. sectioned at 4-6 pm. and stained by hematoxylin and eosin (H&E). The tissues were evaluated by the study pathologist: lesions were graded as slight. mild, moderate, or marked depending upon the severity and the areas involved. Cafcufations. Weekly compound consumption (data not shown) by each animal was calculated by using the nominal concentration of methidathion in the diet, the mean daily food consumption. and the body weight for the week. These weekly individual animal compound consumption values were used to calculate the weekly group means and the grand mean for the group during the 1-year period. The grand mean compound consumption for each dose group, not shown under Results, is stated in the Abstract. Individual animal weekly food consumption (g/day) was calculated by taking the mean of the two measurements performed for each dog during the week. These individual values were then used to calculate the group mean for the week as well as the grand mean for the group during the study. Statistical analysis. Quantitative continuous variables were analyzed statistically by a one-way analysis of variance followed by Dunnett’s t test. The probability of type 1 error (a) was set at 0.05. Statistical test results at the 0.0 1 level of significance were also noted. Statistical analyses of bile acid concentrations at 12 months (Table 1) were not performed due to the small number of samples in the control groups (one female and two males).
RESULTS Survival, Body Weight, and Food Consumption
All dogs survived in good condition. There were no clinical signs in treated dogs which are characteristic of those asso-
METHIDATHION
TOXICITY
ciated with organophosphate exposure. Wet coat on the forefoot, which might have resulted from salivation, was noted in some treated dogs on a few occasions. Overall, neither salivation, dacryorrhea, nor wet coat on the forefoot was seen frequently or in a dose-related manner. There were no treatment-related effects on body weight (Fig. 1) or body weight gain (data not shown) in either sex. Mean weekly body weights of the treated males or females were not statistically different from those of the controls. Mean body weight gain (in 52 weeks) was 3.3, 3.7, 4.2, 5.2, and 3.7 kg for males receiving 0.5, 2, 4, 40, or 140 ppm, versus 4.0 kg in controls. Body weight gains by the treated females, at corresponding dose levels, were 3.5, 3.8, 1.2,4.2, and 3. I kg versus 2.5 kg for the controls. Food consumption was not affected in treated females and no statistical differences were noted at any time point (Fig. 2). In males, food consumption was reduced in the group
309
IN DOGS
-
i
0e 150 100
01 0
,
4
,
8
,
12
,
16
,
Control 0.5 ppm
2 wm
4 wm 40 ppm 140 ppm
,
20
24
,
28
,
32
,
,
,
,
,
36
40
44
48
52
I 36
I 40
1 44
I 4.9
I 52
WEEKS
350 300
250 6 ,D 200 i
vl‘ E
0e 150
-
i
100
t e ol8 0 f
-
Control 0.5 aom
4 ‘2 351
-
40 ppm 140 ppm
0.5 2
ppm
wm
4 pm 40 ppm 140 ppm
M-
O
0
4
1
Ii
I 16
zb
i4
I 28
I 32
WEEKS
FIG. 2. Food consumption for beagle dogs given methidathion in diet for 1 year. Each data point represents the mean of four animals for male dogs (top) or female dogs (bottom).
2 1$ 01, 0
Control
-
,, 8
4
12
, 16
, 20
, 24
, 28
, 32
) 36
, 40
, 44
,, 48
52
WEEKS
given the 140-ppm diet: significantly lower values were noted during Weeks 5-8, 11,2 1,29, and 44. The grand mean daily food consumption was 320, 329, 310, 343, 323, and 277 g, for males receiving 0,0.5,2,4,40, or 140 ppm; corresponding values were 288, 296, 287, 302, 294, and 289 g for females. Clinical Laboratory --
Control 0.5 ppm 2 wm 4 wm 40 ppm 140 ppm
-
0’
b
,
,
,
,
,
,
,
,
I
I
,
I
f
4
8
12
16
20
24
28
32
36
40
44
48
52
WEEKS
FIG. I. Body weight for beagle dogs given methidathion in diet for 1 year. Each data point represents the mean of four animals for male dogs (top) or female dogs (bottom).
Tests
No treatment-related effects on hematology, coagulation, or urinalysis were observed (data not shown). However, in both the 40- and the 140-ppm males and females, there were clinical chemistry changes which indicated liver dysfunction. These included moderate to markedly elevated activities of serum AST, ALT, ALP, and SDH as well as increased bile acids concentrations in both sexes (Table 1). In females, increased serum GGT activity and decreased serum albumin were also noted at 240 ppm. The elevated enzyme activities were seen at all intervals examined (only 3- and 12-month data are shown in Table 1). The increases in enzyme activities for the 140-ppm males and females at termination (per-
CHANG,
310
WALBERG,
AND CAMPBELL
TABLE 1 Clinical Chemistry Parameters” from Beagle Dogs Given Methidathion Dietary level (pm)
Time (months)
Albumin k/dl)
Total bilirubin hddl)
ALP (U/liter)
AST (U/liter)
in Diet for 1 Year
ALT (U/liter)
GGT (U/liter)
SDH (U/liter)
Bile acids (PM/liter)
5+2 5tl 5il 5?0 6+l 6+1 6+l 6+l
5*1 4fl 9k2 6+3 14*4* I I ?I 3** 20 Ii 9** 13 * 5**
NA 0.9 NA 1.4 + 0.9 NA 12.4 ? 1 I.5 NA 9.1 + 2.3
5kl 5+1 5t-I 6&l 7+ I* 7-+2 7f I* 8k5
6kl 5kl 9k2 6+l I5 +6* I3 f 4** I4 + 5* IO i 3**
NA 2.4 NA 3.6 i 0.9 NA II.1 * 5.5 NA 15.1 + 7.7
Males 0 4 40 140
3 I2 3 I2 3 12 3 I2
3.2 I!I 0.1 2.9 f 0. I 3.1 f 0.1 3.0 + 0.2 3.1 +- 0.1 2.9 + 0. I 3.1 * 0.1 2.8 + 0.1
0.1 0.2 0.1 0.2 0.1 0.2 0.2 0.3
+ f i f + if +
0.1 0.0 0.1 0.0 0.0 0.1 0.1* 0.1*
175 i 46 l26k 21 1ss* 32 99 i 34 465 f 14** 297 + 38* 500 i 175** 371 +- 152**
26 23 26 23 33 29 35 32
k 4 ?I 3 f 6 +3 f 5 f 7 + 4* + 4*
19* l5rt_ 41 + 4Ok I58 f 134 + I64 + 140 +
3 4 17 16 42** 37** 67** 70**
Females 0 4 40 140
3 I2 3 I2 3 I2 3 12
3.4 tr 0.3 3.2 * 0.2 3.2 * 0.1 3.1 * 0.1 3.0 ?I 0.2* 3.0 + 0.1 2.9 k 0.2* 2.9 f 0.2s
0.1 0.2 0.1 0.2 0.2 0.3 0.2 0.3
f f t k + f k +
0.0 0. I 0.0 0. I 0.1* 0.1 o.o* 0.1
179* l52k l84k l9Ok 503 2 353 + 615 f 623 +
38 81 64 40 76** 120 290*+ 461*
25 k 2 24 + 2 25 f 3 ..A +- 3 37 29 + 5 28 + 6 41 k8* 35 4 7*
17+ 5 l7f 5 41 ?23 26+ I 130 AZ26** I26 + 42** I57 i 65** I34 * 58**
u Mean + SD of four animals for all parameters at all dose levels except the bile acids (only one female and two males at 0 ppm: four animals in all treated groups). Statistical analysis for bile acids not performed. * Significantly different from control: p < 0.05 by Dunnett’s I test. ** Significantly different from control: p G 0.01 by Dunnett’s f test. No&>. NA. not available.
centage of controls, M/F) were 29414 10% for ALP, 1391146% for AST, 933/788% for ALT, and 325/200% for SDH. In both sexes, the serum total bilirubin concentration was only marginally increased and there were no significant changes in serum cholesterol level (data not shown). Cholinesterase Activities Brain ChE activity, measured in a homogenized fraction from the vermis of the cerebellum or the cerebral hemisphere, was generally depressed at 140 ppm in both sexes. The magnitude of the depression in ChE activity from the control was slightly greater with the cerebellar fraction with statistical significance noted in both sexes. In the 140-ppm males and females, cerebellar ChE activity was reduced 27 and 22%, respectively, from control levels. Cerebral ChE activity was reduced 17% in both sexes at 140 ppm but was significantly different from controls in females only. RBC ChE was depressed at 240 ppm in males and at 140 ppm in females; statistical significance was noted at 140 ppm (Table 2). At 12 months, the mean RBC ChE activity of the 140-ppm animals was inhibited by approximately 76% as compared to the controls. Although RBC activities of the 40-ppm males were not statistically different from control, the activities were reduced by 20-30% during the study and thus the inhibition was considered toxicologically significant.
Serum ChE activities of the treated groups were not inhibited (relative to the control) throughout the study. In vitro incubation of serum or whole blood samples from control dogs with methidathion showed inhibition of RBC or serum ChE in a concentration-dependent manner (2020,000 PM, or 6-6000 pg/ml) (Fig. 3). In addition, RBC ChE activity was inhibited to a greater extent than serum ChE by methidathion between 20 and 2000 PM. Liver Weight and Pathology There were no treatment-related effects on organ weights, including the absolute and relative liver weights (Table 3). The only treatment-related gross pathology findings were the dark red, discolored livers seen in some dogs receiving a40 ppm (Table 3). Moderate to marked cholestasis was noted in all males and females at 40 and 140 ppm, being more severe at the 140-ppm level and in females (Table 3). Cholestasis was characterized histologically by bile plugs in distended bile canaliculi, bile ductules, and bile ducts and was most pronounced in the centrilobular zone in all liver lobes examined. Mild chronic inflammation of the liver, characterized by infiltration of lymphocytes, was also observed at 40 ppm (one male and three females) and 140 ppm (one female).
METHIDATHION
TOXlCITY
TABLE 2 Cholinesterase Activity” from Beagle Dogs Given Methidathion Dietary level (w-N
Time (months)
Serum ChE (U/liter)
311
IN DOGS
in Diet for 1 Year
RBC ChE (U/liter RBC)
Brain ChEb (U/g tissue)
Brain ChE’ (U/g tissue)
1405 rfr 203 1385 rt 262 1535 k 392 1565 f 475 980 + 298 1105 f 302 185 -+ 130** 315 f 123**
NA 2.14 + 0.20 NA 2.07 f 0.17 NA 2.04 + 0.01 NA 1.57 + 0.16**
NA 1.53 rt_0.06 NA 1.60 f 0.19 NA 1.52 20.16 NA 1.29 f 0.08
NA 2.15 f 0.19 NA 2.29 + 0.20 NA 2.06 + 0.17 NA 1.68 + 0.30*
NA 1.57 + 0.12 NA 1.62 f 0.17 NA 1.58 _+0.08 NA 1.3 1 f 0.09*
Males 0 4 40 140
3 12 3 12 3 12 3 12
1906k 61 1969 +_ 66 1884 k 328 1975 f 338 1940+ 80 2028 1+_167 1925 f 444 1952 + 485
Females 0 4 40 140
3 12 3 12 3 12 3 12
1796 1839 2179 2382 2265 2378 2154 2537
f + k f + f k k
250 203 337 542 445 567 425 675
1445 1285 1895 1715 1245 1220 245 310
f k f + f f k i
562 420 428 462 232 198 171** 101**
a Mean f SD of four animals per sex per dose level. b Measured in homogenized fractions from the vermis of the cerebellum. c Measured in homogenized fractions from the right cerebral hemisphere. * Significantly different from control; p < 0.05 by Dunnett’s t test. ** Significantly different from control; p < 0.0 1 by Dunnett’s t test. N&e. NA, not available.
DISCUSSION Although methidathion is an organophosphate insecticide, there were no adverse clinical signs attributable to inhibition of ChE activity. All dogs survived in good condition. The body weights and weight gains of treated dogs were similar to those in the controls although males given the 140-ppm diet generally consumed less food. The highest dietary concentration, 140 ppm, caused inhibition of RBC and brain ChE in both sexes; the inhibition was approximately 76% for RBC ChE (12 months) and 2227% for brain ChE as compared to controls. However, serum ChE activity was comparable to the controls throughout the study. This observation is interesting as serum ChE is usually more readily inhibited than RBC ChE when exposures to many organophosphorus compounds occur (Tietz, 1986). The ChE assay procedure used in this study is capable of detecting both the “true” ChE (also called acetylcholinesterase, normally present in RBC, lung, spleen, nerve endings, and gray matter of the brain) and “pseudo”-ChE (acylcholine acylhydrolase or butyryl ChE, normally found in liver, pancreas, heart, and white matter of the brain and serum). It is thus unlikely that the lack of inhibition was due to insensitivity of the analytical procedure. Also considered unlikely
was that an inhibition might have occurred prior to the first scheduled measurement at 3 months; data from the 90-day study (Chang et al., 199 1) showed no inhibition after 8 weeks of exposure. Excluding the two preceding possibilities, this leaves for consideration the steady state (synthesis and degradation) and the sensitivity of serum ChE to methidathion in vivo. Serum ChE concentration has been shown to be an indicator of liver function as its synthesis is parallel to that of albumin by the liver (Vorhaus and Kark, 1953). While decreased serum ChE activity was seen in humans with acute hepatitis, liver cirrhosis, or carcinoma, normal levels were seen in chronic hepatitis, mild cirrhosis, and obstructive jaundice patients whereas those with nephrotic syndrome had slightly elevated serum ChE activity (Vorhaus and Kark, 1953; Wittaker, 1986; Tietz, 1986). Since the kidney was not a target organ in this study and serum albumin levels in dogs with cholestasis were either unchanged or slightly decreased, an increased ChE synthesis by the liver is questionable. On the other hand, little is known about serum ChE synthesis under diseased liver conditions in dogs, including the total amount, the various variants of ChE and esterases, and their sensitivity to the inhibitors. Results from the in vitro study showed a concentrationdependent inhibition of both serum and RBC ChE by meth-
CHANG,
312
WALBERG.
AND CAMPBELL
707 z s
60-
:
50-
20lo0
2
I
3
t111r1
4 56789’
,
10
2
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3
I1llll
4 56789’
100
Methidothion
2
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3
Concentration
I11111
4 56789’
1000 (uM)
I
2
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3
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4 56789’
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10000
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FIG. 3. Inhibition of serum (0) and RBC (u) ChE by methidathion in vitro. Each data point represent the mean of two samples. Data from “serum + methanol” were similar to those of serum alone.
idathion and greater inhibition of the RBC ChE than serum ChE at equimolar concentrations. This suggests that the methidathion concentrations reached in vivo in the treated dogs might not have been sufficiently high to inhibit the serum ChE activity. Assuming a one-compartment distribution with little or no accumulation, the blood concentration of methidathion in the 140-ppm dog would be 57 pg/ ml (or 4.7 mg/82 ml, based on the fact that a mature beagle dog has a blood volume of 82 ml/kg; Andersen and Schalm, 1970). At 60 pg/ml (or 200 PM) in vitro, methidathion inhibited RBC ChE by 40% and serum ChE by 18% (Fig. 3). These values are surprisingly similar to the in viva data from the 140-ppm dog, which showed 76% inhibition of RBC ChE and no inhibition for serum ChE. The lack of serum ChE sensitivity to methidathion in vivo also appears to be species specific (Table 4). Among the four species tested for the chronic toxicity of methidathion, the rat was the most sensitive to the anti-cholinergic effects. In rats, inhibition of serum ChE occurred at 2 mg/kg and was accompanied by prominent clinical signs of toxicity. Serum ChE from the mouse appeared to be the least sensitive. Whether assays on brain ChE should utilize tissue from the specific regions of the brain or the whole brain has been discussed for some time within the scientific community. The uncertainty stems from the facts that the distribution of ChE in the brain varies from region to region and that brain ChE also exists in different forms with differing sensitivities to inhibitors (Silver, 1974; Meneguz et al., 198 1; Cook et al., 1989). At present, there are no standardized procedures for taking samples to measure representative brain ChE. With rodents, half or whole brain can be ho-
mogenized and analyzed. However, with larger laboratory animals, the specific anatomic sections taken for analysis vary from laboratory to laboratory. Cohen et al. ( 1985) suggested that homogenates be made from different brain sections and combined into a composite homogenate in direct proportion to the wet weights of each section. To see if regional or whole brain ChE activity was more indicative of methidathion exposure (or toxicity) in beagle dogs, brain ChE in this study was determined in two distinct areas for comparisons, the vermis of the cerebellum and the right cerebral hemisphere. The results showed an inhibition of ChE in the cerebellum slightly greater than that in the cerebral hemisphere. This was not unexpected as any regional inhibitory effect that methidathion might have had in the right cerebral hemisphere might have been diluted by the larger amount of tissue present, resulting in a higher ChE activity per gram of tissue. Nevertheless, ChE activity from both the cerebellum and the cerebral hemisphere of the 140-ppm dogs illustrated a minimum inhibitory effect. This was consistent with the clinical observations which did not indicate any functional or behavioral deficits in these dogs. Besides the RBC and brain ChE, liver was clearly the target organ in dogs exposed to methidathion. Moreover, liver toxicity was seen at levels (340 ppm) at which none or minimal inhibition of RBC ChE occurred. The liver toxicity was indicated by clinical chemistry results as well as by necropsy and histopathology findings. Conventional diagnostic tests used to assess hepatocellular dysfunction in dogs include serum concentrations of albumin, cholesterol, urea, ammonia, and coagulation factors whereas biliary function and flow are assessed by serum concentrations of total bilirubin
METHIDATHION
TOXICITY
313
IN DOGS
TABLE 3 Final Body Weight, Relative Liver Weight and Liver Pathology in Beagle Dogs Given Methidathion Dietary level bvm)
Final body weight” (kg)
Relative liver weighta (% body weight)
in Diet for 1 Year
Incidence of gross discoloration
Incidence of cholestasis
Incidence of chronic inflammation
O/4 O/4 l/4 214
O/4 O/4 4146 4/4b
O/4 O/4 114 O/4
O/4 O/4 214 314
O/4 O/4 414' 414'
O/4 O/4 314 114
Males 0 4 40 140
i + 12.1 f 10.3 f 10.8 10.8
1.3
2.12 2.56 2.45 2.62
1.1 1.3
1.5
i f f k
0.45
0.11 0.27 0.39
Females 0 4 40 140
8.1 9.5 10.5 9.5
k rt + *
0.1
3.01 f 0.52 2.94 f 0.15 2.43 + 0.25 2.82 f 0.54
1.6 2.1 1.7
a Mean ? SD of four animals at all dose levels taken before necropsy. b Severity grades were 3 moderate and 1 mild at 40 ppm and 2 marked, 1 moderate, and I mild at 140 ppm. ’ Severity grades were 4 moderate at 40 ppm and 3 marked and 1 moderate at 140 ppm.
and activities of ALT, AST, SDH, GGT, and ALP (Loeb, 1989; Eckersall and Nash, 1983). In recent years, elevated serum bile acids concentration has been used increasingly to diagnose hepatobiliary disease in the dog (Center et al., 1985; Hauge and Abdelkader, 1984). As shown in Table 1, the serum bile acid concentration and activities of ALT and
TABLE 4 Species Comparison of Sensitivity to Methidathion
Effects
Effect levels (mg/kg) Effects Serum ChE inhibition RBC ChE inhibition Brain ChE inhibition Liver pathology
Mouse”
Ratb
Dog’
Monkeyd’
IS 7.5’ 16.1
2
NA
2 2
4.7 4.7
7.59
NA
1.4
NAd 1.0 NA NA
10’ 5
5 -
Note. IS, increased significantly; NA, not affected. a Mice were administered methidathion in diet at 0.3, 3, 10, 50, and 100 ppm for 23 months. The equivalent dosages were 0,0.46, 7.5, and 16.1 mg/ kg/day (Goldenthal et al., 1986; Quest et al., 1990). b Rats were given methidathion in diet at 4,40, and 100 ppm for 2 years. The doses were equivalent to 0, 0.2. 2. and 5 mg/kg/day (Yau et al., 1986; Quest et al., 1990). c Present study. d Monkeys, 5-7/sex/dose were given methidathion via stomach tube at 0, 0.25, or 1.O mg/kg/day for 2 years (Coulston, 197 1). e Four monkeys per dose were given methidathion at 5 or 10 mg/kg daily for 16 months. Two of the four monkeys from each dose group died within a few weeks whereas the others survived (Cot&ton, 1973). fThis value applies to the female mice. The effect level for males was 16.1 m/kg. RThis value applies to the male mice. The effect level for females was 16.1 mg/kg.
ALP were elevated in dogs receiving 240 ppm. The observations of grossly dark, discolored livers at 40 and 140 ppm also suggested cholestasis. In a previous study in which beagle dogs were given methidathion for 90 days, cholestasis was also seen (Chang et al., 199 1). The livers of dogs exposed to 140 ppm methidathion for 1 year in this study showed more severe cholestasis than the livers of dogs administered 140 ppm for 90 days (data not shown). In contrast, treatmentrelated changes in clinical chemistry data did not increase with time in the present study; the effects were maximal at 3 months. Females appeared to have slightly more severe cholestasis than the males; this was seen microscopically by more deposits of pigment and reflected by the slightly higher levels of serum bile acids, GGT, and ALP activities. In addition, females were observed to have slightly higher incidences of chronic inflammation of the liver. The inflammation, perivascular or periportal and characterized by infiltration of lymphocytes, was limited to the 40- and 140-ppm groups, in which cholestasis was seen and did not correlate with the severity of the cholestasis. However, it was not seen in any of the control animals and was considered a treatment-related effect. The liver inflammation might have resulted from necrosis. Necrosis was also indicated by the slight but significantly elevated serum SDH activity in the 40- and 140-ppm groups. Overall, female dogs received slightly higher dosages of methidathion than the males (4.9 versus 4.5 mg/kg/day in males at 140 ppm; 1.4 versus 1.3 mg/kg/day in males at 40 ppm). Compared to the rat, mouse, and monkey, the dog is the species most sensitive to the hepatotoxicity of methidathion (Table 4). Whereas no liver toxicity was detected in the rat (Yau et al., 1986; Quest et al., 1990) or monkey (Coulston
314
CHANG. WALBERG,
et al., 197 1; Coulston,
1973), liver effects were seen in dogs and mice. In the mouse, the liver pathology included biliary stasis, bile duct hyperplasia, cholangiofibrosis, and chronic hepatitis. In addition, increased liver weights and liver tumors were seen in male mice (Quest et al., 1990; Goldenthal et al., 1986). The liver toxicity, not elicited by many other organophosphorus compounds, might have resulted from the thiadiazole ring moiety, which could possibly form hydrazine/hydrazide in the metabolic pathway. No mechanistic study results are available to answer the question. However. the similarity in responses between the mouse and the dog in the liver effect and lack of serum ChE inhibition is interesting and may warrant further investigation. In conclusion, the hepatobiliary toxicity seen in this study, which followed the GLP guidelines, is consistent with that observed in the 1967 study on methidathion. The overall NOEL for the present study was judged to be 4 ppm (0.15 mg/kg) based on the hepatic findings. This NOEL is slightly higher than that obtained in the previous study (0.08 mg/ kg). Additionally this study demonstrates that beagle dogs can tolerate doses nearly four times greater than the highest dose used in the previous study, that is, 4.9 mg/kg/day versus 1.35 mg/kg/day (Hayes, 1982). ACKNOWLEDGMENTS We thank the EHC staff from the vivarium. pharmacy, analytical chemistry. clinical chemistry, and necropsy/histopathology laboratories for their excellent technical support during the course of the study. Special thanks are extended to T. Bell, J. Chase, M. Gilman, and L. O’Donnell for their valuable contributions. The critical review of this manuscript by the various CIBA-GEIGY toxicology groups (Basel, Switzerland; Greensboro, NC; and Farmington, CT) was deeply appreciated.
REFERENCES Andersen, A. C.. and Schalm, D. W. (1970). Cardiovascular system/hematology. In The Beagle as an Experimental Dog (Andersen, A. C., and Good, L. S., Eds.), p. 278. The Iowa Univ. Press, Ames, IA. Center, S. A., Baldwin, B. H., Erb. H. N., and Tennant, B. C. (1985). Bile acid concentrations in the diagnosis of hepatobiliary disease in the dog. J. Am. Vet. Med. A 187, 935. Chang, J. C. F., Pavkov, K. L., Campbell, W. R., and Wyand, D. S. (199 1). 90-day oral toxicity study with methidathion in beagle dogs. Toxicologist 11, 159. CIBA-GEIGY Corp. (199 1). Sample Label Guide. Agricultural Division, Greensboro, NC. Cohen, S. D., Williams, R. A., Killinger, J. M., and Freudenthal, R. I. (1985). Comparative sensitivity of bovine and rodent acetylcholinesterase to in vitro inhibition by organophosphate insecticides. Toxicol. Appl. Pharmacol. 81,452-459.
Cook, W. O., Dellinger, J. A., Sir&, S. S., Dalhem, A. M., Carmichael, W. W., and Beasley, V. R. (1989). Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon. Toxicol. Lett. 49, 29-34.
AND CAMPBELL Coulston, F., et al. (197 I). Two-Year Safety Evaluation Study in Rhesus Monkeys with GS-1300.5 Technical. Institute of Experimental Pathology and Toxicology, Albany Medical College, Albany, NY. [Sponsored by the CIBA-GEIGY Corp., Greensboro, NC] Coulston, F. (1973). Evaluating toxicological data as regards environmental significance. In Environmental Quality and Safety. Global Aspects of Chemistry, Toxicology and Technology as applied to the Environment, Vo! I!. Georg. Thieme/Academic Press, Stuttgart/New York. Eckersall, P. D., and Nash, A. S. ( 1983). Isoenzymes of canine plasma alkaline phosphatase: An investigation using isoelectric focusing and related to diagnosis. Rex Vet. Sci. 34, 310-3 14. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. ( 196 1). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Environmental Protection Agency (EPA) (1988). Peer Review of Methidathion. Office of Pesticide Programs, Health Effects Division, Washington, DC. EPA (I 989). Guidance for the Registration of Pesticide Products containing Methidathion as the Active Ingredient. Office of Pesticide Programs, Health Effects Division, Washington, DC. Food and Agricultural Organization of the United Nations, World Health Organization (FAO/WHO). (1973). Evaluations ofSome Pesticide Residues in Foods, World Health Organization Technical Report Series, No. 2. Goldenthal, E. I., et al. (1986). 2-Year Dietary Oncogenicity (and Toxicity) Study with Methidathion Technical in Mice. Study performed by the International Research and Development Corp. for CIBA-GEIGY Corp., Greensboro, NC. Hauge, J. G., and Abdelkader, S. V. (1984). Serum bile acids as an indicator of liver disease in dogs. Acta Vet. Stand. 25,495-503. Hayes, W. J., Jr. (1982). Pesticides Studies in Man, pp. 371-372. Williams & Wilkins, Baltimore, MD. Johnston, C. D. (1967). GS-13005 Evaluation by a 2-Year Feeding Study in dogs. 4OW, Woodard Research Corp., Herndon, VA. [Sponsored by the CIBA-GEIGY Corp.. Greensboro, NC] Loeb, W. F. (1989). The dog. In The Clinical Chemistry of Laboratory Animals. (Loeb, W. F.. and Quimby, F. W., Eds.), pp. 47-58. Pergamon, Elmsford, NY. Meneguz, A., Bisso, G. M., and Michalek, K. H. (1981). Regional differences in brain soluble acetylcholinesterases and its molecular forms after acute poisoning by isoflurophate in rats. Clin. Toxicol. 18, 1443-145 I. Quest, J. A., Copley, M. P., Hamernik, K. L., Rinde, E., Fisher, B., Eng!er, R., Burnam, W. L., and Fenner-Crisp, P. A. (1990). Evaluation of the carcinogenic potential of pesticides. Reg. Toxicol. 12, I !7- 126. Silver, A. (1974). The biology of cholinesterases. In Frontiers of Biology, Vol. 36, North-Holland. Amsterdam. Tietz, N. W. (1986). Cholinesterase. In The Textbook of Clinical Chemistry, pp. 746-75 I Saunders, Philadelphia, PA. Vorhaus, L. J., and Kark, R. M. (1953). Serum cholinesterase in health and disease. Am. J. Med. 14, 707-719. Wittaker, M. (1986). Cholinesterase: Monographs in Human Genetics, Vol. 11, Karger. Basel, Switzerland. Yau, E. T., et al. (1986). Methidathion 2-Year Oral Oncogenicity and Toxicity Study in Albino Rats (MIN 832001). CIBA-GEIGY Corp., Pharmaceutical Division. Summit, NJ. [Sponsored by the CIBA-GEIGY Corp., Greensboro, NC]
AND
APPLIED
TOXICOLOGY
19, 307-3
14 (1992)
One-Year Dietary Toxicity Study with Methidathion JANE C. F. CHANG,*” *Environmental
Health Center, Agricultural and tAnimal Health Division,
JAMES A. WALBERG,*
AND WILLIAM
in Beagle Dogs
R. C,uN?BELLt
Division, CIBA-GEIGY Corporation, 400 Farmington Avenue, Farmington, CIBA-GEIGY Corporation, P.O. Box 18300, Greensboro, North Carolina
Connecticut 27419
06032;
Received December 12, 199 1; accepted April 9, 1992
the U.S. population.
The current acceptable daily intake
One-Year Dietary Toxicity Study with Methidathion in Beagle (ADI), or the reference dose, was estimated to be 0.001 mg/ Dogs. CHANG, J. C. F., WALBERG, J. A., AND CAMPBELL, kg/day by the EPA. The AD1 was established on the basis W. R. (1992). Fundam. Appl. Toxicol. 19,307-3 14.
The purposeof this study wasto determinethe chronic toxicity of methidathion, an organophosphate insecticide, in dogs. Groups of beagledogs,four/sex/dose, were fed methidathion at constant dietary concentrations of 0, 0.5, 2, 4, 40, or 140 ppm for 1 year. The equivalent daily dosageswere approximately 0, 0.02, 0.07, 0.15, 1.4, and 4.7 mg/kg. There were no deaths or adverseclinical signsassociatedwith the treatment. Weekly body weights and weight gains were not affected. Mean daily food consumption wasreducedin male dogsgiven the 140-ppmdiet. Major treatment-related effects were cholestasis,chronic inflammation in the liver, and cholinesterase(ChE) inhibition. The liver effectswereindicated by grossand microscopicpathologic findings aswell asmoderate increasesin serumbile acids and enzyme activities (alanine aminotransferase,aspartateaminotransferase,sorbitol dehydrogenase, and alkaline phosphatase) in all dogsreceiving >40 ppm. RBC ChE wasinhibited in males at 340 ppm and in femalesat 140ppm. Brain ChE wasinhibited in both sexesat 140 ppm; the magnitude of inhibition relative to control wasslightly greater with the cerebellarfraction than with the cerebral fraction. Serum ChE was not affected at any doselevel. In conclusion, liver was the target organ in beagle dogsgiven a40 ppm (equivalentto 1.4 mg/kg/day) methidathion in diet for I year. The no-observable-effectlevel was4 ppm (0.15 mg/kg/day) for both liver cholestasisand ChE inhibition. o 1992
of results of a 2-year chronic toxicity study in dogs and a safety factor of 100 (EPA, 1988, 1989). In the previous study (Johnston, 1967), beagle dogs were provided for 2 years with methidathion technical in diet at a concentration of 0, 4, 16, or 64 ppm, 6 days/week (200 g of food daily except on Saturdays when 400 g was fed and on Sundays when no test diets were provided). All dogs survived and remained in good condition throughout the study. Major findings at the high dose, 64 ppm, included marked elevations of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities, retention of sodium sulfobromophthalein, and increased pigment in liver and nephrons. A slight inhibition of RBC ChE was also seen at 64 ppm whereas no inhibitory effects on plasma or brain ChE were noted. The no-observable-effect level was judged to be 4 ppm or 0.08 mg/kg (FAO/WHO, 1973; Hayes, 1982). The purpose of the present study was to determine the toxicity of methidathion in beagle dogs after continuous exposure in diet for 1 year. The daily dosage of methidathion (mg/kg) received by dogs given the highest dietary concentration, 140 ppm, was approximately four times higher than that used in the previous study. This study was conducted
to satisfy regulatory requirements by the California Department of Food and Agriculture to update the data base. The EPA FIFRA guidelines on nonrodent chronic toxicity studies (Pesticide Assessment Guidelines, Part 158, Section 83-l) Methidathion (GS-13005,S-2,3-dihydro-5-methoxy-2oxo- 1,3,4-thiadiazol-3-yl-methyl O,O-dimethyl phospho- 1984) and EPA-FIFRA GLP Standards (40 CFR, Part 160) rodithioate) is an organophosphorus insecticide. The chem- were followed. Our results demonstrate that the major in beagle dogs are hepaical is marketed by the CIBA-GEIGY Corp. under the trade chronic toxicities of methidathion but not inname of Supracide 2E Insecticide-Miticide for use on certain tobiliary dysfunction and RBC ChE inhibition citrus fruits, almonds, pecans, walnuts, prunes, safflowers, hibition of serum or brain ChE. Society of Toxicology.
grain sorghum, alfalfa, artichoke, tobacco, nursery stock, and severalother crops (CIBA-GEIGY Corp., 1991; EPA, 1989).
Based on residue tolerances established for methidathion in food and feed crops, the average anticipated residue contribution (ARC) was established at 0.000935 mg/kg/day for
METHODS Test substance. Technical methidathion (GS-1300&S-2,3-dihydro5-methoxy-2-oxo-l,3,4-thiadiazol-3-yl-methyl-O,O-dimethyl phosphorodithioate) was supplied by the CIBA-GEIGY Corp., Agricultural Division (Greensboro, NC). The test material, a solid with a purity of 96%. was stored at 4°C.
’ To whom correspondence should be addressed. 307
0272-0590/92 $5.00 Copyright 0 1992 by the Society of Toxicology. All rights of reproduction in any form reserved.
308
CHANG,
WALBERG,
Animals. Laboratory-reared beagle dogs were obtained from Marshall Farms (North Rose, NY) and maintained in compliance with the U.S. Animal Welfare Act (1985). The dogs were housed individually in stainless steel cageswith approximately 8 ft’ of floor space. The animal room was provided with at least I5 air changes per hour with the temperature maintained at 19-24°C and relative humidity at 40-60%. Fluorescent lighting was provided on a 12-hr light/dark cycle. During the quarantine/acclimation period, dogs were provided with approximately 350 g/day of Purina Certified Canine Diet No. 5007 kibble during the first few days: thereafter, Purina Certified Canine Diet No. 5007 ground meal was provided. The feeding periods were limited to 4 hr daily (midday). Local water was provided ad libitmn by an automatic watering system and in water bowls during feeding hours. After a quarantine period of 28 days, dogs were assigned to the study groups such that all groups had similar mean body weight at study start. Littermates were not assigned to the same group. All dogs were approximately 5 months old at the study start. Test diets. Methidathion technical was used as received without adjustment for purity. The bulk technical material, upon receipt, was melted at xSO”C, aliquoted into small jars. and stored at 4°C until used. To prepare the test diet, the aliquoted technical material was melted, mixed with corn oil, and incorporated into a portion of Purina Certified Canine Diet No. 5007 ground meal. The resulting mixture was further diluted with ground meal in a Patterson-Kelly twin-shell blender to achieve the desired dietary concentrations. The control diet consisted of ground meal blended with corn oil in amounts equivalent to those used for the 140-ppm blends (0.01% final concentration). The test diets were prepared approximately biweekly. All test diets were stored at 4°C until scooped into food bowls. Methidathion was determined to be stable in bulk dietary mixtures (0.5 and 4 ppm) at 4°C for 6 weeks and in food bowls at 25°C for 5 days. The concentration and homogeneity of methidathion in test diet mixtures met the specifications for most blends analyzed. The grand means of the analytical concentrations of the test diets were 0.48, 1.88. 3.67. 37.7. and 133.4 ppm for the nominal concentration of 0.5, 2. 4. 40, and 140 ppm, respectively. The test diets (350 g/dog) were provided for approximately 4 hr daily. Afterward. unconsumed feed was removed. Observations and measurements. The dogs were observed at least twice daily (AM and PM; before and after feeding) for general appearance, behavior, signs of toxicity. and mortality. General physical examinations were performed weekly. In addition, detailed physical examinations, which included measurements of heart rate, respiration. and temperature as well as examination of sensory/motor systems,were performed by the clinical veterinarian at pretest and once every 3 months during the study. Complete ophthalmologic examinations were performed at pretest and before study termination. Body weight was measured weekly. Daily food consumption by the individual animal was measured twice per week for the first I3 weeks and twice monthly thereafter. Complete hematology, clinical chemistry, and urinalysis were performed at pretest and at 3.6, and I2 months. Fecal analyses were performed at pretest and at study termination. Blood samples for hematology and clinical chemistry were obtained from the jugular veins of dogs fasted for at least I8 hr prior to sample collection. Urine samples were collected overnight using a collection tray. Hematology parameters (hematocrit. hemoglobin, platelet count. white and red blood cell counts) were determined by an Ortho ELT-8/ds counter (Ortho Diagnostic System, Inc., Westwood, MA). Differential leukocyte counts were performed manually. Prothrombin time and activated partial thromboplastin time were determined by using Coag-A-Mate (Akzo, Oklahoma City, OK). Clinical chemistry tests (serum ChE, bile acids, aspartate aminotransferase (AST), ALT, ALP. y-glutamyltransferase (GGT). sorbitol dehydrogenase (SDH). creatine phosphokinase, blood urea nitrogen. cholesterol. triglycerides, total bilirubin, total protein, albumin. globulin, creatinine, glucose. calcium, inorganic phosphate, sodium, potassium. and chloride) were performed on an IL Monarch 2000 Chemistry System (Instrumentation Laboratory, Lexington, MA). Test reagents used included the IL Test Reagents (all parameters except bile acids, SDH, and ChE), the
AND CAMPBELL Sigma Diagnostics Reagents (bile acids, SDH) and the Boehringer-Mannheim Diagnostics Reagents (serum and brain ChE). Urinalyses (pH, protein, glucose, ketone. bilirubin, blood, urobilinogen, specific gravity) were performed on an Ames Clini-Tek 10 Urine Chemistry Analyzer (Miles Diagnostics Division. Elkart, IN). RBC ChE was analyzed by the Baker Centrifichem 500 Analyzer (ScronoBaker Instruments Corp., Pleasantsville, NY) based on the Ellman’s method (Ellman rf al.. 196 1). Blood samples were collected in EDTA anti-coagulated tubes. RBC hemolysates were prepared by adding 1 part of saline-washed, packed RBC to 19 parts of 1% Triton X- 100 in saline. The sulthydryl groups in the RBC hemolysate were removed by preincubation with dithiobisnitrobenzoic acid (final concentration. 3 X 10e4 M) at room temperature for 15 min. Subsequently. the substrate, acetylthiocholine (final concentration, 5 X 10m4M in 0. I M phosphate buffer, pH 8.0) was added and the reaction was allowed to proceed at 37°C for 30 sec. The formation of the colored endproduct, thionitrobenzoic acid, was measured at 405 nm. Both serum and brain ChE were determined by using the IL Monarch 2000 Chemistry Systemand the Boehringer-Mannheim Diagnostic Reagents. which are also based on the Ellman’s method. Serum samples were used as obtained. Brain tissuesobtained from animals at necropsies were immediately placed in ice-cold 0. I M phosphate-buffered saline, pH 8.0 (l/IO. w/v). and homogenized with a Brinkman Polytron. The homogenized fractions were next centrifuged at 4°C at 7000g for IO min and the supernatant fractions were aliquoted for analysis. Brain ChE from two homogenized fractions were determined. One homogenized fraction was prepared from the vermis of the cerebellum whereas the other was from the right cerebral hemisphere. The assaytemperature was 30°C for both serum and brain ChE. To determine the sensitivity of serum and RBC ChE to methidathion irr vitro. serum or whole blood from one control male and one control female dog was separately incubated with methidathion (dissolved in methanol: final concentration, 2,20,200.2000, or 20,000 FM) at 37°C for 2 hr. Thereafter, plasma was separated from the RBC and ChE activity in either RBC, plasma, or serum was analyzed. Serum or whole blood samples incubated with or without methanol served as controls. The dogs were terminated by iv phentobarbital injection followed by exsanguination, Complete necropsy examinations were performed and complete tissue collections were made. Organ weights were taken for adrenals. brain. heart, kidneys, liver, ovaries, pituitary, testes, thyroid, and spleen. Tissues were trimmed, embedded in paraffin. sectioned at 4-6 pm. and stained by hematoxylin and eosin (H&E). The tissues were evaluated by the study pathologist: lesions were graded as slight. mild, moderate, or marked depending upon the severity and the areas involved. Cafcufations. Weekly compound consumption (data not shown) by each animal was calculated by using the nominal concentration of methidathion in the diet, the mean daily food consumption. and the body weight for the week. These weekly individual animal compound consumption values were used to calculate the weekly group means and the grand mean for the group during the 1-year period. The grand mean compound consumption for each dose group, not shown under Results, is stated in the Abstract. Individual animal weekly food consumption (g/day) was calculated by taking the mean of the two measurements performed for each dog during the week. These individual values were then used to calculate the group mean for the week as well as the grand mean for the group during the study. Statistical analysis. Quantitative continuous variables were analyzed statistically by a one-way analysis of variance followed by Dunnett’s t test. The probability of type 1 error (a) was set at 0.05. Statistical test results at the 0.0 1 level of significance were also noted. Statistical analyses of bile acid concentrations at 12 months (Table 1) were not performed due to the small number of samples in the control groups (one female and two males).
RESULTS Survival, Body Weight, and Food Consumption
All dogs survived in good condition. There were no clinical signs in treated dogs which are characteristic of those asso-
METHIDATHION
TOXICITY
ciated with organophosphate exposure. Wet coat on the forefoot, which might have resulted from salivation, was noted in some treated dogs on a few occasions. Overall, neither salivation, dacryorrhea, nor wet coat on the forefoot was seen frequently or in a dose-related manner. There were no treatment-related effects on body weight (Fig. 1) or body weight gain (data not shown) in either sex. Mean weekly body weights of the treated males or females were not statistically different from those of the controls. Mean body weight gain (in 52 weeks) was 3.3, 3.7, 4.2, 5.2, and 3.7 kg for males receiving 0.5, 2, 4, 40, or 140 ppm, versus 4.0 kg in controls. Body weight gains by the treated females, at corresponding dose levels, were 3.5, 3.8, 1.2,4.2, and 3. I kg versus 2.5 kg for the controls. Food consumption was not affected in treated females and no statistical differences were noted at any time point (Fig. 2). In males, food consumption was reduced in the group
309
IN DOGS
-
i
0e 150 100
01 0
,
4
,
8
,
12
,
16
,
Control 0.5 ppm
2 wm
4 wm 40 ppm 140 ppm
,
20
24
,
28
,
32
,
,
,
,
,
36
40
44
48
52
I 36
I 40
1 44
I 4.9
I 52
WEEKS
350 300
250 6 ,D 200 i
vl‘ E
0e 150
-
i
100
t e ol8 0 f
-
Control 0.5 aom
4 ‘2 351
-
40 ppm 140 ppm
0.5 2
ppm
wm
4 pm 40 ppm 140 ppm
M-
O
0
4
1
Ii
I 16
zb
i4
I 28
I 32
WEEKS
FIG. 2. Food consumption for beagle dogs given methidathion in diet for 1 year. Each data point represents the mean of four animals for male dogs (top) or female dogs (bottom).
2 1$ 01, 0
Control
-
,, 8
4
12
, 16
, 20
, 24
, 28
, 32
) 36
, 40
, 44
,, 48
52
WEEKS
given the 140-ppm diet: significantly lower values were noted during Weeks 5-8, 11,2 1,29, and 44. The grand mean daily food consumption was 320, 329, 310, 343, 323, and 277 g, for males receiving 0,0.5,2,4,40, or 140 ppm; corresponding values were 288, 296, 287, 302, 294, and 289 g for females. Clinical Laboratory --
Control 0.5 ppm 2 wm 4 wm 40 ppm 140 ppm
-
0’
b
,
,
,
,
,
,
,
,
I
I
,
I
f
4
8
12
16
20
24
28
32
36
40
44
48
52
WEEKS
FIG. I. Body weight for beagle dogs given methidathion in diet for 1 year. Each data point represents the mean of four animals for male dogs (top) or female dogs (bottom).
Tests
No treatment-related effects on hematology, coagulation, or urinalysis were observed (data not shown). However, in both the 40- and the 140-ppm males and females, there were clinical chemistry changes which indicated liver dysfunction. These included moderate to markedly elevated activities of serum AST, ALT, ALP, and SDH as well as increased bile acids concentrations in both sexes (Table 1). In females, increased serum GGT activity and decreased serum albumin were also noted at 240 ppm. The elevated enzyme activities were seen at all intervals examined (only 3- and 12-month data are shown in Table 1). The increases in enzyme activities for the 140-ppm males and females at termination (per-
CHANG,
310
WALBERG,
AND CAMPBELL
TABLE 1 Clinical Chemistry Parameters” from Beagle Dogs Given Methidathion Dietary level (pm)
Time (months)
Albumin k/dl)
Total bilirubin hddl)
ALP (U/liter)
AST (U/liter)
in Diet for 1 Year
ALT (U/liter)
GGT (U/liter)
SDH (U/liter)
Bile acids (PM/liter)
5+2 5tl 5il 5?0 6+l 6+1 6+l 6+l
5*1 4fl 9k2 6+3 14*4* I I ?I 3** 20 Ii 9** 13 * 5**
NA 0.9 NA 1.4 + 0.9 NA 12.4 ? 1 I.5 NA 9.1 + 2.3
5kl 5+1 5t-I 6&l 7+ I* 7-+2 7f I* 8k5
6kl 5kl 9k2 6+l I5 +6* I3 f 4** I4 + 5* IO i 3**
NA 2.4 NA 3.6 i 0.9 NA II.1 * 5.5 NA 15.1 + 7.7
Males 0 4 40 140
3 I2 3 I2 3 12 3 I2
3.2 I!I 0.1 2.9 f 0. I 3.1 f 0.1 3.0 + 0.2 3.1 +- 0.1 2.9 + 0. I 3.1 * 0.1 2.8 + 0.1
0.1 0.2 0.1 0.2 0.1 0.2 0.2 0.3
+ f i f + if +
0.1 0.0 0.1 0.0 0.0 0.1 0.1* 0.1*
175 i 46 l26k 21 1ss* 32 99 i 34 465 f 14** 297 + 38* 500 i 175** 371 +- 152**
26 23 26 23 33 29 35 32
k 4 ?I 3 f 6 +3 f 5 f 7 + 4* + 4*
19* l5rt_ 41 + 4Ok I58 f 134 + I64 + 140 +
3 4 17 16 42** 37** 67** 70**
Females 0 4 40 140
3 I2 3 I2 3 I2 3 12
3.4 tr 0.3 3.2 * 0.2 3.2 * 0.1 3.1 * 0.1 3.0 ?I 0.2* 3.0 + 0.1 2.9 k 0.2* 2.9 f 0.2s
0.1 0.2 0.1 0.2 0.2 0.3 0.2 0.3
f f t k + f k +
0.0 0. I 0.0 0. I 0.1* 0.1 o.o* 0.1
179* l52k l84k l9Ok 503 2 353 + 615 f 623 +
38 81 64 40 76** 120 290*+ 461*
25 k 2 24 + 2 25 f 3 ..A +- 3 37 29 + 5 28 + 6 41 k8* 35 4 7*
17+ 5 l7f 5 41 ?23 26+ I 130 AZ26** I26 + 42** I57 i 65** I34 * 58**
u Mean + SD of four animals for all parameters at all dose levels except the bile acids (only one female and two males at 0 ppm: four animals in all treated groups). Statistical analysis for bile acids not performed. * Significantly different from control: p < 0.05 by Dunnett’s I test. ** Significantly different from control: p G 0.01 by Dunnett’s f test. No&>. NA. not available.
centage of controls, M/F) were 29414 10% for ALP, 1391146% for AST, 933/788% for ALT, and 325/200% for SDH. In both sexes, the serum total bilirubin concentration was only marginally increased and there were no significant changes in serum cholesterol level (data not shown). Cholinesterase Activities Brain ChE activity, measured in a homogenized fraction from the vermis of the cerebellum or the cerebral hemisphere, was generally depressed at 140 ppm in both sexes. The magnitude of the depression in ChE activity from the control was slightly greater with the cerebellar fraction with statistical significance noted in both sexes. In the 140-ppm males and females, cerebellar ChE activity was reduced 27 and 22%, respectively, from control levels. Cerebral ChE activity was reduced 17% in both sexes at 140 ppm but was significantly different from controls in females only. RBC ChE was depressed at 240 ppm in males and at 140 ppm in females; statistical significance was noted at 140 ppm (Table 2). At 12 months, the mean RBC ChE activity of the 140-ppm animals was inhibited by approximately 76% as compared to the controls. Although RBC activities of the 40-ppm males were not statistically different from control, the activities were reduced by 20-30% during the study and thus the inhibition was considered toxicologically significant.
Serum ChE activities of the treated groups were not inhibited (relative to the control) throughout the study. In vitro incubation of serum or whole blood samples from control dogs with methidathion showed inhibition of RBC or serum ChE in a concentration-dependent manner (2020,000 PM, or 6-6000 pg/ml) (Fig. 3). In addition, RBC ChE activity was inhibited to a greater extent than serum ChE by methidathion between 20 and 2000 PM. Liver Weight and Pathology There were no treatment-related effects on organ weights, including the absolute and relative liver weights (Table 3). The only treatment-related gross pathology findings were the dark red, discolored livers seen in some dogs receiving a40 ppm (Table 3). Moderate to marked cholestasis was noted in all males and females at 40 and 140 ppm, being more severe at the 140-ppm level and in females (Table 3). Cholestasis was characterized histologically by bile plugs in distended bile canaliculi, bile ductules, and bile ducts and was most pronounced in the centrilobular zone in all liver lobes examined. Mild chronic inflammation of the liver, characterized by infiltration of lymphocytes, was also observed at 40 ppm (one male and three females) and 140 ppm (one female).
METHIDATHION
TOXlCITY
TABLE 2 Cholinesterase Activity” from Beagle Dogs Given Methidathion Dietary level (w-N
Time (months)
Serum ChE (U/liter)
311
IN DOGS
in Diet for 1 Year
RBC ChE (U/liter RBC)
Brain ChEb (U/g tissue)
Brain ChE’ (U/g tissue)
1405 rfr 203 1385 rt 262 1535 k 392 1565 f 475 980 + 298 1105 f 302 185 -+ 130** 315 f 123**
NA 2.14 + 0.20 NA 2.07 f 0.17 NA 2.04 + 0.01 NA 1.57 + 0.16**
NA 1.53 rt_0.06 NA 1.60 f 0.19 NA 1.52 20.16 NA 1.29 f 0.08
NA 2.15 f 0.19 NA 2.29 + 0.20 NA 2.06 + 0.17 NA 1.68 + 0.30*
NA 1.57 + 0.12 NA 1.62 f 0.17 NA 1.58 _+0.08 NA 1.3 1 f 0.09*
Males 0 4 40 140
3 12 3 12 3 12 3 12
1906k 61 1969 +_ 66 1884 k 328 1975 f 338 1940+ 80 2028 1+_167 1925 f 444 1952 + 485
Females 0 4 40 140
3 12 3 12 3 12 3 12
1796 1839 2179 2382 2265 2378 2154 2537
f + k f + f k k
250 203 337 542 445 567 425 675
1445 1285 1895 1715 1245 1220 245 310
f k f + f f k i
562 420 428 462 232 198 171** 101**
a Mean f SD of four animals per sex per dose level. b Measured in homogenized fractions from the vermis of the cerebellum. c Measured in homogenized fractions from the right cerebral hemisphere. * Significantly different from control; p < 0.05 by Dunnett’s t test. ** Significantly different from control; p < 0.0 1 by Dunnett’s t test. N&e. NA, not available.
DISCUSSION Although methidathion is an organophosphate insecticide, there were no adverse clinical signs attributable to inhibition of ChE activity. All dogs survived in good condition. The body weights and weight gains of treated dogs were similar to those in the controls although males given the 140-ppm diet generally consumed less food. The highest dietary concentration, 140 ppm, caused inhibition of RBC and brain ChE in both sexes; the inhibition was approximately 76% for RBC ChE (12 months) and 2227% for brain ChE as compared to controls. However, serum ChE activity was comparable to the controls throughout the study. This observation is interesting as serum ChE is usually more readily inhibited than RBC ChE when exposures to many organophosphorus compounds occur (Tietz, 1986). The ChE assay procedure used in this study is capable of detecting both the “true” ChE (also called acetylcholinesterase, normally present in RBC, lung, spleen, nerve endings, and gray matter of the brain) and “pseudo”-ChE (acylcholine acylhydrolase or butyryl ChE, normally found in liver, pancreas, heart, and white matter of the brain and serum). It is thus unlikely that the lack of inhibition was due to insensitivity of the analytical procedure. Also considered unlikely
was that an inhibition might have occurred prior to the first scheduled measurement at 3 months; data from the 90-day study (Chang et al., 199 1) showed no inhibition after 8 weeks of exposure. Excluding the two preceding possibilities, this leaves for consideration the steady state (synthesis and degradation) and the sensitivity of serum ChE to methidathion in vivo. Serum ChE concentration has been shown to be an indicator of liver function as its synthesis is parallel to that of albumin by the liver (Vorhaus and Kark, 1953). While decreased serum ChE activity was seen in humans with acute hepatitis, liver cirrhosis, or carcinoma, normal levels were seen in chronic hepatitis, mild cirrhosis, and obstructive jaundice patients whereas those with nephrotic syndrome had slightly elevated serum ChE activity (Vorhaus and Kark, 1953; Wittaker, 1986; Tietz, 1986). Since the kidney was not a target organ in this study and serum albumin levels in dogs with cholestasis were either unchanged or slightly decreased, an increased ChE synthesis by the liver is questionable. On the other hand, little is known about serum ChE synthesis under diseased liver conditions in dogs, including the total amount, the various variants of ChE and esterases, and their sensitivity to the inhibitors. Results from the in vitro study showed a concentrationdependent inhibition of both serum and RBC ChE by meth-
CHANG,
312
WALBERG.
AND CAMPBELL
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60-
:
50-
20lo0
2
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3
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,
10
2
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3
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4 56789’
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Methidothion
2
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I11111
4 56789’
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FIG. 3. Inhibition of serum (0) and RBC (u) ChE by methidathion in vitro. Each data point represent the mean of two samples. Data from “serum + methanol” were similar to those of serum alone.
idathion and greater inhibition of the RBC ChE than serum ChE at equimolar concentrations. This suggests that the methidathion concentrations reached in vivo in the treated dogs might not have been sufficiently high to inhibit the serum ChE activity. Assuming a one-compartment distribution with little or no accumulation, the blood concentration of methidathion in the 140-ppm dog would be 57 pg/ ml (or 4.7 mg/82 ml, based on the fact that a mature beagle dog has a blood volume of 82 ml/kg; Andersen and Schalm, 1970). At 60 pg/ml (or 200 PM) in vitro, methidathion inhibited RBC ChE by 40% and serum ChE by 18% (Fig. 3). These values are surprisingly similar to the in viva data from the 140-ppm dog, which showed 76% inhibition of RBC ChE and no inhibition for serum ChE. The lack of serum ChE sensitivity to methidathion in vivo also appears to be species specific (Table 4). Among the four species tested for the chronic toxicity of methidathion, the rat was the most sensitive to the anti-cholinergic effects. In rats, inhibition of serum ChE occurred at 2 mg/kg and was accompanied by prominent clinical signs of toxicity. Serum ChE from the mouse appeared to be the least sensitive. Whether assays on brain ChE should utilize tissue from the specific regions of the brain or the whole brain has been discussed for some time within the scientific community. The uncertainty stems from the facts that the distribution of ChE in the brain varies from region to region and that brain ChE also exists in different forms with differing sensitivities to inhibitors (Silver, 1974; Meneguz et al., 198 1; Cook et al., 1989). At present, there are no standardized procedures for taking samples to measure representative brain ChE. With rodents, half or whole brain can be ho-
mogenized and analyzed. However, with larger laboratory animals, the specific anatomic sections taken for analysis vary from laboratory to laboratory. Cohen et al. ( 1985) suggested that homogenates be made from different brain sections and combined into a composite homogenate in direct proportion to the wet weights of each section. To see if regional or whole brain ChE activity was more indicative of methidathion exposure (or toxicity) in beagle dogs, brain ChE in this study was determined in two distinct areas for comparisons, the vermis of the cerebellum and the right cerebral hemisphere. The results showed an inhibition of ChE in the cerebellum slightly greater than that in the cerebral hemisphere. This was not unexpected as any regional inhibitory effect that methidathion might have had in the right cerebral hemisphere might have been diluted by the larger amount of tissue present, resulting in a higher ChE activity per gram of tissue. Nevertheless, ChE activity from both the cerebellum and the cerebral hemisphere of the 140-ppm dogs illustrated a minimum inhibitory effect. This was consistent with the clinical observations which did not indicate any functional or behavioral deficits in these dogs. Besides the RBC and brain ChE, liver was clearly the target organ in dogs exposed to methidathion. Moreover, liver toxicity was seen at levels (340 ppm) at which none or minimal inhibition of RBC ChE occurred. The liver toxicity was indicated by clinical chemistry results as well as by necropsy and histopathology findings. Conventional diagnostic tests used to assess hepatocellular dysfunction in dogs include serum concentrations of albumin, cholesterol, urea, ammonia, and coagulation factors whereas biliary function and flow are assessed by serum concentrations of total bilirubin
METHIDATHION
TOXICITY
313
IN DOGS
TABLE 3 Final Body Weight, Relative Liver Weight and Liver Pathology in Beagle Dogs Given Methidathion Dietary level bvm)
Final body weight” (kg)
Relative liver weighta (% body weight)
in Diet for 1 Year
Incidence of gross discoloration
Incidence of cholestasis
Incidence of chronic inflammation
O/4 O/4 l/4 214
O/4 O/4 4146 4/4b
O/4 O/4 114 O/4
O/4 O/4 214 314
O/4 O/4 414' 414'
O/4 O/4 314 114
Males 0 4 40 140
i + 12.1 f 10.3 f 10.8 10.8
1.3
2.12 2.56 2.45 2.62
1.1 1.3
1.5
i f f k
0.45
0.11 0.27 0.39
Females 0 4 40 140
8.1 9.5 10.5 9.5
k rt + *
0.1
3.01 f 0.52 2.94 f 0.15 2.43 + 0.25 2.82 f 0.54
1.6 2.1 1.7
a Mean ? SD of four animals at all dose levels taken before necropsy. b Severity grades were 3 moderate and 1 mild at 40 ppm and 2 marked, 1 moderate, and I mild at 140 ppm. ’ Severity grades were 4 moderate at 40 ppm and 3 marked and 1 moderate at 140 ppm.
and activities of ALT, AST, SDH, GGT, and ALP (Loeb, 1989; Eckersall and Nash, 1983). In recent years, elevated serum bile acids concentration has been used increasingly to diagnose hepatobiliary disease in the dog (Center et al., 1985; Hauge and Abdelkader, 1984). As shown in Table 1, the serum bile acid concentration and activities of ALT and
TABLE 4 Species Comparison of Sensitivity to Methidathion
Effects
Effect levels (mg/kg) Effects Serum ChE inhibition RBC ChE inhibition Brain ChE inhibition Liver pathology
Mouse”
Ratb
Dog’
Monkeyd’
IS 7.5’ 16.1
2
NA
2 2
4.7 4.7
7.59
NA
1.4
NAd 1.0 NA NA
10’ 5
5 -
Note. IS, increased significantly; NA, not affected. a Mice were administered methidathion in diet at 0.3, 3, 10, 50, and 100 ppm for 23 months. The equivalent dosages were 0,0.46, 7.5, and 16.1 mg/ kg/day (Goldenthal et al., 1986; Quest et al., 1990). b Rats were given methidathion in diet at 4,40, and 100 ppm for 2 years. The doses were equivalent to 0, 0.2. 2. and 5 mg/kg/day (Yau et al., 1986; Quest et al., 1990). c Present study. d Monkeys, 5-7/sex/dose were given methidathion via stomach tube at 0, 0.25, or 1.O mg/kg/day for 2 years (Coulston, 197 1). e Four monkeys per dose were given methidathion at 5 or 10 mg/kg daily for 16 months. Two of the four monkeys from each dose group died within a few weeks whereas the others survived (Cot&ton, 1973). fThis value applies to the female mice. The effect level for males was 16.1 m/kg. RThis value applies to the male mice. The effect level for females was 16.1 mg/kg.
ALP were elevated in dogs receiving 240 ppm. The observations of grossly dark, discolored livers at 40 and 140 ppm also suggested cholestasis. In a previous study in which beagle dogs were given methidathion for 90 days, cholestasis was also seen (Chang et al., 199 1). The livers of dogs exposed to 140 ppm methidathion for 1 year in this study showed more severe cholestasis than the livers of dogs administered 140 ppm for 90 days (data not shown). In contrast, treatmentrelated changes in clinical chemistry data did not increase with time in the present study; the effects were maximal at 3 months. Females appeared to have slightly more severe cholestasis than the males; this was seen microscopically by more deposits of pigment and reflected by the slightly higher levels of serum bile acids, GGT, and ALP activities. In addition, females were observed to have slightly higher incidences of chronic inflammation of the liver. The inflammation, perivascular or periportal and characterized by infiltration of lymphocytes, was limited to the 40- and 140-ppm groups, in which cholestasis was seen and did not correlate with the severity of the cholestasis. However, it was not seen in any of the control animals and was considered a treatment-related effect. The liver inflammation might have resulted from necrosis. Necrosis was also indicated by the slight but significantly elevated serum SDH activity in the 40- and 140-ppm groups. Overall, female dogs received slightly higher dosages of methidathion than the males (4.9 versus 4.5 mg/kg/day in males at 140 ppm; 1.4 versus 1.3 mg/kg/day in males at 40 ppm). Compared to the rat, mouse, and monkey, the dog is the species most sensitive to the hepatotoxicity of methidathion (Table 4). Whereas no liver toxicity was detected in the rat (Yau et al., 1986; Quest et al., 1990) or monkey (Coulston
314
CHANG. WALBERG,
et al., 197 1; Coulston,
1973), liver effects were seen in dogs and mice. In the mouse, the liver pathology included biliary stasis, bile duct hyperplasia, cholangiofibrosis, and chronic hepatitis. In addition, increased liver weights and liver tumors were seen in male mice (Quest et al., 1990; Goldenthal et al., 1986). The liver toxicity, not elicited by many other organophosphorus compounds, might have resulted from the thiadiazole ring moiety, which could possibly form hydrazine/hydrazide in the metabolic pathway. No mechanistic study results are available to answer the question. However. the similarity in responses between the mouse and the dog in the liver effect and lack of serum ChE inhibition is interesting and may warrant further investigation. In conclusion, the hepatobiliary toxicity seen in this study, which followed the GLP guidelines, is consistent with that observed in the 1967 study on methidathion. The overall NOEL for the present study was judged to be 4 ppm (0.15 mg/kg) based on the hepatic findings. This NOEL is slightly higher than that obtained in the previous study (0.08 mg/ kg). Additionally this study demonstrates that beagle dogs can tolerate doses nearly four times greater than the highest dose used in the previous study, that is, 4.9 mg/kg/day versus 1.35 mg/kg/day (Hayes, 1982). ACKNOWLEDGMENTS We thank the EHC staff from the vivarium. pharmacy, analytical chemistry. clinical chemistry, and necropsy/histopathology laboratories for their excellent technical support during the course of the study. Special thanks are extended to T. Bell, J. Chase, M. Gilman, and L. O’Donnell for their valuable contributions. The critical review of this manuscript by the various CIBA-GEIGY toxicology groups (Basel, Switzerland; Greensboro, NC; and Farmington, CT) was deeply appreciated.
REFERENCES Andersen, A. C.. and Schalm, D. W. (1970). Cardiovascular system/hematology. In The Beagle as an Experimental Dog (Andersen, A. C., and Good, L. S., Eds.), p. 278. The Iowa Univ. Press, Ames, IA. Center, S. A., Baldwin, B. H., Erb. H. N., and Tennant, B. C. (1985). Bile acid concentrations in the diagnosis of hepatobiliary disease in the dog. J. Am. Vet. Med. A 187, 935. Chang, J. C. F., Pavkov, K. L., Campbell, W. R., and Wyand, D. S. (199 1). 90-day oral toxicity study with methidathion in beagle dogs. Toxicologist 11, 159. CIBA-GEIGY Corp. (199 1). Sample Label Guide. Agricultural Division, Greensboro, NC. Cohen, S. D., Williams, R. A., Killinger, J. M., and Freudenthal, R. I. (1985). Comparative sensitivity of bovine and rodent acetylcholinesterase to in vitro inhibition by organophosphate insecticides. Toxicol. Appl. Pharmacol. 81,452-459.
Cook, W. O., Dellinger, J. A., Sir&, S. S., Dalhem, A. M., Carmichael, W. W., and Beasley, V. R. (1989). Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon. Toxicol. Lett. 49, 29-34.
AND CAMPBELL Coulston, F., et al. (197 I). Two-Year Safety Evaluation Study in Rhesus Monkeys with GS-1300.5 Technical. Institute of Experimental Pathology and Toxicology, Albany Medical College, Albany, NY. [Sponsored by the CIBA-GEIGY Corp., Greensboro, NC] Coulston, F. (1973). Evaluating toxicological data as regards environmental significance. In Environmental Quality and Safety. Global Aspects of Chemistry, Toxicology and Technology as applied to the Environment, Vo! I!. Georg. Thieme/Academic Press, Stuttgart/New York. Eckersall, P. D., and Nash, A. S. ( 1983). Isoenzymes of canine plasma alkaline phosphatase: An investigation using isoelectric focusing and related to diagnosis. Rex Vet. Sci. 34, 310-3 14. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. ( 196 1). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Environmental Protection Agency (EPA) (1988). Peer Review of Methidathion. Office of Pesticide Programs, Health Effects Division, Washington, DC. EPA (I 989). Guidance for the Registration of Pesticide Products containing Methidathion as the Active Ingredient. Office of Pesticide Programs, Health Effects Division, Washington, DC. Food and Agricultural Organization of the United Nations, World Health Organization (FAO/WHO). (1973). Evaluations ofSome Pesticide Residues in Foods, World Health Organization Technical Report Series, No. 2. Goldenthal, E. I., et al. (1986). 2-Year Dietary Oncogenicity (and Toxicity) Study with Methidathion Technical in Mice. Study performed by the International Research and Development Corp. for CIBA-GEIGY Corp., Greensboro, NC. Hauge, J. G., and Abdelkader, S. V. (1984). Serum bile acids as an indicator of liver disease in dogs. Acta Vet. Stand. 25,495-503. Hayes, W. J., Jr. (1982). Pesticides Studies in Man, pp. 371-372. Williams & Wilkins, Baltimore, MD. Johnston, C. D. (1967). GS-13005 Evaluation by a 2-Year Feeding Study in dogs. 4OW, Woodard Research Corp., Herndon, VA. [Sponsored by the CIBA-GEIGY Corp.. Greensboro, NC] Loeb, W. F. (1989). The dog. In The Clinical Chemistry of Laboratory Animals. (Loeb, W. F.. and Quimby, F. W., Eds.), pp. 47-58. Pergamon, Elmsford, NY. Meneguz, A., Bisso, G. M., and Michalek, K. H. (1981). Regional differences in brain soluble acetylcholinesterases and its molecular forms after acute poisoning by isoflurophate in rats. Clin. Toxicol. 18, 1443-145 I. Quest, J. A., Copley, M. P., Hamernik, K. L., Rinde, E., Fisher, B., Eng!er, R., Burnam, W. L., and Fenner-Crisp, P. A. (1990). Evaluation of the carcinogenic potential of pesticides. Reg. Toxicol. 12, I !7- 126. Silver, A. (1974). The biology of cholinesterases. In Frontiers of Biology, Vol. 36, North-Holland. Amsterdam. Tietz, N. W. (1986). Cholinesterase. In The Textbook of Clinical Chemistry, pp. 746-75 I Saunders, Philadelphia, PA. Vorhaus, L. J., and Kark, R. M. (1953). Serum cholinesterase in health and disease. Am. J. Med. 14, 707-719. Wittaker, M. (1986). Cholinesterase: Monographs in Human Genetics, Vol. 11, Karger. Basel, Switzerland. Yau, E. T., et al. (1986). Methidathion 2-Year Oral Oncogenicity and Toxicity Study in Albino Rats (MIN 832001). CIBA-GEIGY Corp., Pharmaceutical Division. Summit, NJ. [Sponsored by the CIBA-GEIGY Corp., Greensboro, NC]
