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Toxicologic studies on diethyl-1-(2,4-dichlorophenyl)-2-chlorovinyl phosphate
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
17, 323-336 (1970)
Toxicologic Studies Diethyl-I-(2,4-DichlorophenyI)-2Chlorovinyl
on Phosphate]
ANTHONY M. AMBROSE, PAUL S. LARSON, JOSEPH F. BORZELLECA, AND GORDON R. HENNIGAR, JR.* Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219, and Department of Pathology, College of Medicine, State University of New York, Brooklyn, New York 11203 Received May 19,1969
Toxicologic Studies on Diethyl-1-(2,4-Dichlorophenyl)-2-Chlorovinyl Phosphate. AMBROSE, ANTHONY M., LARSON, PAUL S., BORZELLECA, JOSEPH F., and HENNIGAR, GORDON R., JR. (1970). Toxicol. Appl. Pharmacol. 17,323-336.Studiesof diethyl-1-(2,4-dichlorophenyl)-2-chlorovinyl phosphate: GC-4072, Supona, Chlorfenvinphos, SD 7859, ENT 24969, a new organophosphatepesticide, were undertaken in several animal speciesto characterizetoxicity and assess safety. Measurementsof acute toxicity by different routes of administration gave the following LDSOvalues(mg/kg): po 9.66 for rats, 300(estimated) for rabbits, >12000for dogs;iv, 6.6for rats and 50.5for dogs.The undiluted form anda 49.1% GC-4072-xylolemulsifiableformulation applieddermally to rabbits gave LD50 valuesof 400 and 1087,respectively.Hensreceiving multiple sublethal dosesip displayed no delayed neurologic effects or histologiclesions. Rats fed dietary levelsranging from 3 to 1000ppm and dogsfed l-1000 ppm for periodsup to two years showedno clinical signsof intoxication, but therewasa significantand variable decrease in plasmaand/or red blood cell cholinesteraseactivity, with reversaltrends. Multigeneration studiesin rats fed dietary levelsof 30, 100,or 300ppm showedno effect on gestation,but therewereprogressiveeffectson fertility, viability, and lactation indices.No grossabnormalitieswerefound in fetuses born dead or alive, or in rats autopsiedwith fetusesin situ. Food consumption, growth, hemograms,urinary findings, histopathology, and liver function testsin dogswere within normal limits. Diethyl-1-(2,4-dichlorophenyl)-2-chlorovinyl
phosphate3 (GC-4072)4, an organophos-
phate ester, is a pesticidal agent proposed for use in the control of insect pests (ticks, fleas) on dogs and livestock; as a residual surface spray for house flies, ticks, and fleas; as a larvicide for flies; for certain root maggots of vegetable crops and insectsattacking 1Supportedby a grantfrom Allied ChemicalCorporation,GeneralChemicalDivision,Agricultural ChemicalDevelopment,Morristown,New Jersey07960. * Presentaddress: Departmentof Pathology,MedicalCollegeof SouthCarolina,Charleston,South Carolina29401. 3 Also referredto asChlorfenvinphos, Supona,SD7859,ENT 24969. 4 GC-4072is thecommonnameandU.S. patent(No. 3,003,916) of Allied ChemicalCorporation, Morristown,New Jersey07960. 323
324
AMBROSE ET/IL.
potato foliage; and for the control of a wide variety of other insect pests. In dairy cattle following single dermal application of a spray containing 32P-labeled (X-4072, in concentrations adequate for insect control, Chamberlain and Hopkins (1962) have reported that the compound is rapidly absorbed and eliminated, maximum blood concentrations appearing in 2 hours, with low urinary concentrations at 1 week after treatment. Under comparable conditions residuesof the compound have been found in the milk of dairy cows in 5 hours, gradually diminishing to zero in IO-12 days(Roberts et al., 1961),and Ivey et al. (1966)have reported low residuesof the compound in various fatty tissuesof beef cattle. The residueswere eliminated in 28 days or less.In lambs and ewes dipped in aqueous emulsions and in 3-month-old calves and yearlings treated with aqueousemulsion sprays, at concentrations or application rates in excessof recommended usage,blood cholinesteraselevels were depressedbut returned rapidly to pretreatment levels (Pickering, 1965). Studies were undertaken: to characterize acute, subacute, and chronic toxicity; to evaluate possible dermal irritation and neurotoxicity; to determine effects on blood cholinesteraseactivity and on reproduction. Technical grade GC-4072 (96 %) used in these studies, except where otherwise indicated, is a light amber-colored liquid with a density of 1.345 and a boiling point of 167170°C (0.5 mmHg). It is soluble in most organic solvents, edible vegetable oils and propylene glycol, and sparingly soluble in water. The structural formula is shown in Fig. 1. 0 C2H50&)-C: C,H,O’
CHCl Cl
FIG. 1. Structural formula of GC-4072.
METHODS
Anticholinesterase Activity In vitro anticholinesterase activity of GC-4072 was determined manometrically on freshly drawn human blood plasma and red blood cells according to the method of DuBois and Mangum (1947).
Single LD50 Determinations Oral. Determined by gastric intubation, following overnight fast, in male albino rats5 (110 to 220 g) in groups of 10 at each of four dosesranging from 5 to 20 mg/kg, in male albino rabbits weighing 1.4 to 3.0 kg, in groups of 10each at five dosesranging from 160 to 400 mg/kg, and in 13 adult male and female mongrel dogs, at dosesof 1, 3, 6, or 12 g/kg. To rats and rabbits GC-4072 was administered as a 1% or 10 % (w/v) solution in peanut oil, respectively, and was given undiluted to dogs and followed by 10 ml corn oil. ’ Wistar strain (Albino Farms, Red Bank, New Jersey), used throughout.
TOXICOLOGIC
STUDlES
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Intravenous. Determined in groups of 10 male albino rats (145-240 g) at doses of 2.5, 3.5, 5.0, 10, 20, or 25 mg/kg, and in 18 mongrel dogs (7.5-18.5 kg) at doses of 25, 35, 50, 65, 75, or 100 mg/kg. Both species received GC-4072 as a solution (w/v) in Lipornul,‘j 1% for rats and 10% for dogs. Dermal. Four groups each consisting of 10 male albino rabbits weighing approximately I .9 kg each were tested for local irritation and systemic dermal toxicity with each of the following preparations : (1) undiluted 96 % technical GC-4072 was applied by gentle massage with the butt end of a test tube, to the closely clipped unabraded skin of the back of rabbits in doses ranging from 200 to 673 mg/kg; and (2) an emulsifiable formulation, containing 49.1% technical GC-4072 (92.5 %) by weight, in a solvent mixture consisting of 45.5 % xylol and 5.4 % mixed emulsifiers, in doses ranging from I .25 to 2.5 ml/kg (equivalent to approximately 588-l 177 mg/kg of GC-4072) was introduced directly underneath a rubber girdle fitted snugly around the closely clipped unabraded skin of the trunk of immobilized rabbits. After 24 hours’ exposure, the cuffs were removed and any unabsorbed material was removed with thumb sponges. In both experiments, the condition of the skin was observed daily for 14 days. The LD50 values, where possible, for the three routes of administration, were calculated by the minimum approximate chi-square method of Berkson (1955). Subchronic Toxicity Studies Neurotoxicity. Durham et al. (1956) have suggested that all new compounds with the organophosphorus grouping as well as pesticidal agents having anticholinesterase activity should be screened for possible delayed paralytic or neurotoxic effects. Since certain organophosphorus esters have been shown to produce paralytic effects and myelin degeneration in chickens (Durham et al., 1956; Davies et al., 1960; Lancaster, 1960; Baron and Johnson, 1964), experiments with chickens were undertaken to determine whether GC-4072 could produce this syndrome. Accordingly, 26 White Leghorn hens, approximately 16 months old and weighing 1.5-1.9 kg each, were treated daily for 10 days, or until death, by ip injection of GC-4072 in 20 % ethanol-80 % propylene glycol. The number of hens used at each dose, is indicated within parentheses as follows: 0 (5), 100 (6), 150 (3), 200 (4), 300 (2) mg/kg. Two other groups, of 3 hens each, were similarly treated with 100 or 200 mg/kg GC-4072, but in addition received atropine sulfate (1 mg/kg) to lessen the effects of excessive acetylcholine accumulation. All doses were contained in not more than 1.5 ml of solvent mixture. Daily observations were made during treatment and for 20 additional days at which time all surviving hens were autopsied. Brain, segments of the spinal cord and sciatic nerve were taken, fixed in neutralized 10 “/, formalin and examined histologically. Twelve-week studies. In a preliminary (12-week) study, six groups of weaning (28days) albino rats,5 10 of each sex per group, individually housed, were placed on control finely ground basic stock diet’ for 5 weeks. Similarly four groups of mongrel dogs consisting of two of each sex per group, individually housed, were placed on a stock diet consisting of 87 % ground basal ration,’ 12 % corn oiL9 and 1 % U.S.P. cod liver oil, prepared fresh daily. At feeding time the feed was mixed with an equal weight of water. 6 Lipomul@ I.V., The Upjohn Company, Kalamazoo, Michigan. ’ Wayne Lab-Blox, Allied Mills, Chicago, Illinois. 8 Basal Ration f/d@; A Prescription Diet @, Hill Packing Company, Topeka, Kansas. 9 Mazola@ oil, Corn Products Company, New York, New York.
326
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During this period plasma and red blood cell cholinesterase (ChE) values, determined by a micromodification of the electrometric procedure of Michel(l949), were obtained weekly on all dogs and on 5 rats of each sex from each group. The same rats were used each week insofar as possible. At the start of the sixth week, rats were fed the basic diet containing 0,3, 10, 30, 100, or 1000 ppm of CC-4072, and the dogs were fed diets containing, 1, 10, 100, or 1000 ppm GC-4072, respectively, for 12 weeks. All diets were prepared fresh each week. At 1, 2, 4, 6, 8, 10, and 12 weeks plasma and red blood cell ChE values were obtained, as above, for 5 rats of each sex on each diet and for all dogs. At the end of week 12, rats in excess of 5 of each sex per diet, not used for following ChE activity, were sacrificed as well as 1 dog of each sex for each diet; remaining rats and dogs were returned to the control basic diet (reversal of ChE effect) and ChE activity values were obtained after 1 and 4 weeks for rats and at 1, 2,4, and 8 weeks for dogs. Terminally, all surviving rats and dogs were sacrificed. Organ-to-body weight ratios were determined for liver, kidneys, heart, spleen, and testes for rats on control and GC-4072 diets for 12 weeks and after withdrawal for 5 weeks. Rats were individually weighed once each week and food consumption was determined over 3-day periods during weeks 4 and 12. Dogs were weighed weekly, and food consumption was measured daily. Histopathologic studies of hematoxyhn-eosin stained paraffin sections of formalin-fixed tissues were made on 5 female and 3 male rats on the control diet, on 2 female and 3 male rats on 100 ppm GC-4072 diet for 12 weeks, and on all dogs. Tissues studied included: heart, lungs, liver, kidney, urinary bladder, spleen, stomach, small and large intestine, skeletal muscle, skin, bone marrow, pancreas, thyroid, adrenal, gonad, pituitary, brain, and in dogs the spinal cord. Body weight changes and organ-to-body weight ratios for rats were evaluated by analysis of variance and Duncan (1955) multiple range and multiple Ftests. ChE values were evaluated by Student t test (Snedecor, 1957), at the 95 y0 probability level, against appropriate controls. Chronic Toxicity
Studies in Rats
Using littermate distribution, four matched groups, each consisting of 30 weanling albino rats5 (28 days) of each sex, culled to a narrow starting weight range, were placed on dietary levels of 0, 10,30, or 100 ppm GC-4072 prepared fresh each week. An additional group of 30 weanling rats of each sex, not littermates of the above, was placed on 300 ppm. The stock diet was that referred to above. All rats were individually housed and were weighed weekly. Food consumption was measured over 3-day periods at the end of 1, 3, 6, and 12 months. Hematologic determinations (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex at each dietary level at 3-month intervals. Urinary tests for reducing substances (Benedict, 19081909) and protein (Shevky and Stafford, 1923) were made at the same times on urine samples pooled by sex and dietary level. Blood ChE activity (red blood cells and plasma) was determined on 4 rats of each sex for each dietary level at 1, 4, 8, and 12 weeks, and at 3-month intervals thereafter. At 13 weeks, at least 4 rats of each sex on each of the diets were sacrificed for histopathologic study. Survivors among the remaining rats were sacrificed during the 104th week, with the exception of male rats on 300 ppm diet which were sacrificed during the 95th week. At each autopsy period organ weights were obtained for heart, spleen, kidneys, liver, and testes. Rats that died during
TOXICOLOGIC
STUDIES
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327
the experiment and male rats terminated at 95 weeks were autopsied, but their organ weights are not included in organ-to-body weight calculations. All rats sacrificed in moribund condition and those autopsied at 13, 95, or 104 weeks were examined grossly and microscopically. Tissues subjected to histopathologic examination were the same as in the preliminary study. Body weight changes at various time intervals and organ-to-body weight ratios at 13 weeks and at the end of the 2-year study were evaluated by analysis of variance and the Duncan test (1955). Hematologic data were evaluated by comparison to control values, and blood ChE values by Student t test for each test period. Reproduction and Lactation Studies. To determine stress effects of GC-4072 on gestation and lactation, successive generation studies were undertaken. For this study, 28-day-old, littermate rats, within but not between sexes, were separated into 4 groups of 30 rats of each sex to constitute the parent (F/O) generation. Mean body weight and weight range were similar for all groups. One group (0 ppm), was fed the basic diet’ and the others basic diet containing 30, 100, or 300 ppm of GC-4072. Rats were individually housed and had free access to water and diet. Weekly body weight records were made, except during mating, through weaning of litters. After 11 weeks of treatment, 20 females from each diet group were transferred to individual breeding cages, and each was mated with a male of the same diet group for the F/l a generation. Male rats were rotated to a different female on each of three successive 7-day periods. On the 20th mating day all males were removed. Records were maintained of mating, number of pregnancies, litters, pups in litter at 1, 5, and 21 days (weaning), and the total weight of the litter at weaning. Litters containing more than 10 siblings were randomly culled to 10 on day 5. All surviving F/la siblings were sacrificed and autopsied at weaning. To ensure that all F/O parent generation rats for each dietary level were approximately the same age to produce F/lb litters, they were remated, as above, about 10 days after weaning of the last F/la litter. Observations recorded were the same as those described for F/la litters. At weaning of F/b litters, surviving parent (F/O) rats were sacrificed and autopsied. Where available, 30 F/lb offspring, from as many litters as possible, of each sex from each dietary level were continued on the parent diet for 11 weeks, at which time 20 rats of each sex, where available, within each group, were separated and mated to produce F/2a and F/2b generations. The same procedure as with F/lb generation was followed with Fj2b offsprings to produce F/3a and F/3b generation rats. All matings with F/lb and Fj2b generation rats were made with male and female rats from different litters. Blood plasma and red blood cell ChE values were obtained for F/2b parent generation rats fed 0, 30, or 100 ppm diets (30 weeks) after weaning of F/3b litters, and for F/3b generation siblings fed 0 or 30 ppm diets for 3 weeks after weaning. At least 10 rats of each sex, from each diet group were used to determine these parameters, except for F/2b rats on 100 ppm diet; only 6 females and 3 males were available. Following weaning of F/3b generation rats, surviving Fj2b generation female rats fed 30 or 100 ppm diets were cross-mated with rats on 0 ppm diet. The few surviving male rats fed 100 ppm diet did not participate in this study. Observations recorded for F/3c generation were the same as described above. Survivors in these two generations were sacrificed and autopsied. Tissues from F/3b
328
AMBROSE
ET
AL.
generation rats were submitted for histopathologic same as those described in the subacute study.
study. Tissues included were the
Chronic Oral Toxicity Studies in Dogs In the 2-year feeding studies 16 purebred beagle dogs, approximately 6 months of age, were divided into 4 groups each consisting of two dogs of each sex. Mean body weight and weight range were similar. A control group was placed on the basal diet previously described, and the other 3 groups were placed on the basal diet containing 30, 200, or 1000 ppm GC-4072. Prior to treatment the dogs were immunized against distemper, infectious hepatitis and leptospirosis and were treated for intestinal parasites. Food consumption was measured daily, and body weight was recorded weekly. The following were obtained on all dogs at start and at 3-month intervals thereafter: urine concentrations of reducing substances and protein, and hematologic determinations (hemoglobin, hematrocrit, and total and differential leukocyte counts). Red blood cell and plasma ChE values were obtained for all dogs at 0, 1, 4, 8, and 12 weeks, and at 3-month intervals thereafter. Bromosulfalein (BSP) retention, serum glutamic-oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SAP), and blood urea nitrogen (BUN) values were obtained at 2 years. All surviving dogs were autopsied and organs examined grossly. Organ weights and organ-to-body weight ratios were obtained for heart, spleen, kidneys, liver, and testes. Tissues submitted for histopathologic study were the same as those in the subacute study. Body weight changes at representative time intervals, and organ-to-body weight ratios were evaluated by analysis of variance and the Duncan test (1955) at the 95 % probability level. Blood ChE values were evaluated by Student t test by comparison to control values at each time interval. Other data such as food consumption, hematologic data, liver function tests, and BUN were evaluated by comparison with the control values. RESULTS
AND
DISCUSSION
In vitro incubation of GC-4072 with human plasma and red blood cells produced direct inhibition of ChE activity. Most sensitive was the plasma. A concentration of 1 x IO-’ M GC-4072 produced 50 % inhibition of plasma ChE which was comparable to that produced by an equimolar concentration of paraoxon. On red blood cells, 50 % inhibition was obtained with 4.9 x lo-’ M GC-0472 as compared to 1.6 x lo-’ for paraoxon. Single Dose Toxicity Orally, the LD50 f SD values were 9.66 mg/kg f 1.35 for rats; approximately 300 for rabbits, and >12000 for dogs. By iv administration the values were 6.6 & 0.58 for rats and 50.5 & 4.5 for dogs. By dermal application on rabbits the values were 400 ?X52 for undiluted GC-4072 and 1087 % 23 for the emulsifiable concentrate. The effects produced by GC-4072 in all animals and by all routes of administration were essentially the same. Deaths occurring within 12 hours were usually preceded by the characteristic signs of ChE inhibition (salivation, lacrimation, muscle fasciculation, diarrhea, and in dogs emesis). After single dermal applications of undiluted GC-4072 to rabbits, no apparent signs
TOXICOLOGIC
STUDIES
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cc-4072
329
of skin irritation were observed. However, in rabbits treated with the emulsifiable formulation cutaneous irritation appeared within 3 days and was characterized by erythema, desquamation, and dry and leathery skin. Two weeks following exposure the skin of all surviving rabbits appeared normal, following sloughing of the coriaceous integument. Postmortem examination of visceral and thoracic organs of all animals dying or sacrificed 14 days after treatment was essentially normal. Subchronic Toxicity Studies
Immediately following each daily ip injection of GC-4072, with or without atropine, the hens showed typical signs of acute anticholinesterase intoxication at all doses, inability to stand, salivation, and retching. Deaths occurred at all doses with or without atropine, the highest frequency occurring with the first injection. Atropine did not protect against the lethal effects of GC-4072. The 8 hens surviving (5 at 100,2 at 150, and 1 at 200 mgjkg) were observed daily for 20 days following the last injection, and none showed any delayed neurotoxic effects. Histopathologic studies of myelin and Nisslstained sections of the brain, spinal cord, and sciatic nerve from 5 control hens and 8 surviving hens treated with GC-4072 for 10 consecutive days failed to reveal any of the neurologic lesions suggesting demyelination or neural damage which are characteristic of certain organophosphate esters (DFP, Merphos, dialkylfluoridates, etc.). In the 1Zweek preliminary studies in rats given O-1000 ppm and dogs given l-1000 ppm GC-4072 diets, no significant effect was observed on mortality or food consumption. In rats, growth was significantly depressed in both sexes fed 1OOOppm; upon withdrawal of GC-4072 from the diet for 4 weeks a slight reversal was noted. In dogs no effect on growth was noted. Blood ChE values for plasma and red blood cells were significantly depressed for rats fed 30, 100, or 1000 ppm diets, sporadically for rats given the 10 ppm diet, and not at all for rats fed the 3 ppm diet. Following withdrawal of GC-4072 from the diet, recovery of plasma ChE was complete within 1 week at all dietary levels, except for female rats formerly fed the 1000 ppm diet, in which it was complete at 4 weeks. Red blood cell ChE recovered completely within 4 weeks for all groups except for male rats formerly fed 100 or 1000 ppm diets. In dogs given CC-4072 diets for 12 weeks, statistically significant depression in plasma ChE values occurred at all dose levels, while significantly depressed red blood cell values appeared sporadically. Upon withdrawal of GC-4072 from the diet for 8 weeks, recovery trends were apparent for plasma but not for red blood cell ChE. Organ-to-body weight ratios for heart, spleen, kidneys, liver, and testes for male rats fed any dietary level were not significantly different from control values. The spleento-body weight ratios of female rats fed 30 or 100 ppm and the kidneys of rats fed 30 ppm and sacrificed at 12 weeks, were significantly decreased. After withdrawal of GC-4072 from the diet for 4 weeks no significant difference was noted. Gross and microscopic pathologic findings, except for minor lesions customarily found in rats, were essentially negative. In dogs histopathologic findings were negative. Chronic Toxicity
Studies in Rats
Body weight gains for female rats fed dietary levels of 100 or 300 ppm became significantly depressed by the 26th week, continuing so until near termination of the study,
330
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ET AL.
at which point differences from controls though still present were not statistically significant. The same dietary levels had no effect on growth of male rats. On survival. a clearcut effect of the test material did not appear. Food consumption determinations indicated no consistent trends which could be related to GC-4072 in the diet. Hematologic examinations and urinary tests for reducing substances and protein gave comparable results for rats on the various dietary levels of GC-4072 and the controls at all test periods. Blood ChE values at representative intervals, are summarized in Table 1 for plasma, and in Table 2 for red blood cells. Significant depression in plasma and red blood cell ChE occurred in both sexes at all dietary levels of GC-4072, except for male rats on 10 ppm during the second year. No signs of intoxication were noticeable in any of the rats. Organ-to-body weight ratios for heart, spleen, kidney, liver, and testes for rats sacrificed after 13, 95, or 104 weeks on the respective diets, were not significantly different from the controls, except for a lower spleen ratio for female rats on the 300 ppm diet for 13 weeks, not confirmed at two years, and a higher liver ratio for male rats on the 100 ppm dietary level of GC-4072 for 104 weeks. No effect on the liver was apparent in male rats on 300 ppm sacrificed at 95 weeks. Histopathologic findings on rats sacrificed after 13,95, or 104 weeks on the respective diets were essentially negative except for the normal incidence of bronchiectasis, murine pneumonia, and disseminated granulomatous inflammatory processes which appeared in approximately equal frequency in both the control and experimental rats. In none of the rats on the various dietary levels of GC-4072 was there any evidence of liver necrosis or other change that might be expected of a chlorinated compound that is hepatotoxic. Reproduction and Lactation Studies. Body weights for each of the three female parent generation (F/O, F/l, F/2) rats receiving 30, 100, or 300 ppm dietary treatment with GC-4072 before mating and at weaning of their respective litters tended to show somewhat lower body weights than the controls. Average percent lower mean body weights for F/O generation were 3, 5, and 11%; for F/l generation 9,2, and 19 %; and for F/2 generation 14 and lo%, respectively. Male rats on these levels of GC-4072, and of these generations showed a comparable decrease in body weight. No offspring on the 300 ppm diet survived beyond the F/l generation. The effects on reproduction of adding GC-4072 to the diet of rats through three generations are summarized in Table 3. No significant effect on fertility occurred in F/O generation rats in two successive matings; however, in the F/lb generation, fertility was decreased by the 100 and 300 diets, and in the Fj2b generation decreased fertility also appeared in rats fed 30 ppm. On gestation no effect was noted at any diet level in any generation. Both the Viability and Lactation Indices were affected by the 100 and 300 ppm diets. On a subsequent cross mating of twenty Fj2b female rats fed the 30 ppm diet with males on the control diet, and 19 female rats fed the control diet with male rats fed the 30ppm diet, the Fertility Indices were 25 and 42, respectively. Vaginal smear studies in both the controls and rats given the 30 ppm diet showed no clear differences that would provide a reason for these low indices. Blood plasma and red blood cell ChE values obtained for Fj2b generation rats fed 30 and 100 ppm diets for 30 weeks and for F/3b generation rats fed 30 ppm diet for 3 weeks following weaning, were depressed by approximately the same order of
0.052 zt 0.005b
300
zt 0.031 zt 0.057b zt 0.046b k 0.008b
0.077 f 0.038b
0.295 0.183 0.106 0.079
1
0.084 0.128b 0.035b 0.017b
0.052 * 0.010”
k k i zt
SUPPLEMENTS
* 0.186 f 0.091b f 0.030b I!Z0.013 (4)’
0.039 xt 0.022b
0.782 0.292 0.171 0.079
0.084 f O.OIOb 0.070 f 0.012b 0.047 + 0.007b
0.092 zt 0.027b 0.047 & 0.012b 0.049 * 0.036b 0.551 0.379 0.186 0.090
0.187 f 0.060 0.149 f 0.015
26 Wk
0.239 * 0.063 0.120 * 0.046b
13 Wk
DIETARY
OF
zt 0.071 + 0.059” i 0.029b zk 0.030b
0.091 & 0.023b
0.666 0.306 0.152 0.109
0.086 i 0.024b 0.090 f 0.024b 0.084 zt 0.026b
0.252 & 0.052 0.152 i 0.02@
52 Wk
Delta pH units (mean & SD)
FOR RATS RECEIVING
@Mean of 5 rats per group, unless otherwise indicated within parentheses. b Value differs significantly from control, P .G0.05.
zt 0.048 zt 0.028b zt 0.015b zt 0.012b
0.237 0.124 0.105 0.080
0 10 30 100
0.066 i 0.015b 0.062 f 0.014b 0.052 f 0.006b
0.081 zt 0.008b 0.072 zt 0.014b 0.051 i 0.004b
Female
0.173 f 0.018 0.084 i 0.004b
0.186 zt 0.026 0.103 zt 0.014b
0 10 30 100 300
4Wk
(ChE) VALUES
1Wk
CHOLINESTERASE
Male
Sex
PLASMA
Dietary level (PPm)
BLOOD
TABLE FOR
f f i f
0.128 0.026b 0.017b 0.012b 0.041 f 0.005b
0.530 0.187 0.084 0.068
f f i i
0.141 (4) 0.058b 0.044b 0~)48~ 0.103 f 0.058 (3)b
0.528 0.224 0.155 0.101
0.093 i 0.013b 0.105 f 0.021b -
0.233 xt 0.052 (4) 0.218 * 0.021 (4)
104Wk
104 WEEKS
0.080 + 0.012” 0.059 zt 0.025b 0.044 i 0.007b
0.218 zt 0.059 0.172 zt 0.031
78 Wk
GC-4072
8 4 ;=:
2 s is
d E 8 a 5
0.113 0.136 0.117 0.101 0.111
0.138 0.122 0.106 0.103 0.106
0 10 30 100 300
0.170 0.136 0.099 0.089 0.086
f f + h *
0.019 0.009b 0.007b 0.003 (4)b 0.007b
zt 0.012 (4) dc 0.007b * 0.009b sc 0.011 (4)b f 0.007 (4)b
2 DIETARY
0.283 0.256 0.213 0.185 0.175
i f zt !c iz
0.014 0.034 0.023b 0.008b 0.016b
0.190 0.147 0.122 0.111 0.097
0.156 0.141 0.111 0.109 0.112 * 0.009 + 0.014b zk O.OO8b +z 0.009 (4)b 5 0.008b
i 0.010 * O.OIOb &O.Ollb + 0.007b f 0.009’
OF
0.188 0.147 0.111 0.116 0.105
i 0.015 zt 0.004b k 0.004b i 0.012b xt 0.006b
0.182 + 0.015 0.136i0.014b 0.125 f 0.01 3b 0.118~0.010b 0.105 + 0.011 b
52 Wk
SUPPLEMENTS
Delta pH units (mean f SD) 13 Wk 26 Wk
FOR RATS RECEIVING
0.272 & 0.039 0.258 + 0.025 0.182*0.012b 0.176 + O.OIOb 0.194 & 0.022b
(ChE) VALUES,
4Wk
0.135 0.104 0.090 0.090 (4) 0.096
(3) (4) (3)
f 0.020 + 0.019 +z 0.016b * O.OIOb h O.OIOb
+ 0.022 i 0.012 * 0.002 zlz 0.013 f 0.011
1 Wk
CHOLINESTERASE
a Mean of 5 rats/group, unless otherwise indicated within parentheses. b Value differs significantly from control, P G 0.05. c Value at week 91.
Female
Male
Sex
____-
CELL
Dietary level (ppm) 0 10 30 100 300
RED BLOOD
TABLE FOR
0.199 i 0.003 (4) 0.175 T 0.037 0.125 zt 0.012’ 0.126 t 0.010” 0. I 11 i 0.012 (3)
0.095 f 0.007b 0.085 i 0.008b
f 0.007 (4) f0.021 (4) + 0.036’ t 0.015’ + 0.009b.C 0.142 dz 0.006 0.106 f 0.009b 0.103 i 0.007b
0.189 0.176 0.141 0.132 0.075
104 Wk _~~~~~~
104 WEEKS
0.120 + 0.005 0.114+0.004 0.099 zk 0.01 5b 0.084*0.012” 0.085 f 0.008”
78 Wk
GC-4072
Mated
20 20 20 20 19 20 19 20
20 20 11 7 20 19 10 6
20 20 7 20 20 7
0 30 100 300 0 30 100 300
0 30 100 300 0 30 100 300
0 30 100 0 30 100
Fo/Fla ‘Fib
F /FZa lb\&
Fzb / F 3a “bb
18 9 1 14 6 1
14 12 4’ 2 16 15 5 2
17b 18 20 17 15 20 19 17
Pregnant
18 9 1 14 6 1
14 12 3 2 16 15 5 2
16 18 20 17 15 20 19 17
Whelped
of rats
161 95 10 125 74 3
116 71 22 13 137 139 32 8
157 193 174 149 133 193 165 119
Born alive
3
7 1 2 19 0 0
4 17 1 1 14 3 4 7
1 5 13 8 8 17 12 18
Born dead
-~
of pups _ _
19 12 0 13 8 0
5 2 0 0 14 11 0 0
93 66 17 10 129 129 13 0 149 91 10 118 67 0
15 23 12 0 17 25 0 2
-152 188 140 96 130 185 55 39
Alive day 5 Discard“
Total number
101 69 9 84 40 0
87 62 15 0 112 111 10 0
137 160 102 63 103 114 27 19
Weaned
~
GC-4072
36 35 27 41 34 0
31 33 34 0 35 31 34 0
34 32 29 28 34 32 34 29
;“g;’
Mean
71 84 90 75 61 0
78 90 68 0 91 86 31 0
97 94 63 42 89 68 16 16
Survival (%)
Weanlings
RATS ON VARIOUS DIETARYLEWLSOF THROUGHTHREEGENERATIONS
0 Number of pups discarded on fifth day to reduce litter size to 10. b One rat died with 14 fetuses in utero. c One rat died with 5 fetuses in utero. d Fertility index = (pregnancies/matings) 100. e Gestation index = (litters cast/pregnancies) 100. f Viability index = (pups surviving 5 days/pups born alive) 100. g Lactation index = (pups weaned/live pups on day 5 less discards) 100.
Generation
--~-
Diet level (ppm)
Number
SUMMARYOFREPRODUCTIONDATAFOR TWOMATINGS
TABLE
90 45 14 70 30 14
70 60 36 29 80 79 50 33
85 90 100 85 79 100 100 85
~__ F.I.d
FOR
100 100 100 100 100 100
100 100 75 100 100 100 100 100
!ii 100 100 100 100 100 100 100
G.I.’
93 97 100 94 91 0
80 93 77 77 94 93 41 0
97 95 80 64 98 96 33 33
V.T.f
Indices (%)
78 87 90 80 68 0
99 97 88 0 97 94 77 0
100 97 80 66 91 71 49 51
L.Ig
AMBROSE ETAL.
334
magnitude as those observed in rats in chronic studies of the same time intervals and dietary levels. The effects of GC-4072 in the diet of rats on other parameters are summarized as follows : no certain effect of diet was observed on the average number of siblings born per litter; the average weaning body weights showed no consistent pattern for the 30 and 100ppm diets, but appear to be depressedin the only generation surviving to weaning on the 300 ppm diet. No gross abnormalities were noted in the newborn in any generation. Surviving F/2b generation rats (18 female and 19 male fed control diet, 18femalesand 18 malesfed 30 ppm diet, and 6 female and 2 malesgiven 100 ppm diet) were sacrificed, and the gonads and the uteri and fallopian tubes of the females were submitted to careful histopathologic study. Follicle formation was evident, ova were seenwithin the follicles, and there was a substantial number of corpora lutea in every section studied. The mucosa of the fallopian tubes and the endometrium of the uterus showed no deviation from the control group. In the testesof males, no evidence of deTABLE 4 BLED /(PLASMA, RED BLOOD CELLS) CHOLINESTERASE VALUES FOR BEAGLEDOGSONDIETARYSUPPLEMENTSOF GC-4072 FOR~O~WEEKS Delta pH units (mean f SD)
Sample Plasma
Weeks 0 1 4 8 12 26 39 52 65 78 91 104
Red blood cells
0 1 4 8 12 26 39 52 65 79 91 104
0 wm
30 pm
200r-vm
1000ppm
0.459* 0.044 0.478xt 0.096 0.450& 0.091 0.4683 0.111 0.484 0.439 0.244 0.460 0.248 0.239 0.094 0.131 0.093 0.100 0.160
f 0.058 iz 0.038 zk 0.135 zk 0.066 f 0.082 f 0.045 f 0.009 zt 0.029 * 0.041 * 0.037 zt 0.097’
0.249 0.108 0.129 0.279 0.189 0.158 0.088 0.116 0.088 0.084 0.090
f 0.040* * 0.070* 7t 0.044 i 0.022* + 0.059 f 0.047* zk 0.006 xk 0.022 * 0.030 & 0.025 h 0.017
0.135 0.084 0.096 0.149 0.099 0.091 0.085 0.110 0.055 0.063 0.068
zt 0.033”
0.116 IIZ0.026*
f 0.01lb 0.059i 0.007* + 0.020 +z 0.052* i 0.014” f 0.028* i 0.004 & 0.010 It 0.037 zt 0.015 & 0.023
0.058 0.080 0.066 0.064 0.090 0.106 0.071 0.053 0.073
i 0.014* zt O.OlS* & 0.012* f 0.012’ & 0.008 * 0.010 i 0.021 rt 0.014 zk 0.014
0.179+ 0.034 0.2095 0.024 0.223* 0.017 0.180& 0.011 0.209 0.151 0.165 0.267 0.173 0.143 0.153 0.148 0.179 0.166 0.152
+z 0.048 +c 0.024 zt 0.013 zt 0.030 rt 0.010 zt 0.009 zt 0.024 zt 0.017 * 0.008 * 0.017 zk 0.022’
0.191 0.129 0.145 0.238 0.158 0.144 0.156 0.146 0.183 0.155 0.138
* 0.041 i 0.041 zk 0.024 xk 0.045 k 0.025 zk 0.014 i 0.030 & 0.014 zt 0.024 zk 0.012 5 0.034
0.225 0.155 0.168 0.256 0.180 0.155 0.175 0.155 0.196 0.131 0.176
& 0.019 h 0.044 f 0.007 + 0.017 zt 0.004 zt 0.008 zk 0.019 zt 0.017 i 0.016 zk 0.029 i 0.021
0.190 0.113 0.106 0.181 0.133 0.121 0.151 0.144 0.144 0.130 0.135
zk 0.009’ h O.OlS* * 0.014* rt 0.017* zt 0.038 zt 0.013 + 0.024 i 0.015 z!t 0.023* zk 0.032 zt 0.053
’ Average of 4 dogs, 2 of each sex, unless otherwise indicated. Dietary level given as parts per million (ppd. ’ Value differs significantly from control, P < 0.05. ’ Average of 3 dogs.
TOXICOLOGIC
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335
pressed spermatogenesiswas found, the basement membranes of the tubules were contiguous, there was no atrophy or hypertrophy of the interstitial cells (Leydig cells), and the blood vesselswere normal. Chronic Toxicity Studies in Dogs One dog fed the control basal diet was sacrificed in a moribund state in the 97th week, all other dogs survived the two-year feeding in apparent good health. Body weight data indicated no adverse effects, and food consumption wascomparable at all dietary levels of GC-4072. Hematologic data and urine concentrations of reducing substancesand protein, obtained at frequent intervals, were within normal limits throughout the test period. Plasma and red blood cell ChE values obtained at intervals during this study are summarized in Table 4. Plasma ChE levels were significantly depressedat all dietary levels of GC-4072 through the first 39 weeks. Red blood cell ChE activity was consistently and significantly depressedduring the first 12 weeksonly in dogs fed the 1000ppm diet. It was depressedagain during the 78th week. Liver function data (BSP, SGOT, SAP) and BUN obtained at the end of the 2-year feeding period showed no adverse trends. Organ-to-body weight ratios showed no significant difference between control dogs and dogs on the various dietary levels of GC-4072 for 2 years. Histopathologic findings indicated no lesions attributable to GC-4072.
In two year study in rats and dogs and reproduction studies in rats, no increasein the incidence of tumors was observed. ACKNOWLEDGMENTS
The authorswish to expresstheir appreciationto the following personsfor their assistance with certain aspectsof the experimentalwork reported: Dr. NelsonF. Young of the Division of Clinical Pathology, M.C.V. for cholinesterase determinations;Dr. JosephJ. McPhillips, for in vitro anticholinesterase tests;Dr. E. M. Crawford for veterinary serviceson dogs: Amalyle M. Lea, Patricia W. Small, GeorgeB. Werthan, Edwin H. Talley, Jr., John S. Nicholas,and Edward J. Baker for technical assistance. REFERENCES
R. L., and JOHNSON, H. (1964).Neurological disruption produced in hens by two organophosphateesters.Brit. J. Pharmacol.23, 295-304. BENEDICT, S. R. (19081909). A reagent for detection of reducing sugars.J. Biol. Chem.5, BARON,
485-488.
J. (1955).Estimateof the integrated normal curve by minimum normit chi-square with particular referenceto bio-assay.J. Am. Statist. Assoc.50, 529-549. CHAMBERLAIN, W. F., and HOPKINS, D. E. (1962). Absorption and elimination of General Chemical4072applied dermally to cattle. J. Econ. Entomol.55, 86-88. DAVIES, D. R., HOLLAND, P., andRUMENS, M. J. (1960).The relationshipbetweenthe chemical structure and neurotoxicity of alkyl organophosphorouscompounds.Brit. J. Pharmacol.
BERKSON,
15, 271-278.
K. P., and MANGUM,G. H. (1947).Effect of hexaethyl tetraphosphateon choline esterasein vitro and in vivo. Proc. Sot. Exptl. Biol. Med. 64, 137-139. DUNCAN, D. B. (1955). Multiple range and multiple F tests.Biometrics11, l-42. DUBOIS,
336
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ET AL.
W. F., GAINES, T. B., and HAYES, JR., W. T. (1956). Paralytic and related effects of certain organic phosphorous compounds. A.M.A. Arch. Ind. Health 13, 326-330. IVEY, M. C., CLABORN, H. V., HOFFMAN, R. A., GRAHAM, 0. H., PALMER, J. S., and RADELEFF, R. D. (1966).Residueof Shell compound4072in body tissuesof sprayedcattle. J. Econ. DURHAM,
Entomol. 59, 379-382.
M. C. (1960).A note on the demyelinationproducedin hensby dialkylfluoridates. 15, 279-281. MICHEL, H. 0. (1949).An electrometricmethod for the determination of red blood cell and plasmacholinesteraseactivity. J. Lab. Clin. Med. 34, 1564-1568. OSER, B. L., and OSER, M. (1956).Nutritional studieson rats on dietscontaininghigh levelsof partial esteremulsifiers.II. Reproduction and lactation. J. Nutr. 60, 489-505. PICKERING, W. R. (1965). The acute toxicity of chlorfenvinphos to sheepand cattle when applieddermally. Vet. Record 77 (39), 1140-l 144. ROBERTS, R. H., RADELEFF, R. D., and CLABORN, H. V. (1961).Residuesin milk of dairy cows sprayedwith P3’ labelledGeneralChemicalGC-4072.J. Econ. Entomol. 54,1053-1054. SHEVKY, M. C., and STAFFORD, D. D. (1923).A clinical method for estimationof protein in urine and other body fluids. Arch. Internal Med. 32, 222-225. SNEDECOR, G. W. (1957).Statistical Methods, pp. 4547. Iowa Univ. Press,Ames, Iowa. LANCASTER,
Brit. J. Pharmacol.
AND
APPLIED
PHARMACOLOGY
17, 323-336 (1970)
Toxicologic Studies Diethyl-I-(2,4-DichlorophenyI)-2Chlorovinyl
on Phosphate]
ANTHONY M. AMBROSE, PAUL S. LARSON, JOSEPH F. BORZELLECA, AND GORDON R. HENNIGAR, JR.* Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219, and Department of Pathology, College of Medicine, State University of New York, Brooklyn, New York 11203 Received May 19,1969
Toxicologic Studies on Diethyl-1-(2,4-Dichlorophenyl)-2-Chlorovinyl Phosphate. AMBROSE, ANTHONY M., LARSON, PAUL S., BORZELLECA, JOSEPH F., and HENNIGAR, GORDON R., JR. (1970). Toxicol. Appl. Pharmacol. 17,323-336.Studiesof diethyl-1-(2,4-dichlorophenyl)-2-chlorovinyl phosphate: GC-4072, Supona, Chlorfenvinphos, SD 7859, ENT 24969, a new organophosphatepesticide, were undertaken in several animal speciesto characterizetoxicity and assess safety. Measurementsof acute toxicity by different routes of administration gave the following LDSOvalues(mg/kg): po 9.66 for rats, 300(estimated) for rabbits, >12000for dogs;iv, 6.6for rats and 50.5for dogs.The undiluted form anda 49.1% GC-4072-xylolemulsifiableformulation applieddermally to rabbits gave LD50 valuesof 400 and 1087,respectively.Hensreceiving multiple sublethal dosesip displayed no delayed neurologic effects or histologiclesions. Rats fed dietary levelsranging from 3 to 1000ppm and dogsfed l-1000 ppm for periodsup to two years showedno clinical signsof intoxication, but therewasa significantand variable decrease in plasmaand/or red blood cell cholinesteraseactivity, with reversaltrends. Multigeneration studiesin rats fed dietary levelsof 30, 100,or 300ppm showedno effect on gestation,but therewereprogressiveeffectson fertility, viability, and lactation indices.No grossabnormalitieswerefound in fetuses born dead or alive, or in rats autopsiedwith fetusesin situ. Food consumption, growth, hemograms,urinary findings, histopathology, and liver function testsin dogswere within normal limits. Diethyl-1-(2,4-dichlorophenyl)-2-chlorovinyl
phosphate3 (GC-4072)4, an organophos-
phate ester, is a pesticidal agent proposed for use in the control of insect pests (ticks, fleas) on dogs and livestock; as a residual surface spray for house flies, ticks, and fleas; as a larvicide for flies; for certain root maggots of vegetable crops and insectsattacking 1Supportedby a grantfrom Allied ChemicalCorporation,GeneralChemicalDivision,Agricultural ChemicalDevelopment,Morristown,New Jersey07960. * Presentaddress: Departmentof Pathology,MedicalCollegeof SouthCarolina,Charleston,South Carolina29401. 3 Also referredto asChlorfenvinphos, Supona,SD7859,ENT 24969. 4 GC-4072is thecommonnameandU.S. patent(No. 3,003,916) of Allied ChemicalCorporation, Morristown,New Jersey07960. 323
324
AMBROSE ET/IL.
potato foliage; and for the control of a wide variety of other insect pests. In dairy cattle following single dermal application of a spray containing 32P-labeled (X-4072, in concentrations adequate for insect control, Chamberlain and Hopkins (1962) have reported that the compound is rapidly absorbed and eliminated, maximum blood concentrations appearing in 2 hours, with low urinary concentrations at 1 week after treatment. Under comparable conditions residuesof the compound have been found in the milk of dairy cows in 5 hours, gradually diminishing to zero in IO-12 days(Roberts et al., 1961),and Ivey et al. (1966)have reported low residuesof the compound in various fatty tissuesof beef cattle. The residueswere eliminated in 28 days or less.In lambs and ewes dipped in aqueous emulsions and in 3-month-old calves and yearlings treated with aqueousemulsion sprays, at concentrations or application rates in excessof recommended usage,blood cholinesteraselevels were depressedbut returned rapidly to pretreatment levels (Pickering, 1965). Studies were undertaken: to characterize acute, subacute, and chronic toxicity; to evaluate possible dermal irritation and neurotoxicity; to determine effects on blood cholinesteraseactivity and on reproduction. Technical grade GC-4072 (96 %) used in these studies, except where otherwise indicated, is a light amber-colored liquid with a density of 1.345 and a boiling point of 167170°C (0.5 mmHg). It is soluble in most organic solvents, edible vegetable oils and propylene glycol, and sparingly soluble in water. The structural formula is shown in Fig. 1. 0 C2H50&)-C: C,H,O’
CHCl Cl
FIG. 1. Structural formula of GC-4072.
METHODS
Anticholinesterase Activity In vitro anticholinesterase activity of GC-4072 was determined manometrically on freshly drawn human blood plasma and red blood cells according to the method of DuBois and Mangum (1947).
Single LD50 Determinations Oral. Determined by gastric intubation, following overnight fast, in male albino rats5 (110 to 220 g) in groups of 10 at each of four dosesranging from 5 to 20 mg/kg, in male albino rabbits weighing 1.4 to 3.0 kg, in groups of 10each at five dosesranging from 160 to 400 mg/kg, and in 13 adult male and female mongrel dogs, at dosesof 1, 3, 6, or 12 g/kg. To rats and rabbits GC-4072 was administered as a 1% or 10 % (w/v) solution in peanut oil, respectively, and was given undiluted to dogs and followed by 10 ml corn oil. ’ Wistar strain (Albino Farms, Red Bank, New Jersey), used throughout.
TOXICOLOGIC
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325
Intravenous. Determined in groups of 10 male albino rats (145-240 g) at doses of 2.5, 3.5, 5.0, 10, 20, or 25 mg/kg, and in 18 mongrel dogs (7.5-18.5 kg) at doses of 25, 35, 50, 65, 75, or 100 mg/kg. Both species received GC-4072 as a solution (w/v) in Lipornul,‘j 1% for rats and 10% for dogs. Dermal. Four groups each consisting of 10 male albino rabbits weighing approximately I .9 kg each were tested for local irritation and systemic dermal toxicity with each of the following preparations : (1) undiluted 96 % technical GC-4072 was applied by gentle massage with the butt end of a test tube, to the closely clipped unabraded skin of the back of rabbits in doses ranging from 200 to 673 mg/kg; and (2) an emulsifiable formulation, containing 49.1% technical GC-4072 (92.5 %) by weight, in a solvent mixture consisting of 45.5 % xylol and 5.4 % mixed emulsifiers, in doses ranging from I .25 to 2.5 ml/kg (equivalent to approximately 588-l 177 mg/kg of GC-4072) was introduced directly underneath a rubber girdle fitted snugly around the closely clipped unabraded skin of the trunk of immobilized rabbits. After 24 hours’ exposure, the cuffs were removed and any unabsorbed material was removed with thumb sponges. In both experiments, the condition of the skin was observed daily for 14 days. The LD50 values, where possible, for the three routes of administration, were calculated by the minimum approximate chi-square method of Berkson (1955). Subchronic Toxicity Studies Neurotoxicity. Durham et al. (1956) have suggested that all new compounds with the organophosphorus grouping as well as pesticidal agents having anticholinesterase activity should be screened for possible delayed paralytic or neurotoxic effects. Since certain organophosphorus esters have been shown to produce paralytic effects and myelin degeneration in chickens (Durham et al., 1956; Davies et al., 1960; Lancaster, 1960; Baron and Johnson, 1964), experiments with chickens were undertaken to determine whether GC-4072 could produce this syndrome. Accordingly, 26 White Leghorn hens, approximately 16 months old and weighing 1.5-1.9 kg each, were treated daily for 10 days, or until death, by ip injection of GC-4072 in 20 % ethanol-80 % propylene glycol. The number of hens used at each dose, is indicated within parentheses as follows: 0 (5), 100 (6), 150 (3), 200 (4), 300 (2) mg/kg. Two other groups, of 3 hens each, were similarly treated with 100 or 200 mg/kg GC-4072, but in addition received atropine sulfate (1 mg/kg) to lessen the effects of excessive acetylcholine accumulation. All doses were contained in not more than 1.5 ml of solvent mixture. Daily observations were made during treatment and for 20 additional days at which time all surviving hens were autopsied. Brain, segments of the spinal cord and sciatic nerve were taken, fixed in neutralized 10 “/, formalin and examined histologically. Twelve-week studies. In a preliminary (12-week) study, six groups of weaning (28days) albino rats,5 10 of each sex per group, individually housed, were placed on control finely ground basic stock diet’ for 5 weeks. Similarly four groups of mongrel dogs consisting of two of each sex per group, individually housed, were placed on a stock diet consisting of 87 % ground basal ration,’ 12 % corn oiL9 and 1 % U.S.P. cod liver oil, prepared fresh daily. At feeding time the feed was mixed with an equal weight of water. 6 Lipomul@ I.V., The Upjohn Company, Kalamazoo, Michigan. ’ Wayne Lab-Blox, Allied Mills, Chicago, Illinois. 8 Basal Ration f/d@; A Prescription Diet @, Hill Packing Company, Topeka, Kansas. 9 Mazola@ oil, Corn Products Company, New York, New York.
326
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During this period plasma and red blood cell cholinesterase (ChE) values, determined by a micromodification of the electrometric procedure of Michel(l949), were obtained weekly on all dogs and on 5 rats of each sex from each group. The same rats were used each week insofar as possible. At the start of the sixth week, rats were fed the basic diet containing 0,3, 10, 30, 100, or 1000 ppm of CC-4072, and the dogs were fed diets containing, 1, 10, 100, or 1000 ppm GC-4072, respectively, for 12 weeks. All diets were prepared fresh each week. At 1, 2, 4, 6, 8, 10, and 12 weeks plasma and red blood cell ChE values were obtained, as above, for 5 rats of each sex on each diet and for all dogs. At the end of week 12, rats in excess of 5 of each sex per diet, not used for following ChE activity, were sacrificed as well as 1 dog of each sex for each diet; remaining rats and dogs were returned to the control basic diet (reversal of ChE effect) and ChE activity values were obtained after 1 and 4 weeks for rats and at 1, 2,4, and 8 weeks for dogs. Terminally, all surviving rats and dogs were sacrificed. Organ-to-body weight ratios were determined for liver, kidneys, heart, spleen, and testes for rats on control and GC-4072 diets for 12 weeks and after withdrawal for 5 weeks. Rats were individually weighed once each week and food consumption was determined over 3-day periods during weeks 4 and 12. Dogs were weighed weekly, and food consumption was measured daily. Histopathologic studies of hematoxyhn-eosin stained paraffin sections of formalin-fixed tissues were made on 5 female and 3 male rats on the control diet, on 2 female and 3 male rats on 100 ppm GC-4072 diet for 12 weeks, and on all dogs. Tissues studied included: heart, lungs, liver, kidney, urinary bladder, spleen, stomach, small and large intestine, skeletal muscle, skin, bone marrow, pancreas, thyroid, adrenal, gonad, pituitary, brain, and in dogs the spinal cord. Body weight changes and organ-to-body weight ratios for rats were evaluated by analysis of variance and Duncan (1955) multiple range and multiple Ftests. ChE values were evaluated by Student t test (Snedecor, 1957), at the 95 y0 probability level, against appropriate controls. Chronic Toxicity
Studies in Rats
Using littermate distribution, four matched groups, each consisting of 30 weanling albino rats5 (28 days) of each sex, culled to a narrow starting weight range, were placed on dietary levels of 0, 10,30, or 100 ppm GC-4072 prepared fresh each week. An additional group of 30 weanling rats of each sex, not littermates of the above, was placed on 300 ppm. The stock diet was that referred to above. All rats were individually housed and were weighed weekly. Food consumption was measured over 3-day periods at the end of 1, 3, 6, and 12 months. Hematologic determinations (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex at each dietary level at 3-month intervals. Urinary tests for reducing substances (Benedict, 19081909) and protein (Shevky and Stafford, 1923) were made at the same times on urine samples pooled by sex and dietary level. Blood ChE activity (red blood cells and plasma) was determined on 4 rats of each sex for each dietary level at 1, 4, 8, and 12 weeks, and at 3-month intervals thereafter. At 13 weeks, at least 4 rats of each sex on each of the diets were sacrificed for histopathologic study. Survivors among the remaining rats were sacrificed during the 104th week, with the exception of male rats on 300 ppm diet which were sacrificed during the 95th week. At each autopsy period organ weights were obtained for heart, spleen, kidneys, liver, and testes. Rats that died during
TOXICOLOGIC
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327
the experiment and male rats terminated at 95 weeks were autopsied, but their organ weights are not included in organ-to-body weight calculations. All rats sacrificed in moribund condition and those autopsied at 13, 95, or 104 weeks were examined grossly and microscopically. Tissues subjected to histopathologic examination were the same as in the preliminary study. Body weight changes at various time intervals and organ-to-body weight ratios at 13 weeks and at the end of the 2-year study were evaluated by analysis of variance and the Duncan test (1955). Hematologic data were evaluated by comparison to control values, and blood ChE values by Student t test for each test period. Reproduction and Lactation Studies. To determine stress effects of GC-4072 on gestation and lactation, successive generation studies were undertaken. For this study, 28-day-old, littermate rats, within but not between sexes, were separated into 4 groups of 30 rats of each sex to constitute the parent (F/O) generation. Mean body weight and weight range were similar for all groups. One group (0 ppm), was fed the basic diet’ and the others basic diet containing 30, 100, or 300 ppm of GC-4072. Rats were individually housed and had free access to water and diet. Weekly body weight records were made, except during mating, through weaning of litters. After 11 weeks of treatment, 20 females from each diet group were transferred to individual breeding cages, and each was mated with a male of the same diet group for the F/l a generation. Male rats were rotated to a different female on each of three successive 7-day periods. On the 20th mating day all males were removed. Records were maintained of mating, number of pregnancies, litters, pups in litter at 1, 5, and 21 days (weaning), and the total weight of the litter at weaning. Litters containing more than 10 siblings were randomly culled to 10 on day 5. All surviving F/la siblings were sacrificed and autopsied at weaning. To ensure that all F/O parent generation rats for each dietary level were approximately the same age to produce F/lb litters, they were remated, as above, about 10 days after weaning of the last F/la litter. Observations recorded were the same as those described for F/la litters. At weaning of F/b litters, surviving parent (F/O) rats were sacrificed and autopsied. Where available, 30 F/lb offspring, from as many litters as possible, of each sex from each dietary level were continued on the parent diet for 11 weeks, at which time 20 rats of each sex, where available, within each group, were separated and mated to produce F/2a and F/2b generations. The same procedure as with F/lb generation was followed with Fj2b offsprings to produce F/3a and F/3b generation rats. All matings with F/lb and Fj2b generation rats were made with male and female rats from different litters. Blood plasma and red blood cell ChE values were obtained for F/2b parent generation rats fed 0, 30, or 100 ppm diets (30 weeks) after weaning of F/3b litters, and for F/3b generation siblings fed 0 or 30 ppm diets for 3 weeks after weaning. At least 10 rats of each sex, from each diet group were used to determine these parameters, except for F/2b rats on 100 ppm diet; only 6 females and 3 males were available. Following weaning of F/3b generation rats, surviving Fj2b generation female rats fed 30 or 100 ppm diets were cross-mated with rats on 0 ppm diet. The few surviving male rats fed 100 ppm diet did not participate in this study. Observations recorded for F/3c generation were the same as described above. Survivors in these two generations were sacrificed and autopsied. Tissues from F/3b
328
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AL.
generation rats were submitted for histopathologic same as those described in the subacute study.
study. Tissues included were the
Chronic Oral Toxicity Studies in Dogs In the 2-year feeding studies 16 purebred beagle dogs, approximately 6 months of age, were divided into 4 groups each consisting of two dogs of each sex. Mean body weight and weight range were similar. A control group was placed on the basal diet previously described, and the other 3 groups were placed on the basal diet containing 30, 200, or 1000 ppm GC-4072. Prior to treatment the dogs were immunized against distemper, infectious hepatitis and leptospirosis and were treated for intestinal parasites. Food consumption was measured daily, and body weight was recorded weekly. The following were obtained on all dogs at start and at 3-month intervals thereafter: urine concentrations of reducing substances and protein, and hematologic determinations (hemoglobin, hematrocrit, and total and differential leukocyte counts). Red blood cell and plasma ChE values were obtained for all dogs at 0, 1, 4, 8, and 12 weeks, and at 3-month intervals thereafter. Bromosulfalein (BSP) retention, serum glutamic-oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SAP), and blood urea nitrogen (BUN) values were obtained at 2 years. All surviving dogs were autopsied and organs examined grossly. Organ weights and organ-to-body weight ratios were obtained for heart, spleen, kidneys, liver, and testes. Tissues submitted for histopathologic study were the same as those in the subacute study. Body weight changes at representative time intervals, and organ-to-body weight ratios were evaluated by analysis of variance and the Duncan test (1955) at the 95 % probability level. Blood ChE values were evaluated by Student t test by comparison to control values at each time interval. Other data such as food consumption, hematologic data, liver function tests, and BUN were evaluated by comparison with the control values. RESULTS
AND
DISCUSSION
In vitro incubation of GC-4072 with human plasma and red blood cells produced direct inhibition of ChE activity. Most sensitive was the plasma. A concentration of 1 x IO-’ M GC-4072 produced 50 % inhibition of plasma ChE which was comparable to that produced by an equimolar concentration of paraoxon. On red blood cells, 50 % inhibition was obtained with 4.9 x lo-’ M GC-0472 as compared to 1.6 x lo-’ for paraoxon. Single Dose Toxicity Orally, the LD50 f SD values were 9.66 mg/kg f 1.35 for rats; approximately 300 for rabbits, and >12000 for dogs. By iv administration the values were 6.6 & 0.58 for rats and 50.5 & 4.5 for dogs. By dermal application on rabbits the values were 400 ?X52 for undiluted GC-4072 and 1087 % 23 for the emulsifiable concentrate. The effects produced by GC-4072 in all animals and by all routes of administration were essentially the same. Deaths occurring within 12 hours were usually preceded by the characteristic signs of ChE inhibition (salivation, lacrimation, muscle fasciculation, diarrhea, and in dogs emesis). After single dermal applications of undiluted GC-4072 to rabbits, no apparent signs
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329
of skin irritation were observed. However, in rabbits treated with the emulsifiable formulation cutaneous irritation appeared within 3 days and was characterized by erythema, desquamation, and dry and leathery skin. Two weeks following exposure the skin of all surviving rabbits appeared normal, following sloughing of the coriaceous integument. Postmortem examination of visceral and thoracic organs of all animals dying or sacrificed 14 days after treatment was essentially normal. Subchronic Toxicity Studies
Immediately following each daily ip injection of GC-4072, with or without atropine, the hens showed typical signs of acute anticholinesterase intoxication at all doses, inability to stand, salivation, and retching. Deaths occurred at all doses with or without atropine, the highest frequency occurring with the first injection. Atropine did not protect against the lethal effects of GC-4072. The 8 hens surviving (5 at 100,2 at 150, and 1 at 200 mgjkg) were observed daily for 20 days following the last injection, and none showed any delayed neurotoxic effects. Histopathologic studies of myelin and Nisslstained sections of the brain, spinal cord, and sciatic nerve from 5 control hens and 8 surviving hens treated with GC-4072 for 10 consecutive days failed to reveal any of the neurologic lesions suggesting demyelination or neural damage which are characteristic of certain organophosphate esters (DFP, Merphos, dialkylfluoridates, etc.). In the 1Zweek preliminary studies in rats given O-1000 ppm and dogs given l-1000 ppm GC-4072 diets, no significant effect was observed on mortality or food consumption. In rats, growth was significantly depressed in both sexes fed 1OOOppm; upon withdrawal of GC-4072 from the diet for 4 weeks a slight reversal was noted. In dogs no effect on growth was noted. Blood ChE values for plasma and red blood cells were significantly depressed for rats fed 30, 100, or 1000 ppm diets, sporadically for rats given the 10 ppm diet, and not at all for rats fed the 3 ppm diet. Following withdrawal of GC-4072 from the diet, recovery of plasma ChE was complete within 1 week at all dietary levels, except for female rats formerly fed the 1000 ppm diet, in which it was complete at 4 weeks. Red blood cell ChE recovered completely within 4 weeks for all groups except for male rats formerly fed 100 or 1000 ppm diets. In dogs given CC-4072 diets for 12 weeks, statistically significant depression in plasma ChE values occurred at all dose levels, while significantly depressed red blood cell values appeared sporadically. Upon withdrawal of GC-4072 from the diet for 8 weeks, recovery trends were apparent for plasma but not for red blood cell ChE. Organ-to-body weight ratios for heart, spleen, kidneys, liver, and testes for male rats fed any dietary level were not significantly different from control values. The spleento-body weight ratios of female rats fed 30 or 100 ppm and the kidneys of rats fed 30 ppm and sacrificed at 12 weeks, were significantly decreased. After withdrawal of GC-4072 from the diet for 4 weeks no significant difference was noted. Gross and microscopic pathologic findings, except for minor lesions customarily found in rats, were essentially negative. In dogs histopathologic findings were negative. Chronic Toxicity
Studies in Rats
Body weight gains for female rats fed dietary levels of 100 or 300 ppm became significantly depressed by the 26th week, continuing so until near termination of the study,
330
AMBROSE
ET AL.
at which point differences from controls though still present were not statistically significant. The same dietary levels had no effect on growth of male rats. On survival. a clearcut effect of the test material did not appear. Food consumption determinations indicated no consistent trends which could be related to GC-4072 in the diet. Hematologic examinations and urinary tests for reducing substances and protein gave comparable results for rats on the various dietary levels of GC-4072 and the controls at all test periods. Blood ChE values at representative intervals, are summarized in Table 1 for plasma, and in Table 2 for red blood cells. Significant depression in plasma and red blood cell ChE occurred in both sexes at all dietary levels of GC-4072, except for male rats on 10 ppm during the second year. No signs of intoxication were noticeable in any of the rats. Organ-to-body weight ratios for heart, spleen, kidney, liver, and testes for rats sacrificed after 13, 95, or 104 weeks on the respective diets, were not significantly different from the controls, except for a lower spleen ratio for female rats on the 300 ppm diet for 13 weeks, not confirmed at two years, and a higher liver ratio for male rats on the 100 ppm dietary level of GC-4072 for 104 weeks. No effect on the liver was apparent in male rats on 300 ppm sacrificed at 95 weeks. Histopathologic findings on rats sacrificed after 13,95, or 104 weeks on the respective diets were essentially negative except for the normal incidence of bronchiectasis, murine pneumonia, and disseminated granulomatous inflammatory processes which appeared in approximately equal frequency in both the control and experimental rats. In none of the rats on the various dietary levels of GC-4072 was there any evidence of liver necrosis or other change that might be expected of a chlorinated compound that is hepatotoxic. Reproduction and Lactation Studies. Body weights for each of the three female parent generation (F/O, F/l, F/2) rats receiving 30, 100, or 300 ppm dietary treatment with GC-4072 before mating and at weaning of their respective litters tended to show somewhat lower body weights than the controls. Average percent lower mean body weights for F/O generation were 3, 5, and 11%; for F/l generation 9,2, and 19 %; and for F/2 generation 14 and lo%, respectively. Male rats on these levels of GC-4072, and of these generations showed a comparable decrease in body weight. No offspring on the 300 ppm diet survived beyond the F/l generation. The effects on reproduction of adding GC-4072 to the diet of rats through three generations are summarized in Table 3. No significant effect on fertility occurred in F/O generation rats in two successive matings; however, in the F/lb generation, fertility was decreased by the 100 and 300 diets, and in the Fj2b generation decreased fertility also appeared in rats fed 30 ppm. On gestation no effect was noted at any diet level in any generation. Both the Viability and Lactation Indices were affected by the 100 and 300 ppm diets. On a subsequent cross mating of twenty Fj2b female rats fed the 30 ppm diet with males on the control diet, and 19 female rats fed the control diet with male rats fed the 30ppm diet, the Fertility Indices were 25 and 42, respectively. Vaginal smear studies in both the controls and rats given the 30 ppm diet showed no clear differences that would provide a reason for these low indices. Blood plasma and red blood cell ChE values obtained for Fj2b generation rats fed 30 and 100 ppm diets for 30 weeks and for F/3b generation rats fed 30 ppm diet for 3 weeks following weaning, were depressed by approximately the same order of
0.052 zt 0.005b
300
zt 0.031 zt 0.057b zt 0.046b k 0.008b
0.077 f 0.038b
0.295 0.183 0.106 0.079
1
0.084 0.128b 0.035b 0.017b
0.052 * 0.010”
k k i zt
SUPPLEMENTS
* 0.186 f 0.091b f 0.030b I!Z0.013 (4)’
0.039 xt 0.022b
0.782 0.292 0.171 0.079
0.084 f O.OIOb 0.070 f 0.012b 0.047 + 0.007b
0.092 zt 0.027b 0.047 & 0.012b 0.049 * 0.036b 0.551 0.379 0.186 0.090
0.187 f 0.060 0.149 f 0.015
26 Wk
0.239 * 0.063 0.120 * 0.046b
13 Wk
DIETARY
OF
zt 0.071 + 0.059” i 0.029b zk 0.030b
0.091 & 0.023b
0.666 0.306 0.152 0.109
0.086 i 0.024b 0.090 f 0.024b 0.084 zt 0.026b
0.252 & 0.052 0.152 i 0.02@
52 Wk
Delta pH units (mean & SD)
FOR RATS RECEIVING
@Mean of 5 rats per group, unless otherwise indicated within parentheses. b Value differs significantly from control, P .G0.05.
zt 0.048 zt 0.028b zt 0.015b zt 0.012b
0.237 0.124 0.105 0.080
0 10 30 100
0.066 i 0.015b 0.062 f 0.014b 0.052 f 0.006b
0.081 zt 0.008b 0.072 zt 0.014b 0.051 i 0.004b
Female
0.173 f 0.018 0.084 i 0.004b
0.186 zt 0.026 0.103 zt 0.014b
0 10 30 100 300
4Wk
(ChE) VALUES
1Wk
CHOLINESTERASE
Male
Sex
PLASMA
Dietary level (PPm)
BLOOD
TABLE FOR
f f i f
0.128 0.026b 0.017b 0.012b 0.041 f 0.005b
0.530 0.187 0.084 0.068
f f i i
0.141 (4) 0.058b 0.044b 0~)48~ 0.103 f 0.058 (3)b
0.528 0.224 0.155 0.101
0.093 i 0.013b 0.105 f 0.021b -
0.233 xt 0.052 (4) 0.218 * 0.021 (4)
104Wk
104 WEEKS
0.080 + 0.012” 0.059 zt 0.025b 0.044 i 0.007b
0.218 zt 0.059 0.172 zt 0.031
78 Wk
GC-4072
8 4 ;=:
2 s is
d E 8 a 5
0.113 0.136 0.117 0.101 0.111
0.138 0.122 0.106 0.103 0.106
0 10 30 100 300
0.170 0.136 0.099 0.089 0.086
f f + h *
0.019 0.009b 0.007b 0.003 (4)b 0.007b
zt 0.012 (4) dc 0.007b * 0.009b sc 0.011 (4)b f 0.007 (4)b
2 DIETARY
0.283 0.256 0.213 0.185 0.175
i f zt !c iz
0.014 0.034 0.023b 0.008b 0.016b
0.190 0.147 0.122 0.111 0.097
0.156 0.141 0.111 0.109 0.112 * 0.009 + 0.014b zk O.OO8b +z 0.009 (4)b 5 0.008b
i 0.010 * O.OIOb &O.Ollb + 0.007b f 0.009’
OF
0.188 0.147 0.111 0.116 0.105
i 0.015 zt 0.004b k 0.004b i 0.012b xt 0.006b
0.182 + 0.015 0.136i0.014b 0.125 f 0.01 3b 0.118~0.010b 0.105 + 0.011 b
52 Wk
SUPPLEMENTS
Delta pH units (mean f SD) 13 Wk 26 Wk
FOR RATS RECEIVING
0.272 & 0.039 0.258 + 0.025 0.182*0.012b 0.176 + O.OIOb 0.194 & 0.022b
(ChE) VALUES,
4Wk
0.135 0.104 0.090 0.090 (4) 0.096
(3) (4) (3)
f 0.020 + 0.019 +z 0.016b * O.OIOb h O.OIOb
+ 0.022 i 0.012 * 0.002 zlz 0.013 f 0.011
1 Wk
CHOLINESTERASE
a Mean of 5 rats/group, unless otherwise indicated within parentheses. b Value differs significantly from control, P G 0.05. c Value at week 91.
Female
Male
Sex
____-
CELL
Dietary level (ppm) 0 10 30 100 300
RED BLOOD
TABLE FOR
0.199 i 0.003 (4) 0.175 T 0.037 0.125 zt 0.012’ 0.126 t 0.010” 0. I 11 i 0.012 (3)
0.095 f 0.007b 0.085 i 0.008b
f 0.007 (4) f0.021 (4) + 0.036’ t 0.015’ + 0.009b.C 0.142 dz 0.006 0.106 f 0.009b 0.103 i 0.007b
0.189 0.176 0.141 0.132 0.075
104 Wk _~~~~~~
104 WEEKS
0.120 + 0.005 0.114+0.004 0.099 zk 0.01 5b 0.084*0.012” 0.085 f 0.008”
78 Wk
GC-4072
Mated
20 20 20 20 19 20 19 20
20 20 11 7 20 19 10 6
20 20 7 20 20 7
0 30 100 300 0 30 100 300
0 30 100 300 0 30 100 300
0 30 100 0 30 100
Fo/Fla ‘Fib
F /FZa lb\&
Fzb / F 3a “bb
18 9 1 14 6 1
14 12 4’ 2 16 15 5 2
17b 18 20 17 15 20 19 17
Pregnant
18 9 1 14 6 1
14 12 3 2 16 15 5 2
16 18 20 17 15 20 19 17
Whelped
of rats
161 95 10 125 74 3
116 71 22 13 137 139 32 8
157 193 174 149 133 193 165 119
Born alive
3
7 1 2 19 0 0
4 17 1 1 14 3 4 7
1 5 13 8 8 17 12 18
Born dead
-~
of pups _ _
19 12 0 13 8 0
5 2 0 0 14 11 0 0
93 66 17 10 129 129 13 0 149 91 10 118 67 0
15 23 12 0 17 25 0 2
-152 188 140 96 130 185 55 39
Alive day 5 Discard“
Total number
101 69 9 84 40 0
87 62 15 0 112 111 10 0
137 160 102 63 103 114 27 19
Weaned
~
GC-4072
36 35 27 41 34 0
31 33 34 0 35 31 34 0
34 32 29 28 34 32 34 29
;“g;’
Mean
71 84 90 75 61 0
78 90 68 0 91 86 31 0
97 94 63 42 89 68 16 16
Survival (%)
Weanlings
RATS ON VARIOUS DIETARYLEWLSOF THROUGHTHREEGENERATIONS
0 Number of pups discarded on fifth day to reduce litter size to 10. b One rat died with 14 fetuses in utero. c One rat died with 5 fetuses in utero. d Fertility index = (pregnancies/matings) 100. e Gestation index = (litters cast/pregnancies) 100. f Viability index = (pups surviving 5 days/pups born alive) 100. g Lactation index = (pups weaned/live pups on day 5 less discards) 100.
Generation
--~-
Diet level (ppm)
Number
SUMMARYOFREPRODUCTIONDATAFOR TWOMATINGS
TABLE
90 45 14 70 30 14
70 60 36 29 80 79 50 33
85 90 100 85 79 100 100 85
~__ F.I.d
FOR
100 100 100 100 100 100
100 100 75 100 100 100 100 100
!ii 100 100 100 100 100 100 100
G.I.’
93 97 100 94 91 0
80 93 77 77 94 93 41 0
97 95 80 64 98 96 33 33
V.T.f
Indices (%)
78 87 90 80 68 0
99 97 88 0 97 94 77 0
100 97 80 66 91 71 49 51
L.Ig
AMBROSE ETAL.
334
magnitude as those observed in rats in chronic studies of the same time intervals and dietary levels. The effects of GC-4072 in the diet of rats on other parameters are summarized as follows : no certain effect of diet was observed on the average number of siblings born per litter; the average weaning body weights showed no consistent pattern for the 30 and 100ppm diets, but appear to be depressedin the only generation surviving to weaning on the 300 ppm diet. No gross abnormalities were noted in the newborn in any generation. Surviving F/2b generation rats (18 female and 19 male fed control diet, 18femalesand 18 malesfed 30 ppm diet, and 6 female and 2 malesgiven 100 ppm diet) were sacrificed, and the gonads and the uteri and fallopian tubes of the females were submitted to careful histopathologic study. Follicle formation was evident, ova were seenwithin the follicles, and there was a substantial number of corpora lutea in every section studied. The mucosa of the fallopian tubes and the endometrium of the uterus showed no deviation from the control group. In the testesof males, no evidence of deTABLE 4 BLED /(PLASMA, RED BLOOD CELLS) CHOLINESTERASE VALUES FOR BEAGLEDOGSONDIETARYSUPPLEMENTSOF GC-4072 FOR~O~WEEKS Delta pH units (mean f SD)
Sample Plasma
Weeks 0 1 4 8 12 26 39 52 65 78 91 104
Red blood cells
0 1 4 8 12 26 39 52 65 79 91 104
0 wm
30 pm
200r-vm
1000ppm
0.459* 0.044 0.478xt 0.096 0.450& 0.091 0.4683 0.111 0.484 0.439 0.244 0.460 0.248 0.239 0.094 0.131 0.093 0.100 0.160
f 0.058 iz 0.038 zk 0.135 zk 0.066 f 0.082 f 0.045 f 0.009 zt 0.029 * 0.041 * 0.037 zt 0.097’
0.249 0.108 0.129 0.279 0.189 0.158 0.088 0.116 0.088 0.084 0.090
f 0.040* * 0.070* 7t 0.044 i 0.022* + 0.059 f 0.047* zk 0.006 xk 0.022 * 0.030 & 0.025 h 0.017
0.135 0.084 0.096 0.149 0.099 0.091 0.085 0.110 0.055 0.063 0.068
zt 0.033”
0.116 IIZ0.026*
f 0.01lb 0.059i 0.007* + 0.020 +z 0.052* i 0.014” f 0.028* i 0.004 & 0.010 It 0.037 zt 0.015 & 0.023
0.058 0.080 0.066 0.064 0.090 0.106 0.071 0.053 0.073
i 0.014* zt O.OlS* & 0.012* f 0.012’ & 0.008 * 0.010 i 0.021 rt 0.014 zk 0.014
0.179+ 0.034 0.2095 0.024 0.223* 0.017 0.180& 0.011 0.209 0.151 0.165 0.267 0.173 0.143 0.153 0.148 0.179 0.166 0.152
+z 0.048 +c 0.024 zt 0.013 zt 0.030 rt 0.010 zt 0.009 zt 0.024 zt 0.017 * 0.008 * 0.017 zk 0.022’
0.191 0.129 0.145 0.238 0.158 0.144 0.156 0.146 0.183 0.155 0.138
* 0.041 i 0.041 zk 0.024 xk 0.045 k 0.025 zk 0.014 i 0.030 & 0.014 zt 0.024 zk 0.012 5 0.034
0.225 0.155 0.168 0.256 0.180 0.155 0.175 0.155 0.196 0.131 0.176
& 0.019 h 0.044 f 0.007 + 0.017 zt 0.004 zt 0.008 zk 0.019 zt 0.017 i 0.016 zk 0.029 i 0.021
0.190 0.113 0.106 0.181 0.133 0.121 0.151 0.144 0.144 0.130 0.135
zk 0.009’ h O.OlS* * 0.014* rt 0.017* zt 0.038 zt 0.013 + 0.024 i 0.015 z!t 0.023* zk 0.032 zt 0.053
’ Average of 4 dogs, 2 of each sex, unless otherwise indicated. Dietary level given as parts per million (ppd. ’ Value differs significantly from control, P < 0.05. ’ Average of 3 dogs.
TOXICOLOGIC
STUDIES
ON
cc-4072
335
pressed spermatogenesiswas found, the basement membranes of the tubules were contiguous, there was no atrophy or hypertrophy of the interstitial cells (Leydig cells), and the blood vesselswere normal. Chronic Toxicity Studies in Dogs One dog fed the control basal diet was sacrificed in a moribund state in the 97th week, all other dogs survived the two-year feeding in apparent good health. Body weight data indicated no adverse effects, and food consumption wascomparable at all dietary levels of GC-4072. Hematologic data and urine concentrations of reducing substancesand protein, obtained at frequent intervals, were within normal limits throughout the test period. Plasma and red blood cell ChE values obtained at intervals during this study are summarized in Table 4. Plasma ChE levels were significantly depressedat all dietary levels of GC-4072 through the first 39 weeks. Red blood cell ChE activity was consistently and significantly depressedduring the first 12 weeksonly in dogs fed the 1000ppm diet. It was depressedagain during the 78th week. Liver function data (BSP, SGOT, SAP) and BUN obtained at the end of the 2-year feeding period showed no adverse trends. Organ-to-body weight ratios showed no significant difference between control dogs and dogs on the various dietary levels of GC-4072 for 2 years. Histopathologic findings indicated no lesions attributable to GC-4072.
In two year study in rats and dogs and reproduction studies in rats, no increasein the incidence of tumors was observed. ACKNOWLEDGMENTS
The authorswish to expresstheir appreciationto the following personsfor their assistance with certain aspectsof the experimentalwork reported: Dr. NelsonF. Young of the Division of Clinical Pathology, M.C.V. for cholinesterase determinations;Dr. JosephJ. McPhillips, for in vitro anticholinesterase tests;Dr. E. M. Crawford for veterinary serviceson dogs: Amalyle M. Lea, Patricia W. Small, GeorgeB. Werthan, Edwin H. Talley, Jr., John S. Nicholas,and Edward J. Baker for technical assistance. REFERENCES
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