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Toxicologic studies on 3′,4′-dichloropropionanilide
TOXlCOLOGYASDAPPLlEDPHARMACOLOGY23,650-659(1972)
Toxicologic
Studies
on 3’,4’-DichloropropionanilidelJ
ANTHONY M. AMBROSE,PAUL S. LARSON, JOSEPHF. BORZELLECAANDGORDON R. HENNIGAR,JR. Depurtment of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219, and Department of Pathology, Medical University of South Carolina Charleston, South Carolina 29401 Received March 27,1972
Toxicologic Studieson 3’,4’-Dichloropropionanilide.A~ROSE,ANTHONY M., LARSON, PAUL S., BOIUELLECA, JOSEPH F. and HENNIGAR, GORDON R., JR. (1972).Toxicol. Appl. Pharmacol. 23,650~659. Studieson 3’,4’-dichloropropionanilide (DCPA), a selectiveherbicide, are presented.For rats and dogsthe singleestimatedpo LD50 f SD values are 1384h 99 mg/kg and 1217& 8 mg/kg, respectively. In subchronic (13 wk) studiesin rats on 0, 100, 330, 1000,3300, 10,000and 50,000ppm diets, survival of rats on the 50,000ppm diet wasaffected,and at 10,000and 3300ppm, growth and feed consumptionwere depressed.Polychromatophilia wasobservedin rats on the 1000and 330ppm diets; the effect wasmarked in rats on the 10,000and 3300 ppm diets. Significant decreasesin hemoglobinvalues were found betweencontrols and the 3 highestdiet levelsfor 13 wk. In a chronic (2 yr) study in rats on 0, 100, 400 and 1600ppm diets, significantdecreasein growth and increasein organ-to-body weightsratio for spleen,liver of females,and testesof ratson the 1600ppmdiet werenoted. No dose-relatedeffects were noted on hemograms,except for significant decreases in hemoglobinvaluesfor femaleson the 1600ppm diet, urinary findingsand histopathology. In a 2 yr study in dogson 0,100,600 and 4000 ppm diets, no adversetrends were noted on survival, hematology, urinary findings, liver function tests,organ-to-body weight ratios (with the exception of the heart of dogson the 4000ppm diet) andhistopathology.However, body weight of dogs on 4000 ppm diets was significantly depressed.In multigeneration studiesin rats on 0, 100, 300 and 1000 ppm diets, no apparent deleteriouseffectsor adversetrends werenoted. Histopathologic studies on F/3b weanlingsrevealed no lesions.In summary, this study establishes 400 ppm asthe overall apparent no-effect level. 3’,4’-Dichloropropionanilide (DCPA), a selective herbicide used as a postemergence treatment, has found wide usage,both nationally and internationally, in the control of weedsin rice fields. Liver homogenates from rats, mice, rabbits and dogs were found to contain an enzyme (acylamidase) which hydrolyzed DCPA to 3’,4’-dichloroaniline (DCA) (Williams and Jacobson, 1966). Soil microorganisms appear to transform DCPA to DCA, and DCA presumably condenses to 3,3’,4,4’-tetrachloroazobenzene (TCAB) 1Supportedby a grant from the Rohm and HaasCompany,Philadelphia,Pennsylvania19105, who alsosuppliedthe DCPA usedin this study. 2Stam@ (trademarkof the Rohmand HaasCompany),referredto in the literature,asPropanil, Propanid,DPA, Stam, FW-734 and in commercialformulationscontainingDCPA referredto: StamF-34,Surcopur,Rogue,StamLV-10. Copyright 0 1972 by Academic Press, Inc. 650 All rights of reproduction in any form reserved.
TOXICITY
651
OF DICHLOROPROPIONANILlDE
(Bartha and Pramer, 1967). In rice plants DCPA is metabolized to DCA and complexedDCA (Still, 1968) and to DCA-lignin complexes (Yih et al., 1968). In both of the latter studies no evidence was found that TCAB was one of the metabolites of DCPA in rice. Bartha and Pramer (1970) present a detailed review and discussion of aniline-based (acylanilide) herbicides. In the present report, studies are presented on the acute, subchronic and chronic physiologic effects of DCPA in rats and dogs, and on reproduction and Iactation in rats. METHODS Material. Technical grade DCPA used in this study contained 97% 3,bdichloropropionanilide, 0.9 % dichloroaniline and 2 % unknowns. It is a dark brown crystalline material with a slight aromatic odor, melting point 92-93°C and a vapor pressure of 0.001 mm at 50°C. It is relatively insoluble in water (0.02 %) but readily soluble in most organic solvents and corn oil. The chemical structure of DCPA is shown in Fig. 1. Cl
I
I0
-
Cl
/
FIG. 1. Structural formula of DCPA (Stam@) 3’,4’-dichloropropionanilide.
Acute Oral Toxicity
The acute po toxicity of DCPA was determined in albino rats3 and in dogs by gastric intubation of a 10 y0 (w/v) solution in corn oil. All animals were fasted overnight prior to dosing, and observed daily for 7 days following treatment. The LD50 values were estimated by the method of Berkson (1955) on 7 day survivors. Rats. Ten male rats (average weight 140 g) were used in each of 4 dose groups to establish the LD50. Doses ranged from 1 to 2 g/kg. Dogs. Mongrel dogs (average weight 8 kg) of both sexes were used in groups of 5 for 0.5, 1.0 and 1.5 g/kg. To prevent emesis, morphine sulfate, 20 mg/kg, was administered SC1 hr before dosing. Subchronic Oral Toxicity Rats. In a preliminary 3 mo study, young albino ratq3 culled to a narrow range of
starting weights averaging 94 g for males and 85 g for females, in groups of 10 of each sex were placed on each of the following dietary concentrations of DCPA: 0, 100, 333, 1000, 3300, 10,000 and 50,000 ppm. Finely ground commercial meal4 served as 3 Wistar derived, obtained from Albino Farms, Red Bank, New Jersey. 4 Purina Dog Chow Kibbled Meal, Ralston Purina Company, St. Louis, Missouri.
652
AMBROSE ET AL.
the basic diet into which was incorporated, with thorough mixing, weighed amounts of finely ground DCPA to yield the desired dietary concentrations. Diets were prepared fresh each week and fed ad libitum. All rats were individually caged and weighed weekly. Feed consumption data were obtained over a 3 day period during week 13; hematologic values (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex on each dietary concentration during week 13; urinary tests for glucose and protein were made during the same period on urines pooled as to sex and dietary levels of DCPA. Survivors were sacrificed after 3 mo on diet, and organ-to-body weight ratios were determined for heart, spleen, kidneys, liver and testes. Histopathologic studies were made on the following tissues, preserved in 10 % formalin: heart, lung, liver, pituitary, kidney, spleen, stomach, large and small intestine, urinary bladder, bone marrow (rib, femur and vertebrae), muscle, skin, brain, thyroid, adrenal, pancreas and gonad from all 3 mo survivors, excepting rats on the 100 ppm diet. Body weight changes, organ-to-body weight ratios and hemoglobin values were statistically evaluated by the Duncan (1955) multiple range and multiple F test. Dogs. In a preliminary (4 wk) study, mongrel dogs, two of each sex, individually housed, were placed on each of the following dietary concentrations of DCPA: 0, 2000, 10,000 and 50,000 ppm. The stock diet consisted of 87% ground basal diet,5. 12% corn oil6 and 1% USP cod liver oil, prepared fresh daily. To the stock diet were added, with thorough mixing, amounts of DCPA calculated to yield the above concentrations. At feeding time, the feed was moistened with an equal weight of water. Feed consumption was measured daily over a 2 hr period, and the dogs were weighed weekly. Chronic Oral Toxicity (2 yr)
Rats. Using littermate distribution, young albino rats (Wistar-derived)3 were separated into four groups of 50 rats each, balanced as to sex and initial body weight, and placed on one of the following dietary concentrations of DCPA: 0, 100, 400 and 1600 ppm. Finely ground commercial meal4 served as the basic diet into which was incorporated, with thorough mixing, a weighed amount of DCPA to yield the 1600 ppm diet. Lower dietary concentrations were prepared by suitable dilution with the basic diet. Diets were prepared fresh each week and fed ad libitum. Rats were individually caged. Body weights were obtained weekly. Feed consumption data were obtained over 3 day periods at the end of 1,3,6, 12 and 24 mo. Hematologic values (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex on each dietary concentration at 3 mo intervals. Urinary tests for reducing substances and proteins were made at 3 mo intervals on urines pooled for sex and diet. Hemoglobin data obtained at 3, 6, 12 and 24 mo were evaluated by the Duncan test (I 955). Body weight changes at various time intervals and organ-to-body weight ratios for heart, liver, kidneys, spleen and testes, obtained at sacrifice of all 2 yr survivors, were also evaluated by the Duncan test. Tissues preserved in 10% formalin, for histopathologic study on rats sacrificed at term, and those dying or sacrificed in extremis. 5 Basal Ration f/dm; A Prescription Diet @,Hill Packing Company, Topeka, Kansas. 6 Mazola@ Oil, Corn Products Company, New York, New York.
TOXICITY
OF DICHLOROPROPIONANILlDE
653
in which no detectable autolysis had set in, were the same as those in the subchronic study described above. Dogs. Two purebred beagle dogs of each sex, approximately 6 mo old, were placed on each of the following dietary concentrations of DCPA: 0, 100, 600 and 3000 ppm #(changed to 4000 ppm at start of week 5). The stock diet used and its formulation are described under the subchronic dog studies above. To the basic diet was added. with thorough mixing, an amount of DCPA to yield the highest dietary concentration. The lower concentrations were prepared by suitable dilution with the basic diet. Prior to being placed on diet, the dogs were immunized against distemper, infectious hepatitis and leptospirosis, and treated as needed for intestinal parasites. The following were obtained on all dogs at start and at 3 mo intervals thereafter: hematologic values (hematocrit, hemoglobin, total and differential leukocyte counts), and urinary tests for reducing substances and protein. Bromosulfalein (BSP) retention, serum glutamic-oxaloacetic acid transaminase (SGOT) and serum alkaline phosphatase (SAP) values were obtained at 24 mo. At term (2 yr), all dogs were sacrificed. Organ weights were obtained and organ-tobody weight ratios were determined for heart, spleen, kidneys, liver and testes. Tissues preserved in lop/; formalin and subjected to histopathologic study included heart. lung, liver, kidney, spleen, stomach, large and small intestine, urinary bladder, skin. skeletal muscle, mesenteric lymph nodes, bone marrow, brain, pituitary. thyroid, pancreas, adrenal and gonads. Reproduction-Rats
A three-generation study was undertaken in albino rats3 in order to determine stress effects of DCPA on reproduction and lactation. Littermate rats, 28 days old, within but not between sexes, separated into 4 groups of 50 rats each and balanced for sex and initial body weight, were individually caged to constitute the F/O generation. One group was placed on each of the following dietary concentrations of DCPA: 0. 100, 300 and 1000 ppm. The stock diet used and incorporation of DCPA in the diet are described under chronic rat studies. After 11 wk on the above dietary regimens, 20 females on each diet were transferred to individual breeding cages, and each was mated with a male on the same dietary concentration of DCPA for the F/la generation. Male rats within each group were rotated to a different female on each of 3 successive 7 day periods. Prior to parturition, male rats were removed from breeding cages. Records were kept of mating, number of pregnancies, number of litters cast, siblings in litters at I, 5, and 21 days (weaning) and total body weight of the litter at weaning. Litters containing more than 10 were randomly reduced to this number on day 5. Indices calculated for each generation were : FI (fertility) = (pregnancies/matings) x 100; GI (gestation) = (litters cast/pregnancies) x 100; VI (viability) = (live pups at day S/live pups born) x 100; LI (lactation) =~ (weaned/live pups - discards on day 5) x 100. All surviving F/la rats at weaning were autopsied. At approximately 10 days after weaning of last F/la litters, F/O rats were remated as above to produce F/lb litters. Following weaning of F/lb rats, all surviving F/O generation rats were autopsied. Twenty-five F/lb rats of each sex from each diet concentration were continued on their respective parental diets for 11 wk, at which time 20 rats of each sex within each
654
ET AL.
AMBROSE
diet group were mated, and the same procedure followed as with the F/O generation, to produce F/2a and Fj2b litters. At weaning of Fj2b rats, all parent F/lb rats were autopsied. F/2b rats were continued through the same procedure as with the F/lb generation through production and weaning of F/3a and F/3b generation litters. Histopathologic studies were performed on F/3b weanlings, 10 of each sex, from each diet concentration. Tissues examined were the same as those described in the 2 yr study.
RESULTS
AND DISCUSSION
Acute Oral Toxicity
The LD50 + SD values were 1384 f 99 mg/kg for rats and 1217 1.8 mg/kg for dogs, as determined by the method of Berkson (1955). In both rats and dogs deaths occurred over a 3 day period. Toxicity was characterized by central nervous system depression, which appeared within 12 hr following gastric intubation. Gross autopsy findings were negative. Subchronic Oral Toxicity Rats. In the 3 mo study, survival in all groups excepting rats on the 50,000 ppm diet was unaffected. In rats on 3300 and 10,000 ppm diets, growth (weight) with time was depressed, and feed consumption, as measured during week 13, was lower by more than 10 % of the respective controls. Hematologic findings in rats on 330 and 1000 ppm diets showed some increase in polychromatophilia as compared to controls. At 3300 and 10,000 ppm dietary concentrations this became quite marked, together with appearance of immature forms of red cells not normally found in peripheral blood.
TABLE 1 ORGAN-TO-BODY WEIGHT ~‘,~‘-DI~HLoR~PRo~I~~L~NILIDI~
Sex
Diet cont. (wm>
Female
0
Ratio of organ-to-body weight (g/kg f SD) No. of Heart rats
3300
9
10,OOO
10
3.18 f 0.48“
4.10
0 loo
10 9
330
10 10 10 9
2.49 &I.22 2.43 IIC0.23 2.52 * 0.16 2.67 rt 0.34 2.59 & 0.42 2.88 f- 0.47”
1.53 f0.85 1.31 i 0.22 1.39 k 0.24 1.58 * 0.22 2.64 f 0.60” 4.04 f 0.77”
330 1OOO
Male
1OOO
3300 10,OOO differs
10 10
Spleen
2.77 f 0.31 2.80&0.41 2.77 f 0.24 2.94 * 0.27 2.95 ho.42
100
’ Value
RATIO FOR RATS RECEIVING IN THEIR DIET FOR 3 MONTHS
10 10
significantly
from
Kidney
Liver
1.67 f 0.38 1.77 zt 0.34 1.91 zk0.20 2.05 f 0.27” 3.16 rtO.46”
6.32 f 0.65 6.78 zt0.56 7.05 * 0.66 6.49 * 0.65 7.26 xtO.57”
30.7 f 4.5 34.9 54.3 35.6 f 3.4 33.3 f 5.1 36.2 f 5.2”
6.79 zk 0.61
46.5
6.61 ht.72 6.33 i 0.36 7.10 + 0.30 6.56 r!z0.52 6.65 zt 0.54 7.27 rt 0.77
35.7 ht.8
f 0.94”
control, p < 0.05.
Testes
xk 4.8”
8.26f 1.20 f 0.92 x!z0.94 f 1.31 f 0.63 f 2.33“
3.10% 3.2 7.88 36.8 i 2.6 7.72 36.5 +c6.2 8.79 37.1 * 5.7 9.84 39.3 f 4.9 14.01
TOXICITY
OF DICHLOROPROPIONANILIDE
655
656
AMBROSE ET AL.
Hemoglobin values of both sexes on 1000,330O and 10,000 ppm diets were significantly decreased (p < 0.05). These values were 15.6 & 1.1 for the controls and 13.5 & 1.9, 13.4 & 1.1, and 12.7 + 0.9 for the three dietary levels of DCPA, respectively. From these observations, it is concluded that there is a definite indication of hemolytic anemia at the 3300 and 10,000 ppm dietary levels of DCPA. Semiquantitative tests for urinary sugar and protein showed no effects of treatment. Organ-to-body weight ratio data are summarized in Table 1. Statistically significant higher ratios values from controls were found for heart of rats on the 10,000 ppm diet, spleen of females on the 1000 ppm and higher diets, and for males on the 3300 and 10,000 ppm diets. Kidney ratios were significantly elevated only in females on the 3300 ppm diets. Liver ratio for females only on the 3300 and 10,000 ppm diets, and testes ratios for rats on the 10,000 ppm diet were significantly elevated. Increases in spleen size are in line with the hematologic appearance of hemolytic anemia, but the other findings may be nonspecific accompaniments of depressed growth. Histopathologic examination revealed no lesions that might be attributed to DCPA, except for a few instances of hydropic change in the liver cells accompanied by bile nephrosis in females on the 10,000 ppm diet. Dogs. Mongrel dogs in the 4 wk study: only dogs on the 10,000 and 50,000 ppm diets showed effects on decreased growth and feed consumption. Dogs on 2000 ppm showed no effects. Chronic Oral Toxicity Rats. Data on body weight and mortality at representative intervals for rats are summarized in Table 2. Increased mortality occurred only in males on the 1600 ppm diet, and this appeared only after 20 mo. Rats of both sexeson the 1600 ppm diet showed significant depression in body weight from onset to end. Rats on other dietary concentrations showed no significant departure in body weight from controls. Feed consumption data obtained at 4, 13, 26, 52 and 104 wk showed no definite trends with increasing dietary concentrations of DCPA. Hematologic values, except for rats on the 1600 ppm TABLE 3 AVERAGES OF ORGAN-TO-BODY WEIGHT RATIO DATA OF RATS RECEIVING 3’,4’-DICHL~R~PR~PI~N~NILIDE IN THEIR DIET FOR Two YEARS Diet
Ratio of organ-to-body
weight (g/kg =L SD)
cont. No. of Sex
Female
(ppm)
0 100 400 1600
Male
0 loo 400 1600
Rats
1.5 10 12 12 17 15 11 5
Heart 3.2 3.2 3.4 3.4 2.6 2.7 2.6 3.1
& i i * f f j, rt
0.4 0.4 0.5 0.5 0.3 0.3 0.3 0.6
Spleen 1.8 2.2 2.3 3.3 1.7 1.9 2.0 2.4
4z 0.3 * 0.6 i 0.7” 5 0.8” f 0.5 f 0.7 i 0.6 f 0.3”
a Value differs significantly from control, p < 0.05.
Kidney 8.1 8.0 7.5 8.3 6.9 7.2 7.5 7.5
f * f f i f It It
1.6 1.4 1.5 1.3 1.3 0.9 0.9 0.9
Liver 32.2 34.1 32.1 40.5 30.9 33.0 34.7 32.1
f * f It & rt h f
Testes 4.5 4.3 6.5 8.6” 6.8 7.5 5.7 5.0
6.1 6.2 6.5 8.5
f 1.4 zk 0.9 + 1.2 zk 1.4”
4 4 4 4
0 loo 600 4000
7.7 7.8 7.8 7.9
i 1.2 i 0.8 zt 0.0 zk 2.0
Initial -__~. 14.98 14.70 14.40 11.55
i It i *
(mean * SD) Terminal .--~
0 Two dogs of each sex per diet level. 6 Value differs significantly from control, p G 0.05.
No. of dogs” -_____~
Diet cont. (w-4
Body weight (g)
2.5 1.3 0.7 0.9
AND ORGAN-TO-BODY 3’,4’-DICHLOROPROPIONANILIDE
AVERAGE BODY WEIGHTS
TABLE
6.69 7.75 7.17 7.51
zt It + t
0.53 1 .oo 1.27 0.16’
Heart 6.93 3.99 6.18 7.21
zk 1.52 * 1.41* * 1.28 -c 2.02
Spleen 4.81 5.05 5.29 6.11
zt 1.15 + 1.57 dz 1.05 _L 0.75
27.78 24.78 28.40 26.93
f i I h
Liver
weight (g/kg I SD) Kidney
Dons RECEIVING YEARS
OF
Ratio of organ-to-body
WEIGHT RATIO DATA IN THEIR DIET FOR TWO
4
5.5 5.9 3.5 3.0
-
z F ii
54 Fl 2 $ c;r ij F g $ g 3 0
*
658
AMBROSE
ET AL.
diet, were within the range of rats on the control diet. Statistically significant (p < 0.05) decreases in hemoglobin values were found for females only on the 1600 ppm diet at 3,6, 12 and 24 mo. The hemoglobin values for females at each period were, respectively: 15.4 f 1.2, 16.1 & 1.0, 15.7 % 0.4 and 14.0 * 0.7 for the controls, as compared to 14.3 + 1.1,13.6 % 0.8, 13.2 & 0.6, and 13.4 & 0.4 for rats on the 1600 ppm diet. Urinary tests for reducing substances and protein were within the range for control rats. Organ-to-body weight ratio data are summarized in Table 3. Significantly higher ratios were found for spleen of rats of both sex, for liver of female rats and for testes of rats on the 1600 ppm diet. Histopathologic findings revealed no lesions in kind or incidence that were not found in the control animals. Dogs. Beagle dogs on 0, 100, 600 and 4000 ppm DCPA survived the 2 yr feeding period. Significantly depressed weight gains were evident in dogs on 4000 ppm from about week 6, and persisted throughout the rest of the study period. Feed consumption data for dogs on 4000 ppm for each test period indicated greater consumption on a g/kg body weight and a decrease in feed efficiency. Hematologic values and urinary tests for reducing substances and protein ranged within normal limits throughout the test period. The BSP, SGOT and SAP tests made during the twenty-fourth month showed no statistically significant differences from the controls. Organ-to-body weight ratio data are summarized in Table 4. There is no indication of a specific effect of DCPA on a target organ. Histopathologic findings revealed no pathologic changes resulting from the ingestion of DCPA for 2 yr. Reproduction
Studies in Rats
In the three-generation reproduction study, body weights for parent generation rats at mating and at weaning of respective litters were not adversely affected. Data on reproduction performance through three generations showed no deleterious effects in any of the parameters recorded for rats on 0,100,300 and 1000 ppm diet levels of DCPA. No gross abnormalities were found in fetuses born dead or alive or in rats autopsied with fetuses in situ. Histopathologic examination of F/3b generation weanlings revealed no lesions, in kind or incidence, attributable to DCPA. In conclusion, under conditions of the experimental procedures, the highest overall apparent no-effect level established by this study was 400ppm in the diet of rats and dogs.
ACKNOWLEDGMENTS The authors are indebted to Patricia A. Weil, James E. Baker and Edwin H. Talley, Jr. for technical assistance, and to Dr. E. M. Crawford (deceased) for veterinary service on dogs.
REFERENCES BARTHA, R. and PRAMER,D. (1967). Pesticide transformation
to aniline and azo compounds
in soil. Science 156, 1617-1618. BARTHA, R. and PRAMER, D. (1970). Metabolism of acylanilide herbicides. Adoan. Appl. Microbial. 13, 317-341. BERKSON, J. (1955). Estimate of the integrated normal curve by minimum normit chi-square with particular reference to bio-assay. J. Amer. Statist. Ass. 50, 529-549.
TOXICITY
OF DICHLOROPROPIONANILIDE
659
DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. STILL, G. G. (1968).Metabolism of 3,4-dichloropropionanilidein plants: The metabolicfate of 3,4-dichloroanilinemoiety. Science 159,992-993. WILLIAMS, C. H. and JACOBSON, K. H. (1966).An acylamidasein mammalianliver hydrolyzing the herbicide3,4-dichloropropionanilide.Toxicol. Appl. Pharmacol. 9, 495-500. YIH, R. Y., MCRAE,D. H. and WILSON, H. F. (1968).Metabolism of 3’,4’-dichloropropionanilide: 3,4-dichloroaniline-lignincomplex in rice plants. Science 161, 376-377.
Toxicologic
Studies
on 3’,4’-DichloropropionanilidelJ
ANTHONY M. AMBROSE,PAUL S. LARSON, JOSEPHF. BORZELLECAANDGORDON R. HENNIGAR,JR. Depurtment of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219, and Department of Pathology, Medical University of South Carolina Charleston, South Carolina 29401 Received March 27,1972
Toxicologic Studieson 3’,4’-Dichloropropionanilide.A~ROSE,ANTHONY M., LARSON, PAUL S., BOIUELLECA, JOSEPH F. and HENNIGAR, GORDON R., JR. (1972).Toxicol. Appl. Pharmacol. 23,650~659. Studieson 3’,4’-dichloropropionanilide (DCPA), a selectiveherbicide, are presented.For rats and dogsthe singleestimatedpo LD50 f SD values are 1384h 99 mg/kg and 1217& 8 mg/kg, respectively. In subchronic (13 wk) studiesin rats on 0, 100, 330, 1000,3300, 10,000and 50,000ppm diets, survival of rats on the 50,000ppm diet wasaffected,and at 10,000and 3300ppm, growth and feed consumptionwere depressed.Polychromatophilia wasobservedin rats on the 1000and 330ppm diets; the effect wasmarked in rats on the 10,000and 3300 ppm diets. Significant decreasesin hemoglobinvalues were found betweencontrols and the 3 highestdiet levelsfor 13 wk. In a chronic (2 yr) study in rats on 0, 100, 400 and 1600ppm diets, significantdecreasein growth and increasein organ-to-body weightsratio for spleen,liver of females,and testesof ratson the 1600ppmdiet werenoted. No dose-relatedeffects were noted on hemograms,except for significant decreases in hemoglobinvaluesfor femaleson the 1600ppm diet, urinary findingsand histopathology. In a 2 yr study in dogson 0,100,600 and 4000 ppm diets, no adversetrends were noted on survival, hematology, urinary findings, liver function tests,organ-to-body weight ratios (with the exception of the heart of dogson the 4000ppm diet) andhistopathology.However, body weight of dogs on 4000 ppm diets was significantly depressed.In multigeneration studiesin rats on 0, 100, 300 and 1000 ppm diets, no apparent deleteriouseffectsor adversetrends werenoted. Histopathologic studies on F/3b weanlingsrevealed no lesions.In summary, this study establishes 400 ppm asthe overall apparent no-effect level. 3’,4’-Dichloropropionanilide (DCPA), a selective herbicide used as a postemergence treatment, has found wide usage,both nationally and internationally, in the control of weedsin rice fields. Liver homogenates from rats, mice, rabbits and dogs were found to contain an enzyme (acylamidase) which hydrolyzed DCPA to 3’,4’-dichloroaniline (DCA) (Williams and Jacobson, 1966). Soil microorganisms appear to transform DCPA to DCA, and DCA presumably condenses to 3,3’,4,4’-tetrachloroazobenzene (TCAB) 1Supportedby a grant from the Rohm and HaasCompany,Philadelphia,Pennsylvania19105, who alsosuppliedthe DCPA usedin this study. 2Stam@ (trademarkof the Rohmand HaasCompany),referredto in the literature,asPropanil, Propanid,DPA, Stam, FW-734 and in commercialformulationscontainingDCPA referredto: StamF-34,Surcopur,Rogue,StamLV-10. Copyright 0 1972 by Academic Press, Inc. 650 All rights of reproduction in any form reserved.
TOXICITY
651
OF DICHLOROPROPIONANILlDE
(Bartha and Pramer, 1967). In rice plants DCPA is metabolized to DCA and complexedDCA (Still, 1968) and to DCA-lignin complexes (Yih et al., 1968). In both of the latter studies no evidence was found that TCAB was one of the metabolites of DCPA in rice. Bartha and Pramer (1970) present a detailed review and discussion of aniline-based (acylanilide) herbicides. In the present report, studies are presented on the acute, subchronic and chronic physiologic effects of DCPA in rats and dogs, and on reproduction and Iactation in rats. METHODS Material. Technical grade DCPA used in this study contained 97% 3,bdichloropropionanilide, 0.9 % dichloroaniline and 2 % unknowns. It is a dark brown crystalline material with a slight aromatic odor, melting point 92-93°C and a vapor pressure of 0.001 mm at 50°C. It is relatively insoluble in water (0.02 %) but readily soluble in most organic solvents and corn oil. The chemical structure of DCPA is shown in Fig. 1. Cl
I
I0
-
Cl
/
FIG. 1. Structural formula of DCPA (Stam@) 3’,4’-dichloropropionanilide.
Acute Oral Toxicity
The acute po toxicity of DCPA was determined in albino rats3 and in dogs by gastric intubation of a 10 y0 (w/v) solution in corn oil. All animals were fasted overnight prior to dosing, and observed daily for 7 days following treatment. The LD50 values were estimated by the method of Berkson (1955) on 7 day survivors. Rats. Ten male rats (average weight 140 g) were used in each of 4 dose groups to establish the LD50. Doses ranged from 1 to 2 g/kg. Dogs. Mongrel dogs (average weight 8 kg) of both sexes were used in groups of 5 for 0.5, 1.0 and 1.5 g/kg. To prevent emesis, morphine sulfate, 20 mg/kg, was administered SC1 hr before dosing. Subchronic Oral Toxicity Rats. In a preliminary 3 mo study, young albino ratq3 culled to a narrow range of
starting weights averaging 94 g for males and 85 g for females, in groups of 10 of each sex were placed on each of the following dietary concentrations of DCPA: 0, 100, 333, 1000, 3300, 10,000 and 50,000 ppm. Finely ground commercial meal4 served as 3 Wistar derived, obtained from Albino Farms, Red Bank, New Jersey. 4 Purina Dog Chow Kibbled Meal, Ralston Purina Company, St. Louis, Missouri.
652
AMBROSE ET AL.
the basic diet into which was incorporated, with thorough mixing, weighed amounts of finely ground DCPA to yield the desired dietary concentrations. Diets were prepared fresh each week and fed ad libitum. All rats were individually caged and weighed weekly. Feed consumption data were obtained over a 3 day period during week 13; hematologic values (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex on each dietary concentration during week 13; urinary tests for glucose and protein were made during the same period on urines pooled as to sex and dietary levels of DCPA. Survivors were sacrificed after 3 mo on diet, and organ-to-body weight ratios were determined for heart, spleen, kidneys, liver and testes. Histopathologic studies were made on the following tissues, preserved in 10 % formalin: heart, lung, liver, pituitary, kidney, spleen, stomach, large and small intestine, urinary bladder, bone marrow (rib, femur and vertebrae), muscle, skin, brain, thyroid, adrenal, pancreas and gonad from all 3 mo survivors, excepting rats on the 100 ppm diet. Body weight changes, organ-to-body weight ratios and hemoglobin values were statistically evaluated by the Duncan (1955) multiple range and multiple F test. Dogs. In a preliminary (4 wk) study, mongrel dogs, two of each sex, individually housed, were placed on each of the following dietary concentrations of DCPA: 0, 2000, 10,000 and 50,000 ppm. The stock diet consisted of 87% ground basal diet,5. 12% corn oil6 and 1% USP cod liver oil, prepared fresh daily. To the stock diet were added, with thorough mixing, amounts of DCPA calculated to yield the above concentrations. At feeding time, the feed was moistened with an equal weight of water. Feed consumption was measured daily over a 2 hr period, and the dogs were weighed weekly. Chronic Oral Toxicity (2 yr)
Rats. Using littermate distribution, young albino rats (Wistar-derived)3 were separated into four groups of 50 rats each, balanced as to sex and initial body weight, and placed on one of the following dietary concentrations of DCPA: 0, 100, 400 and 1600 ppm. Finely ground commercial meal4 served as the basic diet into which was incorporated, with thorough mixing, a weighed amount of DCPA to yield the 1600 ppm diet. Lower dietary concentrations were prepared by suitable dilution with the basic diet. Diets were prepared fresh each week and fed ad libitum. Rats were individually caged. Body weights were obtained weekly. Feed consumption data were obtained over 3 day periods at the end of 1,3,6, 12 and 24 mo. Hematologic values (hematocrit, hemoglobin, and total and differential leukocyte counts) were obtained on 5 rats of each sex on each dietary concentration at 3 mo intervals. Urinary tests for reducing substances and proteins were made at 3 mo intervals on urines pooled for sex and diet. Hemoglobin data obtained at 3, 6, 12 and 24 mo were evaluated by the Duncan test (I 955). Body weight changes at various time intervals and organ-to-body weight ratios for heart, liver, kidneys, spleen and testes, obtained at sacrifice of all 2 yr survivors, were also evaluated by the Duncan test. Tissues preserved in 10% formalin, for histopathologic study on rats sacrificed at term, and those dying or sacrificed in extremis. 5 Basal Ration f/dm; A Prescription Diet @,Hill Packing Company, Topeka, Kansas. 6 Mazola@ Oil, Corn Products Company, New York, New York.
TOXICITY
OF DICHLOROPROPIONANILlDE
653
in which no detectable autolysis had set in, were the same as those in the subchronic study described above. Dogs. Two purebred beagle dogs of each sex, approximately 6 mo old, were placed on each of the following dietary concentrations of DCPA: 0, 100, 600 and 3000 ppm #(changed to 4000 ppm at start of week 5). The stock diet used and its formulation are described under the subchronic dog studies above. To the basic diet was added. with thorough mixing, an amount of DCPA to yield the highest dietary concentration. The lower concentrations were prepared by suitable dilution with the basic diet. Prior to being placed on diet, the dogs were immunized against distemper, infectious hepatitis and leptospirosis, and treated as needed for intestinal parasites. The following were obtained on all dogs at start and at 3 mo intervals thereafter: hematologic values (hematocrit, hemoglobin, total and differential leukocyte counts), and urinary tests for reducing substances and protein. Bromosulfalein (BSP) retention, serum glutamic-oxaloacetic acid transaminase (SGOT) and serum alkaline phosphatase (SAP) values were obtained at 24 mo. At term (2 yr), all dogs were sacrificed. Organ weights were obtained and organ-tobody weight ratios were determined for heart, spleen, kidneys, liver and testes. Tissues preserved in lop/; formalin and subjected to histopathologic study included heart. lung, liver, kidney, spleen, stomach, large and small intestine, urinary bladder, skin. skeletal muscle, mesenteric lymph nodes, bone marrow, brain, pituitary. thyroid, pancreas, adrenal and gonads. Reproduction-Rats
A three-generation study was undertaken in albino rats3 in order to determine stress effects of DCPA on reproduction and lactation. Littermate rats, 28 days old, within but not between sexes, separated into 4 groups of 50 rats each and balanced for sex and initial body weight, were individually caged to constitute the F/O generation. One group was placed on each of the following dietary concentrations of DCPA: 0. 100, 300 and 1000 ppm. The stock diet used and incorporation of DCPA in the diet are described under chronic rat studies. After 11 wk on the above dietary regimens, 20 females on each diet were transferred to individual breeding cages, and each was mated with a male on the same dietary concentration of DCPA for the F/la generation. Male rats within each group were rotated to a different female on each of 3 successive 7 day periods. Prior to parturition, male rats were removed from breeding cages. Records were kept of mating, number of pregnancies, number of litters cast, siblings in litters at I, 5, and 21 days (weaning) and total body weight of the litter at weaning. Litters containing more than 10 were randomly reduced to this number on day 5. Indices calculated for each generation were : FI (fertility) = (pregnancies/matings) x 100; GI (gestation) = (litters cast/pregnancies) x 100; VI (viability) = (live pups at day S/live pups born) x 100; LI (lactation) =~ (weaned/live pups - discards on day 5) x 100. All surviving F/la rats at weaning were autopsied. At approximately 10 days after weaning of last F/la litters, F/O rats were remated as above to produce F/lb litters. Following weaning of F/lb rats, all surviving F/O generation rats were autopsied. Twenty-five F/lb rats of each sex from each diet concentration were continued on their respective parental diets for 11 wk, at which time 20 rats of each sex within each
654
ET AL.
AMBROSE
diet group were mated, and the same procedure followed as with the F/O generation, to produce F/2a and Fj2b litters. At weaning of Fj2b rats, all parent F/lb rats were autopsied. F/2b rats were continued through the same procedure as with the F/lb generation through production and weaning of F/3a and F/3b generation litters. Histopathologic studies were performed on F/3b weanlings, 10 of each sex, from each diet concentration. Tissues examined were the same as those described in the 2 yr study.
RESULTS
AND DISCUSSION
Acute Oral Toxicity
The LD50 + SD values were 1384 f 99 mg/kg for rats and 1217 1.8 mg/kg for dogs, as determined by the method of Berkson (1955). In both rats and dogs deaths occurred over a 3 day period. Toxicity was characterized by central nervous system depression, which appeared within 12 hr following gastric intubation. Gross autopsy findings were negative. Subchronic Oral Toxicity Rats. In the 3 mo study, survival in all groups excepting rats on the 50,000 ppm diet was unaffected. In rats on 3300 and 10,000 ppm diets, growth (weight) with time was depressed, and feed consumption, as measured during week 13, was lower by more than 10 % of the respective controls. Hematologic findings in rats on 330 and 1000 ppm diets showed some increase in polychromatophilia as compared to controls. At 3300 and 10,000 ppm dietary concentrations this became quite marked, together with appearance of immature forms of red cells not normally found in peripheral blood.
TABLE 1 ORGAN-TO-BODY WEIGHT ~‘,~‘-DI~HLoR~PRo~I~~L~NILIDI~
Sex
Diet cont. (wm>
Female
0
Ratio of organ-to-body weight (g/kg f SD) No. of Heart rats
3300
9
10,OOO
10
3.18 f 0.48“
4.10
0 loo
10 9
330
10 10 10 9
2.49 &I.22 2.43 IIC0.23 2.52 * 0.16 2.67 rt 0.34 2.59 & 0.42 2.88 f- 0.47”
1.53 f0.85 1.31 i 0.22 1.39 k 0.24 1.58 * 0.22 2.64 f 0.60” 4.04 f 0.77”
330 1OOO
Male
1OOO
3300 10,OOO differs
10 10
Spleen
2.77 f 0.31 2.80&0.41 2.77 f 0.24 2.94 * 0.27 2.95 ho.42
100
’ Value
RATIO FOR RATS RECEIVING IN THEIR DIET FOR 3 MONTHS
10 10
significantly
from
Kidney
Liver
1.67 f 0.38 1.77 zt 0.34 1.91 zk0.20 2.05 f 0.27” 3.16 rtO.46”
6.32 f 0.65 6.78 zt0.56 7.05 * 0.66 6.49 * 0.65 7.26 xtO.57”
30.7 f 4.5 34.9 54.3 35.6 f 3.4 33.3 f 5.1 36.2 f 5.2”
6.79 zk 0.61
46.5
6.61 ht.72 6.33 i 0.36 7.10 + 0.30 6.56 r!z0.52 6.65 zt 0.54 7.27 rt 0.77
35.7 ht.8
f 0.94”
control, p < 0.05.
Testes
xk 4.8”
8.26f 1.20 f 0.92 x!z0.94 f 1.31 f 0.63 f 2.33“
3.10% 3.2 7.88 36.8 i 2.6 7.72 36.5 +c6.2 8.79 37.1 * 5.7 9.84 39.3 f 4.9 14.01
TOXICITY
OF DICHLOROPROPIONANILIDE
655
656
AMBROSE ET AL.
Hemoglobin values of both sexes on 1000,330O and 10,000 ppm diets were significantly decreased (p < 0.05). These values were 15.6 & 1.1 for the controls and 13.5 & 1.9, 13.4 & 1.1, and 12.7 + 0.9 for the three dietary levels of DCPA, respectively. From these observations, it is concluded that there is a definite indication of hemolytic anemia at the 3300 and 10,000 ppm dietary levels of DCPA. Semiquantitative tests for urinary sugar and protein showed no effects of treatment. Organ-to-body weight ratio data are summarized in Table 1. Statistically significant higher ratios values from controls were found for heart of rats on the 10,000 ppm diet, spleen of females on the 1000 ppm and higher diets, and for males on the 3300 and 10,000 ppm diets. Kidney ratios were significantly elevated only in females on the 3300 ppm diets. Liver ratio for females only on the 3300 and 10,000 ppm diets, and testes ratios for rats on the 10,000 ppm diet were significantly elevated. Increases in spleen size are in line with the hematologic appearance of hemolytic anemia, but the other findings may be nonspecific accompaniments of depressed growth. Histopathologic examination revealed no lesions that might be attributed to DCPA, except for a few instances of hydropic change in the liver cells accompanied by bile nephrosis in females on the 10,000 ppm diet. Dogs. Mongrel dogs in the 4 wk study: only dogs on the 10,000 and 50,000 ppm diets showed effects on decreased growth and feed consumption. Dogs on 2000 ppm showed no effects. Chronic Oral Toxicity Rats. Data on body weight and mortality at representative intervals for rats are summarized in Table 2. Increased mortality occurred only in males on the 1600 ppm diet, and this appeared only after 20 mo. Rats of both sexeson the 1600 ppm diet showed significant depression in body weight from onset to end. Rats on other dietary concentrations showed no significant departure in body weight from controls. Feed consumption data obtained at 4, 13, 26, 52 and 104 wk showed no definite trends with increasing dietary concentrations of DCPA. Hematologic values, except for rats on the 1600 ppm TABLE 3 AVERAGES OF ORGAN-TO-BODY WEIGHT RATIO DATA OF RATS RECEIVING 3’,4’-DICHL~R~PR~PI~N~NILIDE IN THEIR DIET FOR Two YEARS Diet
Ratio of organ-to-body
weight (g/kg =L SD)
cont. No. of Sex
Female
(ppm)
0 100 400 1600
Male
0 loo 400 1600
Rats
1.5 10 12 12 17 15 11 5
Heart 3.2 3.2 3.4 3.4 2.6 2.7 2.6 3.1
& i i * f f j, rt
0.4 0.4 0.5 0.5 0.3 0.3 0.3 0.6
Spleen 1.8 2.2 2.3 3.3 1.7 1.9 2.0 2.4
4z 0.3 * 0.6 i 0.7” 5 0.8” f 0.5 f 0.7 i 0.6 f 0.3”
a Value differs significantly from control, p < 0.05.
Kidney 8.1 8.0 7.5 8.3 6.9 7.2 7.5 7.5
f * f f i f It It
1.6 1.4 1.5 1.3 1.3 0.9 0.9 0.9
Liver 32.2 34.1 32.1 40.5 30.9 33.0 34.7 32.1
f * f It & rt h f
Testes 4.5 4.3 6.5 8.6” 6.8 7.5 5.7 5.0
6.1 6.2 6.5 8.5
f 1.4 zk 0.9 + 1.2 zk 1.4”
4 4 4 4
0 loo 600 4000
7.7 7.8 7.8 7.9
i 1.2 i 0.8 zt 0.0 zk 2.0
Initial -__~. 14.98 14.70 14.40 11.55
i It i *
(mean * SD) Terminal .--~
0 Two dogs of each sex per diet level. 6 Value differs significantly from control, p G 0.05.
No. of dogs” -_____~
Diet cont. (w-4
Body weight (g)
2.5 1.3 0.7 0.9
AND ORGAN-TO-BODY 3’,4’-DICHLOROPROPIONANILIDE
AVERAGE BODY WEIGHTS
TABLE
6.69 7.75 7.17 7.51
zt It + t
0.53 1 .oo 1.27 0.16’
Heart 6.93 3.99 6.18 7.21
zk 1.52 * 1.41* * 1.28 -c 2.02
Spleen 4.81 5.05 5.29 6.11
zt 1.15 + 1.57 dz 1.05 _L 0.75
27.78 24.78 28.40 26.93
f i I h
Liver
weight (g/kg I SD) Kidney
Dons RECEIVING YEARS
OF
Ratio of organ-to-body
WEIGHT RATIO DATA IN THEIR DIET FOR TWO
4
5.5 5.9 3.5 3.0
-
z F ii
54 Fl 2 $ c;r ij F g $ g 3 0
*
658
AMBROSE
ET AL.
diet, were within the range of rats on the control diet. Statistically significant (p < 0.05) decreases in hemoglobin values were found for females only on the 1600 ppm diet at 3,6, 12 and 24 mo. The hemoglobin values for females at each period were, respectively: 15.4 f 1.2, 16.1 & 1.0, 15.7 % 0.4 and 14.0 * 0.7 for the controls, as compared to 14.3 + 1.1,13.6 % 0.8, 13.2 & 0.6, and 13.4 & 0.4 for rats on the 1600 ppm diet. Urinary tests for reducing substances and protein were within the range for control rats. Organ-to-body weight ratio data are summarized in Table 3. Significantly higher ratios were found for spleen of rats of both sex, for liver of female rats and for testes of rats on the 1600 ppm diet. Histopathologic findings revealed no lesions in kind or incidence that were not found in the control animals. Dogs. Beagle dogs on 0, 100, 600 and 4000 ppm DCPA survived the 2 yr feeding period. Significantly depressed weight gains were evident in dogs on 4000 ppm from about week 6, and persisted throughout the rest of the study period. Feed consumption data for dogs on 4000 ppm for each test period indicated greater consumption on a g/kg body weight and a decrease in feed efficiency. Hematologic values and urinary tests for reducing substances and protein ranged within normal limits throughout the test period. The BSP, SGOT and SAP tests made during the twenty-fourth month showed no statistically significant differences from the controls. Organ-to-body weight ratio data are summarized in Table 4. There is no indication of a specific effect of DCPA on a target organ. Histopathologic findings revealed no pathologic changes resulting from the ingestion of DCPA for 2 yr. Reproduction
Studies in Rats
In the three-generation reproduction study, body weights for parent generation rats at mating and at weaning of respective litters were not adversely affected. Data on reproduction performance through three generations showed no deleterious effects in any of the parameters recorded for rats on 0,100,300 and 1000 ppm diet levels of DCPA. No gross abnormalities were found in fetuses born dead or alive or in rats autopsied with fetuses in situ. Histopathologic examination of F/3b generation weanlings revealed no lesions, in kind or incidence, attributable to DCPA. In conclusion, under conditions of the experimental procedures, the highest overall apparent no-effect level established by this study was 400ppm in the diet of rats and dogs.
ACKNOWLEDGMENTS The authors are indebted to Patricia A. Weil, James E. Baker and Edwin H. Talley, Jr. for technical assistance, and to Dr. E. M. Crawford (deceased) for veterinary service on dogs.
REFERENCES BARTHA, R. and PRAMER,D. (1967). Pesticide transformation
to aniline and azo compounds
in soil. Science 156, 1617-1618. BARTHA, R. and PRAMER, D. (1970). Metabolism of acylanilide herbicides. Adoan. Appl. Microbial. 13, 317-341. BERKSON, J. (1955). Estimate of the integrated normal curve by minimum normit chi-square with particular reference to bio-assay. J. Amer. Statist. Ass. 50, 529-549.
TOXICITY
OF DICHLOROPROPIONANILIDE
659
DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. STILL, G. G. (1968).Metabolism of 3,4-dichloropropionanilidein plants: The metabolicfate of 3,4-dichloroanilinemoiety. Science 159,992-993. WILLIAMS, C. H. and JACOBSON, K. H. (1966).An acylamidasein mammalianliver hydrolyzing the herbicide3,4-dichloropropionanilide.Toxicol. Appl. Pharmacol. 9, 495-500. YIH, R. Y., MCRAE,D. H. and WILSON, H. F. (1968).Metabolism of 3’,4’-dichloropropionanilide: 3,4-dichloroaniline-lignincomplex in rice plants. Science 161, 376-377.