Lack of Carcinogenicity of Chlorpyrifos Insecticide in a High-Dose, 2-Year Dietary Toxicity Study in Fischer 344 Rats

Barry L. Yano*,1, John T. Young* and Joel L. Mattsson{dagger}

* Health and Environmental Research Laboratories, The Dow Chemical Company, Midland, Michigan, 48674; and {dagger} Global Toxicology, Dow AgroSciences, Indianapolis, Indiana 46268

Received March 10, 1999; accepted August 4, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorpyrifos (CPF) was administered daily in the feed to evaluate toxicity and oncogenicity potential in male and female Fischer 344 rats, according to U.S. EPA guidelines. Doses for the 2-year study were based on findings in a 13-week feeding study in which lower body weights, urinary perineal staining, adrenal cortical vacuolization, and inhibition (slightly more than 60%) of brain cholinesterase (ChE) occurred at 15 mg/kg/day. The high dose in the subsequent 2-year study was 10 mg/kg/day, with lower doses of 0, 0.05, 0.1, or 1.0 mg/kg/day chosen to define dose-response patterns. Rats given 10 mg/kg/day for 2 years were healthy and there was no evidence of premature deaths. Mild toxicity occurred only in rats given 10 mg/kg/day and consisted of perineal urine soiling in females and a 6–8% body-weight decrease in males. Males given 10 mg/kg/day also had increased adrenal weights and vacuolation of the adrenal zona fasciculata. ChE was considered a measure of exposure. Plasma, RBC, and brain ChE activities were inhibited in rats given 10 mg/kg/day, and the plasma and RBC ChE activities were inhibited in rats given 1.0 mg/kg/day. Chronic exposure to 0.1 mg/kg/day was considered a threshold exposure level for inhibition of plasma ChE. Rats given 10 mg/kg/day, considered a maximum-tolerated dose, had approximately 60% chronic inhibition of brain ChE. This group had similar numbers and types of neoplasms as control rats. Consequently, CPF was not carcinogenic at dose levels up to 10 mg/kg/day.

Key Words: chlorpyrifos; subchronic toxicity; chronic toxicity, carcinogenicity; cholinesterase inhibition; adrenal cortical vacuolation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorpyrifos (CPF) is an organophosphate (OP) insecticide used for the control of various insect pests. Estimates of human environmental exposure to CPF have been made from published measurements of urinary trichloropyridinol (a primary urinary metabolite of CPF) from a randomly selected group of 1000 adults in the U.S. (Hill et al., 1995Go), and from human CPF pharmacokinetic data (Nolan et al., 1984Go). These data indicated that the 50th percentile exposure of the population was about 0.1 µg/kg, while the highest level of exposure (100th percentile) was approximately 1.5 µg/kg. The U.S. EPA has established the reference dose (RfD) for CPF as 3 µg/kg/day, which is a level at or below which daily aggregate exposure over a lifetime will not pose appreciable risks to human health.

Rat single-dose oral LD50 values for CPF range from 118–270 mg/kg for males and 82–174 mg/kg for females (McCollister et al., 1974Go) indicating moderate acute toxicity. Clinical signs in rats following acute to chronic oral administration have generally been attributed to an inhibition of cholinesterase (ChE) activity. In a previously conducted 2-year rat dietary chronic toxicity/oncogenicity study (McCollister et al., 1974Go), rats were given CPF in the diets at concentrations formulated to provide 0, 0.01, 0.03, 0.10, 1.0, and 3.0 mg/kg/day. There were no clinical signs of toxicity, alterations in body/organ weight, gross or histologic effects at any dose level. Plasma and red blood cell (RBC) ChE activities were decreased in rats given 1.0 or 3.0 mg/kg/day for 12 or 24 months, while brain ChE activity was lower only in rats given 3.0 mg/kg/day for 12 or 24 months. No significant differences in ChE activity were observed in rats given <=0.1 mg/kg/day. The numbers and types of tumors were similar for control and CPF-treated rats, indicating that CPF was not carcinogenic to rats under the conditions of the study.

This paper highlights the results from 13-week and 2-year dietary chronic toxicity and oncogenicity studies that were conducted to provide more current data defining the toxicity and oncogenic potential of CPF at higher dose levels than previously evaluated, consistent with the U.S. EPA guidelines (Environmental Protection Agency, 1982Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test material.
Chlorpyrifos (O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate-CAS# 002921-88-2), technical grade DursbanTM F insecticide, identified as AGR214637, was supplied by The Dow Chemical Co., Midland, MI. Test material analyses indicated a purity of 95.7% by gas chromatography with electron capture detection.

Study design.
In the 13-week study, groups of 10 rats/sex/dose were given feed formulated to provide 0, 0.1, 1.0, 5.0, or 15 mg CPF/kg body weight (bw)/day. The high-dose was expected to cause a decrease in bw, clinical signs of ChE depression, and decreases in plasma, RBC, and brain ChE activities (Breslin et al., 1996Go). Intermediate- and low-dose levels were selected to demonstrate a dose-response relationship for ChE inhibition.

Based on results of the 13-week study, groups of 60 rats/sex/dose in the two-year study were given feed formulated to provide 0, 0.05, 0.1, 1.0, or 10 mg CPF/kg bw/day. Ten randomly selected rats/sex/dose were necropsied at 12 months. The remaining 50 rats/sex/dose were on test for an additional year, or until they died or were euthanized in a moribund condition.

Test species and animal husbandry.
Male and female CDF Fischer 344 rats, approximately 28 days old, were obtained from Charles River Laboratories, Inc., Kingston, NY. Animals were individually housed in suspended stainless-steel cages with wire bottoms. Animal rooms were maintained at approximately 13 air changes/h, 22°C temperature, 40–60% relative humidity, and a 12-h light:dark cycle. Rats were fed Purina Certified Rodent Chow #5002 (Richmond, IN mill of Ralston Purina Co., St. Louis, MO) ad libitum in glass feed-jars with stainless-steel partial covers. Municipal water was provided ad libitum via a pressure-activated automatic watering system. Rats were stratified by weight and randomly assigned to experimental groups after acclimation to the laboratory environment. The animal care program of this laboratory has been accredited by the Association for Assessment and Accreditation of Laboratory Care International (AAALAC).

Test diets.
Dietary formulations of CPF were prepared weekly from pre-mixes in which CPF was dissolved in acetone and the solution was added to ball-milled rodent chow. CPF was stable in rodent chow for at least 42 days, and was homogeneously distributed within the diets. Concentrations of CPF in rodent chow, determined by gas chromatography using electron capture detection, ranged from 72 to 111% of the targeted concentration, with a mean of 95%, for the 13-week study, and 86–120% of the targeted concentration, with a mean of 102%, for the 2-year study, indicating that the rats closely received the targeted CPF dosages.

Clinical observations, feed consumption, and body weights.
All rats were observed at least once daily for general appearance, behavior, signs of toxicity, moribundity, mortality, and feed wastage. Rats in the 13-week study were also evaluated in a blinded manner, using a functional observational battery (FOB), on test days 8, 30, and 87. The FOB included, but was not limited to, an evaluation of unusual body position, activity level, and coordination of movement and gait. In addition, unusual or bizarre behavior such as head-flicking, head-searching, compulsive biting or licking, self-mutilation, circling, and walking backwards were noted. Animals were watched for the presence of convulsions, tremors, increased lacrimation and/or red-colored tears, increased salivation, piloerection, pupillary dilatation/constriction, unusual respiration, diarrhea, excessive or diminished urination, vocalization; hind-limb grip strength, and abnormal sensory function (audition and pain perception). Rats in the 2-year study were clinically examined by the laboratory veterinarian at least once weekly beginning after the 6th month. All animals were palpated for external masses before the study, prior to the 12-month necropsy, and monthly thereafter.

Feed consumption and body weights were measured weekly for the first 3 months and monthly thereafter.

Clinical pathology.
Blood for hematology was obtained from the orbital sinuses of non-fasted rats that were lightly anesthetized with methoxyflurane, at the end of the 13-week study and at 6, 12, 18, and 24 months in the 2-year study. Packed cell volume (IEC Hematocrit Mbyte centrifuge), hemoglobin concentration, RBC, white blood cell (WBC), and platelet counts were determined (Ortho ELT-8, Ortho Instruments, Westwood, MA). A differential WBC count of 100 cells, as well as RBC, WBC, and platelet morphology were manually assessed on blood smears from all rats.

Analyses of RBC and plasma ChE activities were determined at 7 and 13 weeks (13-week study) and at 6, 12, 18, and 24 months (2-year study). Brain ChE activity was measured from one-half of the brain obtained at the scheduled necropsies (13 weeks, 12 months, and 24 months) by a photometric method (Boehringer Mannheim Corp., Indianapolis, IN). In this method, a unit of ChE activity was defined as the amount of enzyme that catalyzed the transformation of 1 µmol of substrate (5-mM acetylthiocholine) per min at 37°C.

Blood for serum chemistry determinations was obtained from the severed cervical blood vessels following methoxyflurane anesthesia (13-week study) or from the orbital sinus at 6, 12, 18, and 24 months (2-year study) from fasted rats. The following parameters were measured in the 13-week study: alkaline-phosphatase, alanine-aminotransferase, and aspartate-aminotransferase activities; and total bilirubin, urea nitrogen, glucose, total protein, albumin, and globulin (total protein minus albumin) (Gilford 203-S Clinical Analyzer, Gilford Instrument Laboratories, Oberlin, OH). The same parameters were also measured at 6, 12, 18, and 24 months, with the exception that total bilirubin and aspartate-aminotransferase activity were determined only at 12 and 24 months. In addition, creatine phosphokinase activity and cholesterol, phosphorus, chloride (Gilford), calcium, sodium, and potassium (Ion Specific Electrode, Gilford) were also determined at 6, 12, 18, and 24 months.

A urinalysis was conducted on all rats at the end of the 13-week study and on 10 rats/sex/dose at 6, 12, 18, and 24 months (2-year study). The urinalysis consisted of a determination of the specific gravity (Goldberg refractometer, American Optical Co., Keene, OH), and semi-quantitative estimates were made of pH, protein, glucose, ketones, bilirubin, occult blood, and urobilinogen (Multistix/Ames Co., Div. Miles Laboratories, Elkhart, IN). A microscopic examination of the sediment from a pooled-group urine sample was made from each dose level.

Gross pathology and organ weights.
A complete gross examination of tissues was conducted on all animals by a veterinary pathologist. The following tissues were weighed during necropsy in the 13-week study: brain, liver, kidneys, testes, ovaries, heart, and thymus. These same tissues, excluding the heart and thymus but including the adrenals, were weighed at the scheduled necropsies in the 2-year study.

The following tissues were collected and preserved in neutral, phosphate-buffered 10% formalin: adrenal glands, aorta, bone, bone marrow, brain, caecum, cervix, coagulating glands, epididymides, esophagus, eyes, gross lesions, Harderian glands, heart, kidneys, large intestine, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissue, mesenteric lymph node, mesenteric tissue, nasal tissues, ovaries, oviducts, pancreas, parathyroid glands, pituitary gland, prostate, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tibial nerve, tongue, trachea, urinary bladder, uterus, and vagina.

Histopathology.
A histopathologic examination was performed on all tissues collected during the necropsies from control and high-dose rats and from any rat, irrespective of dose level, that did not survive until the scheduled necropsy. Tissues evaluated from the low- and intermediate-dose groups consisted of liver, kidneys, adrenal glands, and gross lesions (13 weeks and 12 months), and the liver, kidneys, spleen, lungs, testes, pituitary, thyroid/parathyroid glands, adrenal glands, and gross lesions (24 months). Tissues were sectioned approximately 6 microns thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Statistical analyses.
Descriptive statistics (means and standard deviations) were reported for feed consumption and WBC-differential counts. Body weights, clinical chemistry parameters, ChE activity, hematologic parameters, absolute and relative organ weights, and urine specific gravity were evaluated by Bartlett's test for equality of variances (Winer, 1971Go). Based upon the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960Go) or non-parametric analysis of variance (ANOVA) (Hollander and Wolfe, 1973Go). This was followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction for multiple comparisons (Miller, 1966Go). Statistical outliers were identified by a sequential test (Grubbs, 1969Go), but were not routinely excluded, except for feed consumption values used to adjust dietary concentrations of CPF.

Differences in mortality patterns were tested by the Gehan-Wilcoxon procedure for all animals scheduled for the 2-year necropsy. Histopathologic observations were statistically analyzed in the 2-year study, but not from the 13-week study. The incidences of specific histopathologic observations were first tested for linearity, using ordinal spacing of the doses for tissues that were examined from all animals and doses. If linearity was not rejected, the data were then tested for a dose-response relationship using the Cochran Armitage trend test. If the trend was statistically significant, or if significant deviation from linearity was found, incidences for each dose were compared to that of the control using a pairwise Chi-square test with Yates correction. Statistical analysis was limited to the pairwise comparison of control and high-dose groups, for tissues that were evaluated only from control and high-dose rats. When multiple grades of a histologic observation were present, the most advanced grade (or the 2 most advanced when frequencies were low), and the total number of animals with any grade of the observation, were analyzed to determine exacerbation of commonly occurring lesions. Observations from tissues or organs from the low- or mid-doses, which were examined only because of an observed gross lesion, were not analyzed statistically, and no analyses were performed on metastatic tumors.

The nominal alpha levels used were as follows: Bartlett's test = 0.01 (Winer, 1971Go), parametric ANOVA = 0.10 (Steel and Torrie, 1960Go), non-parametric ANOVA = 0.10 (Hollander and Wolfe, 1973Go), Dunnett's Test = 0.05, 2-sided (Winer, 1971Go), Wilcoxon rank-sum test = 0.05, 2-sided (Hollander and Wolfe, 1973Go), Bonferroni correction (Miller, 1966Go), outlier test = 0.02, 2-sided (Grubbs, 1969Go), Gehan-Wilcoxon = 0.05 (Breslow, 1970Go), Chi-square test for linearity = 0.01, trend test = 0.02, 2-sided (Armitage, 1971Go), and the pairwise comparison, Yates Chi-square test = 0.05, 2-sided (Fleiss, 1981Go).

Previous studies have shown depression of ChE activity in plasma, RBC, and brain of rats administered CPF in the diet. Since similar effects were postulated for this experiment, ChE activities were subjected to an alpha = 0.05 1-sided-hypothesis test, rather than 2-sided exploratory analysis.

Because numerous measurements were compared statistically within the same group of animals, the overall rate of false positive (Type I) errors was unknown and may have been much greater than the cited alpha levels suggested. As a consequence, the final interpretation of data in this study considered the results of statistical analyses, as well as other factors such as dose-response relationships, repeatability, and whether the results were plausible in light of other biological findings.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overall, male and female rats in both the 13-week and 2-year studies remained in good health, in spite of very significant inhibition of blood and brain ChE. A large number of dependent variables were examined, and the salient findings are summarized in Table 1Go.


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TABLE 1 Summary of Treatment-Related Findings
 
Thirteen-Week Study
In-life observations.
All rats survived until the scheduled necropsy. All treated rats had a normal appearance and behavior, except for perineal soiling. Female rats given 15 mg/kg/day had a low incidence of urinary perineal soiling on days 8 (1 of 10 rats) and 30 (3 of 10); while seven of 10 male and eight of 10 female rats given 15 mg/kg/day had perineal soiling on day 87. Some perineal soiling also occurred on day 87 in 2 of 10 females given 1.0 or 5.0 mg/kg/day. Urinary perineal soiling was attributed to treatment, because of the high incidence in the high-dose rats and an absence in the control and 0.1 mg/kg/day rats. Urinary soiling also was noted in a 13-week neurotoxicity screening study, in female rats given 15 mg/kg/day (3 of 10 rats) and 5.0 mg/kg/day (1 of 10), but not in those given 1.0 mg/kg/day, 0.1 mg/kg/day or in controls (Mattsson et al., 1996Go). It should be noted, however, that urinary soiling is sometimes seen in control rats, and low-incidence findings of urinary soiling may be background events and not treatment related.

Body weights of male rats given 15 mg/kg/day were 4-6% lower than the controls and were statistically identified. Male rats given 5.0 mg/kg/day also had body weights that were slightly lower than the controls during the last month of the study. However, these differences were minor, not statistically identified, and were interpreted as not treatment related. There were no significant differences in body weights of males given <=1.0 mg/kg/day. Female rats from the control and all dose levels had a transient decrease in body weight of unknown etiology on test day 49. There were no other remarkable effects on body weight of female rats at any dose level. Feed consumption was not significantly affected in either sex at any dose level.

Clinical pathology.
The ChE activity for male and female rats given 0, 0.1,1.0, 5.0, or 15 mg/kg/day was similar across time (weeks 7 and 13), and has been averaged across time for purposes of illustration (Fig. 1Go). ChE activity of male and female rats given 15 mg/kg/day was significantly decreased in plasma (81 and 95%), RBC (46 and 55%), and brain (62 and 65%) compared to the controls (inhibition = 0%). Similar depression of plasma (77 and 94%), RBC (45 and 49%), and brain (40%) ChE activity also occurred in male and female rats given 5.0 mg/kg/day. Male and female rats given 1.0 mg/kg/day had significantly lower plasma (54 and 81%) and/or RBC (24 and 36%) ChE activity; however, there was no significant inhibition of brain ChE activity. Slightly lower plasma ChE activity (6 and 12%) was present in rats given 0.1 mg/kg/day, but was statistically significant only for females at 7 weeks. In context with the plasma ChE data from the 2-year study, the slight decrease in plasma ChE activity at 0.1 mg/kg/day was interpreted as probably due to treatment. No treatment-related effects were noted for RBC or brain ChE activities of rats given 0.1 mg/kg/day.



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FIG. 1. ChE activity from the 13-week study. Percentage decrease or increase in plasma, red blood cell (RBC) and brain cholinesterase activity, relative to controls, for males and females. Data are an average of inhibition of plasma and RBC activity at 7 and 13 weeks of exposure, and brain ChE inhibition at 13 weeks of exposure. *Statistically significantly different from control. Notation (a) indicates lower levels of plasma ChE activity were attributed to treatment, although inhibition was sometimes significant and sometimes not.

 
A number of other parameters in rats given CPF, such as a 5% lower RBC count or slightly lower alkaline phosphatase activity (data not presented), were sometimes statistically different from the controls. However, they were interpreted as not biologically significant because these alterations were minor, within the historical control range, or lacking a dose-response relationship.

Gross pathology and organ weights.
Seven of 10 female rats given 15 mg/kg/day had urine soiling of the perineal region. One of 10 males given 15 mg/kg/day and one of 10 females given 5.0 mg/kg/day also had urine perineal soiling. A few organ weight parameters were statistically identified as different from the controls (data not presented). However, these differences were interpreted as not being treatment related, because the values were within the historical control range, lacked a dose-response relationship, or were secondary to lower body weights.

Histopathology.
The adrenal glands of male rats given 5.0 or 15 mg/kg/day had a very slight, slight, or moderate degree of vacuolation of the parenchymal cells in the zona fasciculata of the adrenal cortex (Table 2Go). This vacuolation was in excess of the amount normally observed in rats of this age and strain, and histologically appeared to be consistent with the accumulation of lipid (fatty change). These differences were dose related and were interpreted to be treatment related. Similar changes were not observed in the adrenal glands of males given <=1.0 mg/kg/day or females at any dose level. There were no other microscopic observations in any other tissues that were attributed to CPF.


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TABLE 2 Thirteen-week Study: Histopathologic Treatment-Related Adrenal Gland Observations
 
Two-Year Study
In-life observations.
The mortality rate for rats given 0, 0.05, 0.1, 1.0, or 10 mg/kg/day for up to 2 years was 30, 50, 36, 48, and 28% for males and 30, 22, 26, 18,and 28% for females. The differences in mortality were not statistically different from the controls and were interpreted to be the result of normal variability.

In general, treated rats appeared and behaved similarly to the control rats. The only exception was a small amount of urine staining of the perineal region in 30–40% of the females given 10 mg/kg/day during the 6th to the 20th month of the study, which was attributed to CPF. Females given <=1.0 mg/kg/day and males from all dose levels did not have treatment-related clinical signs.

Body weights of male rats given 10 mg/kg/day were 6–8% lower than the controls throughout the study, were consistently statistically identified, and were interpreted to be treatment related. Body weights of females given 10 mg/kg/day were 2–4% lower than the controls from approximately test-day 60 through most of the first year. The body weights of these females were not different from the controls during the remainder of the study and therefore were not attributed to treatment. Body weight differences were not apparent for male or female rats given <=1.0 mg/kg/day. Feed consumption was not significantly affected in either sex at any dose level.

Clinical pathology.
For purposes of illustration (Fig. 2Go), the ChE activities for male and female rats given 0, 0.05, 0.1, 1.0, or 10 mg/kg/day were combined by sampling time and were normalized to the control levels (controls = 0%). Plasma and RBC ChE activities of male and female rats given 10 mg/kg/day were decreased approximately 75 and 87%, and 29 and 23%, respectively. Brain ChE activities of male and female rats given 10 mg/kg/day were decreased approximately 56 and 58%. Similar, but less pronounced depressions of plasma ChE (58 and 70%) also occurred in male and female rats given 1.0 mg/kg/day. RBC ChE activities were depressed 26 and 13% for males and females, respectively, given 1.0 mg/kg/day. All of these differences from control were clearly due to treatment.



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FIG. 2. ChE activity from the 2-year study. Percentage decrease or increase in plasma, red blood cell (RBC), and brain cholinesterase activity, relative to controls, for males and females. Data are an average of inhibition of plasma and RBC ChE at 6, 12, 18, and 24 months of exposure, and brain ChE at 12 and 24 months of exposure. *Statistically significantly different from control. Notation (a) indicates lower levels of plasma ChE activity at 0.1 mg/kg/day were attributed to treatment, although inhibition was sometimes significant and sometimes not.

 
Statistical analyses of brain ChE for rats exposed to 0.05, 0.1, or 1.0 mg/kg/day presented a more confusing picture. After 12 months of treatment, in increasing order of dose level, male brain ChE activity was 6%, 7%, or 9% less than controls and all were statistically significant. Thus, although dosage increased 20-fold, the levels of ChE activity were virtually the same. After 24 months of exposure, male-brain ChE activity for these 3 dosage levels was slightly greater than control (+2%, 0%, +3%; not statistically significant). In context with data from the 13-week study, where males given 1.0 mg/kg/day had brain ChE activity 2.5% less than control (virtually the same as control), the overall conclusion was that the statistically significant findings at 0.05, 0.1, and 1.0 mg/kg/day were spurious. It was thought unlikely that exposure to CPF significantly inhibited male brain ChE at these dosage levels. There was no significant difference from control in female brain ChE activity at 0.05 or 0.1 mg/kg/day. At 1.0 mg/kg/day, the 12-month brain ChE value was statistically significant (5% less activity than control), while the 24-month activity was not statistically significant (4% less activity than control). In the 13-week study, 1.0 mg/kg/day female brain ChE activity was 1% less than control. Again, due to the consistently very small differences from control, it was concluded that 1.0 mg/kg/day female brain ChE activity was within normal variation and not due to treatment.

Minor decreases in plasma ChE (range of 2 to 20% less than control) occurred in rats given 0.1 mg/kg/day, and only 2 of the 8 statistical analyses (2 sexes, 4 time-periods) were significant. However, the consistency of the data between the chronic and 13-week studies and between the sexes lead to the conclusion that these minor differences in activity were probably due to treatment and reflected a threshold level of exposure. There were no significant differences in RBC ChE at 0.05 or 0.1 mg/kg/day, or in plasma ChE at 0.05 mg/kg/day.

As in the 13-week study, a number of other parameters were sometimes statistically different from the controls (data not presented). However, they were interpreted not to be biologically significant because these alterations were minor, within the historical control range, or lacked a dose-response relationship.

Gross pathology and organ weights.
There were no treatment-related gross pathologic observations in rats necropsied at 12 or 24 months, at any dose level. Males and females given 10 mg/kg/day for 12 months had increased adrenal gland weights that were statistically identified frequently and were attributed to CPF. Adrenal weights, compared to control and in ascending order by dosage level, were: for males, 100% (control), 100%, 102%, 102%, and 117% (high dose was significantly heavier); for females 100% (control), 96%, 102%, 102% and 109% (high dose was significantly heavier). The adrenal gland weights of males and females from the lower dose levels were unaffected. Adrenal weights from 24 months were extremely variable due to intervening tumors and other pathology, and were not useful for evaluation. Other differences in organ weights of rats given 10 mg/kg/day for 12 and 24 months were secondary to slightly lower body weights or to random variability in organ weights.

Histopathology.
Adrenal cortical vacuoles in the zona fasciculata were seen in control, low- and mid-dose rats at both 1 and 2 years (Table 3Go). It was almost entirely in males, and it ranged in severity from very slight (most of the rats) to moderate (a small number of rats). This pattern of vacuolation was regarded as normal background. In contrast, male rats exposed to 10 mg/kg/day had an increase in severity of the lesion by one grade, such that most animals had a slight rather than a very slight degree of vacuolation. The severity of the lesion did not progress from 12 to 24 months of treatment. This effect was consistent with the accumulation of lipid (fatty change) and was interpreted to be treatment-related.


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TABLE 3 Two-year Study: Treatment-Related Histopathologic Observations
 
Male rats given 10 mg/kg/day for 24 months had a lower severity of spontaneous geriatric glomerulonephropathy (Table 3Go). The number of male rats with moderate to severe glomerulonephropathy was 28/50 for controls and 13/50 at the high dose; this was statistically significant by a post-hoc z-test of proportions (p = 0.002). Male and female rats given 10 mg/kg/day for 24 months also had a 30% lower incidence of hepatic biliary hyperplasia, which also was statistically significant.

A tabulation of all primary neoplasms that differed between control rats and high-dose rats is presented in Table 4Go. The table was reduced about 40% by its not listing tumors that occurred at a low but identical incidence in control and high-dose rats (0 vs. 0, 1 vs. 1, or 2 vs. 2) or that occurred only in low- or mid-dose rats. There were no significant increases or decreases in the incidences of any tumors for males or females at any dose level; however, high-dose rats tended to have fewer neoplasms than controls. In addition, the total number of primary, benign, or malignant tumors was comparable in all groups (Table 5Go) and was consistent with the tumor types normally seen in rats of this strain.


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TABLE 4 Two-year Study: Primary Neoplasms that Differed between Control and High-Dose Rats
 

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TABLE 5 Two-year Study: Summary of Primary Neoplastic Histopathologic Observations
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Only minor signs of OP intoxication, i.e., perineal urine soiling, occurred in rats given 1.0 to 15 mg/kg/day for 13 weeks, and in female rats given 10 mg/kg/day for up to 2 years. Male rats given 15 mg/kg/day for 13 weeks and 10 mg/kg/day for up to 2 years also had 4–6% and 6–8% decreases in body weight, respectively. Because cholinergic toxicity is mediated via excessive inhibition of acetyl cholinesterase (Mileson et al., 1998Go), ChE activity was evaluated as a measure of internal exposure. A significant inhibition of plasma, RBC, and brain ChE activities occurred in rats given 5.0 or 15 mg/kg/day for 13 weeks and rats given 10 mg/kg/day for up to 2 years. A significant inhibition of plasma and RBC ChE activities also occurred in rats given 1.0 mg/kg/day for 13 weeks and for up to 2 years. It appeared that the threshold for inhibition of plasma ChE was about 0.1 mg/kg/day.

Mortality rate was not affected in rats given CPF for up to 2 years, compared to the controls. Rats given 10 mg/kg/day for up to 2 years had increased adrenal weights and male rats had vacuolation of the cells of the adrenal zona fasciculata. Similar adrenal vacuolations did not occur in female rats, and were not noted in mice (Warner et al., 1980Go), dogs (McCollister et al., 1974Go), or cats (Hooser et al., 1988Go). These findings led to a conclusion that the lesion may be male-rat specific. The vacuoles had no recognized toxicological significance and occurred only at the high dose of 10 mg/kg/day. The vacuoles did not progress in severity from 1 to 2 years of exposure, and the incidence of male rats with vacuoles was actually less at 2 years (roughly 100% at 1 year, and 50% at 2 years; Table 3Go). No treatment-related vacuoles occurred at 1.0 mg/kg/day.

Adrenal weights were increased about 17% over controls in male rats at 1 year of exposure to 10 mg/kg/day, and adrenal weights of female rats treated at 10 mg/kg/day were about 9% greater than controls. No differences in adrenal weights were apparent at 1.0 mg/kg/day in either sex. Given the lack of adverse health effects after 2 years of exposure to 10 mg/kg/day, the functional consequences of the increased adrenal weights and vacuoles appear to have been minimal or absent. The no-observed-effect level for adrenal effects was 1.0 mg/kg/day, based on adrenal weight and vacuole effects at 5.0 or 10 mg/kg/day.

There was a significant decrease in the severity of chronic renal disease in male rats given 10 mg/kg/day, and a decreased incidence of biliary hyperplasia in male and female rats given 10 mg/kg/day. The relationship of CPF exposure to these decreases in normal geriatric diseases is unknown, but may be related to minor indirect effects such as the slight decreases in body weight. Overall, however, life expectancy and tumor incidences were not measurably affected.

In a previously conducted 2-year oncogenicity study, rats were given 0, 0.01, 0.03, 0.1, 1.0, or 3.0 mg CPF/kg body weight/day for up to 2 years (McCollister et al, 1974Go). Significant depressions in plasma, RBC, and brain ChE activities occurred in rats given 3.0 mg/kg/day, while rats given 1.0 mg/kg/day only had depressions in plasma and RBC ChE activities. Significant treatment-related effects in ChE activity were not observed in rats given 0.1 mg/kg/day. Treatment-related effects were not observed in body weights, clinical observations, or organ weights, and the incidence of non-neoplastic and neoplastic observations was similar between control and CPF-treated groups. The results from the chronic toxicity/oncogenicity study reported herein, which was conducted at approximately 3-fold higher concentrations of CPF, substantiated the findings of the earlier chronic toxicity/oncogenicity study that CPF was not carcinogenic. Similarly, no indications of carcinogenicity were observed in CD-1 mice that were given CPF at targeted concentrations of 0, 0.5, 5.0, or 15 mg/kg/day for up to 2 years (Warner et al, 1980Go). These carcinogenicity studies in rats and mice are also consistent with the lack of genotoxicity observed in in vitro studies (bacterial gene mutation assays, an assay for chromosomal aberrations in cultured mammalian cells, and an assay for DNA damage/repair in rat hepatocytes) and in an in vivo assay for cytogenetic damage in the mouse bone marrow (Gollapudi et al., 1995Go).

A number of other OP pesticides have also been evaluated for carcinogenic potential in rats and/or mice and include: coumaphos (NCI, 1979), diazinon (NCI, 1979), dioxathion (NCI, 1978), dichlorvos (NCI, 1977 and NTP, 1989), dimethoate (NCI, 1977), fenthion (NCI, 1979), malathion (NCI, 1978), methyl parathion (NCI, 1979), parathion (NCI, 1979), triamiphos (Verschuuren and Kroes, 1974Go), and tetrachlorvinphos (NCI, 1978). Seven of these 11 OP pesticides did not demonstrate evidence of carcinogenicity; however, a carcinogenic response was observed in a corn oil gavage study with dichlorvos, and dietary studies evaluating fenthion, parathion, and tetrachlorvinphos. In the dichlorvos study, an increased incidence of pancreatic exocrine adenomas (males and females), mononuclear leukemia (males), and mammary fibroadenomas (females) were observed in rats; while papillomas on the nonglandular stomach were noted in mice (male and female). Fenthion induced sarcomas, fibrosarcomas, and rhabdomyosarcomas in male mice, with no carcinogenic effect noted in female mice or male and female rats. Parathion induced an increased incidence of adrenal cortical adenomas in male and female rats, with no carcinogenic effect in mice. Lastly, tetrachlorvinphos induced C-cell adenomas of the thyroid, and adrenal cortical adenomas in female rats, and neoplastic nodules and hepatocellular carcinomas in female and male mice, respectively.

Possible carcinogenicity was identified in 4 of the above 11 OPs. Although clinical toxicity of the pesticidal OPs is mediated via inhibition of ChE, the lack of carcinogenicity in 7 of 11 studies, and the lack of a consistent target organ tumor among the other 4 OPs suggests a lack of similarity of tumor mechanism. In addition, at least some of the tumor findings are likely to be falsely positive, since the overall statistical false-positive rate for mouse and rat cancer bioassays is quite high (nearly 50%; Haseman et al., 1986Go).

In summary, the data obtained from the 13-week and 2-year studies indicated that minor urine staining, reduced body weights, and significant depressions of plasma, RBC, and brain ChE activities occurred in rats given the highest doses of CPF. Minor adrenal gland vacuolation occurred in male rats given the highest-dose levels for 13 weeks and for 2 years. Rats given the highest-dose level for up to 2 years also had decreases in the severity of two non-neoplastic spontaneous lesions. Lifetime exposure to the highest-dose level did not affect the mortality rate or cause a treatment-related increase in the incidence of neoplasms in any tissue.


    NOTES
 
1 To whom correspondence should be addressed at The Dow Chemical Company, Health & Environmental Research Laboratories, 1803 Building, Midland, MI, 48674. Fax: (517) 638-9863. E-mail: blyano{at}dow.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Armitage, P. (1971). Statistical Methods in Medical Research. John Wiley, New York.

Breslin, W. J., Liberacki, A. B., Dittenber, D. A., and Quast, J. F. (1996). Evaluation of the developmental and reproductive toxicity of chlorpyrifos in the rat. Fundam. Appl. Toxicol. 29, 119–130.[ISI][Medline]

Breslow, N. (1970). A generalized Kruskal-Wallis test for comparing samples subject to unequal patterns of censorship. Biometrika 57, 579–594.[ISI]

Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision F., Hazard Evaluation: Human and Domestic Animals. National Technical Information Service Report PB83–153916.

Fleiss, J. L. (1981). Statistical Methods for Rates and Proportions. John Wiley & Sons, New York.

Gollapudi, B. B., Mendrala, A. L. and Linscombe, V. A. (1995). Evaluation of the genetic toxicity of the organophosphate insecticide chlorpyrifos. Mutat. Res. 342, 25–36.[ISI][Medline]

Grubbs, F. E. (1969). Procedures for detecting outlying observations in samples. Technometrics 11, 1–21.[ISI]

Haseman, J. K., Winbush, J. S., and O'Donnell, Jr., M. W. (1986). Use of dual control groups to estimate false positive rates in laboratory animal carcinogenicity studies. Fundam. Appl. Toxicol. 7, 573–584.[ISI][Medline]

Hill, R. H., Jr., Head, S. L., Baker, S., Gregg, M., Shealy, D. B., Bailey, S. L., Williams, C. C., Sampson, E. J., and Needham, L. L. (1995). Pesticide residues in urine of adults living in the United States: Reference range concentrations. Environ. Res. 71, 99–108.[Medline]

Hollander, M., and Wolfe, D. A. (1973). Nonparametrical Statistical Methods. John Wiley, New York.

Hooser, S. B., Beasley, V. R., Sundberg, J. P., and Harlin, K. (1988). Toxicologic evaluation of chlorpyrifos in cats. Am. J. Vet. Res. 49, 1371–1375.[ISI][Medline]

Mattsson, J. L., Wilmer, J. W., Shankar, M. R., Berdasco, N. M., Crissman, J. W., Maurissen, J. P., and Bond, D. M. (1996). Single-dose and 13-week repeated-dose neurotoxicity screening studies of chlorpyrifos insecticide. Food Chem. Toxicol. 34, 393–405.[ISI][Medline]

McCollister, S. B., Kociba, R. J., Humiston, C. G., McCollister, D. D., and Gehring, P. J. (1974). Studies of the acute and long-term oral toxicity of chlorpyrifos (0,0-diethyl-0-(3,5,6-trichloro-2-pyridinyl)phosphorothioate). Food Cosmet. Toxicol. 12, 45–61.[ISI][Medline]

Mileson, B. E., Chambers, J. E., Chen, W. D., Dettbarn, W., Erich, M., Eldefrawi, A. T. et al. (1998). Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41, 8–20.[Abstract]

Miller, R. G., Jr. (1966). Simultaneous Statistical Inference. McGraw-Hill., New York

National Cancer Institute (1977). Bioassay of dimethoate for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 4, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 77-804.

National Cancer Institute (1977). Bioassay of dichlorvos for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 10, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 77-810.

National Cancer Institute (1978). Bioassay of malathion for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 24, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 78-824.

National Cancer Institute (1978). Bioassay of tetrachlorvinphos for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 33, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 78-833.

National Cancer Institute (1978). Bioassay of dioxathion for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 125, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 78-1380.

National Cancer Institute (1979). Bioassay of parathion for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 70, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 79-1320.

National Cancer Institute (1979). Bioassay of methyl coumaphos for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 96, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 79-1346.

National Cancer Institute (1979). Bioassay of fenthion for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 103, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 79-1353.

National Cancer Institute (1979). Bioassay of diazinon for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 137, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 79-1392.

National Cancer Institute (1979). Bioassay of methyl parathion for possible carcinogenicity. NCI Carcinogenesis Technical Report Series 157, National Technical Information Service (NTIS), Springfield, VA (DHEW Publication (NIH) 79-1713.

National Toxicology Program (NTI) (1989). Toxicology and carcinogenesis studies of dichlorvos in F344/N rats and B6C3F1 mice (gavage studies). National Toxicology Program Technical Report Series 342, NIH Publication 89-2598.

Nolan, R. J., Rick, D. L., Freshour, N. L., and Saunders, J. H. (1984). Chlorpyrifos: Pharmacokinetics in human volunteers. Toxicol. Appl. Pharmacol. 73, 8–15.[ISI][Medline]

Steel, R. G. D., and Torrie, J. H. (1960). Principles and Procedures of Statistics with Special Reference to the Biologic Sciences. McGraw-Hill, New York.

Verschuuren, H.G. and Kroes, R. (1974). Triamiphos: Long-term toxicity and three-generation reproduction studies in rats. Toxicology 2, 327–338.[ISI][Medline]

Warner, S. D., Gerbig, C. G., Strebing, R. J., and Molello, J. A. (1980). Results of a two-year toxicity and oncogenicity study of chlorpyrifos administered to CD–1 mice in the diet. Report of The Dow Chemical Company, Midland, MI.

Winer, B. J. (1971). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York: