* Global Health, Environmental Safety and Regulatory, Dow AgroSciences, L. L. C., 9330 Zionsville Road, Indianapolis, Indiana 46268;
Health and Environmental Research Laboratories, The Dow Chemical Company, Midland, Michigan 48674;
Jefferson Medical College, Philadelphia, Pennsylvania 19107
Received August 30, 1998; accepted April 28, 1999
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ABSTRACT |
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Key Words: TCP; organophosphates; chlorpyrifos; rats; rabbits; developmental toxicity.
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INTRODUCTION |
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The acute oral toxicity of CPF is considered moderate, with acute oral LD50 values in rats ranging from 118 to 245 mg/kg (McCollister et al., 1974). Multigeneration reproduction studies have been conducted in rats using chlorpyrifos, and developmental toxicity studies have been conducted in mice, rats, and rabbits. In an oral developmental toxicity study in CD-1 mice, dose levels ranging from 0.1 to 25 mg/kg/day were administered on gestation days 615. Severe maternal toxicity, including mortality, clinical signs of cholinesterase inhibition, and decreased maternal body weight, were seen at 25 mg/kg/day. The only effects on fetuses from treated mice were decreased body measurements (weight and length) and an increased incidence of minor skeletal variants in this high-dose group (Deacon et al., 1980
). In a developmental toxicity study in rats, dose levels ranging from 0.1 to 15 mg/kg/day of chlorpyrifos were used. Clinically, cholinergic signs (salivation, urination, and tremors) were seen, and body weights and weight gains were decreased in maternal animals given 15 mg/kg/day. However, there were no adverse fetal effects in rats (Breslin et al., 1996
). In a developmental toxicity study in rabbits, gavage doses of 1140 mg/kg/day were administered on gestation days 719. Plasma cholinesterase activity was reduced in all treatment groups, with reduced feed consumption and decreased body weight gain seen at 140 mg/kg/day. Developmental effects were limited to decreased fetal body weights and slight delays in skeletal ossification only in the highest dosage group (EPA, 1998
). There was no indication of any teratogenic effect in mice, rats, or rabbits
A developmental neurotoxicity study has also been conducted using Sprague-Dawley rats in which dams were exposed by gavage from gestation day (GD) 6 through postnatal day 10 to dosages of 0, 0.3, 1, or 5 mg/kg/day (Maurissen et al., manuscript in preparation). At 5 mg/kg/day, dams showed clinical signs of toxicity (muscle fasciculations, hyperactivity), and decreased survival promptly after birth and delayed maturation were noted in the pups, but no effects were evident at lower dosages. Despite the delayed maturation, learning and memory were unimpaired and there were no signs of CNS or any other structural anomalies in the offspring. Using the same dosage regimen as the developmental neurotoxicity study, Mattsson et al. (1999) demonstrated that in dams given 5 mg CPF/kg/day, concentrations of TCP in the blood of fetuses on GD 20, 4 h postdosing, were similar to blood levels in the dams (18002000 ng/g), although blood CPF levels in dams were only 108 ng/g, with even lower CPF blood levels in fetuses (46 ng/g).
TCP has a low intrinsic toxicity and has no cholinesterase activity. The oral LD50 in rats is approximately 800 mg/kg, whereas the dermal LD50 in rabbits is greater than 2000 mg/kg. The liver and kidneys have been identified as the primary target organs in rats following 4-week dietary exposure to the sodium salt of TCP at dose levels of 120 mg/kg/day and greater (Unreported data, The Dow Chemical Company). Due to the rapid detoxification of CPF to TCP (Racke, 1993) resulting in exposure primarily to TCP, and because residues of TCP are found in humans as well as in a variety of animal species exposed to chlorpyrifos, the potential of TCP to produce developmental effects was evaluated as part of the commitment of Dow AgroSciences to product stewardship. Dose levels of TCP used in this study were as much as 3- to 18-fold higher on a molar basis than respective rat and rabbit developmental toxicity studies conducted with the parent compound, chlorpyrifos.
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MATERIALS AND METHODS |
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Groups of 3234 bred female rats were given 0, 50, 100, or 150 mg TCP/kg/day by oral gavage at a dose volume of 4 ml/kg on GD 615. Dose volumes were adjusted daily based on individual body weights. All animals were observed twice daily for signs of treatment-related effects. Maternal body weights were recorded on GD 0, daily on GD 616, and on GD 21. Feed and water consumption were measured at 3-day intervals beginning on GD 6. At Cesarean section on GD 21, a complete gross postmortem examination was performed, and the maternal liver, kidneys, and gravid uterus were weighed. The number of corpora lutea, and number and position of implantations, resorptions, and live or dead fetuses were recorded. Uteri with no visible implantations were stained with a 10% solution of ammonium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions. Each fetus was individually identified, weighed, sexed, and given a gross examination for external malformations/ variations including observation for palatal defects. All fetuses were euthanized by CO2 asphyxiation, and approximately one-half of the fetuses in each litter were evaluated for visceral malformations/variations (Staples, 1974
). The heads of fetuses selected for visceral examination were removed, placed in Bouin's fixative, and subsequently sectioned and examined for craniofacial defects (Wilson, 1965
). All fetuses were then eviscerated and processed; the ossified skeletal structures were stained with alizarin red S (Dawson, 1926
) and examined for skeletal alterations.
Rabbits.
Stock supplies of female New Zealand White rabbits, approximately 5 months of age, were obtained (Hazleton-Dutchland, Inc., Denver, PA), examined upon receipt in the laboratory by a veterinarian for health status, and acclimated to laboratory conditions for at least 3 weeks. The animal rooms of the facility were regulated for rabbits to maintain temperature at 6872°F, relative humidity at 4060%, and a 12-h light:12-h dark photocycle. A priming dose of 50 IU of human chorionic gonadotropin (hCG; W. A. Butler) was administered by IV injection to all females on test 3 weeks prior to insemination. Adult females, approximately 6 months old, weighing 34714222 grams were artificially inseminated (Gibson et al., 1966) with fresh semen collected from bucks of the same strain, and the day of insemination was considered GD 0. Upon insemination, rabbits were given an intravenous injection of 100 IU of hCG and randomized by body weight into the 4 groups using a computer-generated procedure. Animals were housed individually in cages with wire floors and identified using uniquely coded alphanumeric metal ear tags. Animals were maintained on 8 oz/day of Certified Laboratory Rabbit Chow No. 5322 (Purina Mills, Inc., St. Louis, MO). Municipal tap water was available ad libitum.
Groups of 16 inseminated adult female New Zealand White rabbits were administered TCP at dose levels of 0, 25, 100, or 250 mg/kg/day by oral gavage once daily on GD 719 of gestation at a dose volume of 2 ml/kg body weight. All animals were observed daily for treatment-related alterations in behavior or demeanor. Body weights were recorded on GD 0, daily during the dosing period, and on GD 20 and 28. Dose volumes were adjusted daily based on individual body weights. On GD 28, rabbits were euthanized by an intravenous injection of T-61 Euthanasia Solution (American Hoechst Corporation, Somerville, NJ), and a gross necropsy was performed. The weights of the maternal liver with gallbladder, kidneys, and gravid uterus, and any obvious gross pathologic alterations were recorded. The number of corpora lutea, and number and position of implantations, resorptions, and live or dead fetuses were recorded. Uteri with no visible implantations were stained with a 10% solution of ammonium sulfide (Kopf et al., 1964), and examined for evidence of early resorptions. Each fetus was individually identified, weighed, sexed, and given a gross examination for external malformations/variations to include observation for palatal defects. All fetuses were euthanized and examined by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974
). This examination also included a fresh examination of the brain. All fetuses were then preserved in alcohol, eviscerated, cleared, stained with alizarin red-S (Dawson, 1926
) and examined for skeletal alterations.
Statistical evaluation.
Maternal body weights and body weight gains, fetal body weights, and organ weights (absolute and relative) were evaluated by Bartlett's test for equality of variances (Winer, 1971). Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960
) or nonparametric (Hollander and Wolfe, 1973
) analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test (Winer, 1971
) or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973
) with Bonferroni's correction (Miller, 1966
), respectively, was performed. Descriptive statistics (means and standard deviations) were reported for feed and water consumption (rats only). Statistical evaluation of the frequency of preimplantation loss, resorptions, and fetal alterations among litters and the fetal population was performed using a censored Wilcoxon test (Haseman and Hoel, 1974
) with Bonferroni's correction. The number of corpora lutea and implants, as well as litter size, were evaluated using a nonparametric ANOVA followed by the Wilcoxon Rank-Sum test with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (Siegel, 1956
). Fetal sex ratios were analyzed using a binomial distribution test. Nonpregnant animals were excluded from the appropriate analyses. Statistical outliers were identified using a sequential method (Grubbs, 1969
), but except for feed and water consumption, were not excluded unless justified by sound scientific reasons unrelated to treatment. The level of statistical significance was set a priori at
= 0.05.
Good Laboratory Practice.
These studies complied with Good Laboratory Practice Standards (EPA, 1983), and were conducted according to the procedures outlined in the Pesticide Assessment Guidelines, Subdivision F- Hazard Evaluation: Human and Domestic Animals (NTIS, 1984
). The laboratory is fully accredited by the Association for Accreditation of Laboratory Animal Care, Internation (AALAC).
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RESULTS |
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Rabbits
As in rats, there were no clinical signs of toxicity noted in rabbits administered TCP during gestation (data not shown). Overall body weight gain during the treatment period in females given 250 mg/kg/day was significantly depressed relative to the control group (Table 4), though there were no statistically significant differences in mean body weights or body weight gains over discrete intervals (i.e., GD 710, 1013, 1316, or 1620) among rabbits in any dose group. In fact, females at this dose level lost approximately 1.7% of their GD 7 body weight over the course of the treatment period; all other groups gained weight, with the controls gaining approximately 3.5%. There were no significant differences in absolute or relative organ weights among rabbits administered TCP (data not shown).
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DISCUSSION |
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The incidence of CNS malformations (hydrocephalus and severely dilated cerebral ventricles) in rabbits in the 100 and 250 mg/kg/day dose groups initially suggested a possible relationship to treatment with TCP. However, there were no other indications of any adverse fetal effects in this study to support an association with treatment. An examination of historical control data revealed that in the 3 years prior to the conduct of this study, only one CNS anomaly had been noted among control rabbits in our laboratory; that being hydrocephaly. However, an increase in the spontaneous incidence of CNS malformations in the control rabbit population was observed in rabbit teratology studies conducted in our laboratory in the 57 years surrounding this study. In five studies conducted subsequent to this study, a total of 11 control fetuses with these same CNS anomalies were reported (5 having severely dilated lateral ventricles and 6 hydrocephalus), 4 of which occurred in a single study with a control size of 132 fetuses (3.0%), and another 3 in a second study with a control group of 138 fetuses. Figure 2 depicts the incidence, by year of reporting, of CNS malformations noted in control rabbit populations in our laboratory. As can be seen in this figure, a cluster of hydrocephalus/dilated cerebral ventricles appeared in control fetuses in 19871988, the years immediately surrounding the conduct of the study described herein. This historical database includes evaluation of 878 control fetuses from 198 litters in this 2- year period, with individual study incidences of 0%2.2% of control fetuses with CNS malformations.
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The CNS malformations seen in this study also need to be viewed in a broader context. These CNS anomalies were very specific in nature, with no indication of any other CNS anomalies such as exencephalus or spinal bifida to suggest neural tube development as a unique target. Also, in the present study, there were no increases in malformations in any other organ system, and no evidence of any delayed maturation such as depressed fetal body weights or skeletal ossification to suggest any adverse effect. Even with classic teratogenic agents such as thalidomide and its association with limb defects, numerous other malformations were observed (Shepard, 1998). It should also be noted that there was no indication of any treatment-related CNS malformations in fetal rabbits exposed to a dosage as high as 140 mg CPF/kg/day, a molar equivalent dose level of ~80 mg TCP/kg/day roughly equivalent to the mid-dose level used in the current study with TCP (EPA, 1998
). Thus, the lack of any additional effects in rabbit fetuses from this study, the lack of any evidence of CNS anomalies in rabbits following exposure to CPF, and the historical control incidence both within our laboratory as well as in others support the conclusion that the CNS anomalies observed were most likely unassociated with treatment.
The dose levels at which the current studies were conducted with TCP were, on a molar equivalent basis, ~18-fold higher than the dose levels used to evaluate the parent material, chlorpyrifos, in rodent studies, and greater than 3-fold higher than the CPF level used in the developmental toxicity study in rabbits. TCP lacks the phosphate moiety of chlorpyrifos and does not inhibit cholinesterase activity, and it is this functional group that is responsible for the higher toxicity associated with the parent compound. Nonetheless, the lack of any significant developmental toxicity with TCP is consistent with the results of studies with chlorpyrifos. As noted previously, chlorpyrifos is rapidly and extensively hydrolyzed to TCP in mammalian systems. The results of the studies reported herein indicate that the TCP metabolite of chlorpyrifos has no intrinsic developmental toxicity and that the metabolism of chlorpyrifos to TCP represents an effective detoxification pathway that protects against developmental effects.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (317) 337-4557. E-mail: trhanley{at}dowagro.com.
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