4
Methods for Toxicity Testing

THE PURPOSE OF THIS CHAPTER is to familiarize the reader with the testing that is currently conducted by a manufacturer prior to and during the process of submitting a petition to register a pesticide. Codified toxicologic evaluation of potential pesticides has been a requirement in the United States for approximately 50 years. The testing requirements and guidelines continue to evolve based on new science. This chapter identifies the current testing that is pertinent to the young animal and young human as well as aspects of testing that are needed to fill the data gaps to better  ensure the protection of infants and children. The current testing guidelines can be found in Pesticide Assessment Guidelines issued by the Environmental Protection Agency (EPA, 1991a,b).

Data, including those derived from toxicity testing, crop residue analyses, environmental fate testing, and ecotoxicology testing, are generated by the manufacturer of a pesticide to meet the mandatory requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) for pesticide registration. Although these data are essential to the EPA's registration process, other data generated by EPA itself, as well as by other government institutions and academia, are considered in the registration decision-making process.

EPA has issued 194 registration standards on 350 chemicals used as active and inert ingredients in pesticide products. These standards are published by EPA and are intended to upgrade and update the data base on a previously registered pesticide or class of pesticide products. They call for additional studies in the areas of toxicity testing, crop residue analyses, environmental fate, and ecotoxicology testing. This testing must be conducted within an EPA-mandated time frame to allow for the continued



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Pesticides in the Diets of Infants and Children 4 Methods for Toxicity Testing THE PURPOSE OF THIS CHAPTER is to familiarize the reader with the testing that is currently conducted by a manufacturer prior to and during the process of submitting a petition to register a pesticide. Codified toxicologic evaluation of potential pesticides has been a requirement in the United States for approximately 50 years. The testing requirements and guidelines continue to evolve based on new science. This chapter identifies the current testing that is pertinent to the young animal and young human as well as aspects of testing that are needed to fill the data gaps to better  ensure the protection of infants and children. The current testing guidelines can be found in Pesticide Assessment Guidelines issued by the Environmental Protection Agency (EPA, 1991a,b). Data, including those derived from toxicity testing, crop residue analyses, environmental fate testing, and ecotoxicology testing, are generated by the manufacturer of a pesticide to meet the mandatory requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) for pesticide registration. Although these data are essential to the EPA's registration process, other data generated by EPA itself, as well as by other government institutions and academia, are considered in the registration decision-making process. EPA has issued 194 registration standards on 350 chemicals used as active and inert ingredients in pesticide products. These standards are published by EPA and are intended to upgrade and update the data base on a previously registered pesticide or class of pesticide products. They call for additional studies in the areas of toxicity testing, crop residue analyses, environmental fate, and ecotoxicology testing. This testing must be conducted within an EPA-mandated time frame to allow for the continued

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Pesticides in the Diets of Infants and Children TABLE 4-1 End Points for Various Toxicity Studies Study End Points Developmental toxicity Fetus: mortality, growth retardation, skeletal variations, gross external malformations, soft tissue/internal organ defects Female parent: general toxicity Reproductive Toxicity Male parent: general toxicity, effects  on fertility, reproductive organ changes Offspring: effects on viability, sex ratio, growth, behavior Carcinogenicity Tumor development and general toxicity Neurotoxicity Behavior, function, and motor activity deficits; microscopic nervous tissue  changes Mutagenicity Heritable lesions leading to altered phenotypes   SOURCE: EPA, 1984 registration of given product. The list of pesticides for which registration standards have been issued is referred to as List A and can be found in Appendix I of the Federal Register notice of February 22, 1989. Under the FIFRA Amendments of 1988, the data bases on the remaining registered pesticide products are being upgraded in five phases over a 9-year period. The first sections of this chapter describe in detail the present toxicity testing procedures for pesticides in relation to their registered use patterns and EPA's proposed changes or additions to these procedures. The conclusions and recommendation of the committee for further changes and additions to the toxicity testing battery to allow for more adequate consideration of the special testing needs for infants and children are presented. CURRENT METHODS: GENERAL CONSIDERATIONS Toxicity studies are required to assess potential hazards to humans through the acute, subchronic, and chronic exposure of laboratory animals to pesticides. The more specific types of toxicity that are determined include carcinogenicity; developmental (including teratogenicity in offspring) and reproductive toxicity; mutagenicity; and neurotoxicity (Table 4-1). Detailed information on the metabolism or biotransformation of the pesticide is also obtained. Consideration is given to testing individual metabolites in animals, and in or on pesticide-treated plants to which humans could exposed through their diet. The extent of metabolite testing required depends on the level of potential toxicity and environmental persistence of the metabolite. With the exception of

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Pesticides in the Diets of Infants and Children the acute toxicity tests, most tests are conducted to determine the nature of any toxicity that can be produced by repeatedly dosing animals over an extended period. The results enable toxicologists to estimate the safety of a material of humans (Loomis, 1978). Weil (1972) published the following set of guidelines, which reflected a consensus among toxicologists. These should be considered before initiating a toxicity test: Use, wherever practical or possible, one or more species that biologically handle the material qualitatively and/or quantitatively as similarly as possible to man. For this, metabolism, absorption, excretion, storage and other physiological effects might be considered. Where practical, use several dose levels on the principle that all types of toxicologic and pharmacologic actions in man and animals are dose-related. The only exception to this should be the use of a single, maximum dosage level if the material is relatively nontoxic; this level should be a sufficiently large multiple of that which is attainable by the applicable hazard exposure route, and should not be physiologically impractical. Effects produced at higher dose levels (within the practical limits discussed in 2) are useful for delineating mechanism of action, but for any material effect, some dose level exists for man or animal below which this adverse effect will not appear. This biologically insignificant level can and should be set by use of a proper uncertainty factor and competent scientific judgment.… Statistical tests for significance are valid only on the experimental units (e.g., either litters or individuals) that have been mathematically randomized among the dosed and concurrent control groups…. Effects obtained by one route of administration to test animals are not a priori applicable to effects by another route of administration to man. The routes chosen for administration  to test animals should, therefore, be the same as those to which man will be exposed. Thus, for example, food additives for man should be tested by admixture of the material in the diet of animals. In general, Weil's guidelines are considered by EPA in its toxicity testing requirements and subsequent evaluation of results for pesticides. One exception to Weil's points is found in his guideline 3. EPA does not recognize the existence of a dose level at which a carcinogen will not exert its effect. For carcinogens, EPA generally accepts a risk of 10-6, as extrapolated from bioassays using the nonthreshold modification of the linearized multistage model of Armitage and Doll (1954), as adequate for the protection of humans. The selection of animal species for toxicity tests depends on life span,

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Pesticides in the Diets of Infants and Children behavior, availability, and overall costs. EPA recommends using rats for subchronic, chronic, carcinogenicity, and reproduction studies; mice for carcinogenicity studies; and dogs for subchronic and chronic studies. Rats are routinely used for acute oral and inhalation studies and rabbits for eye and skin irritation studies and acute dermal studies. One exception to this is the use of guinea pigs for dermal sensitization testing. The rat and rabbit are recommended for developmental toxicity (teratogenicity) testing. Justification must be provided for the use of species other than those outlined above. The number of animals to be tested in each dose group depends on a number of factors, including the purpose of the experiment, the required sensitivity of the study, the reproductive capacity and the fertility of the species, economic aspects, and the availability of animals (IPCS, 1990). Table 4-2 lists the minimum number of animals required by EPA for some toxicity studies. For the most part, these numbers are consistent with those recommended by the International Program Chemical Safety (IPCS). The selection of dose levels for subchronic studies should be based on the results of acute toxicity testing, on range-finding studies, and on pharmacokinetic (metabolism, including rate in various tissues) data. For subchronic studies, four dose groups of animals should be included: a control group; a low-dose group (a dose that produces no compound related toxicity); a mid-dose group (a dose that elicits some minimal signs of toxicity); and a high-dose group (a dose that results in toxic effects but not in an incidence of fatalities that would prevent a meaningful evaluation; for nonrodents, there should be no fatalities) (EPA, 1984). This same guidance is relevant to chronic toxicity and reproduction studies. For teratology studies, the highest dose tested should elicit some signs of maternal toxicity, but the toxicity should not obscure the results. The one notable exception to this guidance pertains to carcinogenicity studies. The highest dose levels for these studies should be at a maximum tolerated dose (MTD), as determined in 90-day toxicity studies in the appropriate test species and from pharmacokinetic information on the material being tested. The Committee on Risk Assessment Methodology of the National Research Council (NRC) recently examined the criteria for the MTD and other doses used in carcinogenicity studies (NRC, 1993). The EPA has issued its own guidance for the selection of this dose level. Some of the factors to consider in selecting an MTD are: 10% decrement in body weight gain in 90-day study; observation of potential life-threatening lesions during microscopic examination of organs, e.g., liver necrosis; significant inhibition of cholinesterase activity in two biological compartments, such as brain and plasma; and significant signs of anemia or other biologically relevant effects on blood.

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Pesticides in the Diets of Infants and Children TABLE 4-2  Animal Model Requirements in Toxicity Studies   Minimum No. of Animals Required Study Type Males Females Dosing Age at Start of Study Acute oral (rat), dermal, or inhalation (rat) 5 5 Single Young adult Eye and skin irritation (rabbit) 6a   Single Young adult Dermal sensitization (guinea pig) b   Repeated Young adult 21-Day dermal (rat, rabbit, or guinea pig) 5 5 Repeated Rat, 200–300 g; rabbit, 2.0–3.0 kg; guinea pig, 350–450 g 90-Day oral (rat) 10 10 Repeated 6–8 weeks 90-Day inhalation (rat) 10 10 Repeated Young adult 90-Day dermal (rat, rabbit, or guinea pig) 10 10 Repeated Rat, 200–300 g; rabbit, 2.0–3.0 kg; guinea pig, 350–450 g 90-Day or chronic (1 ear) oral (dog) 4 4 Repeated 4–6 months Reproduction (rat)c 20 20 Repeated 8 weeks Teratology Rat   20d Repeated Young adult Rabbit   12d Repeated Young adult Chronic toxicity (1 or 2 year) (rat) 20 20 Repeated 6–8 weeks Oncogenicity (lifetime) (rat and mouse) 50e 50e Repeated 6–8 weeks a Either males or females may be used in this test. b The number of animals used depends on the method used. Several different experimental methods are acceptable. c EPA prefers that one male rat be housed with one female during mating. d Number of pregnant females required. e 50 rats and 50 mice of each sex. SOURCE: EPA, 1984. In general, EPA has set a cap on dosing of 1.0 g/kg/day for toxicity tests other than acute studies. This dose level is referred to as the limit dose and corresponds to approximately 20,000 ppm in the diet of rats, 7,000 ppm in the diet of mice, and 40,000 ppm in the diet of dogs. The duration of exposure for toxicity testing of a pesticide depends on the expected duration of human exposure to the pesticide in practice. The typical length of various toxicity tests and the number of doses administered are shown in Table 4-2. Repeated dosing refers to dosing once per day for the designated number of days. When the material is

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Pesticides in the Diets of Infants and Children given to the test animals in their diet, dosing is usually continuous for 7 days a week. If the material is administered by gavage (oral bolus dose), by dermal application, or by inhalation, doses are frequently given 5 days a week, which is acceptable to EPA because of practical considerations (EPA, 1984). The type of statistical analysis performed on the toxicity data resulting from these studies depends on the type of data under consideration (see, for example, Gad and Weil, 1982, for review). Interpreting the meaning of statistical significance for any particular parameter depends on the dose level at which it was achieved, the biological significance of the finding, and the normal spontaneous occurrence of this finding in the strain and species being tested. For regulatory purposes, the no-observed-effect level (NOEL) is defined as a dose level at which no effects attributable to the pesticide under test can be found. A no-observed-adverse-effect level (NOAEL) can also be determined for each study; however, EPA does not routinely use the NOAEL to regulate pesticide usage. To establish a NOAEL, the toxicologist must determine what is and what is not adverse effect, which can be defined differently by different scientists. For example, effects such as hair loss can be considered adverse by some and not by others. Plasma and red blood cell cholinesterase inhibition can be viewed as either an adverse effect or simply as a market of exposure to a pesticide. EPA uses the NOEL to calculate the acceptable daily intake (ADI) of the pesticide under consideration. More recently, the EPA has replaced the ADI with the reference dose, or RfD. Chronic studies, such as reproduction studies and lasting 1 year or longer in the rat or dog are used for this purpose. EPA does not routinely use the NOEL determined from teratology (developmental toxicity) studies for calculating ADIs because the observed effect are not considered chronic; however, these NOELs can be used to support the calculated ADI. EPA does routinely use developmental toxicity NOELs for other types of risk assessments, such as calculating the risk from acute, daily dietary or occupational exposure or from exposure of homeowners to a developmental toxicant. EPA's toxicity testing requirements for food and nonfood use pesticides have been published in 40 CFR Part 158. In general, for food use chemical with maximum human exposure, the following toxicity tests are required: • acute oral toxicity • acute dermal toxicity • acute inhalation toxicity • primary eye irritation • primary dermal irritation • chronic feeding toxicity • dermal sensitization • acute neurotoxicity • 90-day toxicity • 21-day dermal toxicity • 90-day neurotoxicity study • reproduction study

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Pesticides in the Diets of Infants and Children • carcinogenicity • developmental toxicity • mutagenicity tests • general metabolism study More than 30% of the tests for pesticides submitted to EPA in the past have been rejected. Those rejected must be resubmitted until they are in conformance with EPA criteria before registrations can be granted. The criteria for rejection are summarized in Table 4-3. Some of them fall in the category of regulatory policy; others involve scientific concerns. The most commonly cited reason for noncompliance is lack of characterization of the test material. To improve the quality of testing and incorporate new scientific methods in its testing requirements, EPA is currently revising the 40 CFR Part 158 data requirements for food and nonfood use pesticides. The proposed revisions to these requirements can be found in Table 4-4. ACUTE TOXICITY STUDIES General Description Acute toxicity studies provide information on the potential for health hazards that may arise as result of short-term exposure. Determination of acute oral, dermal, and inhalation toxicity is usually the initial step in evaluating the toxic characteristics of a pesticide. In each of these tests the animal is exposed to the test material only once on 1 day. Together with information derived from primary eye and primary dermal irritation studies (also 1 dose on 1 day), which assess possible hazards resulting from pesticide contact with eyes and skin, these data provide a basis for precautionary labeling and may influence the classification of a pesticide for restricted use. Acute toxicity data also provide information used to determine the need for child-resistant packaging, for protective clothing requirements for applicator, and for calculation of farm worker reentry intervals. A minimum number of animals, usually adults, are used in these studies and only the end points of concern are monitored, i.e., mortality, observable skin or eye effects, dermal sensitization, and observable neurotoxic behavioral changes. One exception is the inclusion of microscopic examination of neural tissues in the newly required acute neurotoxicity study. EPA's Proposed Changes Guideline number 81-1 (EPA, 1984), acute oral study in the rat, would be revised to include special visual system testing, which would be required for all organophosphate pesticide and other pesticides known to affect the visual system.

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Pesticides in the Diets of Infants and Children TABLE 4-3 Summary of EPA Rejection Factors Guideline Rejection Factor Acute Oral Toxicity (81-1) Lack of characterization of the test material Inadequate dose levels to calculate LD50 Acute Dermal Toxicity (81-2) Lack of characterization of the test material Inadequate percentage of body surface area exposed No quality assurance statement Improper number of animals tested per dose group Only one sex tested Omitted source, age, weight, or strain of test animal Acute and 90-Day Inhalation (81-3 and 82-4) Less than 25% of particles were <1 µm; LC50 could not be calculated; highest concentration did not produce toxicity Inadequate reporting of exposure methodology Protocol errors Lack of characterization of the test material Compound preparation Chamber concentration not measured Primary Eye Irritation (81-5) Lack of characterization of the test material Primary Dermal Irritation (81-5) Lack of characterization of the test material No quality assurance statement and/or no Good Laboratory Practice (GLP) statement Improper test material application/preparation Omitted source, age, weight, or strain of test animal Missing individual/summary animal data Dermal Sensitization (81-6) Control problems Dosing level problems Lack of characterization of the test material Unacceptable protocol or other protocol problems Individual animal scorers or data missing Scoring method or other scoring problem Reporting deficiencies or no quality assurance statement 90-Day Feeding—Rodent (82-1(a)) A NOEL was not established Lack of characterization of the test material or incorrectly reported Lack of clinical chemistry and/or lack of histopathology 90-Day Feeding—Nonrodents (82-1(b)) Reporting deficiencies Lack of characterization of the test material A NOEL was not established An investigation parameter missing

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Pesticides in the Diets of Infants and Children Guideline Rejection Factor 90-Day Feeding—Nonrodents (cont.) (82-1(b)) Information on the pilot study and other problems associated with dose level selection An investigational parameter missing Information on the pilot study and other problems associated with dose level selection 21-Day Dermal Toxicity (82-2) Lack of characterization of the test material Raw data analyses incomplete or missing A systemic NOEL was not established Inadequate percentage of body surface area exposed in each dose group Insufficient number of dose levels tested 90-Day Dermal Toxicity (82-3) Lack of characterization of the test material A systemic NOEL was not established Incomplete/missing raw animal data analyses Insufficient number of dose levels tested Poorly controlled test environment Chronic Feeding/ Carcinogenicity—Rats (82-3(a) and (83-2(a)) Missing histopathology information Missing information in study reports MTD was not achieved Missing historical control data Lack of characterization of the test material Deficiencies in reporting the study data Carcinogenicity—Mice (83-2(b)) Histopathology information missing MTD was not achieved Lack of historical control data Information missing in study reports Lack of characterization of the test material Deficiencies in reporting of study data Developmental Toxicity—Rodents (83-3(a)) Missing historical controls Lack of characterization of the test material Information missing or requiring clarification of the laboratories' methods Information missing or requiring clarification of the laboratories' results A NOEL was not established Statistical problems Did not use conventional assessments for skeletal or visceral examinations Developmental Toxicity—Nonrodents (83-3(b)) Clarification of laboratory procedures of interpretation of the data Individual maternal or fetal data missing Missing historical controls Lack of characterization of the test material Excessive maternal toxicity

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Pesticides in the Diets of Infants and Children Guideline Rejection factor Developmental Toxicity—Nonrodents (cont.) (83-3(b)) A NOEL was not established Statistical problems Reproduction (83-4) Information missing from laboratory results Lack of characterization of the test material Information missing or requiring clarification of laboratory methods or results Missing historical controls A NOEL was not established due to effects at the lowest dose tested Low fertility and/or inadequate number of animals were used per dose level A NOEL was not established  in the absence of reproductive effects Metabolism (85-1) Inadequate or missing data on identification of metabolites Improper methodology or dosing regimen Inadequate number of animals were used in the dose groups No individual animal data Improper reporting Inadequate or missing tissue residue analysis data Testing at only one dose level Only one sex of animal used Lack of an intravenous dose group No collection of 14 CO2 Dermal Penetration (85-2) Incomplete/missing data evaluation Improper test material preparation/application Raw data missing and incomplete summary tables No signed quality assurance statement Missing purity or concentration of test material   SOURCE: P. Fenner-Crisp, EPA, personal communication, 1992 The additional acute study proposed in guideline number 81-4 is acute neurotoxicity testing in the rat. This study would be required for all pesticide registrations (food and nonfood) and experimental use permits (EUPs), and it would include assessments of function and activity as well as histopathological (microscopic) examination of selected neural tissue. EPA presently requires that this study be conducted by manufacturers wishing to reregister.

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Pesticides in the Diets of Infants and Children SUBCHRONIC TOXICITY STUDIES General Description Subchronic exposures do not elicit effects that have a long latency period (e.g., carcinogenicity). However, they do provide information on health hazards that may result from repeated exposures to a pesticide over a period up to approximately 30% of the lifetime of a rodent. Subchronic tests also provide information necessary to select proper dose levels for chronic studies, especially for carcinogenicity studies for which an MTD must be selected. According to EPA (1984), rats selected for these studies should be started on the test material shortly after weaning, ''ideally before the rats are 6 and, in any case, not more than 8 weeks old." For dogs, dosing should begin when they are 4 to 6 months of age and "not later than 9 months of age." Most subchronic toxicity studies monitor clinical or behavioral (neurological) signs of toxicity, body weight, food consumption, eye effects, certain plasma or serum and urine parameters, organ weights, and gross and microscopic pathology. Clinical and behavioral signs of toxicity are observed and recorded daily. They can consist of activity, gait, excreta, hair coat, and feeding and drinking patterns. Body weight and food consumption data are routinely recorded throughout the study at intervals (usually weekly) determined by the length of the study. Ophthalmoscopic examinations are conducted at the beginning of the study and, typically, just before it terminates. The laboratory parameters typically examined are summarized in Table 4-5. The results of hematology testing indicate whether, for example, the chemical affects blood cell formation and survival, clotting factors, and platelets. Clinical chemistry and urinalysis results can indicate possible kidney, liver, pancreas, and cardiac function or toxicity as well as any electrolyte imbalance. Urinalysis results can indicate adequacy of kidney, liver, and pancreas function. After necropsy, the weights of certain organs are also recorded. These organs generally include brain, gonads, liver, and kidneys, which are the four required according to EPA testing guidelines (EPA, 1984). If toxicity is known to occur in another organ from previous testing, the weight of this organ should also be reported. For thyroid toxicity, for example, the weight of the thyroids should be recorded. Changes from untreated control animals are generally an indication of potential toxicity in this organ. A complete necropsy is performed after sacrifice or death of the test animal. Generally all tissues are examined, and those saved for microscopic examination are aorta, jejunum, peripheral nerve, eyes, bone marrow, kidneys, cecum, liver, esophagus, colon, lung, ovaries, duodenum,

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Pesticides in the Diets of Infants and Children capability. Such studies may also provide information about the effects of the test substance on neonatal morbidity and mortality and about the meaning of preliminary data for developmental toxicity. EPA requires that the study include a minimum of two generations and that one litter be produced each generation. Dosing of both parents should begin when they are 8 weeks old and continue for 8 weeks prior to mating. Dosing of parental males should continue at least until mating is completed. Dosing of parental females continues through a 3-week mating period and pregnancy and up to the time of weaning 3 weeks after delivery of the pups. Dosing of pups selected for mating to produce the second generation should begin at weaning and continue as discussed above. The dosing and breeding schedule is clarified in the timeline presented in Table 4-6. Parental animals should be observed daily for signs of toxicity. This is especially important for females during pregnancy in order to detect signs of difficult or prolonged parturition. Weights of parental animals are recorded weekly. The duration of pregnancy should be determined from the time evidence of mating was first observed. Each litter should be examined for the number of dead and live pups and for gross abnormalities. Live pups should be individually weighed on days 0 (optional), 4, 7 (optional), 14, and 21 after birth. A complete gross necropsy should be performed on all parental animals, all pups found dead prior to day 21 (weaning), and all weanlings not selected as parental animals for a next generation. Pups culled on day 4 do not have to undergo gross necropsy. Histopathology is required for reproductive and target organs (those known from previous studies to be adversely affected by the test material) for all control and high-dose parental animals and should be conducted on weanling animals (except for those selected as parental animals in the next generation) as described for parental animals (EPA, 1988). EPA's Proposed Changes The addition of a fertility assessment of parental males is recommended by EPA if fertility or reproductive parameters are found to be affected by the test chemical. The parameters to be examined or reported in this assessment include weight of reproductive organs, spermatid count, total cauda epididymal sperm count, assessment of sperm morphology and motility, examination of epididymal fluid for debris and unexpected cell types, and additional histopathology of the testes. A reproduction study (Guideline 83-4; EPA, 1984) could also be required to support nonfood uses if adverse effects on the reproductive system or developmental toxicity are observed in other studies.

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Pesticides in the Diets of Infants and Children TABLE 4-6 Approximate Dosing and Breeding Schedule for a Rat Two-Generation Reproduction Study Weeks of Study P1 F1 F2 0 Dosing of P1 males and females begins     8–14 P1 mating period     11–17 Dosing of P1 males may end at week 25; P1 females are killed     22–23   F1 mating; dosing of F1 males may end at week 40; F1 males killed   25–37   Remaining F1 females are killed F2 born. Litter sizes randomly adjusted to 8 each       F2 offspring are killed   SOURCE: EPA, 1984.

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Pesticides in the Diets of Infants and Children MUTAGENICITY STUDIES General Description A battery of mutagenicity tests is required to assess the potential of each test chemical to affect genetic material. The test selection criteria focus on the test's ability to detect, with appropriate assay methods, the capacity of the chemical to alter genetic material in cells. When mutagenic potential is demonstrated, these findings are considered in the assessment of potential heritable effects in humans, in the weight-of-the-evidence evaluation for carcinogenicity, and in the decision to require submission of a carcinogenicity study if otherwise conditionally required. Mutagenicity results per se are not used by themselves for risk assessment purposes, even when results suggest possible heritable genetic effects in humans. EPA's Proposed Changes EPA has already published changes to the 40 CFR Part 158 data requirements for mutagenicity (EPA, 1984). As described in Pesticide Assessment Guidelines: Subdivision F (EPA, 1984), the original mutagenicity test battery consisted of three assays: one for gene mutations, one for structural chromosome aberrations, and one for other genotoxic effects. Other testing included DNA damage and repair. The revised guidelines would require an initial battery of tests consisting of: Salmonella typhimurium reverse mutation assay; mammalian cells in culture forward gene mutation assay allowing detection of point mutations, large deletions, and chromosome rearrangements; and in vivo cytogenetics. Results derived from these assays could trigger the requirement for further mutagenicity testing. The type of additional required testing would depend on the observed results from the initial battery and other toxicity testing results. For example, testing could involve cytogenetic testing in spermatozoa if other test results suggest that they are targets. GENERAL METABOLISM STUDIES General Description Data from studies on the absorption, distribution, bioaccumulation, excretion, and metabolism of a pesticide may also allow more meaningful evaluation of test results and more appropriate risk assessment (as a result of more meaningful extrapolation from data on animals to humans). Such

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Pesticides in the Diets of Infants and Children data may also aid in designing more relevant toxicology studies. Information on metabolites formed in laboratory animals is also used to determine whether further toxicity testing is required on plant metabolites. If a major metabolite forms in the plant but not in the test animal, separate toxicity testing on the plant metabolite could be necessary. The extent of testing required depends on the level of concern raised by the initial battery of toxicity tests (acute and subchronic studies, one teratology study, and a battery of mutagenicity tests). As presently designed, the metabolism study consists of four separate parts: a single low, intravenous dose of radiolabeled test material (not required if the test material is insoluble in water or normal saline solution); a single low, oral dose of radiolabeled test material; 14 consecutive daily low, oral doses of unlabeled test material followed by a single low dose of radiolabeled material; and single high, oral dose of radiolabeled test material. Selection of the low dose is based on the NOEL. The high dose should elicit some signs of toxicity but not be so high that it results in mortality. The test species of choice is the rat. Urine, feces, and expired air are collected for 7 days after administration of the radiolabeled material or until >90% of the radioactivity is recovered. Bone, brain, fat, testes, heart, kidney, liver, lung, blood, muscle, spleen, residual carcass, and tissues showing pathology in this or prior tests should be examined for radioactivity for all animals except those given the intravenous dose. This is done to determine if the test material or radiolabeled metabolite accumulates in any particular organ and to relate this information to the findings in toxicity studies. In addition, quantities of radiolabeled material in feces, urine, and expired air must be monitored for all dose groups at appropriate intervals up to 7 days after dosing. Furthermore, urinary and fecal metabolites must be identified. EPA's Proposed Changes A metabolism study would also be required when significant adverse effects are observed in toxicology studies, including reproduction and developmental studies (Guideline 85-1; EPA, 1984). EPA is currently rewriting to guidelines for conducting metabolism studies and is including a tiered approach for study design and conduct. NEUROTOXICITY STUDIES General Description Neurotoxicity studies are required to evaluate the potential of each pesticide to adversely affect the structure or function of the nervous

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Pesticides in the Diets of Infants and Children system. The objectives of these studies are to detect and characterize the following: effects on the incidence and severity of clinical signs, the alteration of motor activity, and histopathology in the nervous system following acute, subchronic, and chronic exposures; the potential of cholinesterase inhibiting pesticides and related substances to cause a specific organophosphate-pesticide-type induced delayed neurotoxicity; other neurotoxic effects based on screening studies on certain chemical classes; and effects on organisms exposed prior to birth or weaning. Results from these studies may be used for qualitative and quantitative risk assessment. The guidelines for these studies were published in March 1991 as addendum 10 to the EPA guidelines (EPA, 1991a). EPA's Proposed Changes The changes in the requirements for neurotoxicity testing were described above under ''Acute Toxicity" and "Subchronic Toxicity." SPECIAL TESTING EPA intends to develop better definitions of the conditions under which domestic animal safety (Guideline 85-2; EPA, 1984) testing and visual system studies (Guideline 85-4; EPA, 1984) would be required for all organophosphates and other pesticides shown to affect the visual system. These studies could be of acute, subchronic, or chronic duration, whichever is deemed appropriate for the pesticide under study. Since guidelines have not been formulated for these studies, they will be designed in conjunction with EPA scientists. CONCLUSIONS AND RECOMMENDATIONS Conclusions Current and past studies conducted by registrants are designed primarily to assess pesticide toxicity in sexually mature animals. The protocols for these studies have evolved over several decades and have included some testing paradigms that allow extrapolation to infant and adolescent animals. These studies have produced some valuable information on toxicity and exposure. After reviewing EPA's current and proposed toxicity testing guidelines, however, the committee concluded that current studies do not directly address the following areas:

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Pesticides in the Diets of Infants and Children toxicity of pesticides in neonates and adolescent animals; metabolism of pesticides in neonates and adolescent animals; and exposure during early developmental stages (after the second trimester through adolescence) and sequelae in later life. Recommendations Studies should be redesigned and expanded in scope to elucidate the differences in the metabolism and disposition of pesticides in the infant, adolescent, and young adult. Current metabolism studies are designed to provide information about sex-related differences, metabolic pathways and excretion, bioaccumulation in tissues, and tissue distribution in adult rats. EPA uses the data to determine whether toxicity testing needs to be conducted on individual plant or animal metabolites in addition to the parent compound. The metabolism of pesticides in newborn animals needs to be more thoroughly investigated. Greater knowledge in this area would make it possible to develop computer programs for physiological pharmacokinetic modeling to forecast how information about metabolism in infant animals could be extrapolated to infant humans. The committee realizes that this is a very difficult area of investigation and application. Nevertheless, it urges that such investigations be pursued, since the resulting information could provide more realistic systemic exposure scenarios for risk assessment. A study should be conducted to compare the toxicity of several representative classes of pesticides in both adult and immature animals. Results of such a broad-range study designed to specifically address the infant and young adult animal should indicate whether comparative studies of this nature should routinely be required by EPA. This study should be designed to examine several critical end points in the developing animal, including neural (functional and behavioral), immune, and endocrine systems to cite a few examples. Because the battery of acute toxicity tests now required by EPA is generally performed in adult animals, very little information is available on acute toxicity in immature animals. Such data are important in determining dietary risk to infants and children for acutely toxic pesticides such as organophosphates and carbamates. The committee recognizes that some of these data can be obtained from multigeneration studies if specific observation requirements are added to the current studies. Test animals should be exposed to the chemical of concern early in their lives so the risks of exposure of infants and children to the compound can be more adequately assessed.

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Pesticides in the Diets of Infants and Children The committee recognizes the difficulty in dosing animals during lactation and is aware that testing requirements would have to be modified to accomplish these studies. EPA Guideline 83-5 for a chronic toxicity/carcinogenicity study states that exposure of rats to pesticides should begin at approximately 6 to 8 weeks of age, essentially when they are adolescents (EPA, 1984). Because the effects of the pesticide in the rat are not determined early in its lifetime, chronic toxicity/carcinogenicity studies in adolescent animals may not be representative of the responses of younger animals. Current reproduction studies (Guideline 83-4; EPA, 1984) partially address this period in the life of a rat, but the effects of early exposure are not addressed past 21 days of age for second-generation pups or past the death of the second-generation parents (first-generation pups used for mating to produce the second generation). The protocol does not indicate whether exposure early in life has any impact on the adults or whether continuous exposure from birth to young adulthood influences the severity of the toxicity over a lifetime. FDA has used the multigeneration studies to include the F2A or F3A generation of laboratory animals for direct and indirect food additives (Becci et al., 1982). To obtain lifelong data on rodents for a given pesticide, the committee recommends that the testing guideline for a rat chronic toxicity/carcinogenicity study be modified to include in utero exposure during the last trimester, exposure through the mother's milk, and after weaning, oral exposure through diet. This would mean that weanlings from the F1A or F2A generation would be selected from each dose group and tested throughout their lifetimes (see Table 4-6). In addition to this group, another smaller group of rats from the F1 generation would be killed at 6 months and 1 year and necropsied to examine the same parameters normally measured at the end of a lifetime feeding study. The NTP tested three chemicals using a similar protocol and their standard protocol for an carcinogenicity study. One of the chemicals was ethylenethiourea, which is a breakdown product and metabolite of the ethylenebisdithiocarbamate fungicides and a thyroid toxicant (decreases T3 and T4). In utero exposure did not affect the occurrence of liver tumors in male and female mice, but did result in a sex-dependent increase in the number of malignant thyroid tumors in mice and rats (NTP, 1992). Measurement of the serum thyroid hormones T3 and T4 and serum TSH should be routinely added to the EPA chronic/carcinogenicity study protocol or to the subchronic toxicity protocol for the rat so that adverse effects on thyroid function can be determined earlier. When examining the parameters currently measured in the EPA chronic/ carcinogenicity study, the committee found that endocrine function

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Pesticides in the Diets of Infants and Children was adequately covered for all but the thyroid. Although the thyroid is saved in these studies for microscopic examination and its weight is recorded, the committee believes that changes in the functioning capabilities of this organ can occur regardless of whether there are organ weight or histopathologic changes. If abnormalities are found during histopathologic examination of the spleen, lymph nodes, thymus, and bone marrow, more detailed and specific studies should be conducted on a case-by-case basis relevant to the types of effects initially seen in immune system tests. EPA has developed protocols for immunotoxicity testing for some pesticides that affect the immune system, and the agency is considering developing a generic testing protocol. The committee believes that because the human immune system is one of the most robust of systems in terms of resistance to pesticides or other chemical toxicity, initial evaluation using current histopathologic examination of spleen, lymph nodes, thymus, and bone marrow should be sufficient unless abnormalities are noted. A modified reproductive/developmental toxicity study in the rat is suggested for registration of all food-use pesticides. One set of dams in this study would be dosed continuously with the test material from day 6 of gestation through birth of the pups and until weaning of their offspring at 21 days of age. A developmental assessment would be performed on the pups as described in EPA's recently published developmental neurotoxicity testing guidelines (EPA, 1991a). In addition, a set of pups from each dam would undergo gross and histopathologic examination at day 60 post partum. The second set of dams would be dosed from day 6 of gestation to term; however, these animals would not be allowed to deliver but, rather, would be subjected to cesarean section as in a routine teratology study. The fetuses would be subjected to skeletal and visceral examination, as described for a teratology study (Guideline 83-3) designed to examine the prenatal development of pups. This study design allows a determination of the reversibility of postnatal significance of findings seen in fetuses at the time of cesarean section. EPA has indicated in its proposed changes to Part 158 that a similar study be required; however, the committee recommends that this study be made a requirement for registration of all food-use pesticides. Because neurotoxicity is such an important consideration for the newborn, EPA should continue to revise its published guidelines on developmental and functional neurotoxicity testing as new information emerges from the actual conduct of preregistration studies and from ongoing research in rodent neurotoxicity.

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Pesticides in the Diets of Infants and Children The committee supports EPA's proposed requirement for acute and subchronic neurotoxicity testing for pesticides and encourages the agency to make this a general requirement for all food-use pesticides—not just for organophosphate and carbamate pesticides. New approaches to neurotoxicity testing are described in the report Environmental Neurotoxicology (NRC, 1992). EPA should develop a general guideline for visual system toxicity testing that can be modified and applied on a case-by-case basis. The eye is exquisitely sensitive to changes in glucose metabolism, blood flow, and neuronal function, and several pesticides have been shown to be visual system toxicants (hexachlorophene, naphthalene, 2,4-DNP, and some organophosphates). In the past, scientists have examined the effects of chemicals that may irritate the eye by accidental contact. More recently, however, researchers have been examining the effects of chemicals on specific sections of the visual system, such as the optic nerve, iris, retina, and lens. The guideline proposed by the committee should be applied to species in which this type of testing appears to be appropriate, e.g., EPA has recently considered protocols for visual system testing in dogs. Recent studies indicate that visual system damage may be associated with dietary exposure to some cholinesterase inhibiting compounds. Thus the committee supports EPA's proposed testing (the sensory evoked potential test) of such pesticides for visual system toxicity. However, it does not believe that a single protocol would suffice to cover all classes of compounds because different classes would affect different parts of the visual system. REFERENCES Armitage, P., and R. Doll. 1954. The age distribution of cancer and multi-stage theory of carcinogenesis. Br. J. Cancer 8:1–12. Becci, P.J., K.A. Voss, F.G. Hess, M.A. Gallo, R.A. Parent, K.R. Stevens, and J.M. Taylor. 1982. Long-term carcinogenicity and toxicity study of zearalenone in the rat. J. Appl. Toxicol. 2(5):247–254. EPA (U.S. Environmental Protection Agency). 1984. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals. Revised Ed. November 1984. PB-86-108958. Washington, D.C.: U.S. Environmental Protection Agency. EPA (U.S. Environmental Protection Agency). 1988. FIFRA Accelerated Reregistration Phase 3 Technical Guidance. US EPA 540/09–0784. Washington, D.C.: U.S. Environmental Protection Agency. EPA (U.S. Environmental Protection Agency). 1991a. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals, Carcinogenicity of Ethylene Thiourea [CAS No. 96-45-7] in F/344 Rats and B6C3F1 mice. Addendum 10—Neurotoxicity. Washington, D.C.: U.S. Environmental Protection Agency.

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Pesticides in the Diets of Infants and Children EPA (U.S. Environmental Protection Agency). 1991b. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals Series 84. Addendum 9—Mutagenicity. PB-158394. 540/09-91-122. Washington, D.C.: U.S. Environmental Protection Agency. Gad, S.C., and C.S. Weil. 1982. Statistic for toxicologists. Pp. 273–320 in Principles and Methods of Toxicology, A.W. Hayes, ed. New York: Raven Press. IPCS (International Program on Chemical Safety). 1990. In Environmental Health Criteria 104: Principles for the Toxicological Assessment of Pesticide Residues in Food. Geneva, Switzerland: World Health Organization. Loomis, T.A. 1978 Essentials of Toxicology. Philadelphia, Pa.: Lea & Febiger. NRC (National Research Council). 1992 Environmental Neurotoxicology. Washington, D.C.: National Academy Press. NRC (National Research Council). 1993. Issues in Risk Assessment. Washington, D.C.: National Academy Press. NTP (National Toxicology Program). 1992. NTP Technical Report on the Perinatal Toxicology and Carcinogenesis Studies of Ethylene Thiourea (CAS No. 96-45-7) in F3441N Rats and B6C3F1 Mice (Feed Studies). NTP TR 388. Research Triangle Park, N.C.: National Toxicology Program. Weil, C.S. 1972. Guidelines for experiments to predict the degree of safety of a material for man. Toxicol. Appl. Pharmacol. 21:194–199.

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