Toxicology

Chapter 27

Toxicology Thomas E. Harem, Jr., Angela King-Herbert, and Mary Ann Vasbinder

I. II. III. IV. V.

Introduction ....................................... History .......................................... H u m a n e Use o f A n i m a l s in T o x i c o l o g y ................... T h e Use of the R a t in P h a r m a c o l o g i c Testing: A n O v e r v i e w . . . . . Toxicological Studies ................................ A. A c u t e Toxicity Testing ........................... B. C h r o n i c Studies ................................ 1. C a r c i n o g e n i c i t y Testing ........................ 2. M u t a g e n i c i t y Studies . . . . . . . . . . . . . . . . . . . . . . . . . . 3. R e p r o d u c t i v e Studies . . . . . . . . . . . . . . . . . . . . . . . . . . 4. D e v e l o p m e n t a l Studies . . . . . . . . . . . . . . . . . . . . . . . . . 5. N e u r o t o x i c o l o g y Studies . . . . . . . . . . . . . . . . . . . . . . . . C. I n h a l a t i o n T o x i c o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. P h a r m a c o k i n e t i c s , P h a r m a c o d y n a m i c s , a n d B i o m e t a b o l i s m Studies ............................ VI. C o n s i d e r a t i o n of Variables in Toxicological Testing . . . . . . . . . . . A. R a t Strain or Stock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. W a t e r ........................................ C. Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Intestinal F l o r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. C a g i n g a n d B e d d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Disease Status .................................. G. E n v i r o n m e n t a l E n r i c h m e n t C o n s i d e r a t i o n s .............. VII. H u m a n e E n d p o i n t s a n d Role of A l t e r n a t i v e s in T o x i c o l o g y Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Safety in T o x i c o l o g y E x p e r i m e n t s ....................... IX. C o n c l u s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

THE LABORATORY RAT, 2ND EDITION

804 804 804 805 805 806 806 806 807 807 808 808 809 810 811 811 811 811 812 812 813 813 813 813 813 814

Copyright 9 2006, 1980, Elsevier Inc. All rights reserved.

803

804

THOMAS

I.

E. HAMM,

JR.,

ANGELA

INTRODUCTION

It is impossible in a single chapter to cover the enormous body of information that has resulted from the use of the rat in toxicology. Rather, this chapter will focus on major points concerning the use of the rat in toxicology and provide key references that will allow the reader to delve further into the subject. In any discussion of toxicology, it should be emphasized that the toxicity of any compound can be easily influenced by the design and conduct of the experiment. For example, results of an experiment using one strain or stock of rats, in which experimental variables (diet, water quality, intestinal flora, caging, bedding, etc.) are not defined or controlled cannot be extrapolated with absolute confidence to other species, including humans. Moreover, putative toxicities to humans are most validly defined when a compound causes similar findings in multiple experiments.

II.

KING-HERBERT,

AND

MARYANN

VASBINDER

wastes are regulated under the Resource Conservation and Recovery Act; (4) water pollution is regulated under the Federal Water Pollution Control Act; (5) drinking water is regulated under the Safe Drinking Water Act; and (6) air pollution is regulated under the Clean Air Act (CAA). Two other major governmental agencies were also charged with consumer safety in the United States. The Occupational Safety and Health Administration (OSHA) including the Consumer Products Safety Commission, relied mainly on already established toxicology data; and The National Institutes of Occupational Safety and Health (NIOSH) collected and analyzed data for OSHA. The FDA, EPA and the Organization for Economic Cooperation and Development (OECD) have worked together to define standard toxicology tests. These standards are constantly evolving, and the latest standards, which can be found by contacting respective organizations or visiting their Web sites, must be considered when designing a test that might be used in a regulatory approval process.

HISTORY

III. Toxicology as an organized discipline is relatively new. The first journal expressly dedicated to experimental toxicology, Archiv fur Toxikologie, started publication in Europe in 1930 (Amdur et al., 1991), and the preeminent toxicology organization, the Society of Toxicology, was not founded until 1961 (Hays, 1986). The reader is referred to excellent reviews of the history of toxicology (Gallo and Doull, 1991) and regulatory toxicology (Gad, 2001). Animals have been used in experiments and testing for hundreds of years based on the assumption that they react similarly to experimental conditions as do humans. The use of animals in toxicology was formalized in the 1930s with the passage of the Food Drug and Cosmetic Act in 1938. Basis for this act included a 1933 report describing a severe allergic reaction to Lash-Lure, a synthetic aniline dye for eyebrows and eyelashes (Parascandola, 1991), and a rash of deaths associated with administration of the new wonder drug sulfanilamide that were later attributed to the vehicle of the drug, ethylene glycol. The Food, Drug, and Cosmetic Act gave the U.S. Food and Drug Administration (FDA) the authority to require toxicology tests necessary to assess the safety of food, drugs, medical devices, and cosmetics. In 1970, the U.S. Environmental Protection Agency (EPA) was created. It was given a wide variety of regulatory duties: (1) pesticides are regulated under authority of the Federal Insecticide, Fungicide, and Rodenticide Act: (2) chemicals in commercial production are regulated under the Toxic Substances Control Act; (3) hazardous

H U M A N E USE OF A N I M A L S IN TOXICOLOGY

Many events in the 19th (French, 1975) and 20th centuries lead to current concepts in animal use in toxicology, including the concepts of humane endpoints and the use of alternatives. For example, any experiment, including those in toxicology, should be preceded by a review of the three R's--reduction, refinement and replacement--that were first outlined in The Principles of Humane Experimental Technique (Russell and Burch, 1959). The atrocities of World War II lead to the Nuremberg Code of Ethics of 1947. The Nuremberg Code included the following provision: "'The experiment should be so designed and based on the results of animal experimentation and a knowledge of the natural history of the disease or other problem under study, that the anticipated results will justify the performance of the experiment" (Silverman, 1985). In 1964, the World Medical Association adopted the Declaration of Helsinki, a formal code of ethics for doctors in clinical research. This code was extended in 1973 by the 29th World Medical Assembly. This new code, Helsinki II, included the following provision: "Biomedical Research involving human subjects must conform to generally accepted scientific principles and should be based on adequately performed laboratory and animal experimentation and on a thorough knowledge of the scientific literature" (Silverman, 1985). These ethical standards established that animal experimentation must be conducted before human experimentation when possible.

27.

805

TOXICOLOGY

IV.

T H E U S E OF T H E RAT IN

P H A R M A C O L O G I C TESTING: AN OVERVIEW Drug discovery is a complex process that can take more than 10 years and require hundreds of millions of dollars for the development of a single product. All of the testing before clinical trials requires both in vivo and in vitro testing. The rat is the major rodent species used for in vivo testing during drug development. Drug discovery is rigorous and carefully staged, beginning with the selection of a disease on which to focus. This decision is based on needs in the patient population, expertise and technology within the company, and potential competition and commercial considerations. Once marketing and commercial studies are completed, a commitment can be made to search for a particular "product type." The desired characteristics of that product are outlined, including efficacy, safety, route of administration, frequency of dosing, pricing, and needs of the patient populations. Scientists are then asked to consider the types of biological "targets," which are molecules that may be addressed by drugs to produce a therapeutic effect. A decision is then made to select a particular target class or family of molecules that will be investigated for drug development. A hypothesis is formed, and targets are selected with the hope that a new drug molecule with the proper characteristics can be created. The route of drug administration must be determined in the preclinical phase. Oral formulations include capsules, tablets, or oral gavage. Solutions may be administered intravenously, intraperitoneally, or intramuscularly, but this requires a more limited application to the general human population. Inhalational administration can be accomplished by nebulization or by powder-form or metereddose inhalers. Creams, ointments, lotions, and sprays are the options for topical application. Lastly, preparations for nasal application can be made as solutions or suspensions. Once the route of administration is determined, the vehicle or carrier is decided. Important considerations for selection of vehicles include pH, solubility, viscosity, osmolality, sterility, and biocompatibility with the compound. The volume administered is also critical, and several authors have reviewed these issues (Diel, 2000; Morton, 2000). The next phases require setting up screening systems that are robust and conducive to high-volume, automated tests. The new compounds are analyzed by medicinal chemists to identify molecules that may be useful as a candidate compound. Selected compounds are then expanded into a series of compounds that contain variations of the starting molecule. Eventually, a compound is selected based on in vitro potency, selectivity, and the drug-like properties of the compound.

Synthesis and testing of molecules to create an optimal balance of potency, selectivity, and safety are part of drug optimization. Simultaneously, pharmacological screening for efficacy and potential toxicity are carried out by using in vitro testing. Once chemicals are determined to have characteristics that make them interesting as potential drugs, they are called drug candidates and are slated for in vivo testing. Drug metabolism and pharmacokinetic studies are aimed at understanding the pharmacokinetic and pharmacodynamic nature of compounds. The initial testing typically consists of a single oral dose, administered to rats, with evaluation over a short period of time (less than 72 hours). Samples such as urine, feces, and blood are collected temporally and analyzed for drug or drug metabolites. After the studies are accomplished in rodents, the study is repeated in a second species such as the dog. These studies are the first effort at characterizing the nature of the drug in animals. Target validation studies investigate efficacy and physiologic response of the drug molecule on the target. This work may also require a basic understanding of the mechanism of action of the target. Studies that provide supportive data for the pharmacologic usefulness of a molecule are often called "proof of concept" studies. Safety parameters must be well defined for each chemical entity. Dose-range-finding studies, typically in the rodent and dog, provide a range of safe and efficacious dosages for compounds. Studies describing toxic doses and description of the toxicity are required for drug approval. All of the data collected are then presented to an internal review process for approval, and a request is made for review by an external ethical review committee. These approvals are necessary to gain permission to initiate clinical evaluation in humans. Next the data is submitted to the FDA who must approve the Investigational New Drug (IND), and the plan to initiate clinical studies. Safety studies using animals continue during phase I clinical trials, including chronic toxicity tests, reproductive toxicity, oncogenicity studies, and supplementary pharmacology work. This is a dynamic process and occurs as data are collected and compared across species and the need for information becomes apparent (Marzo, 1997; Roberts, 2001). After successful phase III trials, the new drug application (NDA) is submitted to the FDA with the request for approval to market the new drug.

V.

TOXICOLOGICAL STUDIES

The goal of toxicology is to minimize the adverse effects of compounds in the human population and the

806

THOMAS

E. HAMM,

JR.,

ANGELA

environment. Assessment of chemicals, pharmaceuticals, devices, etc., in animals is the best measure of interactions between complex organ system functions and the test article. Evaluation of compounds in an animal, at doses that approach a maximally tolerated dose, provides an understanding of the consequences of physiologic, pharmacological, and toxicological actions. The FDA has written guidelines for toxicity testing (FDA, 1993b) and international harmonization of tests has been described by the OECD (OECD, 1993), but these guidelines have not yet been adopted. Regulations for both Europe and Japan have also been published (EEC, 1992; PAFSC, 1997). Toxicology studies are conducted as described by Good Laboratory Practice in the United States (FDA, 1978) or by similar standards in other countries.

A.

AcuteToxicityTesting

In acute toxicity testing, a single dose of a test compound is given once to ascertain the adverse effects on the animal. The compound may be administered orally, parenterally, and dermally or by inhalation (Echobichon, 1992). The route of exposure in the test animals usually depends on the usual route of exposure in humans. Typically, rats and mice are the species of choice for acute toxicity testing, with test protocols requiring the use of both sexes. The OECD has established guidelines that recommend that in acute oral toxicity testing, only female rats should be used when there is no indication that males are more sensitive to the compound being tested (OECD, 1998). Range-finding tests with a small number of animals are usually used to determine the relative lethality of a compound. Then larger numbers of animals are used to determine relative toxicity. LDs0 is a dose that is lethal to half of the animals on study. The EDs0 is a measure of the dose for which half of the animals exhibit an effect and half of the animals exhibit no effect. In LDs0 testing, several dose levels of the test compound are used, with one dose per group of animals, and the testing is performed in replicates (Ballantyne, 1999). Typical LDs0 tests require 100 animals or more. In addition to providing the median lethal concentration of a test compound, LDs0 determinations can provide information in terms of the dose-response relationship, clinical signs of toxicity, and gross and histopathologic findings (Hayes, 2001). LDs0 testing for acute lethality is no longer required by the FDA in an effort to reduce the number of animals necessary for study and lessen the number of animals that experience pain and distress. Alternatives to the LDs0 have been described (Sass, 2000; Botham, 2002). The FDA specifically refers to the limit test as an alternative to LDs0 testing. The limit test is used to determine if the toxicity of a compound is above or below a specified dose (FDA,

KING-HERBERT,

AND

MARYANN

VASBINDER

1993b). Five to 10 animals of each sex are exposed to the test compound, and toxic responses are recorded in a given time. Further testing is pursued based on the animal response. Other alternatives to the LDs0 test include the up and down procedure (UPD), fixed dose method (FDM), and the acute toxic class method (ATC). These alternative tests provide information about the toxicity of the compound while minimizing the number of animals on study and number of animal deaths. The UPD test gives a point estimation of the LDs0 using six or seven animals. This test starts with a fixed dose and adjusts the subsequent testing based on the toxic response of the initial test. The UPD is composed of three tests: the primary test, the limit test, and a final test for estimation of the slope and confidence interval for the dose response curve. The F D M avoids using death of animals as an endpoint and relies on observed signs of toxicity at one of a series of fixed dose levels (Botham, 2004). The ATC uses fixed dose levels, but animal mortality serves as an endpoint. Details of these testing strategies can be found on the OECD Web site (http://oecd.org/ehs/test/testlist.htm).

B.

Chronic Studies

Various longer term tests may be conducted. Carcinogenicity, mutagenicity, reproductive toxicology, developmental toxicology, and neurotoxicology will be discussed in the following sections.

1.

CarcinogenicityTesting

Carcinogenicity studies are performed to assess the potential of a compound to cause cancer. The primary species for this testing is the rat; the mouse is used secondarily. The experimental approach to demonstration of carcinogenic potential of a drug must be flexible, but a scheme usually includes one long-term rodent study and either another rodent study in an alternate species or shorter in vivo studies. These shorter investigational studies are performed to provide information about carcinogenesis, including mechanism or insight into induction and promotion of the cancer. Dose dependency and the relationship to carcinogenicity should be evaluated in these studies. This may be accomplished in a number ways. For example, tissues can be collected and evaluated for morphologic and histochemical changes. Biochemical analysis of systemic or tissue hormone levels, enzymes, and growth factors may also be important information in these studies (ICH, 1997). Animal strain, food type, and housing must be taken into consideration when setting up long-term rodent studies. The species must have longevity that meets the

807

27. T O X I C O L O G Y

criterion for the duration of the study. The species must not have background disease or cancers that confound the study. The diet can significantly impact the manifestation of cancer and the longevity of the animals. The National Cancer Institute has recommended guidelines for long-term carcinogenicity studies (NCI, 1976), and a review of the carcinogenesis bioassay test has been published (Hamm, 1985). Carcinogenicity studies in rodents are 2-year studies incorporating exposure to the compound for a significant portion of the lifespan of the animal. Usually, both sexes are used with the route of exposure the same as would be found for human exposure. Groups of approximately 50 randomly assigned animals per exposure concentration (high, mid, and low dosage) are used, along with untreated controls. A complete necropsy and subsequent microscopic examinations are performed on all exposed and control animals on the study, including those that are euthanized for humane reasons before the end of the study. Evaluation of the carcinogenic potential of a drug is multifold. The decision should be based on tumor incidence and latency in the long-term studies, the pharmacokinetics of the compound in the animal compared with humans, and information gained from the ancillary data that may shed light on human disease. This information together forms a "weight of evidence" approach to the scientific interpretation of the data (ICH, 1997). 2.

Mutagenicity Studies

These studies are designed to determine if compounds induce genetic damage. Both in vitro and in vivo tests are used to assess genetic damage directly or indirectly, by various mechanisms. In vivo tests for genotoxicity are important test batteries because they allow examination of the whole animal, including tissue distribution, absorption, metabolism, and excretion of chemicals and their metabolites (Dean et al., 1999). The use of animal models allows investigation into the mechanisms of DNA damage, the relationship between gene structure and cell and tissue function, and the relationship between sequence alteration and disease. Damage to DNA can include frameshifts, insertions, and deletions of DNA. Recombinatorial and numerical chromosome changes have the potential to induce mutagenesis. These changes can be manifested by inheritable disease or cancer (Derelanko and Hollinger, 2002). The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use drafted two key guidance documents for determination of genotoxicity in pharmaceutical testing. The first recommendation, Guidance of Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH, 1995) provides recommendations for in vitro and in vivo

testing and makes recommendations for interpretation of test results. The second document, Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals (ICH, 1997), establishes a standard set of tests for potential genotoxicity and provides guidance on the test limitations. The two primary in vivo tests recommended are the micronucleus and metaphase analysis assays. The micronucleus test screens for chemicals that cause spindle formation and micronuclei (FDA, 2000), which are clumps of cytoplasmic chromatin that are formed when chromosomes are fragmented or fail to be incorporated into the cell nuclei during anaphase of cell division. This leads to abnormal chromosomes, spindle abnormalities, and micronuclear fragmentation. Clastogens are agents that cause double-strand breaks in the chromosomes, which result in the formation of micronuclei. This assay is typically performed in healthy, young adult rats or mice. The micronucleus test is performed by exposing the animal to a chemical by a predetermined route, typically orally or intraperitoneally, over a predetermined time course. At necropsy, bone marrow or peripheral blood is collected and stained. Positive results indicate that a substance is the cause of the formation of micronuclei (Ashby, 1995; FDA, 2000). Another test less frequently used for in vivo testing of compounds is the metaphase analysis assay. Mice or rats are administered a dose of test compound. At predetermined time intervals, colchicine is given to arrest bone marrow cell division in the metaphase stage. A necropsy is performed, and bone marrow cells are evaluated microscopically for chromosomal damage (FDA, 2000). Some of the most promising newer test systems are the transgenic rodent mutation models. Early transgenic animal models were created in the early 1990s, by inserting bacteriophage lambda shuttle vector systems of either Lac I or Lac Z, for creation of the Muta Mouse and the Big Blue assay systems, respectively (Kohler et al., 1991; Provost et al., 1993). The Big Blue assay system has been developed for both rats and mice. This recoverable reporter gene allows the detection of in vivo mutations in various target tissue locations (Turner et al., 2001; Yu et al., 2002). Animal models have also been developed that provide the ability to monitor, recover, and sequence mutations that arise in all mammalian tissues (MacGregor et al., 1998). 3.

Reproductive Studies

The purpose of reproductive toxicology studies is to discover any possible substances that impact reproduction (OECD, 1996). Reproductive toxicology studies are used to identify chemicals or physical agents that impact reproduction for example by interfering with fertility in both sexes (Neubert, 2002). The basic unit of study is the mother, father, and the offspring (Hoyer, 2001). Toxicants

808

THOMAS

E. H A M M ,

JR.,

ANGELA

may affect reproduction as a primary target, causing damage to the reproductive organs, germ cells, or the developing embryo/fetus. Alternatively, indirect toxicity may impact reproduction by mimicking or eliminating sex hormones or affecting hormone receptors. Rats are the preferred rodents used for studying reproductive toxicology. They are suited for this purpose for several reasons. Rats are small and highly fertile and have a short gestation period (21 days) and estrus cycle (4 to 5 days). They reach puberty at 37 to 67 days and are spontaneous ovulators that can reproduce all year. This comparatively short reproductive cycle is desirable for multigenerational studies. The average litter size is six to nine pups, and several litters may be born to one dam in a year. The large litter size allows for intra- and interlitter comparisons that are more statistically powerful. It is also helpful that there exists a large database of knowledge on rat toxicology (Ecobichon, 2002). Guidelines for reproductive toxicity studies have been published by the FDA, (FDA, 1993a, 2000) and by other regulatory bodies (EPA, 1996; OECD, 1996). These flexible guidelines provide a framework for initial testing. Rats of age 5 to 9 weeks are acclimated and randomized by body weight. The goal is to obtain 20 males and 20 pregnant females per group, with high-dose, intermediate-dose, low-dose, and control groups. Males for the trial should be dosed at least 10 weeks before mating to enable examination of the effect of the compound on a full cycle of spermatogenesis. Dosing of females begins at the same time and continues through parturition and weaning of the litter. Offspring should be dosed throughout the postnatal life. Successful mating is determined by daily examination for a copulatory plug and/or vaginal smears. After mating, the male and female pairs are separated. Studies typically continue for two generations, so at least one male and one female from each litter should be randomly selected to produce the next generation (FDA, 2000). Clinical observations are made twice daily, and abnormalities in condition or behavior are recorded. Body weights are recorded weekly, and vaginal smears can provide information regarding estrus cycle length and regularity. Litters are examined, and the number of live, dead, or abnormal pups are noted. Sex and weight of the pups is recorded (OECD, 1996). Necropsies are conducted on any dead or anomalous pups that are found during the study. Pups that are not to be used for breeding are euthanized, and a necropsy examination is performed. Reproductive organs and other tissue are collected for gross and histologic evaluation. Sperm motility, morphology, and number are assessed at necropsy. Review of female reproductive performance is made by a series of indexes. The female fertility, gestation, live-born, weaning, and sex ratio indexes provide population-based data for reproductive performance (FDA, 2000).

4.

KING-HERBERT,

AND

MARYANN

VASBINDER

D e v e l o p m e n t a l Studies

The purpose of developmental toxicological studies (teratology) is to evaluate compounds for their effect on the developing fetus. The rat and rabbit are the preferred species for these studies. These tests may be integrated into the multigenerational tests during evaluation of reproductive toxicity. The test compound is administered to pregnant animals from implantation (day 6 for rats) to the day before parturition (day 20 or 21). Manifestations of toxicity to the fetus are categorized as death, structural anomaly, altered or retarded growth, or functional deficiencies. Studies are monitored in a manner similar to that of the reproductive studies, with clinical observations of the dam conducted twice daily. At necropsy, reproductive tracts and fetuses are collected for counts of live/ dead fetus and for gross and histologic examination of the fetus and reproductive organs. Special stains may be used to facilitate understanding of the mechanism of toxicity (FDA, 2000).

5.

Neurotoxicology Studies

Neurotoxicity has been defined as an adverse change in the structure or function of the nervous system that results from exposure to a chemical, biological, or physical agent. Damage to the nervous system can occur by many different mechanisms. The chemical may affect the nervous system as a primary target or may affect the nervous system indirectly by toxicity to other target organs. Disease of the nervous system may be categorized as neuronopathies, axonopathies, myelinopathies, or neurotransmitter effects (Mandella, 2002). The goals of neurologic testing should include detection of neurotoxic potential and definition of dose range and time-course, as well as give a profile for effect to focus future testing (Moser, 1991). Assessment of neurotoxicity must include the severity, type, and reversibility of the effects. Various governmental bodies have described neurotoxic testing guidelines. The FDA has not identified any formal protocol requirement for systematic neurotoxicity testing for pharmaceutics; requirements are made on a case-by-case basis (Mandella, 2002). The FDA has taken this approach to accommodate the broad spectrum of behavioral, structural, and biochemical changes and to lend flexibility to the testing strategy (Ross, 2002). The guidelines require that neurobehavioral and neuropathologic testing be assessed to describe toxicity and risk. The FDA has published draft guidelines for food additives in the Redbook (FDA, 2000). An ad hoc expert panel described a two-tiered method of testing for assessment of neurotoxic potential of pharmaceuticals. Tier-one testing requires assessment of neurobehavioral changes through clinical observations and testing of motor

27.

809

TOXICOLOGY

activity (Irwin, 1968). The second tier of testing is based on findings from the primary screenings and is to be tailored to evaluate specific toxicities. The OECD and EPA have published other guidelines (EPA, 1998; Haggarty, 1989; OECD, 2000b). The first-tier studies typically follow EPA study guidelines by using 20 (10 male, 10 female), young-adult rats (Mandella, 2002; Ross, 2002). Half of these animals (10) are used for neuropathology. The standard toxicity studies use at least three dose groups and a control. The highest dose is usually the maximum tolerated dose; mid- and low-dose groups are calculated from this dose. Route of exposure may depend on a number of factors, including the human indication for dosing or optimal bioavailability. Acute, single-dose studies are followed by 2- to 4-week, 90-day, and 12-month chronic studies. (EPA, 1998; FDA, 2000). The functional observational battery (FOB) is a testing strategy that was created by the EPA to make quantitative and qualitative measurements of clinical changes. These observations are made in the home-cage, by openfield activity monitoring and by interactive assessments of the rat behavior and neurological function (EPA, 1998). The observers must be well trained to critically evaluate the animals in a consistent manner. It is desired that the same observer be used for evaluation across the course of a study and that observation should be randomized. Variables such as sound, lighting, humidity, temperature, odor, and time of day should be minimized during observations. Defined scales for measuring observation response are to be used. The goal is to describe the test animal's appearance, behavior, and functional integrity. Tests proceed from the least to most interactive tests. Most of these tests do not rely on learned responses or require training or conditioning of the animal. Measurements that may be taken for the FOB are as follows. Autonomic functions can be assessed: ranking of lacrimation and salivation, presence or absence of piloerection and exophthalmos, count of frequency of urination and defecation, pupillary size and responsiveness, degree of palpebral closure, presence, intensity and incidence of seizures, ranking of response to general stimuli, ranking of activity level, assessment of posture and gait, fore-and hind-limb strength, and quantitative measure of foot splay. Sensorimotor responses to stimuli can be assessed by gross deficits. Tail pinch, tail-flick, or hot-plate procedures may test deficits or exaggeration in pain perception. Gross assessments such as body weight, body temperature, vocalization, quality of respiration, and righting ability may also be documented (EPA, 1998). In the evaluation of some compounds, enhanced clinical observations or detailed clinical observations are necessary. This would include all of the observational testing of the FOB without the manipulative aspects (i.e., grip strength, landing foot splay, and motor activity). Motor

activity can be monitored by automated sampling devices. This is typically achieved with video cameras or activity measurement by photodetector beams. The device must be capable of detecting increases and decreases in activity, and each animal must be tested individually. Second-tier testing is generally determined after the initial screening, to characterize the nature of a chemical's toxicity. The tests performed depend on the questions that are generated by the first-tier testing. Typically second-tier tests further study sensorimotor deficits, evaluate cognitive behaviors related to learning and memory, and assess performance of complex tasks. Third-tier tests are usually mechanistic studies that attempt to describe the toxicity on the cellular or molecular level. These studies provide information to understand the basis of the neurotoxicity (Harry, 1995). Routine neuropathologic testing for standard toxicology studies, in which abnormalities in the FOB have not been discovered, includes fixation of tissues with buffered formalin, staining with hematoxylin and eosin stain and examination with light microscopy. Tissue samples taken for evaluation should represent all major regions of the nervous system. When there is reason to believe that neurological damage has occurred, systemic perfusion with fixatives is recommended for tissue preparation. Paraffin and/or plastic embedment is recommended to better preserve tissue for areas of suspected local damage and to improve the resolution of the images. Special staining methods, such as Bodian's or Bielchowsky's silver methods, or immunohistochemistry may be used to better characterize disease processes (O'Donoghue, 1989, EPA, 1998).

C.

Inhalation Toxicology

Inhalation exposure allows the controlled delivery of an airborne compound to the respiratory tract. Inhalation exposures can be acute, in which the duration of exposure is very short; subchronic, a varying interval usually less than 90 days; or chronic, exposure to the airborne compound several hours a day for 1 to 2 years. There are several kinds of exposure systems that facilitate the delivery of the airborne compound. These include the following: whole-body exposure, head-only exposure, nose-only exposure, lung-only exposure (tracheostomy, airway catheters, or intubation), and partial-lung exposure. Each type of exposure system has advantages and disadvantages. Whole-body exposure requires the use of inhalation chambers, which can be quite expensive. Continuous airflow is maintained in the chamber during exposure. Rats are usually housed in cages within the chambers. Although whole-body exposure affords minimal restraint of the animal, inhalation of the aerosolized compound may

810

THOMAS

E. HAMM,

JR.,

ANGELA

be decreased owing to animal avoidance (i.e., rats burying their nose in their fur, thereby decreasing the amount of compound being inhaled). Whole-body exposure also allows exposure to the aerosolized compounds by other methods, such as orally and topically, therefore adding other variables to research results. Head-only exposure provides a more efficient delivery of the aerosolized compound because the animal cannot avoid inhalation other than limiting the number or depth of respirations. The head of the rat is firmly restrained, which may be quite stressful. With nose-only exposure, rats are restrained in tubes that are open at one end to the aerosolized compound. As in the head-only exposure system, the delivery of the aerosolized compound is very direct. Restraint within these tubes is stressful. Rats subjected to even mild restraint have been reported to have increased baseline serum corticosterone concentrations (Pitman, 1988). Rats immobilized in nose-only tubes have been reported to gain significantly less weight than did unrestrained controls (McConnell, 1994). Large control groups and historical data from animals in the restraint device are needed to assist in the interpretation of this data. Lung-only and partial-lung-only exposure bypasses the upper respiratory system and directly instills the aerosolized compound. These types of exposure systems require the rats to be anesthetized during exposure, which may affect the animal's breathing pattern. The anesthetic may also have some interactions with the test compound. Intratracheal instillation has become sufficiently widely used that the Inhalation Specialty Section of the Society of Toxicology has recommendations and guidelines when using this exposure method (Driscoll et al., 2000). DO Pharmacokinetics, P h a r m a c o d y n a m i c s , and

Biometabolism Studies Pharmacokinetics is the study of the absorption, distribution, biotransformation, and excretion of compounds (Ross and Gillman, 1990). Pharmacokinetic parameters such as peak plasma concentrations (Cmax), the time of peak absorption (Tmax), the rate of clearance (CL), biological half-life (T1/2), and the extent of distribution in the body (V) are determined from these studies. Area under the time-concentration curve (AUC) is calculated by plotting the plasma concentration against time and measuring the plotted area. These measurements are used to establish absorption and exposure in circulation. Linearity of exposure, relative to dose, is a key parameter used to define appropriate dose ranges for a drug. Pharmacodynamics is the study of the mechanisms of action and the relationship between concentration and effect (Ross and Gillman, 1990). Maximum serum concentrations and duration in circulation are used as an

KING-HERBERT,

AND

MARYANN

VASBINDER

estimate of drug dosages for humans. Characteristics, including oral bioavailability, and plasma elimination half-life are also assessed. Single-dose and repeated-dose studies are examined to predict steady-state plasma concentrations (Marzo, 1997; White, 2000). Rats with jugular catheters or with jugular and femoral catheters are a primary model for pharmacokinetic and pharmacodynamic studies. Compounds are administered by mouth or through a venous catheter, and blood is collected over time. If rats are housed in metabolic cages, compound excretion may also be characterized by collection of feces and/or urine. Provided that the half-life and clearance rates are acceptable, the animals may be used repeatedly to reduce the number of animals used for studies. Automated blood sampling machines have been created to reduce the manpower required for blood collection and decrease the stress on the animals used in these studies. Computer-driven systems withdraw the blood from a catheter, replace the blood volume with saline or another appropriate solution, and then refrigerate the stored samples for future analysis. The frequency and timing of samples can be programmed for optimal time intervals. These systems incorporate elaborate mechanisms to prevent unwanted twisting or crimping of the catheter, increasing the comfort of the animal while increasing the time of patency of the line (Roberts, 2001). Simultaneous dosing of multiple compounds to a single animal is a technique called N-in-one dosing or cassette dosing. N represents some number of drugs, typically between 1 and 10 that are dosed to a single animal. This method requires fewer animals and results in fewer samples for analysis. This technique, although highly desirable, has inherent limitations. It can be difficult to dose a meaningful quantity of several compounds in one oral administration, and it requires that the chemistry of the compounds be compatible and assumes there are not interactions in the co-administered compounds. The initial assumption is the dose will not be toxic or overwhelm the metabolic capacity of the animal (White, 2000). Positive results must be confirmed in traditional tests. Compounds are evaluated for dosage range, regimen, and metabolic profiles. A study series known as ADME (absorption, distribution, metabolism, and excretion) is used to answer questions about excretion route, plasma and tissue concentrations, extent of metabolism, and administered/excreted balance. Excretion-balance studies using radioactive isotopes allow characterization of compound distribution and excretion over time. After administration of a dose of a radio-labeled compound, urine, feces, and respired air (if it is a suspected route of elimination) are collected. In mass balance studies, the first goal is to determine where the entire compound goes relative to the dose administered. Blood samples are taken

27.

811

TOXICOLOGY

periodically during the study, and the exposure to the compound (radioactivity) is determined. Radioactivity in the carcass can be quantified to determine if compound resides in the tissues during or at the end of the study. Autoradiography is another method used to study whole-body distribution and organ concentration of compounds and their metabolites. Rats are administered a radioactive dose and sacrificed over a pre-selected time course. The sections taken over the time course demonstrate uptake and subsequent decrease over time as the compound is cleared. Rats are used as the species of choice for this work primarily because of their size and the ease of obtaining samples (Marzo, 1997). Bile duct-cannulated rats are frequently used to study biliary excretion and enterohepatic recirculation of compounds and metabolites. Bile can be collected at predetermined time intervals from a biliary cannula. Urine, blood, and feces are collected at the same time interval, allowing for examination of excretion balances and flow. Mass balance of drug can be assessed by adding blood, bile, urine, feces, and final carcass content for total radioactivity (White, 2000).

VI.

C O N S I D E R A T I O N OF VARIABLES IN TOXICOLOGICAL TESTING

Well-designed toxicology experiments always include appropriate control animals. In addition to the main independent variable of the project (compound), many other variables should be considered in experimental design. This is especially relevant to toxicology experiments as interactions between test variables and environmental or animal variables may be present. For example, a specific environmental variable may interact with the test article but have no effect in control animals. Thereby, a variable can increase or decrease toxicity and may cause the approval or disapproval of a compound based on erroneous experimental data. Thus, attempts to control every possible variable are critical to toxicology experiments. Variables to consider include strain or stock of rat, water, diet, intestinal flora, caging and bedding, and disease status.

A.

R a t Strain or S t o c k

The rat has been and is a major animal in toxicology testing. Historically, specific strains and stocks of rats have been used for various tests. Because interpretation of any toxicological test often requires historical control information for comparison, introduction of new strains or stocks should be carefully considered. Moreover, when

working on a specific test, the literature on that test should be reviewed to determine the strain or stock that has been used in the past. To ensure genetic stability, strains or stocks of rats used in toxicology testing should be genetically monitored and bred in a system that minimizes genetic drift over time. The breeding colony should be maintained in a maximum barrier under rigidly controlled conditions of husbandry (see below). Efforts are ongoing to create international standards for the production and monitoring of rats for use in toxicology and pharmacology to minimize variability in test animals. These include those of the Global Alliance for Laboratory Animal Standardization (GALAS, 1993), Charles Rivers Laboratories International Genetic Standard (Charles River, 1999) and the Rat Resource and Research Center (RRRC, 2003). B.

Water

Water contains numerous and constantly changing impurities and can be an important source of variability in experiments. Monitoring the quality is difficult because each analysis can only account for a small number of possible contaminants and only represents the water at the site and on the day the sample was collected. Water can be purified using reverse osmosis systems (Raynor et al., 1984). The reverse osmosis water can then be monitored periodically to ensure the system is operating properly. Purified water can be corrosive so consideration should also be given to water lines, and equipment and systems should be flushed regularly. Systems, which automatically flush lines, are available and recommended. C.

Diet

Diet can be an extremely important source of variability in the results, and standardization of diets has been discussed for many years (Newberne et al., 1978; Coates, 1982). Toxicology experiments have usually been conducted by using diets made from natural ingredients that are either closed formula or open formula. A closedformula diet can contain one of several possible sources of ingredients. Ingredients in these diets are usually those that cost the least or are more readily available at the time of preparation. An open-formula diet is prepared following an established and invariable list of ingredients. Although there is more variability in the ingredients in a closed-formula diet than in an open-formula diet, an open-formula diet still has variability because the ingredients are natural products that may vary from batch to batch. It is generally recommended that rats in toxicology experiments be fed an open-formula diet to minimize dietary variability in experiments (NCI, 1976; AIN, 1977; Newberne et al., 1978; Feron et al., 1980; Hamm, 1980;

812

THOMAS

E. HAMM,

JR.,

ANGELA

Coates, 1982). The National Institutes of Health has used open-formula diets since 1972; the National Cancer Institute/National Toxicology Program, for all bioassays since 1976; and the Chemical Industry Institute of Toxicology Centers for Health Research, since 1981. The American Institute of Nutrition has recommended that the NIH-07 open-formula diet be used for rat studies (AIN, 1977). NIH-31 was developed as an autoclavable alternative to NIH-07; however, it should be noted that NIH-31 is not an autoclavable NIH-07, it is a unique diet. Experiments that require that a particular component of the diet be controlled (e.g., a vitamin deficiency) can be conducted by using purified diets. These diets are composed of refined proteins, carbohydrates, and fats with added vitamin and mineral mixtures. For experiments that require a purified diet, the AIN-93 purified diet (AIN, 1993; Reeves et al., 1993) was developed. The use of purified diets should be carefully pursued as there are reports of complications associated with their use (Bieri, 1980), and these complications can increase with the length of the experiment (Nguyen and Woodard 1980; Hamm et al., 1982; Medinsky et al., 1982). Moreover, the use of different purified diets may affect the toxicity of the compound being studied. As an example, Goldsworthy and colleagues (1986) showed that feeding NIH-07 diet containing 2,6-dinitrotoluene resulted in liver tumors in rats, whereas the same chemical fed in the AIN-76a diet was not carcinogenic. The only known variable in these experiments was the diet. One mechanism for this phenomenon was the effect of diet on intestinal flora (see next section). Collectively, these studies highlight the principle that dietary changes should be made with care. The use of dietary restriction, pair feeding, and other feeding methods also require careful experimental design. The reader is referred to an excellent review of the implications of dietary restriction for the design and interpretation of toxicity and carcinogenicity studies (Hart, 1995).

D.

Intestinal Flora

The influence of intestinal flora on toxicology experiments is one variable that is easily overlooked. An excellent review of this topic is available (Rowland, 1988). The intestinal flora may metabolize xenobiotics more than the host, and different intestinal floras can result in different toxicological endpoints in experiments. The diet may also affect the intestinal flora and subsequently affect compound toxicity. For example, deBethizy et al. (1983) showed that Fischer-344 rats fed Purina 5002 or NIH-07 diets had 135% and 150% higher hepatic covalent binding of 2,6-dinitrotoluene, respectively, compared with that of rats fed a AIN-76A purified diet or AIN-76A diet with added pectin. Changes in betaglucuronidase and

KING-HERBERT,

AND

MARYANN

VASBINDER

nitroreductase activities, enzymes implicated in the activation of 2,6-dinitrotoluene to a toxicant, correlated with the total number of cecal microflora that were associated with feeding of each diet. Goldstein et al. (1984) studied the influence of dietary pectin on intestinal microfloral metabolism and toxicity of nitrobenzene. They found that nitrobenzene induced methemoglobinemia in male Fischer-344 rats fed NIH-07 and was not detectable in rats fed AIN-76a when both groups were exposed to 200 mg/kg of nitrobenzene. Inclusion of 5% pectin in the AIN-76a diet resulted in methemoglobin concentrations comparable to those found in rats fed NIH-07. To control and study the importance of the microflora on a toxic endpoint, one may consider the use of axenic or gnotobiotic rats. Gnotobiotic technology has been used in experimentation for many years (Trexler et al., 1999). More information can be obtained in the gnotobiology chapter of this textbook and from the Association for Gnotobiotics (http.'//www.gnotobiotics.org/). As an example of the use of gnotobiotic rats in toxicology experiments, Mirsalis and colleagues (1982) used axenic Fischer 344 rats maintained in sterile flexible isolators to show the role of the intestinal flora in the genotoxicity of dinitrotoluene. They showed that there was extensive dinitrotolueneinduced DNA repair in rats with an intestinal flora but not in rats that had no intestinal flora, indicating that metabolism by the intestinal flora is a necessary step in the genotoxicity of this compound.

E.

Caging and Bedding

Historically, rats were housed in either wire-bottom or solid-bottom caging. Solid-bottom cages with filter caps changed in hoods and individually ventilated filter-top cages are now commonplace. Filter-top cages exclude infectious organisms, allowing experiments to be completed without this confounding factor. The cages also contain the test chemical and the animal byproducts, resulting in greatly increased worker safety. Use of these cages also allows the use of bedding to absorb the urine, reducing the exposure of the animals to ammonia and other odors. Rats often consume and possibly inhale some of their bedding. The type of bedding that produces the least variability is heat-treated hardwood chips. The hardwood should be manufactured exclusively for this use and should not be a treated byproduct of some other product. Dust and contamination should be minimized. The toxicologist may also wish to analyze for specific contaminants if the test compound is likely to be affected by a specific contaminant in the bedding. Wire floor inserts for solidbottom cages minimize the access of the animals to the bedding below the insert and have been used in some experiments to minimize ingestion of bedding

27.

813

TOXICOLOGY

(Raynor et al., 1983). The Guide for the Care and Use of Laboratory Animals (NRC, 1996) recommends solidbottom caging with bedding for rodents. Therefore, the use of wire-bottom cages or the use of wire inserts in solidbottom caging should be carefully justified and approved by the institutional animal care and use committee. F.

Disease Status

Because it is usually impossible to determine the effect of any disease on the outcome of a particular toxicology study, it is imperative that disease-free animals be used and maintained as disease free for the entire study. A plan to accomplish this in a toxicology laboratory has been published (Hamm et al., 1986). G.

Environmental Enrichment Considerations

The recent International Symposium on Regulatory Testing and Animal Welfare (Richmond et al., 2002a) recommended that rodents be housed in compatible social groups and not housed singly. They also recommended that rodents be provided with environmental enrichment. Whenever an enrichment device for a rat in a toxicology experiment is considered, the possible effect of the device on the results must be taken into account. Anything the rat ingests can affect metabolism; therefore, only devices that cannot be ingested or inhaled should be considered. Careful use of controls and historical data should be incorporated whenever a new variable such as an enrichment device is added to an experiment.

VII.

H U M A N E E N D P O I N T S A N D ROLE OF

ALTERNATIVES IN TOXICOLOGY TESTING Historically, the endpoint of many toxicologic tests was death. Over the course of the past 20 years, an abundance of effort has been placed on the development of humane endpoints and alternatives to death as an endpoint. A thorough review of endpoints in toxicologic testing is beyond the scope of this chapter, but the reader is referred to a number of excellent reviews and position papers including the following: the Society of Toxicology's position paper on the LDs0 and acute eye and skin irritation tests (SOT 1989); the American College of Toxicology's policy statement on the care of animals used in toxicology (ACT, 1992); a review of death as an endpoint (Hamm 1995); the OECD's guidelines on the recognition and assessment of pain and distress and informed decision making for euthanasia (OECD, 2000a); and the Institute for Laboratory Animal Research's journal issues devoted

to humane endpoints in biomedical research and testing (ILAR, 2000) and to regulatory testing and animal welfare (ILAR, 2002). Moreover, the Interagency Coordinating Committee on the Validation of Alternative Methods and its supporting center, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (http:/'/iccvam.niehs.nih.gov/home. htm), are coordinating the development, validation, acceptance, and harmonization of alternative toxicological test methods.

VIII.

SAFETY IN TOXICOLOGY EXPERIMENTS

The reader is referred to several recent texts and manuscripts regarding safety in toxicology experiments: The text Occupational Health and Safety in the Care and Use of Research An#rials (National Research Council, 1997) is an excellent source of information that can be used to develop a safety program. Reviews of chemical hazard control are also provided in Prudent Practices in the Laboratory: Handling and Disposal of Chemicals (NRC, 1995), and chapter 24 of the Laboratoo' Animal Medicine textbook (Hamm, 2002) is devoted to worker safety in the animal facility. The Institute for Laboratory Animal Research has devoted a recent issue of its journal to laboratory safety (ILAR, 2003). All projects should minimize worker exposure and emphasize the use of personnel protective equipment when applicable. Four activities involving animals in toxicology testing are potentially hazardous and, as a result, should have procedures developed to minimize risks. These areas include the following: manipulating the animal's enclosure, mixing of test chemicals into feed, filling food containers with dosed feed, and washing the cage. A discussion of animal enclosure is presented in a previous section. Precautions to consider when mixing chemicals into feed include placement of equipment such as balances and feed blenders into enclosures, such as chemical fume hoods or negative pressure enclosures, that prevent worker exposure. Food containers can be filled in hoods designed to minimize exposure to the personnel, and cage waste can be removed before washing in ~dump stations" that connected to exhaust systems.

IX.

CONCLUSION

The rat has been one of the most important animal models in toxicology. This chapter overviews the types of toxicological studies and provides information about

814

THOMAS

E. H A M M ,

JR., ANGELA

variables that must be considered in designing toxicology experiments such as rat strain, diet, water quality, intestinal flora, and environmental enrichment.

REFERENCES Amdur, M.O., Doull, J., and Klaassen, C.D. (1991). "Casarett and Doull's Toxicology the Basic Science of Poisons," 4th Ed. Pergamon Press, New York. American College of Toxicology [ACT] (1992). Policy statement: care and use of animals in toxicology. J. Am. Coll. Toxicol. 11, 1. American Institute of Nutrition [AIN] (1977). Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107, 1340-1348. American Institute of Nutrition [AIN] (1993). Final report of the AIN ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939-1951. Ashby, J. (1995). Rodent germ cell mutagens and their activity in the rodent bone marrow micronucleus assay. Mutat. Res. 328, 239-241. Ballantyne, B., Marrs, T., and Syversen, T. (1999). "General and Applied Toxicology," 2nd Ed. Grove's Dictionaries, Inc., New York. Bieri, J.G. (1980). Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110, 1726. Botham, P.A. (2002). Acute systemic toxicity. ILAR J. 43, $27-$30. Botham, PA. (2004). Acute systemic toxicity-prospects for tiered testing strategies. Toxicol. In Vitro 18, 227-230. Charles River Laboratories' International Genetic Standard (1999). Charles River Reference Paper, Volume 11, #1. Coates, M. (1982). Workshop on laboratory animal nutrition. ICLAS Bull. 50, 4-11. Dean, S.W., Brooks, T.M., Burlinson, B., Mirsalis, J., Myhr, B., Recio, L., and Thybaud, V., (1999). Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens. Mutagenesis 14, 141-151. deBethizy, J.D., Sherrill, M., Rickert, D.E., and Hamm, Jr., T.E. (1983). Effects of pectin-containing diets on hepatic macromolecular covalent binding of 2,6-Dinitro[3H]toluene in Fischer-344 rats. Toxicol. Appl. Pharmicol. 69, 369-376. Derelanko, M.J. and Hollinger, M.A. (2002). "Handbook of Toxicology." CRC Press, New York. Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M., and Vorstenbosch, C. (2001). A practice guide for the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21, 15-23. Driscoll, K.E., Costa, D.L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., and Schlesinger, R.B. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations. Toxicol. Sci. 55, 24-35. Ecobichon, D.J. (1992). "The Basis of Toxicity Testing." CRC Press, Boca Raton, FL. Ecobichon, D.J. (2002). Reproductive toxicology. In "Handbook of Toxicology," Vol. 1 (M. Derelanko and M. Hollinger, eds.), pp. 371-388. CRC Press, Boca Raton, FL. European Economic Community [EEC] (1992). Council Directive 92/32/EEC, April 30, 1993, amending for the seventh time Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Official Journal of the European Communities 35(no. L-154), pp. 1-29. Feron, V.J., Grice, H.C., Griesemer, R., Peto, R., Agthe, C., Althoff, J., Arnold, D.L., Blumenthal, H., Cabral, J.R., Della Porta, G., Ito, N.,

KING-HERBERT,

AND MARYANN

VASBINDER

Kimmerle, G., Kroes, R., Mohr, U., Napalkov, N.P., Odashima, S., Page, N.P., Schramm, T., Steinhoff, D., Sugar, J., Tomatis, L., Uehleke, H., and Vouk, V. (1980). Basic requirements for long-term assays for carcinogenicity. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. Suppl. 2 Suppl, 21-83. French, R.D. (1975). "'Antivivisection and Medical Science in Victorian Society." Princeton University Press, Princeton, NJ. Gad, S.C (2001). "Regulatory Toxicology," Taylor and Francis, New York. Gallo. M.A., and Doull, J. (1991). History and scope of toxicology. b~ "Casarett and Doull's Toxicology," 4th Ed. (M.A. Amdur, J. Doull, and C.D. Klaassen, eds.), chapter 1. Pergamon Press, NY. Global Alliance for Laboratory Animal Standardization [GALAS] (1993), h t tp :/ / www.galas.org. Goldstein, R.S., Chism, J.P., Sherrill, J.M., and Harem, Jr., T.E. (1984). Influence of dietary pectin on intestinal microfloral metabolism and toxicity of nitrobenzene. Toxicol. Appl. Pharmicol. 75, 547-553. Goldworthy, T.L., Hamm, Jr., T.E., Rickert, D.E., and Popp, J.A. (1986). The effect of diet on 2,6-dinitrotoluene hepatocarcinogenesis. Carcinogenesis 7, 1909-19 l 5. Haggarty, G.C. (1989). Development of tier 1 neurobehavioral testing capabilities for incorporation into pivotal rodent safety assessment studies. J. Am. Coll. Toxicol. 8, 53. Hamm, Jr., T.E. (1980). Proceedings of a Workshop on the Optimal use of Facilities for Carcingenicity/Toxicitv Testing. NTP/NCI Carcinogenesis Testing Program, Boiling Springs, PA. Hamm, Jr., T.E. (1985). Design of the carcinogenesis bioassay. In "Handbook of Carcinogen Testing" (H.A. Milman and E.K. Weisburger, eds.), pp. 252-267, Noyes Publications, Park Ridge, NJ. Hamm, Jr., T.E. (1995). Proposed IACUC guidelines for death as an endpoint in rodent studies. Contempt. Top. Lab. Anita. Med. 34, 69-71. Hamm, Jr., T.E. (2002). Control of biohazards associated with the use of experimental animals. In "Laboratory Animal Medicine," 2nd Ed. (J.G. Fox. L.C. Anderson, F.M. Loew and F.W. Quimby, eds.), pp 1047-1056, Academic Press, New York. Hamm, Jr., T.E., Raynor, T., and Caviston, T. (1982). Unsuitability of the AIN-76A diet for Male F-344 and CD rats and improvement by substituting starch for sucrose. Lab Anita. Sci. 32, 414-415. Hamm. Jr., T.E., Raynor, T.H., and Sherrill, M. (1986). Animal Management Procedures to Avoid or Minimize Disease Problems. In '~Complications of Viral and Mycoplasma Infections in Rodents to Toxicology Research and Testing" (T.E. Hamm, Jr., ed.), pp. 175-184, Hemisphere Publishing Corporation, Washington, DC. Harry, J., O'Donoghue, J.L., and Goldberg, A.M. (1995). Neurotoxicity. In "Screening and Testing Chemicals in Commerce: OTA-BP-ENV-166 Background Paper for U.S. Congress." Office of Technology Assessment, Washington, DC. Hart, R.W., Neumann, D.A., and Robertson, R.T. (1995). "Dietary Restriction Implicat!ons for the Design and Interpretation of Toxicity and Carcinogenicity Studies," ILSI Press, Washington, DC. Hayes. A.W. (2001). "Principles and Methods of Toxicology," 4th Ed. Taylor & Francis, Philadelphia. Hays, H.W. (1986). "Society of Toxicology History." Society of Toxicology, Washington, DC. Hoyer, P.B. (2001). Reproductive toxicology: current and future directions. Biochem. Pharmicol. 62, 1557-1564. Institute of Laboratory Animal Research [ILAR] (2000). Humane endpoints for animals used in biomedical research and testing. ILAR J. 41(2). Institute of Laboratory Animal Research [ILAR] (2002). Regulatory testing and animal welfare. ILAR J. 43(Suppl).

27. T O X I C O L O G Y Institute of Laboratory Animal Research [ILAR] (2003). Occupational safety and health in biomedical research in the laboratory. I L A R J. 44 (1). International Conference on Harmonization [ICH] (1995). "'Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals." International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland. International Conference on Harmonization [ICH[ (1997). "Genotoxicity: A Standard Battery for Genotoxicity Testing for Pharmaceuticals." International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland. Irwin, S. (1968). Comprehensive observational assessment: a systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia 13, 222-257. Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Putman, D.L., Sorge, J.A., and Short, J.M. (1991). Spectra of spontaneous and induced mutations using a lambda ZAP/lacl shuttle vector. Environ. Mol. Mutagen. 18, 316-321. MacGregor, J.T. (1998). Transgenic animal models for mutagenesis studies: role in mutagenesis research and regulatory testing. Environ. Mol. Mutagen. 32, 106-109. Mandella, R.C. (2002). Applied neurotoxicology. In "Handbook of Toxicology," Vol. 1 (M. Derelanko and M. Hollinger, eds.), pp. 371-388. CRC Press, Bocca Raton, FL. Marzo, A. (1997). Clinical pharmacokinetic registration file for NDA and ANDA procedures. Pharmacol. Res. 36, 425-50. McConnell, E.E., Kamstrup, O., Mussellman, R., Hesterberg, T.W., Chevalier, J., Miiller, W.C., and Thevenaz, P. (1994). Chronic inhalation study of size-separated rock and slag wool insulation fibers in Fischer 344/N rats. Inhal. Toxicol. 6, 571-614. Medinsky, M.A., Popp, J.A., Hamm, Jr., T.E., and Dent, J.G. (1982). Development of hepatic lesions in male Fisher-344 rats fed AIN-76A purified diet. Toxicol. Appl. Pharmacol. 62, 111-120 Milman, H.A. and Weisburger, E.K. (1985). "Handbook of Carcinogen Testing." Noyes Publications, Park Ridge, NJ. Mirsalis, J.C., Hamm, Jr., T.E., Sherrill, J.M., and Butterworth, B.E. (1982). The role of intestinal flora in the genotoxicity of dinitrotoluene in rats. Nature 295, 322-323. Morton, D.B., Jennings, M., Buckwell, A., Ewbank, R., Godfrey, C., Holgate, B., Inglis, I., James, R., Page, C., Sarman, I.L., Verschoyle, R., Westall, L., and Wilson, A.B. (2001). Refining procedures for the administration of substances. Lab. Anim. 31, 1-41. Moser, V.C. (1991). Applications of a neurobehavioral screening battery. J. Am. Coll. Toxicol. 10, 661-669. National Cancer Institute [NCI] (1976). "Guidelines for Carcinogen Bioassay in Small Rodents, National Cancer Institute Technical Report Series, No. 1." National Academy Press, Bethesda, MD. National Research Council [NRC] (1995). "Prudent Practices in the Laboratory Handling and Disposal of Chemicals." National Academy Press, Washington, DC. National Research Council [NRC] (1996): "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, DC. National Research Council [NRC] (1997): "Occupational Health and Safety in the Care and Use of Research Animals." National Academy Press, Washington, DC. Neubert, D. (2002). Reproductive toxicology: the science today. Teratog. Carcinog. Mutagen. 22, 159-174. Newberne, P.M., Dieri, J.G., Briggs, G.M., and Nesheim, M.C. (1978). "Control of Diets in Laboratory Animal Experimentation." Institute of Laboratory Animal Resources, Assembly of Life Sciences, National Research Council, National Academy of Sciences, Washington, DC.

815

Nguyen, H.T. and Woodard, T.C. (1980). Intranephronic calcinosis in rats. Am. J. Pathol. 100, 39-55. O'Donoghue, J.L. (1989). Screening for neurotoxicity using a neurologically based examination and neuropathology. J. Am. Coll. Toxicol. 8, 97-115. Organization for Economic Co-operation and Development [OECD] (1993). "Guidelines for the Testing of Chemicals." OECD, Paris. Organization for Economic Co-operation and Development [OECD] (1996). "'Guidelines for Testing of Chemicals: Proposal for Updating Guideline 414: Prenatal Developmental Toxicity Study. Draft Document." OECD, Paris. Organization for Economic Co-operation and Development [OECD] (1998). "Guideline 425: Acute Oral Toxicity-Modified Up and Down Procedure." OECD, Paris. Organization for Economic Co-operation and Development [OECD] (2000a). "Guideline 19: Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation," OECD, Paris. Organization for Economic Co-operation and Development [OECD] (2000b). "'Guideline 20: Regulatory Guidelines for Neurotoxicity Testing, Series on Testing and Assessment." OECD, Paris. Parascandola, J. (1991). The development of the Draize test for eye toxicity. Pharm. Hist. 33, 111-117. Pitman, D.L., Ottenweller, J.E., and Natelson, B.H. (1988). Plasma corticosterone concentrations during repeated presentation of two intensities of restraint stress: chronic stress and habituation. Physiol. Behav. 4, 47-55. Pharmaceutical Affairs and Food Sanitation Council [PAFSC] (1997). Articles 12, 2214, 19-2, 5 and 24, Ministry of Health, Labour and Welfare, Tokyo, Japan. http://www.mhlw.go.jp/english/index. Provost, G.S., Kretz, P.L., Hamner, R.T., Metthews, C.D., Rogers, B.J., Lundberg, K.S., Dycaico, M.M., and Short, J.M. (1993). Transgenic systems for in vivo mutation analysis. Mutat. Res. 288, 133-149. Rat Resource and Research Center [RRRC] (2003). www.nrrc.missouri.edu. Raynor, T.H., Steinhagen ,W.H., and Hamm, Jr., T.E. (1983). Differences in the microenvironment of a polycarbonate caging system: bedding vs. raised wire floors. Lab. Anim. 17, 85-89. Raynor, T.H., White, E.L., Cheplen, J.M., Sherrill J.M., and Hamm, Jr., T.E. (1984). An evaluation of a water purification system for use in animal facilities. Lab. Anon. 18, 45-51. Reeves, P.G., Rossow, K.L., and Lindlauf J. (1993). Development and testing of the AIN-93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. J. Nutr. 123, 1923-1931. Richmond, J., Fletch, A., and Van Tongerloo, R. (2002a): The International Symposium on Regulatory Testing and Animal Welfare: recommendations on best scientific practices for animal care in regulatory toxicology. I L A R J. 43 Suppl, S129-S132. Roberts, S.A.(2001). High-throughput screening approaches for investigating drug metabolism and pharmacokinetics. Xenobiotica 31, 557-589. Ross, E.M. and Gilman, A.G. (1990). Pharmacodynamics: Mechanism of drug action and the relationship between drug concentration and effect. In "The Pharmacological Basis of Therapeutics," 8th Ed. (A.G. Gilman, L.S. Goodman, T.W. Rall, and F. Murad, eds.), chapter 2. Macmillan Publishing Company, New York. Ross, J.F. (2002). Tier 1 neurological assessment in regulated animal safety studies. In"Handbook of Neurology," Vol. 2 (J. Massaro, ed.), pp. 461-505. Humana Press, Totowa, NJ. Rowland, I.R. (1988). "'Role of the Intestinal Flora in Toxicity and Cancer." Academic Press, San Diego, CA. Russell, W.M.S. and Burch, R.L. (1959). "The Principles of Humane Experimental Technique". Charles C. Thomas, Springfield IL.

816

THOMAS

E. HAMM,

JR.,

ANGELA

Sass, N. (2000). Humane endpoints and acute toxicity' testing. ILAR J. 41, 114-123. Silverman, W.A. (1985). "'Human Experimentation" A Guided Step into the Unknown." Oxford University Press, New York. Society of Toxicology [ S O T ] (1989). SOT position paper: comments on the LDs0 and acute eye and skin irritation tests. Fundam. Appl. Toxicol. 134, 621-623. Technical Committee on Alternatives to Animal Testing [TCAAT] (1996). Replacing the Draize eye irritation test: scientific background and research needs. J. Toxicol. Cutaneous Ocul. Toxicol. 15, 211-234. Trexler, P.C. and Orcutt, R.P. (1999). Development of gnotobiotics and contamination control in laboratory animal science, h~ "Fifty Years of Laboratory Animal Science," (C.W. McPherson and S.F. Mattingly. eds.), chapter 16. Sheridan Books, Chelsea, MI. Turner, S.D., Tinwell, H., Piegorsch, W.. Schmezer, P.. and Ashby. J. (2001). The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue rats. Mutagenesis 16, 329-332. U.S. Environmental Protection Agency [EPA] (1996). "'Health Effects Test Guidelines: Prenatal Developmental Toxicity study. Document OPPTS 870.3700. U.S. Government Printing Office. Washington, DC.

KING-HERBERT,

AND

MARYANN

VASBINDER

U.S. Environmental Protection Agency [EPA] (1998). "'Health Effects Test Guidelines: Neurotoxicity Screening Battery," Document OPPTS 870.6200. U.S. Government Printing Office, Washington, DC. U.S. Food and Drug Administration [FDA] (1978). '~Good Laboratory Practice Regulations for Nonclinical Laboratory Studies." U.S. Code of Federal Regulations, Title 21. Part 8. U.S. Food and Drug Administration [FDA] (1993a). International Conference on Harmonization: guideline on detection of toxicity to reproduction for medicinal products. Fed. Reg. 59(183), 48746-48752. U.S. Food and Drug Administration [FDA] (1993b). "New drug evaluation guidance document, refusal to file, July 12, 1993." FDA, Center for Drug Evaluation and Research. Rockville, MD. U.S. Food and Drug Administration [FDA] (2000). "Toxicological Principles for the Safety' of Food and Ingredients, Redbook 2000." FDA. Center for Food Safety and Applied Nutrition, Rockville, MD. White. R.E. (2000). High-throughput screening in drug metabolism and pharmacokinetic support of drug discovery. Annu. Rev. Pharmacol. Toxicol. 40, 133-157. Yu. M.. Jones. M.L.. Gong, M., Sinha, R., Schut, H., and Snyderwine. E.G. (2002). Mutagenicity of 2-amino-l-methyl-6phenylimidazo[4.5]pyridine in the mammary gland of Big Blue rats on high-and-low-fat diets. Carcinogenesis 23, 877-884.