Toxicity Testing for Assessment of Environmental Agents Interim Report (2006) / Chapter Skim
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2 Animal and In Vitro Toxicity Testing
2 Animal and In Vitro Toxicity Testing
Pages 26-70
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From page 26... ...
Those goals, however, are weighed in light of constraints on costs and other resources. The types and extent of human exposure are important considerations in designing toxicity studies for human health risk assessment.
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From page 27... ...
EPA strongly recommends that the lowest dose not produce any evidence of toxicity. The numbers of animals required are defined in each study protocol and range from five rats per sex per dose in 28-day toxicity studies to 10 rats per sex per dose in subchronic studies to 50 rats per sex per dose in carcinogenicity assays.
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From page 28... ...
Thus, the toxicity tests characterized by exposure duration -- acute, subchronic, and chronic -- are reviewed first; these tests are designed to gain an understanding of systemic effects, given various lengths of exposure, and can be used to guide human health risk assessment for those exposure durations. Toxicity tests designed to evaluate specific end points are discussed next and include tests for reproductive and developmental toxicity, neurotoxicity, immunotoxicity, and genotoxicity.
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From page 29... ...
Today, acute toxicity tests are used also to determine dosing regimens for longer-term toxicity tests and to evaluate more fully the effects of acute exposure. Acute testing protocols have evolved over the years to conserve animal use, to minimize the pain and discomfort of the test animals, and to obtain more information on the pathogenesis of toxicity.
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From page 30... ...
As discussed in greater detail in Chapter 6, acute toxicity tests can be redesigned to provide additional information on more subtle effects than lethality and gross clinical signs. One particular end point that has received increasing attention is cardiovascular toxicity, specifically adverse effects on ion channels in the myocardium that lead to abnormalities in the electrocardiogram, namely prolongation of the QT interval.
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From page 31... ...
Acute toxicity tests provide at least one relatively quick and inexpensive tool in testing schemes that screen large numbers of chemicals and identify chemicals that warrant further toxicity testing. Subchronic or Repeated-Dose Toxicity Testing Subchronic studies evaluate the adverse effects of continuous or repeated exposure over a portion of the average life span of experimental animals.
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From page 32... ...
Test guidelines require measurement and evaluation of a number of parameters, including clinical signs (such as changes in skin, fur, eyes, secretions, gait, posture, and response to handling) , motor activity, grip strength, sensory reactivity to stimuli, body weight, food consumption, clinical pathology (clinical chemistry and hematology)
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From page 33... ...
. The purpose of carcinogenicity testing is to determine the cumulative neoplastic effects of repeated daily oral, dermal, or inhalation exposures to various doses of test chemicals over most of the life span of the test species (EPA 1998f)
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From page 34... ...
has conducted over 600 lifetime cancer bioassays and has been at the forefront of developing definitive guidelines for detecting carcinogenic activity in rodents; the carcinogenicity data obtained reside in a public database. EPA guidelines for combined chronic toxicity and carcinogenicity testing (EPA 1998g)
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From page 35... ...
Considerable effort is being devoted to broadening the comparison of tumor data from transgenic mouse strains and strains of mice traditionally used in lifetime bioassays. TOXICITY TESTING CHARACTERIZED BY SPECIFIC END POINT Toxicity testing of most chemicals begins with acute testing, progresses to subchronic testing, and, depending on the results, concludes with chronic testing.
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From page 36... ...
The measurements made provide insight into gonadal function, fertility, pregnancy, parturition, and prenatal and postnatal developmental toxicity. OECD testing guidelines (TG)
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From page 37... ...
, although this test is not included by EPA in its test guidelines. The test chemical is administered to young adult rats of both sexes (generally to breeding pairs)
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The two-generation test is considered the appropriate test for reproductive toxicity, and the OECD test guideline has recently been updated to reflect the scientific state of the art (OECD 2005)
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From page 39... ...
. Neurotoxicity Testing in Standard Toxicity Studies Neurotoxicity evaluations required by EPA and OECD guidelines for standard acute, subchronic, and chronic toxicity tests include detailed clinical observations, functional tests, and histopathology.
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From page 40... ...
Both include detailed clinical observations or a functional observational battery in the home cage and open field; functional tests, including assessments of motor activity, grip strength, and reactivity to sensory stimuli; and neuropathologic examination of perfusion-fixed tissues. Adult neurotoxicity studies require functional tests and clinical observations similar to those in standard toxicity studies but require perfusion-fixed tissues, more frequent measurement of functional tests, and observations to be conducted without knowledge of treatment level.
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From page 41... ...
Developmental Neurotoxicity Studies The developmental-neurotoxicity study protocol (EPA 1998l) is designed to develop data on the potential functional and morphologic hazards to the nervous system in offspring of mothers exposed during pregnancy and lactation.
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From page 42... ...
. Thus, it could be difficult to experimentally reproduce potential environmental exposure to the human fetus in rat developmentalneurotoxicity studies.
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. Specialized Studies for Neurotoxicity Testing EPA has developed six test guidelines for neurotoxicity testing (see Appendix B)
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In summary, a tiered or iterative approach to neurotoxicity testing is being used by OECD and EPA. Initial evaluation of the nervous system can be obtained with standard toxicity studies, which are typically conducted over a range of doses that include the maximum tolerated dose.
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From page 45... ...
If the immune system recognizes the new tissue-associated protein as foreign, an immune response to the tissue is generated and results in an autoimmune response and, if it is persistent, autoimmune disease. Potential immunosuppressive effects are often identified during subchronic toxicity testing.
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From page 46... ...
. Immunotoxicity testing for contact hypersensitivity has been conducted for many years; guinea pigs have been the model of choice.
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From page 47... ...
Animal studies, however, remain a part of test guidelines of EPA, FDA, and other government agencies and organizations. Test methods in genetic toxicology are categorized according to their ability to detect gene or point mutations; chromosomal effects, such as breaks, gaps, translocations, and aneuploidy (loss or gain of one or
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Table 2-2 lists representative genotoxicity tests, which include both in vitro and whole-animal tests, particularly in the case of chromosomal abnormalities. Only the tests that are used most often for regulatory purposes are discussed here.
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i Mouse lymphoma Mouse lymphoma Rat liver primary cellsj L5178Yc L5178Yc Chinese hamster ovary Rodent bone marrow cellsd cellsf Dominant-lethal assayg Heritable-translocation assayh a Maron and Ames 1983.
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In addition, results of in vitro testing can be verified in vivo with the same end point (chromosomal aberrations) , and this is not easily done in the case of gene mutations.
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From page 51... ...
Commonly used in vitro models for assessing chemical toxicity include perfused organ preparations, isolated tissue preparations, singlecell suspensions, and cell-culture systems, such as primary cell cultures and mammalian cell lines. Of these in vitro models, cell-culture systems have been used more often by investigators because they are reliable, reproducible, and relatively inexpensive experimental systems to assess chemical toxicity at the cellular level.
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From page 52... ...
, cultured human and other mammalian cells, such as lymphocytes and fibroblasts, are an integral part of EPA test guidelines for the determination of gene mutation, sisterchromatid exchange, chromosomal aberrations, and unscheduled DNA synthesis. EPA has accepted a battery of in vitro tests to evaluate the high-production-volume chemicals.
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From page 53... ...
Micromass test Assay to evaluate ability of Micromass Inhibition of differentiation, ECVAM substances to inhibit cultures of rat viability, and growth differentiation limb bud cells Embryotoxicity Assay to identify substances that Cultured rat em- Embryo morphology after 48 ECVAM testing in induce malformation that results bryos hours of culture postimplantation in embryo toxicity containing 1-5 rat whole-embryo somites culture In vitro 3T3 NRU Assay to detect the phototoxicity 3T3 adult mouse Neutral red uptake to indicate ECVAM phototoxicity testb induced by the combined action fibroblast cell line cytotoxicity of a chemical and light (Continued)
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From page 54... ...
; testc corrosivity model system dehydrogenase activity measured ICCVAM (screen by formazan production from in tiered-testing MTT strategy) EPISKIN Assay to evaluate skin EPISKIN Cell viability as determined by ECVAM corrosivity testc corrosivity reconstructed reduction of mitochondrial (replacement)
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From page 55... ...
similar to those of rat skin) , and produce a visually detectable change In vitro Assay to select starting doses for BALB/c3T3, Cell survival or viability as ICCVAM cytotoxicity test acute oral toxicity in rodents normal human determined by the cell's ability to (reduction)
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From page 56... ...
2003) have covered the advantages and limitations of various in vitro liver cell systems, including isolated perfused liver, isolated hepatocytes in short-term suspension and primary monolayer culture, various liver cell lines, and liver precision-cut slice cultures.
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From page 57... ...
studies can evaluate the time course of changes in concentrations in the body, persistence of radioactivity after dosing with radiolabeled compounds, or accumulation with multiple dosing. The EPA test guidelines for metabolism and pharmacokinetic studies (EPA 1998y)
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From page 58... ...
The data are intended to aid in understanding mechanisms of toxicity and in determining whether animal toxicity studies are adequate for testing toxicity concerns associated with plant metabolites of the pesticide that might be present on raw agricultural products. The guidelines discuss two tiers of tests: a required core group of studies (tier 1)
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From page 59... ...
. Current test guidelines for metabolism and PK studies will need to be revised to produce data more useful for PBPK model development.
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2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment.
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1998c. Health Effects Test Guidelines OPPTS 870.1300.
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1998k. Health Effects Test Guidelines OPPTS 870.6200.
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1998t. Health Effects Test Guidelines OPPTS 870.5380.
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1998y. Health Effects Test Guidelines OPPTS 870.7485.
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From page 65... ...
2003. Health Effects Test Guidelines OPPTS 870.2600.
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2001. Ciprofloxacin: In vivo genotoxicity studies.
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From page 67... ...
1998. A Retrospective Analy sis of Twelve Developmental Neurotoxicity Studies Submitted to the U.S.
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1996. Combined Repeated Dose Toxicity Study With the Reproduction/ Developmental Toxicity Screening Test.
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2003. Motor activity in developmental neurotoxicity testing: A cross-laboratory comparison of control data.
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From page 70... ...
2004. Learning and memory tests in developmental neurotoxicity testing: A cross-laboratory comparison of control data.
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