methods for toxicologic testing

Appendix C

Methods for Toxicologic Testing *by SRI International

METHODS FOR TOXICOLOGIC TESTING

137

138 ● Environmental Contaminants in Food

scheme, experiments are designed and data arecollected based on expected results such as func-tional systemic changes, teratogenicity, or car-cinogenicity. By the use of an appropriate experi-mental design, several endpoints can be assessedin the same experimental period such as is donein FDA’s three-generation studies (2).

For ease of presentation, this appendix hasbeen subdivided by endpoint into sections on Sys-temic Toxicity, Carcinogenicity, Mutagenicity,Teratology and Effects on Reproduction, Metabo-lism, and Structure-Activity Relationships. Thisappendix is not intended as an exhaustive surveyof all testing methods used, but is meant to give anoverview of those methods most commonly usedtoday by toxicologists.

LocaI and Systemic Toxicity

Some of the fastest and simplest methods fordetermining the toxicities of substances involvethe observation of changes in the structure andfunction of organs and organ systems. Thesemethods generally involve absolute and relativeweight changes, gross and microscopic structuralalterations, and primary and secondary tests fororgan, system, or whole animal function. With ad-vances in the chemical, physiological, and behav-ioral sciences, modifications for testing systemictoxicity have been proposed that make these pro-cedures more sophisticated and relatively compli-cated. Several good texts are available which re-view systemic toxicity [3,4).

Range FindingThe classic determinations of toxicity involve

percent lethal or effective dose, concentration, ortime. These tests may employ any route of expo-sure, the ones chosen usually being based on fac-tors such as chemical and physical properties ofthe agent and potential routes of exposure fromthe environment. The results obtained from thesedeterminations are usually specific for the spe-cies, sex, age, and condition of the organism, andfor the route of exposure and environmental con-ditions before, during, and after exposure. Theendpoints of these tests may be either structuralor functional changes, but they are usually lim-ited to gross effects such as death or narcosis.These tests are primarily used to determine rela-tive toxicities of various agents and for rangefinding for maximum tolerated dosage prelimi-nary to beginning a subacute study. They are notusually used to directly evaluate the hazard.

In general, these tests will employ young adultrats and another mammalian nonrodent species.

Selection of this other species " . . should con-sider such factors as comparative metabolism ofthe chemical and species sensitivity to the toxiceffects of the test substance . . . . “(l) The route ofadministration chosen is that most nearly iden-tical to the potential human exposure. Doses areusually chosen to give results in the 20- to 80-percent lethal or effective range and are usuallyseparated by 0.5 log units (5). Many modificationsof this basic procedure are accepted.

IrritationThe irritation potential of substances is tested

by observation of the reflex behavior of the ani-mal and by direct observation of the site of con-tact with the agent, Attempts to quantitate reflexbehavioral responses, i.e., eye rubbing, regurgita-tion, or shallow breathing, have met with littlesuccess. The simplest protocols for evaluating ir-ritation involve the skin and eyes. Semiquantita-tive systems for scoring skin and eye irritationhave been proposed by several authors (6-12) andinvolve placing the suspected irritant in contactwith the skin or eye of New Zealand White rab-bits. Protocols for skin irritation involve contactwith both intact and abraded skin to differentiatethe agent’s ability to penetrate the skin barrier,and occlusion of the contact site to maximize theresponse. The severity of erythema and edema isscored as the endpoint. Eye irritation studies arecarried out without washing and with washing atvarious intervals to determine the effectiveness ofremoval of the agent to reducing the adverse ef-fect, Opacity, area affected, iris reaction to light,hemorrhage, swelling, and discharge are scored.Results from these tests can vary greatly depend-ing on the method of application of the substance,whether dry or premoistened, etc.

Other potential sites of irritation such as thesensory nerves, respiratory system, urinary sys-tem, and gastrointestinal tract are usually evalu-ated secondarily or through necropsy. Secondaryeffects include shallow breathing, regurgitation,agitation on urination, and eye rubbing, whichare broadly categorized as reflex behavior, andblood in feces, urine, and sputum, or nasal dis-charge, which are more indicative of the primaryirritant effect. These results are not quantifiableby present methodologies. Primary evaluationthrough necropsy is also a qualitative procedureand has not undergone the extent of standardiza-tion and validation that the skin and eye testshave. Other methods for testing irritation such asresistance/compliance tests of the pulmonary sys-tem, direct observation by scope of the esophagus

Appendix C—Methods for Toxicologic Testing ● 739

and gastrointestinal tract, and roentgenographicexamination with and without radio-opaque dyes,have not received wide use as testing techniquesfor Government regulatory purposes.

SensitizationSome substances, although not necessarily pri-

mary irritants, elicit an irritant-type responseafter repeated contact with the organism. Testsfor this sensitization potential involve exposingthe animal to an agent at doses below those neces-sary to produce signs of primary irritation, wait-ing an appropriate interval, and then challengingthe animal with the substance again at a differentsite (11). If the response on retest is substantiallyhigher than the initial response, the agent can beclassified as a sensitizer. Various test methodsand modifications have been proposed for testingsensitizing potential (11, 13-21). Perhaps the mostcommon direct test for sensitization is the guineapig maximization test. In this test, the agent ispresented in Freund’s complete adjuvant whichincreases the response. Studies on the mechanismof sensitization have shown that the agent or ametabolize of it (antigen) may induce the lym-phocytes of the body to form a complex molecule(antibody) which reacts with the antigen to forman antigen-antibody complex. This reaction maybe with circulating free antibody or with lympho-cyte-bound antibody, T h e f o r m a t i o n o f t h eantigen-antibody complex induces the productionand release of histamine and other compoundswhich cause the erythema and edema at the siteof antigen attack, or may cause anaphylaxis if theantigen reaches the blood stream (22,23). Theproblem with testing methods based on thismechanism, such as immunoelectrophoresis ,radioimmunoassay, ring test, hemagglutinationtests, or microphage migration, is that they do notmeasure the actual adverse effect [dermatitis orshock) but measure an indicator response. Themethods are valuable, however, in demonstratingthe presence of antibody capable of producingthese health effects. Several comparative testshave demonstrated that these in vitro techniquesare often more sensitive indicators of the hazardthan the classic in vivo ones (22,23).

Structural EffectsThe basic determination of structural effects

on organs and systems begins with the determina-tion of absolute and relative weight changes.Decreases in absolute body weight or rates ofweight gain for a test with a substance incorpo-rated into the food or water may show either that

the agent is unpalatable or that it is interferingwith the energy balance, the central nervous sys-tem (CNS) regulation of food or water consump-tion, or the motivation of the animal. Althoughsubstances administered by other routes of expo-sure may also interfere with palatability of food,through direct or indirect effects on the sensorynerves, this is less common. Increases in weightmay be caused by a proliferating tumor mass. Theevaluations of structural changes can be obtainedfrom animals exposed during subacute experi-ments.

In general, subchronic or subacute experi-ments are designed to last approximately 10 per-cent of the animals’ lifespan (90 days for rats). Im-mediately preceding and during the experimentalperiod, observations on animals should includerate of growth, food and water consumption, de-meanor, and reflex behavior; blood, urine, andfeces should be collected. During the experimen-tal period, tissue biopsies may be taken for obser-vation of structural changes. These techniquesmay be unrel iable , however , i f a s t ructuralchange is localized and not included in the biopsymaterial, and such manipulation is often notallowed by regulatory testing guidelines. If biop-sies are done, additional animals are required tomaintain the statistical validity of the experiment.At the end of the experimental period, the animalsare sacrificed and the organs are inspected forgross changes, removed and weighed, and pre-served for histologic treatment and microscopicexamination (5). The specific organs and tissuesremoved and examined will depend somewhat onthe expected action of the agent administered(usually perceived from preliminary testing) butshould include at least the brain, liver, kidneys,spleen, heart, testes (and epididymis) or ovaries(and uterus), thyroid, and adrenals.

Changes in organ weights may signal a func-tional change in this organ or in other organs; forexample, an increase in heart weight could be dueto a decrease in oxygen diffusion from the lungs,an increase in adrenal weight could signal ablockage of steroid synthesis within it, etc. Theweights of organs can be directly compared withthose from control animals; however, this often in-troduces an artifact since experimental and con-trol body weights are usually different. It is com-mon practice therefore to determine the relativeweights of the organs in relation to the total bodyweight of the animal. Recently it has been pro-posed that the relative weights should be taken asa function of the animal’s brain weight, the postu-lation being that the brain’s growth curve devi-


ates the least of any tissue in the body. While nor-malization based on this procedure would tend toemphasize changes more than other techniquescurrently in use, it is not yet widely accepted.Tissue dry weight, after desiccation or ashing,has also been used as a tool for determiningmechanisms of growth and metabolic balances(24). This method has a major drawback, how-ever, because it removes the organ from furtherstudies such as microscopic examination.

After gross observation and weighing, theorgans and tissues are preserved for histologicalpreparation and microscopic examination. Themost common methods for the preparation andstaining of individual tissues involve fixing with10-percent buffered formalin solution, embeddingin parafin, and staining with hemotoxylin/eosin,Many pathologists prefer other fixing and embed-ding media, and certain tissues require differentprocedures. There are also special stains forhighlighting different cellular components. Thereis no one best method for preparation and obser-vation of the tissues. The most valuable procedurefrom the pathologists’ viewpoint is to prepare thetissues in a number of ways, which allows com-parison of various aspects such as specific cellu-lar components, nuclei, cell membranes, etc.(25-27).

Special consideration can also be given to tech-niques in histochemistry and electron micro-scopy. These methods are not used routinely intoxicological evaluation and depend on a knowl-edge of the mode of action of the toxic agent. Theycan, however, indicate changes in cellular metab-olism or structure before those changes becomemanifest by the conventional histological proce-dures, and therefore they may be more suitablefor observing changes from agents whose toxici-ties are low or develop slowly. The equipmentnecessary for these techniques is generally moreexpensive than that needed for the more conven-tional microtechnique methods. They are alsomore time consuming and less standardized thanconventional methods. The histochemical meth-ods, although they might be more appropriatelyclassified as tests of organ function, are becomingmore widely accepted with investigators studyingmechanisms of toxic: action.

Functional EffectsFrequently, changes in organ or system func-

tion are observable before any change in struc-ture becomes apparent. The test methods dis-cussed below have generally been adapted fromhuman to animal use, and results are ordinarily

compared with animal control values and are notnecessarily comparable between species, Meth-ods for evaluation of pulmonary function (28), car-diovascular function (29), and brain and neuralactivity (30) have been modified for human andanimal use. These methods include testing ven-tilator flow, resistance, compliance, and gas dif-fusion capacity for the pulmonary system; elec-trical activity of the heart, and blood flow andpressure for the cardiovascular system; electricalactivity of the brain (field and single unit) andmuscles; perception threshold, reflexes, andchronaxy for neural function. The significance ofchanges in brain activity as a determinant of tox-icity is under question at present, however.

Generally, in these evaluations, each animalserves as its own control. Baseline data for eachprocedure is determined prior to administrationof the agent, and any changes in function arenoted during and after administration, since it isimportant to determine whether the agent causesreversible or irreversible changes in function.

Various other methods of testing for organ orsystem function rely on both primary and second-ary parameters. For example, liver function maybe assessed by dye clearance studies (primary) orby analysis of serum enzyme concentrations (sec-ondary). Most of the secondary procedures arenow automated and available through variousclinical laboratories at a reasonable cost, Manyinvestigators, however, still prefer to perform thetests manually, and standard procedures arewell-defined and available in several texts(31-33). Various modifications of these tests forspecific animal systems have been developed andpublished, Tests usually considered appropriateinclude total and differential blood counts, serumenzyme and ion analysis, urinalysis (especially formetabolizes of the agent), and liver and kidneyfunction tests (dye clearance). The value of thesetests is that abnormal results will often precedeobvious structural damage of the organ system inquestion and will be apparent at lower doselevels.

The study of hematologic effects encompasseschanges in the bone marrow as well as those inthe cells of the circulating blood. Observationsare made of the cells and of their absolute andrelative numbers. Specific tests such as dye dilu-tion for blood volume, specific gravity, sedimenta-tion rates, osmotic fragility, hematocrits, or clot-ting time, are not routinely performed but may beindicated. Serial bone marrow biopsies may alsobe performed for hematologic effects; the results

Appendix C—Methods for Toxicologic Test/rig ● 141

give both structural and functional information,but these techniques are also not widely used.

The functions of the liver may be tested forbiliary obstruction (icterus index, alkaline phos-phatase), liver damage (thymol turbidity, plasmaprotein ratios, cholesterol ratio, glucose level,transaminase level, and cholinesterase level), ex-cretory function (bromsulphalein clearance, bili-rubin tolerance), and metabolic function (glucosetolerance, galactose clearance), The most com-mon tests used in toxicology are the serum alka-line phosphatase and serum transaminases, andin some cases a dye clearance (bromsulphalein) orglucose tolerance.

The kidneys are responsible for excretion ofcertain substances, e.g., urea, and for concentra-tion and dilution of urine. Tests for excretion in-volve dyes like phenolsulphonphthalein and alsomeasure such substances as urea and creatinine,Concentration and dilution tests involve measure-ment of urine specific gravity after fasting forvarious periods. These tests are generally notused in toxicology screening studies unless thereis reason to believe the toxicant acts on thekidneys.

The evaluation of these tests may proceed withor without modification of the tissue metabolism.That is, promotors and inhibitors of enzyme sys-tems, e.g., SKF-525A for mixed function oxidase,may be used to enhance the susceptibility of aparticular organ or system to damage from a toxi-cant. This in effect maximizes the response sothat the toxic action can be more readily ob-served.

Many other specific tests are available forevaluating various organs and systems such assperm motility, specific gravity of cerebrospinalfluid. calcium-phosphorus ratios for the skeletalsystem along with tensile strength and compac-tion, epinephrine sensitivity of heart muscle,acetylcholine test of lungs, metabolism of excisedtissue, work and strain measurements of the vari-ous muscle systems, etc.

Behavioral EffectsA recent addition to the field of toxicology has

been behavioral testing. Testing methods havebeen devised for everything from simple percep-tion to complex tasks involving perception, learn-ing, judgment, motivation, and motor activity. Thevalue of the behavioral methods lies in the abilityof the nervous system to respond to toxic agentsa t doses much lower than those necessary to pro-duce “classic” signs of toxicity in the organism.Therefore, these methods are a potential sensitive

indicator of hazard and can be used as an “earlywarning system. ” Several good reviews of behav-ioral toxicology are available (34-37).

Some of these methods rely on newer methodsof analysis such as contingent negative variation(CNV). Some are really an application of preexist-ing principles such as dorsal evoked potentialsand neuromuscular transmission time that havebeen widely used by experimenters in the field ofneurophysiology. Most of these techniques haveonly recently been turned to the evaluation of tox-icity.

At the present time, standardization and vali-dation of behavioral techniques has not been ac-complished. The question often raised by regula-tory agencies is how do you relate an observed be-havioral decrement to an adverse health effect,especially if there is no concurrent structuralchange apparent in the nervous system. Becauseof these factors, behavioral studies are often der-ogated by these agencies when setting exposurelimits for toxicants. Current research is beingconducted, however, under Government con-tracts to answer some of these questions.

Comparison of Short- and Long-TermMethods for Systemic Toxicity

Most of the procedures noted in this section areequally applicable to short- and long-term testing,obvious exceptions being irritation and sensitiza-tion tests. The value of long-term testing for sys-temic toxicity lies in the ability to use low dosesthat do not produce detectable adverse effects ina short time period to see whether bioaccumula-tion and cumulative effects occur. Predictions ofthe effect of bioaccumulation can be made know-ing the effects of short-term high doses, but finalevaluation of the toxicity depends on the long-term effects observed, As will be pointed out inthe metabolism section, toxicants can be poten-tiated or inhibited by the metabolism and relativeaccumulation of the toxic moiety. Without defi-nitely knowing the various metabolic reactions,rates, and probabilities, it is impossible to ac-curately predict toxic effects. High short-termdoses may induce a toxic reaction, such as deathfrom pulmonary edema, that might mask long-term, low-level exposure effects such as livercancer, The differences in short-term and long-term tests for systemic toxicity include the num-ber of interim measurements allowed, the abilityto ascertain the types of effects which mightdevelop only over a long period and the progres-sion or time course of toxic manifestations, and

142 w Environmental Contaminants in Food

the ability to evaluate mechanisms of bioaccumu-lation or adaptation in the organism.

Mutagenicity

Rapid identification of a food contaminant as apossible mutagen is necessary to reduce the po-tential genetic risk to humans who might contactthe contaminant. Mutagenic effects on humansoften cannot be directly detected, and deleteriouseffects on the human gene pool may not becomeapparent for many generations if, for instance,the deleterious effect is due to a recessive gene.Heritable genetic damage in humans may resultfrom any of several types of effects on the geneticmaterial, The two major classes of effects arepoint mutations, which generally affect a singlegene or part of a gene, and more extensive chro-mosomal effects such as gross changes in struc-ture or changes in number.

Only a few tests are available that directlyevaluate genetic effects of exposure of mammalsto chemicals: however, the potential of a chemicalto produce heritable genetic alterations in mancan be evaluated indirectly from its effects on ge-netic material in various biologic test systems, in-cluding micro-organisms, mammalian cell cul-tures, insects, and intact mammals.

For substances that cannot feasibly be elimi-nated from the human environment, it is not suffi-cient to identify the existence of a genetic hazard;quantitative assessment of the risk involved isnecessary for appropriate regulatory activity,such as establishing action levels or tolerancesfor food contaminants.

Mutagenicity testing is also used to prescreenchemicals as an indicator of carcinogenic poten-tial and, less frequently, other toxic effects suchas teratogenicity. This application is based on em-pirical demonstration or correlation between mu-tagenicity and carcinogenicity of chemicals (38)and does not depend on the assumption that thesame mechanism is involved in both types of ef-fect.

Approaches to TestingAs in all toxicological tests, mutagenicity tests

may produce false negatives (a negative resultwhen the substance is actually mutagenic) andfalse positives (a positive result when it is notmutagenic), and correlation between the resultsfrom two test systems may be poor. Ideally, a mu-tagenicity test system should be sensitive enoughto detect any chemical that may cause heritablegenetic damage and its results should be repro-

ducible. Finally, the test results should be quan-titatively applicable to mutagenesis in humans.Since no single test can fulfill these requirementsand none is reliable enough to stand alone as anindicator of mutagenic potential, mutagenicity‘testing should include a variety of systems se-lected to show whether the test substance or itsmetabolize produce any of a range of genetic ef-fects. The test battery approach includes systemsthat will detect several types of gene mutations,chromosomal aberrations, and DNA repair; thus,this approach offers the greatest reliability fordetermining mutagenic potential, Tests that eval-uate effects in intact mammals are essential forpredicting mutagenicity in humans.

Since screening large numbers of chemicals bythe test battery approach (39,40) may be prohibi-tively costly, a hierarchical approach to mutage-nicity testing, known as tier testing (41), has beensuggested. Tier 1 consists of relatively inexpen-sive short-term prescreening tests. These usemicro-organisms or other in vitro systems to de-termine priorities for indepth testing. Substancesthat produce positive results in these tests, aswell as those that are negative but are structur-ally similar to known mutagens or to which thereis a substantial risk of exposure for humans dur-ing or preceding their reproductive years, shouldcontinue into Tier 2.

Tier 2 tests are usually designed to detect sub-stances that are not mutagenic in vitro but aremetabolized to an active form in the intact mam-mal. Tests used at this level may include the domi-nant lethal test, in vivo cytogenetic tests, the host-mediated assay, and body-fluid analysis. Sub-stances that are negative in Tiers 1 and 2 are gen-erally considered safe for use and are given verylow priority for further testing.

Only substances for which it is important to as-sess risk are subjected to Tier 3 testing, designedto permit quantitative evaluation of mutagenic po-tential. Tests used at this level include multigen-eration mammalian studies, such as the heritabletranslocation test, X chromosome loss test, andspecific loci test in mice.

Tier testing may represent an efficient use ofresources in large-scale mutagenicity testing, butthe use of prescreening tests carries serious dis-advantages in determining mutagenic potential, Iftest chemicals are prescreened by a single micro-bial test, the proportion of false negatives may beunacceptably high, and potentially hazardous oruseful substances may escape further testing.The use of two or three tests at this level, in-cluding both micro-organisms and mammalian

Appendix C—Methods for Toxicologic Testing . 143. . . , . . . . . .—- >--

cell cultures with and without activation by mam-malian enzyme systems, may substantially in-crease the reliability of prescreening (96). Never-theless, such cell systems may not approximatemetabolic events in the intact mammal closelyenough to reveal the mutagenic action of somesubstances that are potential human mutagens.

Whatever the testing approach and test sys-tems selected, mutagenicity tests should include apositive control as well as negative (untreatedand solvent) controls. The positive control sub-stance, a known mutagen in animal systems thatis selected for its structural similarity to the testchemical, serves to demonstrate the sensitivity ofthe test organism and the efficacy of the metabol-ic activation system used.

Current Test SystemsChromosomal effects of many substances have

been demonstrated in plants such as Vicia fabaand Tradescantia (42), and the latter organismhas also been used in detection of somatic muta-tion (43). While the genetic events involved (alter-ations in DNA) are the same as those in mammali-an cells, their relevance to human mutagenesishas been questioned because of the major phylo-genetic and physiologic differences betweenplants and animals; thus, a negative result inplants does not indicate that a substance is not amutagen in mammalian systems.

Of the many bacterial species that have beenused to detect point mutations, the most exten-sively employed are the Salmonella typhimuriummutants developed by Ames (44,45). The Amestest uses a series of histidine-requiring mutantstrains that revert to histidine-independence byspecific mechanisms, either base-pair substitu-tions or frameshift mutations, The original strainshave undergone several further modificationsthat increase their sensitivity to mutagens by in-terfering with DNA repair or modifying the cellwall to enhance the penetration of chemicals intothe cell. Bacteria treated with the test chemicalare plated on selective media or cultured in liquidsuspension to determine the number of revert-ants. A reproducible mutation rate twice thespontaneous (control) rate is usually consideredevidence of mutagenic activity.

Because microbial cell systems do not possessthe metabolic capabilities of mammals, they willnot detect chemicals that exert a mutagenic effectthrough metabolic intermediates, Several activat-ing systems have been developed for use with invitro test systems to duplicate the effects of mam-malian metabolism. The most extensively used

means of metabolic activation is the addition ofmicrosomal mixed-function oxidase enzymes, typi-cally from rodent liver homogenates, to metabo-lize the test chemical in vitro, This activating sys-tem is added to the culture medium as part of theAmes testing procedure with S. typhimurium, andit has provided evidence for the mutagenicity ofmany substances that have no direct mutageniceffect on these bacteria (51). Microsomal enzymeactivation is also used with other microbial testsystems (46-48). The major drawbacks of this sys-tem are that it would not detect chemicals metab-olized to mutagenic intermediates by mechanismsother than liver microsomal enzymes, e.g., sub-stances metabolized by the intestinal flora, and itis possible that the in vitro metabolism of thesubstance does not adequately mimic its metabo-lism in the intact organism because of competingreactions. Another drawback is that a standard-ized in vitro activation system has not been de-vised to date.

Another widely used bacterial system is themultipurpose strain of Escherichia coli developedby Mohn and coworkers (49). This strain can beused to measure reverse mutations restoring theability of the bacteria to synthesize the nutrientsarginine and niacin. Forward mutation rates intwo genes controlling galactose metabolism canalso be scored in this strain of E. coli. Use of thistest organism permits the detection of severaltypes of mutation in a single experiment.

Eukaryotic micro-organisms that are used todetect the ability of chemicals to produce pointmutations include haploid strains of the yeastsSaccharomyces cerevisiae and Schizosaccharo-myces pombe (47,5o) and of the ascomycete Neu-rospora crassa (51). A diploid strain of S. cerevi-siae permits detection of chromosomal damageexpressed as mitotic recombination that producesphenotypic color changes (52).

Whole-animal activation mechanisms can cir-cumvent this problem but they are generally muchless sensitive than tests using in vitro activation.In these systems, rodents are exposed to the testchemical by an appropriate route, and the effectof rodent metabolizes on microbial genetic mark-ers is determined by either body-fluid analysis orhost-mediated assay procedures,

In the body-fluid analysis (53-56), the micro-organisms are treated with the urine, blood, orhomogenized tissues of the exposed animals. Cau-tion is necessary in interpreting negative resultsof these studies, in the absence of supplementarypharmacologic data, since even if the chemical ismetabolized to a mutagen, other factors such as


t issue-specif ic act ivat ion and detoxif icat ionmechanisms and the half-life of the compound andits metabolizes may affect test results. In the host-mediated assay (57,58), the micro-organisms areexposed to mammalian metabolic products of thetest substance by being introduced into the perito-neal cavity, circulatory system, or testes of thehost mammal. The host is treated with the testsubstance, and after an appropriate incubationperiod, the indicator organism is removed and ex-amined for mutations.

Genetic damage in micro-organisms can also beassessed indirectly through the use of DNA re-pair-deficient strains of bacteria (44,59). Thesetests organisms and otherwise identical strainsthat have normal ability to repair DNA aretreated with the test substance. Toxic action ofthe test substance produces zones in which bac-terial growth is inhibited, and the difference insize between the inhibition zones in repair-defi-cient and normal strains indicates the extent towhich this toxicity is due to damage to the DNA.

A number of mammalian cell systems in culturehave been developed for detecting point muta-tions, including cell lines derived from mouse lym-phomas, Chinese hamster ovaries and embryos,and human fibroblasts and lymphoblasts (60-63).In addition, gross chromosomal changes such asbreaks, gaps, and rearrangements can be micro-scopically observed in these cells. Stable re-arrangements, such as translocations and inver-sions, are considered evidence of her i tablechanges. The induction of only gaps and breaks isnot regarded as evidence of mutagenicity, be-cause these aberrations often occur as a result ofgeneral cytotoxicity and thus may be present onlyin moribund cells. Like microbial systems, in vitromammalian cell tests can be used in conjunctionwith activation by mammalian enzymes or withwhole-animal activation to permit detection ofmutagenic effects by metabolic products of a testsubstance.

Mammalian cells, including human white bloodcells, are also used to detect chemical damage toDNA by measuring unscheduled DNA synthesis(63). This indirect indicator of genetic damage isevaluated by measuring the uptake of radioactivethymidine for repair of damaged DNA duringthose stages of cell growth when DNA synthesisdoes not normally occur. A similar test withmouse spermatocytes exposed in vitro or in vivodemonstrates effects on DNA in germinal cells(64),

Sister chromatid exchange, a reciprocal ex-change of segments at homologous loci. measured

by autoradiographic methods, has also been usedto examine a variety of chemicals (65,66). Sisterchromatid exchange in various cell systems hasbeen demonstrated following exposure to knownmutagens, but additional work is needed to definethe extent to which results are correlated withmore traditional mutagenicity tests,

The fruit fly Drosophila melanogaster is used ina comprehensive and extensively characterizedmutagenicity test system which can detect alltypes of mutagenic activity at a fraction of thetime and cost of in vivo mammalian testing (67).The large number of genetic markers and knownchromosomal aberrations make it possible toassay a chemical for many types of mutagenic ac-tivity in a single test. The sex-linked recessivelethal test (68) in Drosophila is a very efficientmutagenicity assay, since about 20 percent of theinsect’s genetic material is located in the Xchromosome. Recessive lethal changes caused bypoint or chromosomal mutation can be mappedand in most cases the nature of the change caus-ing the mutation can be determined. This test alsopermits a quantitative assessment of mutagenicactivity. Because of the large size of Drosophilachromosomes, this organism can also be usedreadily to assess meiotic and mitotic recombina-tion, dominant lethality, translocations, and dele-tions. Some indirect mutagens that required meta-bolic activation have been shown to be mutagenicin Drosophila, indicating that these insects have amicrosomal mixed-function oxidase system (69).However, to determine whether Drosophila testresults are useful for risk assessment, more in-formation is needed on how their metabolism offoreign chemicals compares to that in humans. Afew other species of insects have also proven use-ful in mutagenicity testing, including ‘severalspecies of the parasitic wasp, Habrobracon (70).

Clearly the most accurate predictions of muta-genic potential in humans can be drawn fromtests that determine direct genotypic and pheno-typic effects in mammals exposed to the testchemical by routes relevant to human exposures,Several direct mammalian tests exist, but thesehave the disadvantage of detecting only a few ofthe possible types of genetic damage. Chromo-somal damage occurring in vivo can be detectedin several different cell types, such as bone mar-row cells and circulating lymphocytes, and thepresence of micronuclei in red blood cells (7 I).Cytogenetic tests using mammalian lymphocytesand the micronucleus test offer the advantage ofpermitting direct comparison with effects on hu-mans resulting from accidental exposures; how-


ever, these tests demonstrate only effects on so-matic cells and do not provide direct evidence ofheritability.

Cytogenetic changes in mammals can also beevaluated in germinal tissue from the testes. Inthe direct spermatocyte test (72), male mice areexposed to the test substance. After sufficienttime for the treated spermatogonia to reach thespermatocyte stage, they are examined for cyto-genetic abnormalities. This test allows for the ac-tual observation of induced cytologic changes inpremeiotic male germ cells, but it does not permitdetection of effects in postmeiotic cells or theirtransmission to the offspring.

Effects on offspring can be evaluated in mice bythe heritable translocation test (73) and the Xchromosome loss test (74). In the heritable trans-location test, F, male offspring of treated mice aremated to determine sterility, and indication opossible translocation heterozygosity. Chromo-somal effects are then confirmed by cytogeneticanalysis of the germinal cells of the male of offspr-ing. The X chromosome loss test permits thedetection of chromosome loss resulting from non-disjunction in the female, since, unlike somaticchromosome aneuploids, animals of XO genotypeare usually viable, Aneuploidy for the X chromo-some can be detected by genetic markers and con-firmed by cytologic observations.

The dominant lethal assay, usually performedin the rat or mouse, uses fetal loss as an indicatorof induced chromosomal mutations in male ger-minal cells (75). The death of the zygote is assum-ed to result from chromosomal abnormalities inthe sperm of male mice exposed to the test chem-ical. This test is relatively easy to perform and itsresults have been positively correlated with muta-genicity in other animal systems. Preimplantationloss alone is not used as an indication of muta-genicity since it has been found to occur for rea-sons other than chromosomal changes in thesperm. Disadvantages of this test are its relativeinsensitivity and difficulty in clearly distinguish-ing weakly positive results.

Only one test is available at present that candetect heritable gene mutations induced in mam-malian germ cells. In the specific locus assay inmice, forward mutations at seven loci, affectingcharacteristics such as coat and eye color aremated with mice homozygous for recessive allelesat these loci (75). Because such a small number ofloci are involved, this test required the scoring of20,000 to 30,000 offspring at each dose level toproduce reliable results and is therefore verycostly and time consuming.

Carcinogenicity

In the event of massive or long-term environ-mental contamination of food destined for humanconsumption, one of the decisions to be made iswhether the contaminant appears to pose a sig-nificant carcinogenic risk. With this in mind FDAsubmits the candidate compound to the ChemicalSelection Working Group at the National CancerInstitute for consideration under the carcinogenbioassay screening program (76,77).

Prerequisites for a CarcinogenicityStudy

Once the compound has been selected, it isscreened using a chronic or lifetime exposureregimen (76,78,79). However, before the long-termstudy is undertaken, specific toxicologic profilesmust be obtained. Young healthy adult animals ofeach sex and strain to be used in the long-termstudies should be used in the preliminary studies.The animals should be of uniform age and weightand should be tested using the same formulationand route of exposure to be used in the long-termstudies. The first is an acute study designed togain additional information on the acute toxicity,assuming there is a paucity of data on this aspectof toxicity, and to determine the lethality of thetest compound. The duration of this test shouldnot exceed 24 hours and should include at leastthree dose levels determined by a geometric pro-gression. one of the dose levels selected shouldrepresent the highest dose to be used in subse-quent studies. Throughout the investigation, allrelevant clinical signs should be recorded. Ne-cropsies should be performed on a random selec-tion of animals of each sex and strain, and any ab-normal histopathologic changes should be noted.

After the 24-hour study, a 14-day investigationshould be initiated in an effort to ascertain thedoses necessary for the subchronic study, thenext prerequisite investigation for the chronicstudy. This toxicologic study requires five doselevels, with the highest one, estimated from the24-hour acute study, producing no more than 10-percent lethality. The other dose levels shouldrepresent geometric decrements of the highestdose. Animals should be treated with the testsubstance for no more than 14 days, held another24 hours, and then sacrificed for necropsy.Throughout the study, the animals should be ob-served for clinical signs of toxicity. Other toxicitydata, such as those derived from organ functiontests and metabolism studies, are also necessary.


The next toxicity study involves the administra-tion of the test substance for 90 days and is usedas a predictor of the maximum tolerated dose(MTD). This can be defined as the highest dosegiven during a chronic study that can be pre-dicted to not alter the animals’ normal longevityfrom effects other than carcinogenicity, In prac-tice, MTD is considered to be the highest dose thatcauses no more than a 10-percent decrement inweight compared to controls. Five dose levels arerequired in this study, with a minimum of 10 ani-mals of each sex and strain in each dose group.The highest dose level used should be the lowestconcentration that produced any detectable un-toward toxic effects in the 14-day study. The re-maining dose levels should be determined as inthe 14-day study, If the selected dose levels do notproduce a discernible no-effect level, the studyshould be repeated with lower doses.

Carcinogenic BioassayThe chronic study (76,78,79) represents the

essence of the carcinogenicity bioassay. It is usedto determine the carcinogenicity of a compound inmales and females of two mammalian species,usually the rat and mouse. The species selectedare tested throughout their entire lifespan. Eachtest group should consist of a statistically repre-sentative number of animals. The highest selecteddose should represent MTD and the remainingdose levels should be adjusted accordingly. Thereshould be at least one control group, in which theanimals receive only the vehicle used for adminis-tration of the test material, If no vehicle is used,this control group should be untreated but iden-tical in every way to the experimental groups. Inaddition to the concurrent control group, a colonyor historical group should be used for the com-parison of longevity, spontaneous diseases, andspontaneous tumor incidence. The historical con-trol may also be used for statistical comparisons.In some studies, a positive control group that hasbeen treated with a compound structurally simi-lar to the test compound and known to be carcino-genic in the test species may be indicated. How-ever, because of the added risk of handling aknown carcinogen, a positive control group is sel-dom used.

Throughout the study, animals must be ob-served for signs of toxicity. Every animal shouldbe examined carefully each week, Animals shouldbe weighed and food consumption measured. Insome cases, it is desirable to evaluate tissuedistribution and concentration of the substanceor its metabolizes.

The animals in any one test group should besacrificed at an adjusted or prearranged date.However, a group can be terminated earlier ifthere has been high cumulative mortality. Mori-bund animals should be sacrificed immediatelyupon discovery to lessen the likelihood of unob-served deaths and subsequent autolysis or canni-balism. Control groups should be sacrificed ac-cording to the original or adjusted sacrifice date;the later date is preferred. Because of the strongdependency on histopathologic results and toavoid possible criticisms of the study, necropsiesand histopathologic examinations should followstandard procedures required by regulatoryguidelines.

The major drawback to the carcinogenic bio-assay procedure is the time and cost required tocomplete and analyze such a study. If the study isdone properly, however, the results should beconclusive and for the most part indisputable, al-though there still remains the question of extrap-olation of results to the human population.

Short-Term Testing as a Prediction ofCarcinogenicity

Evaluation of carcinogenicity has generally re-lied on the results of long-term animal studies, Touse this kind of testing approach for every sub-stance suspected of being carcinogenic would becost-prohibi t ive and certainly impractical interms of the overall time required to test all sus-pected chemicals. Therefore, short-term tests arebeing developed to identify carcinogenic sub-stances. There has been much criticism concern-ing the comparison of short-term testing resultsfrom different laboratories because of the vary-ing conditions and refinements in techniquespracticed among testing facilities. The protocolsfor these short-term tests, especially those involv-ing mammalian enzyme activation systems, havenot been standardized or validated through inter-laboratory comparative testing procedures; thus,comparisons of the data from one laboratory tothe next have often produced conflicting conclu-sions. In this light, a study conducted in one lab-oratory that compares several short-term testingsystems has added importance in clarifying therelative usefulness of the compared systems,

In a recent study by Dr. Ian Purchase (80), 120organic chemicals (50 known carcinogens and 62noncarcinogens, based on published experimen-tal data) were evaluated for activity in six short-term test systems. These systems included: 1) mu-tation of Salmonella typhimurium (45), 2) celltransformation (81), 3) degranulation of endoplas-

Appendix C—Methods for Toxicologic Testing ● 147.—

mic reticulum (82), 4) sebaceous gland suppres-sion (83), 5) tetrazolium reduction (84), and 6) le-sion formation after subcutaneous implant (85).Four additional tests used in a preliminary studywere found to be insufficiently accurate or sen-sitive to justify a full evaluation. The tests re-jected were transplacental blastomagenesis (86),piperidine alkylation (87], iodine test (88), and theacridine test (89).

Although there were considerable variationsbetween tests in their ability to predict car-cinogenicity, two tests were quite accurate indistinguishing between the known carcinogensand the noncarcinogens. These were the celltransformation test and the bacterial mutationtest, which had accuracies of 94 and 93 percent,respectively. The use of cell transformation andbacterial mutation together provided an advan-tage over the use of either alone, predicting 99.19percent of carcinogens. Not surprisingly, the in-clusion of the other four tests in a screening bat-tery with these two resulted in an improved abili-ty to detect carcinogens (99.97 percent), butgreatly decreased the accuracy and discrimina-tory value of the battery. It is important to notethat all tests generated both false positives andfalse negatives: the percentages for both can bereadily calculated by subtracting the positivepredictability values from 100 percent.

A description of each of the tests, with compar-ative percent accuracies for predicting carcino-genicity as determined by Purchase et al. (80) isas follows:

● Bacterial mutation. The procedures usedwere those of Ames, in which four strains ofS. typhimurium (TA 1535, TA 1538, TA 98, TA100) were tested with each compound in anassay medium containing a metabolic activa-tion system composed of rat liver postmito-chondrial supernatant (S-9 fraction) and co-factors. The overall accuracy of the test inpredicting the carcinogenicity of the com-pounds in this study (80) was 91 percent forcarcinogens and 94 percent for noncarcino-gens. These figures agree with the previouslypublished value of 90 percent by McCann etal. (90) but are considerably higher thanthose published by Heddle and Bruce (91)who found 65 and 81 percent, respectively.

● Cell transformation. The procedures usedwere those of Styles (81) and involved threetypes of mammalian cells—human diploidlung f ibroblasts (WI-38) , human l iver-derived cel ls (Chang), and baby Syrianhamster kidney cells (BHK 21/cl 13). In all

●

●

●

●

assays, the cells were used with and withoutmetabolic activation with S-9 fraction asdescribed previously. Without activation,very few carcinogens transformed hamsteror human cells in the period of study. Withactivation, all cell lines detected carcinogenswith an accuracy of 88 percent or better.Furthermore, by the use of both the hamstercells and either of the human cells, theoverall accuracy was improved to 94 percent(91 percent for carcinogens and 97 percentfor noncarcinogens).Degranulation. The procedure used is that ofWilliams and Rabin (82) and the test is com-monly called the Rabin test. The test meas-ures the loss of ribosomes (degranulation]from isolated rat liver endoplasmic reticu-lum following incubation with the test com-pound. The overall predictive value was 71percent for both carcinogens and noncar-cinogens.Sebaceous gland test. The procedures usedwere those of Bock and Mund (83) where testchemicals were applied directly to the skin ofmice and a depression in the ratio of seba-ceous gland to hair follicles indicates a posi-tive response. The overall predictive value ofthe test was 65 percent (67 percent for car-cinogens and 64 percent for noncarcino-gens).Tetrazolium reduction. The procedures usedwere based on those described by Iversenand Evensen (84). Test solutions were ap-plied directly to the skin of mice and the skinsamples were incubated in tetrazolium redsolution. An increase in the in situ biologicreduction of the colorless tetrazolium to acolored formazan compound indicates a posi-tive response. The overall predictive value ofthis test was 57 percent (40 percent for car-cinogens and 71 percent for noncarcino-gens).Subcutaneous implant. The procedures usedwere essentially - those of - L o n g s t a f f a n dWestwood (85] and involved the subcutane-ous implantation of a filter disc overlaid witha gelatinous suspension of the test c o m-pounds into mice. After 3 months, the sur-rounding tissues were scored for lesions. Theoverall predictive value for this test was 68percent (37 percent for carcinogens and 95percent for noncarcinogens).

While the work of Purchase and associates (80)does present a very salient assessment of themore pertinent in vitro assays, it also points out


the shortcomings of this type of approach. Short-term tests do not use the induction of cancer as anendpoint, but each has a parameter, such as in-duction of a point mutation, that varies with thecarcinogenicity or noncarcinogenicity of the testsubstance. Accordingly, the authors feel thatthese test parameters should not be given agreater weight than that of any other arbitraryresponse, regardless of how biologically signifi-cant any of these tests might appear to be withrespect to the theories of the chemical inductionof cancer. Also, however much generalized datamight be generated to support the predictive ac-curacy of the given test, this accuracy should notbe assumed to apply uniformly to compounds ofevery chemical class,

Teratology and Effects onReproduction

An investigation of a teratogenic agent involvesthe study of congenital malformations other thanthose that are inherited. Teratogens themselvesact as triggers for malformation induction. Mal-formations may include gross, histological, mo-lecular, and behavioral anomalies,

The sensitivity of an animal to a teratogen is de-termined by: 1 ) the period in which the insult is re-ceived during the gestation period (this includesbefore germ layer formation and during embry-ogenesis or organogenesis); 2) the dose and theroute of administration of the compound; 3) pla-cental transfer of the suspect teratogen, includingits lipid volubility, protein binding ability, andmetabolism : and 4) uterine and dietary factors.

A toxicologic profile of a suspect teratogen re-quires an evaluation of potential hazards to re-production and, particularly, to developmentalprocesses that respond to environmental insultthrough mutation, chromosomal aberrations, mi-totic interference, altered nucleic acid synthesis,enzyme inhibition, and altered membrane charac-teristics. This is particularly important in the con-ceptus, embryo, and the neonate where the bio-chemical, morphologic, and physiologic proper-ties change rapidly. Thus risk assessment mustnot only address the teratogenicity of a contami-nant but also its effect on reproduction.

Classical ApproachFor an adequate teratologic assessment (79,92,

93), exposure to the toxicant should parallel asclosely as possible that expected in the humanpopulation. The pharmacologic activity of thecompound as well as its acute and chronic toxici-

ty is also a consideration. For teratogenic studies,the toxicant is usually administered daily on thespecific days of gestation representing the periodof greatest sensitivity. Administration of the testsubstance should begin at or before implantationand should continue throughout the period of ma-jor organogenesis.

The selection of an animal species for evalua-tion of teratogenicity is an important considera-tion. Test protocols currently in use recommendat least two mammalian species, the first being arodent (e. g., mouse. rat, hamster) and a nonrodentmammalian species (e.g., rabbit). One speciesshould be the same as that used in the test forreproductive effects.

In conducting the teratogenic investigation, atleast three dose levels should be used, along withconcurrent control groups. These control groupsshould consist of untreated animals, animalstreated with the vehicle of administration only,and, possibly, animals treated with an agent thatis known to cause the effect that is being in-vestigated. The use of historical or colony con-trols may also be helpful in evaluating the data.Both test and control animals must be young,mature, prima gravida females of uniform age,size, and parity. The control groups should behandled and maintained like the test groups. Thehighest dose level to be considered should pro-duce signs of embryo or fetotoxicity as suggestedby fetal growth retardation and more significant-ly by maternal or fetal mortality: however, mater-nal mortality should not exceed about 10 percent.The other dose levels can be obtained in a de-creasing logarithmic fashion to a suspected no-ob-servable-adverse-effect level. In the classicalteratology study, treatment may be by gavage sothat the administered dose is accurately known. Ithas been shown, however, that use of this route ofadministration may induce anomalies in progenythat are a consequence not of the test compoundbut of the stress of dose administration (94).

Prior to initiating the study, it is important todetermine whether sires and dams have success-fully mated, This includes examinations for thepresence of plugs or evidence of sperm in vaginalsmears. Throughout the study, females should beobserved for behavioral changes, food and waterconsumption, body weight, vaginal bleeding in-dicating possible abortion, and spontaneousdeaths. Females showing signs of aborting or de-livering prematurely should be sacrificed.

Fetuses should be obtained 24 hours before an-ticipated parturition by cesarian section, Damsshould be sacrificed and a complete necropsy per-


test material for at least 120 days. At this pointthey are bred to produce the F, generation.

The types of data to be collected includegrowth and time of delivery for each weanling aswell as overt s igns of toxici ty , the generalbehavior and condition of the mothers, measure-ments of spermatogenesis in all Ff generationmales used to produce the F: generation, littersize, number of stillborn/live births, and any phys-ical or behavioral anomalies.

A statistically valid number of animals (malesand females) obtained from the F, generation andused to produce the F~ generation should be sacri-ficed and examined at the appropriate time, withspecial emphasis on the histopathologic state ofthe reproductive system. In addition, an adequatenumber of weanlings of each sex from each doselevel, including controls, should also be sacrificedand used for histopathologic analyses.

Data derived from the above should be eval-uated for the existence of a relationship betweenexposure and the incidence and severity of ef-fects on reproduction and behavior, tumors, andmortality. The no-observable-adverse-effect levelshould also be determined.

Compared to a teratology investigation, whichrequires about 3 months to complete the exposureand to analyze the results, a three-generationreproduction study represents a considerablylonger expenditure of time, approaching 1.5 yearsbetween the initiation of the study and the com-pletion of the analysis of the gathered data. How-ever, an elementary profile on teratogenicity andreproductive performance can be obtained withina period of a year or less as a part of a continuingl o n g - t e r m t o x i c i t y s t u d y b y a d d i n g t h e a p p r o p r i -a t e n u m b e r o f a n i m a l s a t t h e b e g i n n i n g o f t h elong-term study (2).

Metabolism

Metabolic assessment studies are not directlyused for the assignment of risks, tolerances, or ac-tion levels, These studies are used to determineparameters, such as absorption, distribution,storage, and excretion, that may affect the per-formance of materials in biologic systems, thusenabling the researcher to design testing proto-cols that measure the overall effect of exposure tothe material rather than just measuring a part ofthe biologic response. These protocols can thentake into account such factors as tissue concen-tration, length of time in contact with specificorgans or tissues, ease of reactivity, and the pro-duction of significant (or nonsignificant) changes


in overall body concentrations that may alter theoutcome of a specific testing regimen.

Metabolic study systems are usually centeredaround the concept that the circulatory system isthe major means of transportation of the material,regardless of the route of exposure, Although spe-cific materials may be readily metabolized at ornear the site of entry into the body (e.g., the lungs,skin, and intestines), the majority of materials aretransported unchanged to the liver and then dis-tributed via the circulatory system to other tis-sues and organs. At the same time, some of thematerial may be excreted unchanged in the urine,feces, and air or converted to various metabolizesthat are excreted or bound in the tissues. The pro-portions of the metabolic products that are ex-creted or bound depends on the chemical natureof the original compound, the dose, route of ad-ministration, species, strain, sex, diet, and envi-ronmental factors.

The objective of the metabolic study is to math-ematically evaluate the rates and relative im-portance of these processes in limiting the con-centration of materials in the tissues of the body.For this purpose, the body is usually visualized asa group of pharmacokinetic compartments, a sim-plistic view that surprisingly approximates fairlyaccurately the complex, interdependent proc-esses that actually occur in the body (95). Thesecompartmental models allow the researcher touse measurements of blood concentration as in-direct estimates of tissue concentrations and todetermine the length of time the material remainsunaltered (i. e., the biologic half-life). Rates of ab-sorption from one compartment to another canalso be readily measured, thus giving the re-searcher information on the rate constants of dif-fusion into tissues and on the rates of distributionfrom the blood to various tissues. Clearance orelimination rates can also be determined for theexcretion of the material from the body via thekidneys, gastrointestinal tract, lungs, saliva, andperspiration.

The rate of metabolism of materials depends onmany factors, among the most important of whichare the physiochemical characteristics of themolecule itself. Polar compounds are usually ex-creted very rapidly from biologic systems largelyunchanged because of the chemical activity ofthese compounds, whereas nonpolar compoundssuch as lipids usually remain in the body longerbecause they must be metabolized to polar com-pounds before excretion occurs.

Another important factor in the metabolism ofa compound is its structural resemblance to natu-

rally occurring substances in the body. Foreigncompounds that closely resemble normal bodyconstituents are frequently metabolized by thesame specific enzyme systems that metabolizetheir normally occurring analogues. Most foreigncompounds, however, have no endogenous coun-terpart and must be metabolized by relativelynonspecific enzyme systems. These nonspecificenzymes catalyze many different types of reac-tions leading to a diversity of metabolic products.In general, the nonspecific enzyme reactions canbe categorized into two types, The first includesthe conversion of one functional group intoanother (oxidation of alcohol to aldehyde), thesplitting of neutral compounds to fragments hav-ing polar groups (hydrolysis of esters and amides),or the introduction of polar groups into nonpolarcompounds (hydroxylation). The second type in-cludes the conjugation of the created polar groupwith glucuronate, sulfate, glutathione, or methylgroups to form a soluble, excretable product. Theproduct formed in the first reaction may be eithermore or less toxic than the parent compound ormay possess a different type of toxicity. Often, itis this product that actually causes the toxic ef-fects, including cancer, mutations, cellular necro-sis, hypersensitivity, fetotoxicity, and blood dys-crasias. A portion of the chemically reactivemetabolize formed becomes bound to tissue mac-romolecules, such as cellular proteins, DNA,RNA, glycogens, or lipids. In this way, it disruptsthe normal function of the macromolecule, caus-ing adverse biologic effects. In contrast, the ma-jority of the second type of reaction products areusually either nontoxic or considerably less toxicthan the parent compound,

The metabolism of a foreign material is con-trolled by enzymes, and any factor which affectsthese enzymes also affects the metabolism of thecompound and consequently its toxicity. The me-tabolism of a compound may be inhibited or stimu-lated by the presence of competing substrates.Pretreatment with drugs, steroids, food additives,pesticides, polycyclic hydrocarbons, polycyclicamines, and normal constituents of food can re-sult in an increase in the activity of enzymes thatmetabolize foreign compounds. This increase inenzyme activity differs according to the inducingchemical, but is mediated through an increasedrate of synthesis of enzyme protein, a decreasedrate of turnover of enzyme, or an activation of en-zyme, possibly by changes of structure or con-formation. Because of this phenomenon, chronicadministration of a foreign material may enhance


the activity of the enzymes that catalyze itsmetabolism.

Several types of biologic phenomena are readi-ly studied using a metabolic test system. These in-clude activation, antagonism, synergism, and po-tentiation. Activation has been briefly touched onabove with the description of the process of tox-ification: it involves the rendering of an inactivemolecule into an active molecule, usually throughthe removal or substitution of a neutralizing fac-tor attached to the molecule, but it may also in-volve the direct addition of a constituent to themolecule. One of the most common examples ofactivation, the conversion of a precarcinogen to acarcinogen, is the addition of oxygen molecules topolycyclic aromatic hydrocarbons via the micro-somal NADPH-dependent cytochrome P-450mixed function oxidase enzyme system. This con-version to the oxide produces a carcinogenicagent of considerable potency, whereas the par-ent material does not possess a direct carcino-

APPENDIX

1.2.

3,

4.

5.

6.

7.

8.

9.

10.

11,

12.

13.

14.

15.16.

41 CI+’R 51206 (1976).Food and Drug Administration Advisory Commi[-tee on Protocols for Safety Evaluations: Panel onReproduction Report on Reproduction Studies inthe Safety Evaluation of F’ood Adciit ives and Pest i-icicle Residues ( 1970). Tox, App). Pharm. 16:264.A40dern Trends in Toxic~Jlogy ( 1968). E. Eloyiandand R. Coulding, ecis,, But terworths, London.Methods in ‘1’oxicolog}’ ( 1970). G, E. Pagett, cd.,Davis, Philadelphia.Frawlev, J. P. ( 1955). Fooff Drug Cosmetic L(IW J.1 o: 703.Sm}th, 1{. I+’,, Jr,, an(i (;. P . Carpenter (1944) . J.Jnd. Hyg. (Jnd Toxicd. 26:269.Friedenwt~ld, J. S., W. l“, Hughes, Jr., and H. Her-mann ( 1944). Arch. of Oph th. 31 :279.Carpenter , C. P., and H. F, Smyth, Jr, (1946). A m .J. Ophth. 29: 1363.Draize, J. H., G. Woodard, and H. O. Calvery[ 1944),]. Phc~rm. find Expt. “rherc~peu. 82:377.I)raizc, J. 11., and E. A. Kelly ( 1952). Proc. of theSci. Sec. of Toilet G(MN]S Assoc. 17:1.Ilraize, J. H. ( 1955). Foo{j Drug Cosmetics Low ].10:722.Draize, J. 11. ( 1965). Assoc. of Food and DrugOf ficicds ~)f the U. S., Topeka. Kans., 46.Lansteiner, K., and J. Jacobs (1935). ]. Exp. Med.61: 643.Lansteiner , K,, and J. Jacobs (1936). ]. Exp. Meal,64:625.Buehler, E. V. (1!365). Arch. Derm, 91 :171.Baer, R. L., S. Rosenthal, and C, J. Sims [1956). J.Invest. Derm. 27:249.

genie potential per se. The determination of meta-bolic activation can permit the experienced re-searcher to predict the kind of adverse effect thatis likely to be elicited from the parent molecule,thus enabling the researcher to better design ex-periments to observe these effects.

From a properly designed and well-carried-outmetabolic study, the researcher can gain valuableinsight into the potential toxic actions and bio-logic effects to be expected from a foreign com-pound. If a compound is not absorbed or if thecompound is destroyed or rapidly eliminated fromthe body there is little likelihood of pronouncedtoxic effects. If the principle metabolic productsare polar, conjugation (and thus elimination)rapidly occurs and again there is little potentialfor pronounced toxicity. On the other hand, if me-tabolism produces an activation product, toxicityis enhanced and biologic effects may be pro-nounced.

REFERENCES

17.

18.

19.20.214

22.

23.

24.

25.

26.

27.

28.

29.

30.

31,

32.

Llarzulli, F. N., and Ef. 1. klaibiich ( 1970). J. Soc.Cosmet. Chem. 21 :695.h4aguire. H. C., Jr, ( 1973). ]. Soc. Cosmet. Chern.24: 151.Levine, B. B. ( 1960). ~. Exp. A4efi. 112: 1131.Salvin, S. B. (1965). Fed. Proc. 24:40.Turk, J. L. (1964). Int. Arch. Allergy 24:191.Bio]ogy of the Immune Response ( 1970). P, Abro-noff and hf. LaVie, eds., hlcGraw-Hill, N.Y.Textbook of Immunop(lthologv ( 1976). H. G. hlLLl-ler-Eberhard, cd., Grune and St rat ton, N. Y..Medicul Physiology ( 1968). V. B. hlountcastle, cd.,hfosby, St. Louis.A Textbook of Histology ( 1964). W. Bloom and D.W. Fa~cet t, eds., Saunders, Philadelphia.Pfltholog}r (1971). L$’. A. D. Anderson, cd., Mosbv,St. Louis,Nelson, A. A. [ 1955). Foo(j Drug Cosmetic L~Jw j.10:732,Comroe. J. H., Jr,, R. Il. F“orster II, A. B. Dubois, W.A. Briscoe, and E. Garleson ( 1962). ‘rhe Lung: (lin-ic{d Physiology (lntl Pulm~~n(] r-y Func ti(~n Tests,Yearbook hfedical Publ., (;hicago.Armst rong, hl. L. ( 1974). Ii~[~ctr[)cf)r-( ii(~gr{lms,Yearbook hledical Publ., Chicago.Current Practice of Clinical El[~ct~()(>rlc[?l~h~)l()g-r(lphy ( 1977). D, W’. Klass and D. D. Daly, eds.,Raven.Goodale, R. 11. ( 1965). Clinicul In terpre ta t ion o.fLahor~ltory Tests. Davis, Philadelphia.Henry, R. J. ( 1974). (linic(d (;hernistry: Principlesan{j Techniques, Harper hledical.


33.

34.

35.

36.

37.

38.

39.

40,41.42.

43.

44.

’45.

46.

47.

48.

q:] .

50,

51.

52.53.

54.55.

56.

57,

58,

Amuino , J. S, (1976). Clinical Chemistry: Prin-ciples and Procedures, Little.Xintaras, C., and B. L. Johnson (1976). Essays inToxicology, W, J. Hayes, cd., 7:155, AcademicPress, New York.Ekel, G. J., and W. H. Teichner (1976), A nAnalysis and Critique of Behavioral Toxicology inthe U. S. S. R., 13 HEW (NIOSH) Publication No.77-60, Cincinnati.Behavioral Toxicology (1974). C. Xintaras, B. L.Johnson, and 1, DeGroot, eds. DHEW (NIOSH)Publication No. 74-126, Cincinnati.Weiss, B., J. Brozek, H. Hanson, R. C. Leaf, N. K.Mello, and J. M. Spyker ( 1975). Principles for Evu]-uu ting (Jhemicais in the Environment t, 198 Na-tional Academy of Sciences, Washington, D.C.Alc(; arm, J., and B. h!. Ames ( 1976). Ann. fV. Y.Ac{](I. Sci. 271 :5.Bridges, B. A. ( 1973). Environ. Heo]th Perspect6:22 1,Bridges, B. A. ( 1974). Mu(. H[?s, 26:335.b’lamm, W. G. ( 1974). h’fut. Res. 26:329.Ehrenberg, L. ( 1973). In: Chernicul Mu ta~ens:Princi{j]es (In(i M[~lh(xls for Their Detection, v o l .2. A. }Iollaender, d., Plenum Press, New York.Underbrink, A. E., L. A, Schaierer, and A. H.Sparrow (1973), In: Chemical Mutagens: Princi-ples and Methods for Their Detection, vol. 2, A.Hollaender, cd., Plenum Press, New York.Ames. B. N.. F. 1). Lee, and W. E. Durston (1973).Pr(m. lV(It, Ac(Id. Sci., U.S.A. 70:782.Ames, B, N., J. hlc(hnn, and E. Yamasaki ( 1975).Mut. ~[?S. 31 :347.I.[]ishes, B. A. , and 11. E’. S[ich ( 1973). Biochern.Biophys, Res. [lmlmun. 52:827.Brusick, D. J., :]nd V. W“. Nfavcr ( 1 9 7 3 ) . Env.He({lth Persp[’c[. 6:83.ong, ‘1”-hf, []nd 1{. V. ifal]in~ ( 1 9 7 5 ) . M u t . lies.31 : 195.hlohn. {;.. J. Ell[?nlwrger, :]nd D. hlc~re~or ( 1974).MU (. 1{[]s. 25: 187.Mortimer, R. K., and T. R. Manney (1971). I nChemicul Mutagens: Principles and Methods forTheir Detec~tion, vol. 1, A. Hollaender, cd., PlenumPress, New York.De%rnes. ~’. J., a n d If. V. hl:]lling ( 1 9 7 1 ) . I n :[;hcmi[;(il MU tflgens, Principles (]n(i Meth(xls forTheir Detecti{jn, vol. 1, A. 1 Iollaender, cd., PlenumPress, New York.Z i m m e r m a n l+’. K. ( 1975). Mut RCS. 3 1:71.

Gabri(ige, Ll, C., A. L)eNunzio, and M. S. Legator( 1969).N(ltur[?68:221,Siebert, D. ( 1973). Niut. Res. 17::~07,Durston, M’. ~;., fln(~ Il. N. Ames (1!174). Proc. Nut.Ac(I(I, Sci. USA 71 :737.Commoner, B.. A . J . frith:lv;ithil. and J . H e n r y( 1974). N(lt urc 249:850.Brew en, J. (;., P . Nettesheim, i~nd K . P . J o n e s( 1970), Mu(. 11(:s, 10:645.I,eg[}tor, hf. S., (l nd I{. ~’. hi;} II ing [ 1971 ). In: Chemi[;(li Jlfu t(]gens: Prin(; iples (ln(l Metho(ls for Their

59.

60.

61.

62.

63.

64.

65.66.

67.

68.69.

70,

71.72,

7:3.74.

75.

76.

77.

78.

Detection, vol. 1, A. Hollaender, cd., PlenumPress, New York,Slater, E. E., M. D. Anderson, and H. S. Rosen-kranz (19i’1). Cuncer Res. 31 :97o,Chu, E. H. Y. ( 1971). In: Chemicul Mutagens: Prin-ciples and Methods for Their Detection, vol. 1, A.Hollaencier, ed., Plenum Press, New York.Kao, F-T, and T. T. Puck (1974). Methods Cell BioJ8:23.Cline, D., and J-A F. S. Spector (1975). Mut. Res.31: 17.Regan, J. D., and R. B. Setlow ( 1973). In: ChemicalMutugens: Principles and Methods for Their De-tection, vol. 2, A. Hollaender, cd., Plenum Press,New York.Sega, G, A., J. G. O w e n s , and R. B. Cumming(1!276). Mut. ~t?S. 36: 193.‘1’aylor, J. H. ( 1958). Genetics 43:5 15.Allen, J. W., and S. A. Latt ( 1976). Nature260:449.Abrahamson, S., and E. B. Lewis (1971). In: Chem-ical Mutagens: Principles (]n(f Methods for TheirDetection. vol. 1, A. Hollaender, eci., P l e n u mPress, New York.Vogel. E., and El. Leigh ( 1975), Mut. Hes. 29:383.Casida, J, E. ( 1969). In: Micr-osomes (Infl Drug Oxi-du tion, J. R. Fouts and G. J. hlannering. eds., Aca-demic Press, New York.Smi th , R. 11., and R. ~;, von Borstel ( 1973). In:(jhemic~d Mut(],gens: Principles und Methods forTheir Detection. vol. 2, A. Ilollaender, ed., plenumpress, New York.!+:hmid, W. ( 1975). Mut. R[?s. 31 :9.[,eoll:lrd, A. ( 1 g73), In: Chemical Mutugens: Prin-ciples un[j Meth(~(js for Their Deter; tion, vol. 2, A.}Iollaender, cd., Plenum Press, New York.I,eonard, A. ( 1975). Mut. HCS. 3 I :291.Russell, 1,. B. ( 1976). [n: (11[’mi~;~ll Mut{lgens:Pr inciples (ln(j Meth(xis ff~r Their fletection, vol.4, A. Ilollaencier, cd., Plenum Press, New York.Batem~~n A. J. :~rld S. S. ~PStein ( 1 9 7 1 ) . In:Chemic~lf Mut~lgens: Principles (In(i Metho(is forTheir Detection, vol. 1, A. Ilollaender, cd., PlenumPress, New York.Sontag, J. N1., hf. P, Page, and U. Saffiotti ( 1976).I n : Cui(ielines for Curcin(~gen Bimlss[]y in Sm(dlHo(jen ts. U.S. Department of Health, Educationand Welfare, Public Health Service, National In-sti tutes of [iealth. National Technical In forma-t ion Service PB-264 061, Springfield, Va.Saffiotti, [J., :]nd 11. Autrup ( 1978). In: In Vitro(1] rc;inogenesis; Guide to t h e Liter(lture. A(l-v(lnces (ln~l Lahor(ltory Procedures, U.S. Depart-ment of I Ieal t h, Education and Welfare, Public}iealth Service, National Institutes of Health. Na-tional Technical Information Service PB-281 737,Springfield, Va.(~arcinogenicit y ( 1973). In: The Testing of Chem-ic(ils f~~r t~cll.[;inf~g(~nic;ity, Mu t(lgenic;ity, (Ind ‘I’eru -togenicity, Nlinistry of Ilealth a n d W e l f a r e ,(;anada .

Append/x C—Methods for Toxicologic Test/rig ● 153

79.80.

81.82.

83.

84.

85.

86.

87.

88.

89.

43 (;F’R 37336 ( 1978).Purchase, I. F. H., E. Longstaff, J. Ashby, J. A.Stvies, D. Anderson, P. A. Lefevre, and F, R.Westwo(d ( 1978). Br. /. (hncer (in press).St Vlcs, J. A. ( 1977), Br. ], Cancer 36:558.VL’illiams, I). J., and B. R. Ral)in ( 1971). Nclture232: 102.Bock; h’. 11., and R. Mund ( 1958), (llncer Heseurch18:887.Iverscn, (), }1.. and A. Evensen ( 1962). In: F:XJX>ri-men t~~~ Skin (l] rein ogencsis in Mice, NorwegianIJniversit y Press.I,ongst{]ff. E,, and F. R. L1’estwood ( 1978). [Jnpub-lishe(i.DiPaol:), J. A., R. L. Nelson, P. J. Donovan, and C,11, Ev:)ns ( 197:3). Archs. P(]thol. 95::380.Epstein, J., R, W’. Rosenth:]l, and R. J. F;ss [ 1 9 5 5 ) .Anfll. fllcrn. 27: 1435.Szcnt-GVorg yi, A., 1. Is(:nberg, and S . IJ. Baird( 1960). f%(~c. N(lt. Ac~Ici. Sci. USA. 16: 1444.Szent-Gvorg}’i, A., :]n(i J. hlcLau8hlin (1!361 ). Prf)c.Nflt. Ac[I~l, Sf;i. 11. S.A. 47: 1397.

90,

91.

92.

93.

94.95.

96.

McCann, J., and El. N . Ames ( 1976). Proc. Nat.Acud. Sci. U.S.A. 73:950.IIeddle, J. A., and W . R. Bruce ( 1977). In: Originsof Humun Cflncer. 13(x)k C. Human Risk Assess-ment. H. H. Hiatt, J. D. VL’atson, and J. A, W“insten,eds. Cold Spring Harbor 14abor a torv.‘reratogenicity (1973). In: The ‘1’[?s ting of Chem-iculs for C(1 rcinogcn ici ( 1, M u t(lgf’n ici ty, (ln(i Terf I-tr)genicity, Ministr>r of Ile:]lth an(i L’1’elfarc,Canada.Courtney, K. D., and hi. ( ;h[?rnoff ( 1974). In: ‘~rf]in-ing M(lnuol for 7’er[l t{jl~)g}’. office o f R e s e a r c hand Development, U.S. Environmental ProtectionAgency, Research ‘1’ri:]n~le Park, N.C.Green, E. L. ( 1962). Gcnctif;s. N.Y. 47: 1085.Compartment ts, Pools (~n(i Sj)fJces i n MwliccdPhysiol[~g~ ( 1967). P.-Ii. E. [3ergner ;ind f;. C. ],ush-baugh, eds. U.S. Atomic F;nergy (X)mmission, Divi-sion of ‘1’echnicnl In forma t ion, 0:] k Rid8e, ‘1’enn.Purchase, I. ( 1976). N(It ure 264:624.

methods for toxicologic testing

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