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Public Debate About Pesticidesand Human Health

The public perceives “pesticides” as a unique classof chemicals more “dangerous” than chemicals inprescription and over-the-counter medications andmore “toxic” than chemicals that occur naturally in foodand the environment. Actually, pesticides are intention-ally designed to be toxic to plant, animal, or microbialpests—e.g., weeds, rodents, bacteria, insects, fungi—just as antibiotic drugs are intentionally designed to betoxic to specific disease bacteria. Many natural chemi-cals in our food supply can also be toxic to livingorganisms.

The private sector, academia, and regulatoryagencies worldwide combine expertise to assesspesticide safety and risk potential. Specialists intoxicology, human and veterinary medicine, epidemiol-ogy, chemistry and biochemistry, statistics, physiology,anatomy, pathology, and molecular biology evaluatenumerous studies and other information. Conclusionsdrawn by these scientific communities represent theirbest professional judgment based on many years ofeducation, research, and experience. It is impossible totest or prove safety under every imaginable scenario.However, the overall testing program is comprehen-sive; and it examines the responses to pesticide levelsmuch higher than man or animal would normallyencounter.

The public is expected to place confidence inscientific and regulatory professionals, yet as a societywe are ill-informed on the mandatory, comprehensiveevaluation process that precedes the registration ofevery pesticide product. Innumerable media discus-sions center on the impact of pesticide residues infood, air, or water on past, present, or future humandiseases and disorders. And without the necessarytool—that is, knowledge—to sort out the media hype,we have no basis on which to judge the validity of whatwe hear and read. Our lack of knowledge, coupled withthe misperception of pesticides as an exceptionallydangerous class of chemicals, provides fertile groundfor debate among groups with conflicting opinions onthe benefits and risks of pesticide use.

The debate is polarized, contentious, and specula-tive. The underlying issues center on how “safe” or“dangerous” pesticides are. That’s it. That’s what the

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public wants to know, once and for all: Just tell uswhether or not the benefits of pesticide use justify anyrisks their use might pose! But it’s not that easy.

There exists a common misperception that pesti-cides can be classified as “safe” or “not safe.” But nochemical, either natural (produced by plants or otherorganisms) or synthetic (produced by man), can bedetermined completely safe. Researchers, state andfederal regulators, manufacturers, and public healthofficials continually grapple with the possibility thatpesticides might contribute to the development ofhuman disease or trigger adverse environmentaleffects. The effort to develop conclusive evidence ofsafety is ongoing, but absolute safety can never beguaranteed. The U.S. Environmental Protection Agency(EPA) regulates the pesticide product registrationprocess and mandates studies to determine the condi-tions under which pesticides can be used with minimalrisk and maximum benefit. The findings dictate theconditions under which the product can be marketed.

The public forum often is slanted by an incompleteunderstanding of the principles of toxicology and thepesticide registration process. Addressing publicconcern requires comprehension of the science and theregulatory system; and appreciation for the judgmentsand predictions derived from scientific data is essential.This publication is intended to foster a better under-standing of the role of toxicology in the safety evalua-tion and registration processes.

The Science of Toxicology

Toxicology is the scientific study of the harmfuleffects of chemicals on living organisms: humans,animals, and plants. Toxicological testing evaluateswhether short-term exposure to a pesticide will produceacute effects (e.g., eye and skin irritation, death) andwhether long-term, continual exposure will causechronic effects (e.g., impaired liver function, reproduc-tive abnormalities, cancer).

Toxicological evaluations are conducted withexperimental animals exposed to various levels of thepesticide for various lengths of time, from hours toyears. Results often lead investigators to additionalresearch on the interaction of the pesticide with biologi-cal systems. Understanding the biological mechanismsthat underlie effects observed in animals allows toxi-

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Pesticide and Animal Interaction

cologists and risk assessors to predict the chances ofharm to human populations exposed to the pesticide.

Consideration of exposure levels and effectsproduced at specific doses is essential in determiningtoxicity. Exposure, in and of itself, does not necessarilyproduce harmful effects; for instance, people who areexposed to low levels of pesticides in their food ordrinking water, or through contact at the workplace,usually suffer no harm. But harm is to be expectedwhen people are exposed, accidentally or otherwise, tomuch higher levels that have been shown to produceadverse health effects in laboratory animals.

The dose/response concept is familiar: High dosesare likely to produce detectable injury, while low dosesmay produce little or no injury. For example, ingestingone or two sleeping tablets might have a beneficialeffect, while consuming a bottle of them could be lethal.One glass of beer may not affect an adult, but thesame amount could easily intoxicate a child. A little saltimproves the taste of food, but a lot could causeserious health consequences—even death.

Toxicologists follow the basic premise that allchemicals, both natural and synthetic, can prove toxicat some dose. Those such as table salt cause adverseeffects only at high doses, while others (e.g., cyanide)exhibit toxicity at a very low dose. When toxicologistsdetermine the gradation of effects resulting fromincreasing doses of a pesticide, they have establisheda dose-response for that pesticide.

The duration and magnitude of exposure determinethe severity of the poisoning. In other words, theincrement of time during which exposure to the doseoccurs (duration), plus the size and number of doses(magnitude) combine to determine the severity of thepoisoning. Although the terms dose and exposuresometimes are used interchangeably, they are notsynonymous. Example: A dose of oral medicine isprescribed by the doctor, but the patient is not exposedto the medication unless and until he swallows thedose. A pesticide will trigger an adverse responsewhen a person is exposed long enough to a dose largeenough to cause harm.

It is important when investigating the toxicology of apesticide to understand the effects of the chemical onthe animals—and vice versa. The genetic, physiologi-

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cal, anatomical, and biochemical variability among andwithin animal species helps scientists understand whya pesticide may be highly toxic to rats but nontoxic todogs or people, for example, or why another is toxic torats and mice but not to fish or birds.

Pesticides cannot be categorized as “safe” or“dangerous” to humans merely because they areclassified as substances that kill pests. Each activeingredient has its own unique chemical structure andtoxicological characteristics. Pesticides with very similarchemical structures in many instances produce dra-matically different effects. One chemical may generatea highly toxic effect while another may exhibit notoxicity whatsoever to the same animal at the samedose. Laboratory studies are useful in predicting andexplaining the toxicity of a pesticide.

Effects on animals are determined by the chemicalstructure of the pesticide, its action mechanism, andthe fate of the chemical within the animal. Not allanimals react to all pesticides in the same manner; andresponse can be species- or individual-specific. Thesystem of one animal species may metabolize apesticide to a nontoxic metabolite, whereas that ofanother species may not (species-specific response);and individual animals of a species also can responddifferently (individual-specific response).

Effect of the Chemical on the Animal

Species-Specific

Pesticidal effects often vary with the species ofanimals studied; for instance, one species may exhibitskin irritation and another, liver disease. The degree ofsensitivity may vary, as well; two species may reactdermally, for example, with one exhibiting severesensitivity compared to the other’s mild skin irritation.

Individual-Specific

Individual animals within a species can exhibitdissimilar responses to the same pesticide. Toxiceffects can vary with the size, sex, age, and generalhealth of the test animals.

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An animal’s response to a pesticide may hinge onhow its internal systems “process” the chemical.

Evaluation of the effect of test animals on thepesticide involves consideration of how readily thechemical is absorbed into the animals’ system and howthe system distributes, breaks down, and eventuallyeliminates the pesticide. Animals whose systems retaina pesticide for a long time may exhibit effects not seenin animals whose systems eliminate the chemical morerapidly. In some cases, a chemical may appear toxiconly after being converted by an animal’s system into amore reactive form.

Test animals whose skin does not readily absorb agiven pesticide may exhibit only minimal effects, butthe same animals might react seriously when thecontaminant is administered orally or through inhala-tion.

The fate of a pesticide from its point of contact withand elimination from the body of laboratory animals isstudied to gain an understanding of how humans mightrespond to the same chemical. A pesticide may gainaccess to the circulatory system through dermal,inhalation, or oral exposure and subsequently becarried in blood from the point of entry to variousorgans and tissues. The body’s physiological pro-cesses may store or excrete the toxin or metabolize(alter) its structure, thus modifying its toxicologicaleffects. Body tissue might absorb the pesticide from theblood and store it, or it might release the contaminantback into the bloodstream for elimination in urine orfeces, or through exhalation.

These processes are complex and interactive. Themechanisms by which biological systems handlepesticides determine whether toxic effects will beobserved. Following are biological and physiologicalfactors that influence the interaction between pesticidesand humans.

Effect of the Animal on the Chemical

Each pesticide is unique; pesticide molecules differin their chemical structure, size, shape, stability, andelectronic charge. These differences determine howpesticides function in the human body. For example,the chemical structures of some pesticides make themfat soluble, while those of others dictate water solubil-

Chemical Properties

8

ity; the significance is that the former would be storedfor various lengths of time in fatty tissue, while the lattermight be rapidly eliminated through urination. Theshape and electronic charge of some pesticide mol-ecules allow binding to critical site receptors in cells oron cell membranes. These and other variables oftenlead to significant differences in biological response topesticides.

Absorption Into the Body

Pesticides may enter the human body through the skin (dermal exposure),mouth (oral exposure), and lungs (respiratory exposure). The site of exposure tothe pesticide impacts the rate of absorption into the bloodstream, as well as itsdistribution pattern.

Skin is a natural barrier to many pesticides, but penetration can occur,especially if the skin is disrupted by cuts and abrasions.

Each region of the gastrointestinal (GI) tract—mouth, esophagus, stomach,small and large intestines, colon, and rectum—has its own internal environment

that dramaticallyaffects the absorptionof a pesticide. Somecompounds readilyabsorb in the mouth,while others areabsorbed only in theintestines. Somepesticides simply passthrough the organism(man or animal)without being absorbedat all.

Pesticides inhaledinto the lungs needonly to cross the thinbarrier separating lungtissue from the bloodsupply to gain rapidaccess into the blood-stream.

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The transport of a pesticide within the body dependson whether the pesticide is absorbed through the skin,lungs, or GI tract. Pesticides absorbed in the GI tractenter the bloodstream flowing directly to the liver—themajor site of pesticide metabolism—where they arebroken down soon after absorption. Those that areabsorbed into the bloodstream through the skin orthrough inhalation to the lungs actually circulatethroughout the body before reaching the liver to bebroken down. Therefore, the same pesticide dose maybe more toxic through inhalation than ingestion.

Movement Within the Bloodstream

Uptake by Organs, Tissues, and Cells

Blood is the medium through which the pesticide istransported to organs, tissues, and cells. Uptake bycells is dependent on a pesticide’s physical and chemi-cal properties and the type of cell involved. Therefore,different pesticides may be distributed to differenttissues in the body. Pesticides can enter a tissuepassively by simple diffusion; that is, the pesticide canmove from a high concentration in blood to a lowerconcentration in body tissue. Pesticides may be ac-tively transported by reactions that use energy to “pull”the chemical into the cell.

Pesticides are subjected to chemical alterations byenzymes in the body via a process known as metabo-lism. Metabolism refers to chemical reactions that alterthe structure and the physiochemical properties of thepesticide by adding, removing, or substituting variouschemical components.

Metabolism takes place primarily in the liver, wherecells usually change the original pesticide molecule to aless toxic, more water soluble form which makes thechemical easier to excrete. However, some moleculesare converted to more toxic forms.

Pesticides may accumulate in body tissues, pro-teins, fat, and bone. It is fat-soluble pesticides, prima-rily, that are stored in the body for long periods of time.The depletion of body fat can release them into thebloodstream.

Metabolism Within Cells

Pesticide Storage Sites Within the Body

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Metabolized chemicals in the body ultimately areeliminated by the kidneys, in urine, or carried to theintestine in bile from the liver. Pesticides may bereabsorbed into the bloodstream from the intestinaltract or excreted in fecal material. Pesticides also canbe eliminated through the lungs in expired air or in bodysecretions such as tears, saliva, and milk.

Excretion and Elimination From the Body

The Swiss physician Paracelsus (1493-1541), the father of toxicology, believedthe relationship between dose and response to be inseparable. Paracelsus asked,“What is it that is not poison? All things are poison and nothing is without poison.The right dose differentiates a poison and a remedy.”

These principles, having withstood the test of time, serve as the basis oftoxicology. We all have some understanding of this basic tenet. For instance, twoaspirins may remedy a headache, but a much larger dose—a whole bottle ofaspirin—may be poisonous. Therefore, the question ofhow much exposure (dose amount) can be toleratedbecomes a critical factor in evaluating safety.

The Relationship Between Dose andResponse

The Concept of the Bell Shaped Curve

It is important to discuss sensitivity differencesamong individuals of a single species. Just as peopledo not look alike, they also do not respond in exactly

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the same manner to medicines, pesticides, or otherchemicals. We know, for example, that penicillin is alife saving antibiotic for most people; however, somehypersensitive individuals may exhibit serious sideeffects from it. Certain individuals become intoxicatedafter a few alcoholic drinks, while others can consumeconsiderable quantities of the same drink withoutadverse effects.

People and all living organisms exhibit a broadspectrum of reactions and sensitivities to chemicals.Some people are very resistant to high doses whileothers are very susceptible even to low doses; mostindividuals are somewhere between very resistant andvery susceptible. In fact, when the sensitivities of alarge number of people (or rats) are graphed againstincreasing doses of a drug, a pesticide, or some otherchemical, the resulting distribution curve is shaped likea bell. The far left and right sides of the bell lip repre-sent the small number of people that are either verysusceptible or very resistant to the chemical, and thesensitivities of the remaining majority of people fallbetween those extremes. If a certain dose of a toxicchemical is given to a large number of animals, someshow no effect, some get sick, and some die. Thus, inany toxicological test, we do not expect all animals tobe affected, nor do we expect all animals affected toexhibit the same degree of severity.

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The toxicity of a pesticide is determined by quantify-ing the response of laboratory animals to a series ofincreasing doses. This relationship between adminis-tered dose and animal response is graphically depictedas the dose-response curve. The graph includes themeasured response (e.g., the number or percent ofanimals affected, or the severity of the response) onthe vertical axis and increasing doses of the testchemical on the horizontal axis. For a measuredresponse such as death, the percentage of animals thatdie increases proportionally as the dose increases. Acommon measure used to define toxicity when aboutone-half of the animals die at a certain dose is theLD

50—the lethal dose for 50 percent of the animals

tested. More than 50 percent of the animals die atdoses higher than the LD

50, while fewer or no animals

die at lower doses. Thus, the higher the LD50

dose, theless acutely toxic the pesticide.

A generalized dose-response curve has threedistinct regions:

• no detectable response

• increasing linear response

• plateau (maximum) response

Animals exposed to low doses exhibit no signs oftoxicity. The specific point on the dose-response curvewhere the more susceptible animals are first affected

Understanding the Concept ofDose-Response

LOEL

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by a pesticide dose is termed the threshold level: thelowest dose that produces a measurable response inthe most sensitive animals. The threshold level is thebeginning of the linear response region of the curveand is the demarcation between the “no observedeffect level” (NOEL) and the “lowest observed effectlevel” (LOEL). Increasing the dose beyond the thresh-old level increases not only the proportion of animalsthat show response, but the severity of the effect, aswell. It is this linear region of the dose-responsecurve—increased dose, increased response—that isused to measure, describe, and predict the toxicologi-cal properties of a pesticide.

The third part of the curve, the plateau, begins atthe point where an increase in dose no longer pro-duces an increase in response. The point at which theresponse levels off at the upper end of the dose rangeis known as the maximum effect level. The responsemay plateau at 100 percent because all of the animalstested are affected, or it may plateau at a lower re-sponse level because the remaining unaffected ani-mals are resistant to even the highest dose tested.

The following should be considered when reviewingtoxicology studies and interpreting dose-responserelationships.

• No single dose-response curve can describe theentire range of toxicological responses exhibited by anexperimental animal. Each response (death, cell injury,etc.) is a separate end point and can have a differentdose-response curve.

• End points may be actual observations, such aschanges in animal behavior or food consumptionpatterns; they also may be an indirect indicator oftoxicity. Toxicologists can use measurements of bloodand urine constituents as indicators of toxicity, disease,or deterioration in test animals without having to resortto surgery, x-ray, or whole body scanning. For in-stance, a toxicologist might use changes in bloodenzymes as an indicator of damaged liver cells.

• The dose required to produce a given effect (endpoint) may vary, depending on the pesticide.

• The data used to develop each dose-responsecurve are unique to the organism (test animal) thatreceived the pesticide dose.

• Dose-response curves may differ dramatically,depending on the route of exposure to the pesticide:oral, dermal, or inhalation.

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Using Dose-Response Curves

Dose-response curves provide valuable informationto the toxicologist. Examination of these curves andtheir supporting data provides a basis for comparingpesticide toxicity threshold levels as well as medium(LD50) and maximum effective doses. For example,regulatory agencies categorize pesticides according toLD

50 values which, in turn, are useful in determining

label language for precautionary statements, first aiddirections, packaging restrictions, and transport recom-mendations.

The slope of the dose-response curve is of criticalimportance. A steep curve indicates only a slightdifference between a nontoxic dose and a toxic dose; insuch a case, even a small increase in dose produces asignificant change of effect. Conversely, a somewhatflat dose-response curve indicates that a relativelylarge increase in dose has little effect. A flat dose-response curve indicates a larger margin of safetybetween a nontoxic dose and a toxic dose. Clearly,correct interpretation of toxicological data and validconclusions on the toxicity of a pesticide require an in-depth understanding of the dose-response curve.

Describing Adverse ToxicologicalEffects

Toxicity Described by Exposure Duration

The effects of a pesticide vary with duration ofexposure:

• acute (short-term exposure; a single exposure ormultiple exposures within a very short period of time)

• subchronic (intermediate-term exposure; repeatedexposure over a longer period of time)

• chronic (long-term exposure; repeated exposureover a very long time)

For many pesticides, the response to acute expo-sure can be very different than the response tosubchronic or chronic exposure; that is, a dose admin-istered once may evoke little or no response, whilemultiple exposures (at the same dose) over an interme-diate or long period of time might generate a significantresponse.

A pesticide is said to be acutely toxicity whenadverse effects result from a single exposure, usuallyat a relatively high dose. But it should be noted that

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exposure to the same or lesser doses multiple timeswithin a very short period of time (e.g., 24 hours) also istermed acute. Acute effects in humans often result fromaccidents, such as a child ingesting a pesticide. Suicideattempts and, in some cases, the blatant misuse ofpesticide products, may constitute acute exposure.Anytime a pesticide causes adverse effects followingacute exposure, it is said to exhibit acute toxicity.

Subchronic toxic effects manifest after frequentlyrepeated (e.g., daily) exposure, over weeks or months,to pesticide doses which might produce only minimal orno response to a single, acute exposure. The body isnot allowed time to eliminate the pesticide beforesuccessive exposure, thus resulting in a buildup thattriggers adverse subchronic effects.

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Chronic effects result from continual exposure overa long period of time—a lifetime, for example. Pesti-cides can have cumulative effects on the body, even atdoses so low that no immediate or short-term effectsare apparent. While the body might be able to recoverfrom minimal effects that a single dose or a few lowdoses might cause, it may not be able to recoup totallybetween repeated exposures over a long period oftime. The buildup of the chemical in the host systemeventually becomes recognizable as a chronic effect;and the resulting, cumulative damage can be perma-nent.

The toxicity of a pesticide is described as reversibleif its effects subside or disappear when exposure ends.But in situations where adverse pesticidal effectspersist even when exposure is eliminated, the toxicity isconsidered irreversible.

The toxic effects of some pesticides are reversiblewhen exposure is eliminated, regardless of the dose,while the effects of others may be reversible at low-dose exposures but irreversible at high doses. Toxiceffects sometimes are reversible, initially, but withcontinued exposure become irreversible, the dosenotwithstanding.

Toxicity Characterized as Reversible orIrreversible

Following are examples of pesticide interaction atthe subcellular level.

• Enzymes are proteins that speed up chemicalreactions of specific molecules. A pesticide that inter-feres with an enzymatic process can prevent, slowdown, or speed up a chemical reaction within a cell.Enzymatic interference can lead to a toxic response bythe cell, tissue, organ, or system. For example, acetyl-cholinesterase is an enzyme essential to properfunction of the nervous system; it can be inhibited byorganophosphorous insecticides, leading to nervoussystem toxicity.

• Critical cellular components (e.g., DNA, hormonereceptors, energy producing chemicals, nerve impulse-transmitting chemicals, and cell membrane transportproteins) can interact with pesticides to produceharmful effects. Pesticides may interfere with moleculesthat serve specific purposes. For example: Hemoglobin

Toxicity Characterized by Alterations atthe Subcellular Level

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is a special molecule whose primary function is totransport oxygen in red blood cells. Interfering withhemoglobin so that it does not perform effectively canresult in injury stemming from changes in oxygentransport.

Toxicity often can be described according to theobservable or measurable effect it causes.

• Death is the ultimate toxic effect, occurring whencritical bodily functions are altered or inhibited.

• Irritation is observed when a pesticide affects cellsof the skin or eye; corrosion occurs when the integrityof the outer layer of cells is destroyed. The effectfrequently is referred to as a “burn.” Less severeirritation might appear as redness, swelling, or inflam-mation of the skin. Irritation/corrosion can result from asingle or cumulative exposure.

• Skin sensitization is an allergic reaction; sensitiza-tion requires multiple exposures over a period of time.The initial exposure “sensitizes” the person, andsubsequent exposures cause the individual to react tothe chemical by developing a “rash.” Poison ivy is afamiliar example of a skin sensitizing, natural chemical.

• Mutagenicity (also called genotoxicity) resultsfrom a change in the genetic material of a cell. Thereare two general types: a gene mutation that changesthe DNA genetic code; and a structural mutation thatcauses structural chromosome damage.

A mutagenic compound may produce chromosomalaberrations by modifying the physical structure ornumber of chromosomes; the result is chromosomesthat are fragmented or mismatched, or chromosomesthat fail to undergo cell division.

Gene mutations include the deletion, addition, orsubstitution of the chemical components of DNA, whichcontains all the coded information that allows organ-isms to function. Disruptions in genes or chromosomescan lead to diseases (including cancer) and birthdefects. A mutagen is of concern when it damages eggor sperm cells, enabling the defect to be passed on tosuccessive generations.

• Tumors—also called neoplasms—are abnormalgrowths of tissue; they can be either benign or malig-nant. Most benign tumors are not life-threateningbecause cell division usually is slow and the cells arenoninvasive: They will not spread to surrounding tissue.Malignant tumors divide rapidly, in an uncontrolled

Toxicity Characterized by Effect

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fashion, and spread to other body tissues; this, coupledwith their tendency to intercept nutrients needed byhealthy tissue, thereby destroying it, renders them life-threatening.

Malignant tumors may be one of four cancer types:

• Leukemias are cancers of red blood cells, certainwhite blood cells, and the tissues that produce thesecells.

• Lymphomas are cancers that affect organs of thelymphatic system, such as lymph nodes.

• Sarcomas are cancers of connective tissues suchas bone, muscle, and cartilage.

• Carcinomas are cancers of the internal or externalepithelial tissues.

Toxicological effects often are described accordingto the organ or system that they impact: cardiovascular,respiratory, gastrointestinal, urinary, muscular, skeletal,and dermal effects; central or peripheral nervoussystem and sense organ effects; immune systemeffects; endocrine gland effects; and reproductivesystem effects. Organ effects in experimental animalsoften, though not always, are predictive of the effectsexpected from human exposure to the same pesticide.A toxicological insult to one organ may have indirectrepercussions on other parts of the same system, or onmultiple systems, due to the complex interaction andcoordination of various systems of the body.

Toxicity Described byTarget Organ/System Effects

The Federal Insecticide, Fungicide, and RodenticideAct (FIFRA) requires pesticide manufacturers toconduct and submit to the U.S. Environmental Protec-tion Agency (EPA) an extensive battery of toxicologicalstudies to evaluate the pesticide product. Toxicologicalstudies, in combination with other required tests (e.g.,ecotoxicology, crop residues, and environmental fate),are used by pesticide manufacturers to judge whetherto proceed with the development of a pesticide. EPAscientists use the data to evaluate whether a registra-tion should be granted and, if so, to determine its

Animal Testing Crucial to SafetyEvaluation

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conditions of use; the data also are useful in develop-ing appropriate label language (e.g., permitted uses,use restrictions, warnings, precautions).

Biomedical research has for decades relied onexperimental animals as human surrogates. Thedevelopment of new medicines and the characteriza-tion of safe and effective doses are impossible withoutthe use of laboratory animals specifically bred for thispurpose. The use of animal models for describing thetoxicological properties of pesticides was prescribed byEPA at its inception in the 1970s, and by the Food andDrug Administration (FDA) which preceded it. Regula-tory entities around the world require similar testing.

The use of specially bred research animals intoxicological research is not without controversy. Viewson the ethics of animal use and welfare issues arepolarized, as is scientific dialogue on the relevance ofdata derived from animal models. Animal rights organi-zations contend that experimental use of laboratoryanimals is cruel, unethical, and indefensible. Although,at present, animals cannot be totally eliminated fromthe testing process, pesticide manufacturers andgovernment agencies worldwide have made greatstrides in reducing the number of animals used,eliminating unnecessary experimentation, and ensuringthat animals are housed properly and treated hu-manely.

The reliability of predicting human hazards fromanimal data has long been debated. Many argue thatdrawing conclusions about human safety from animalmodels is fraught with uncertainty. For example,experimental results can be influenced by the choice ofspecies and strain; an animal species that absorbs,metabolizes, or eliminates a pesticide differently fromhumans makes extrapolation to humans less relevant.It is essential that toxicologists use all available knowl-edge, data, and expertise to select the appropriateanimal species, to evaluate the results of experimenta-tion to reach reliable conclusions.

While physiological differences exist betweenhumans and experimental animals, most scientistsagree that the similarities outweigh the differences andthat animals are the only alternative to direct humantesting. There is overall agreement that animal models

The Use of Animals in Toxicity Studies

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generally provide reliable information that helps tosafeguard public health.

The inclusion of laboratory animals as models inpesticide testing programs provides many advantagesto scientists and regulators:

• Animals are the only alternative to human testing.

• Disease-free animals bred for uniformity areavailable commercially at a reasonable cost.

• Laboratory animals can be produced in numberssufficient for toxicological investigations. They can behoused in a relatively small space, have reasonablefood requirements, and are amenable to frequentphysical examinations.

• The historical information background on aparticular animal strain or species—normal responses,disease rates, and tumor frequencies—providesaccuracy in toxicological data interpretation.

• The use of genetically similar individuals (inbredstrains) can lead to more consistent results.

• The relatively short life span of laboratory animalsfacilitates the observation of effects and diseasesassociated with lifetime exposures.

• Certain diseases such as cancer can be predict-ably observed through the use of animals bred specifi-cally for their susceptibility to a disease or for ananticipated response.

• Experimental animals reach sexual maturity at anearly age, have relatively short pregnancies, andproduce large litters; these characteristics facilitate thestudy and evaluation of pesticidal impact on reproduc-tion over multiple generations.

• Test animal routes of entry—oral, dermal, orinhalation—simulate human exposure, yielding arelevant understanding of the chemical fate andproperties of a pesticide inside the body.

• Experiments can be standardized by monitoringand controlling “uncertainties.” For instance, food andwater quality can be monitored and adjusted as re-quired to achieve standardization, and environmentalfactors such as temperature and light can be manipu-lated, as well.

• Historically, regulatory agencies around the worldmake pesticide registration decisions based on theirestablished familiarity with certain test animal species.This standardization of test species, along with experi-mental methodology, forms a database by which tojudge the toxicity of the pesticide being tested.

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Researchers and regulators do not rely on any oneanimal species in conducting safety assessments.Human responses to a pesticide cannot be mimickedexactly or modeled by a single animal species; there-fore, toxicologists must use multiple species—rats,mice, rabbits, guinea pigs, dogs—to predict pesticidetoxicity to humans. Hamsters, monkeys, pigs, chickens,and cats are used less frequently.

Toxicologists repeatedly test the same strain ofanimals to facilitate toxicity comparisons between newand existing pesticides. Animals are purchased fromsources that document the history and purity of thegenetic strain and guarantee the animals to be healthyand disease-free.

Species Commonly Usedin Pesticide Testing Programs

The mouse (Mus musculus) is commonly used inpesticide and pharmaceutical testing; in fact, currentestimates indicate that 70 percent of all animals used intesting programs are mice. Mice are used for pesticidecarcinogenincity tests, predominantly, offering theseadvantages: They are small; they are easy to maintain;and they have relatively short life spans.

Mouse

Strains derived from the Norway rat (Rattusnorvegicus, commonly called the laboratory rat) havebeen used in agricultural and pharmaceutical researchsince the 1850s. The rat is the second most commonexperimental animal, comprising 20 percent of allanimals used, and it offers many of the same advan-tages as mice.

Rat

Albino rabbits (Lepus cuniculus) are used to evalu-ate skin and eye irritation as well as birth defects. Theybreed readily, produce large litters, and are easilyreared in quantity. Their large bodies and eyes facilitateskin and eye exposure studies.

Albino Rabbit

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Guinea Pig

The guinea pig (Cavia porcellus), through decadesof testing, has been a reliable human surrogate inidentifying pesticides that induce skin sensitization—that is, allergies.

Domestic Hen

The nervous system of the domestic hen (Gallusdomesticus) is sensitive to organophosphorus insecti-cides; thus, it is used to evaluate nervous systemtoxicity for this class of pesticides.

Dog

The beagle dog (Canis familiarus) is commonlyused as the nonrodent species of choice. Dogs sharemany physiological properties with man and fullycomplement rodent studies. Their size facilitatesdifficult surgical procedures, and their ample bloodsupply allows larger and more frequent samples to betaken without affecting the animals’ health. Dogs havelonger life spans than laboratory rodents, lending themuseful in pesticide toxicology studies that last a fewmonths to a year, or longer. Some studies have lastedeight or more years.

On page 23 is a summary of the comparison of thebiological and physiological parameters of animals withthose of humans.

Standard Operating Proceduresand Protocols

Scientists conducting toxicological studies tosupport regulatory approval of pesticides in the UnitedStates must comply with commonly accepted scientificstandards, the guidelines of EPA and other regulatoryauthorities, and Good Laboratory Practices (GLP, 40CFR 160). Animal studies also must comply with theAnimal Welfare Act Regulations (9 CFR 3) regardinganimal use and care.

GLP regulations require that each testing facilitydevelop written procedures that describe all aspects ofanimal testing, from start to finish, including datadevelopment, collection, and security. These proce-dures are viewed as the codes of experimental conductand are commonly referred to as Standard Operating

23

Procedures (SOPs). GLP requirements also include thefollowing:

• Develop study protocols prior to testing.

• Ensure that the test pesticide is appropriatelycharacterized as to composition, purity, and stability.

• Train, allocate, and schedule personnel.

• Provide adequate resources and facilities, instru-mentation and equipment, animal care, and archives.

Life Span (years) 2–3 2 6 6 3–5 15 70

Adult Weight (kilograms) .2–1 .02–.04 2.5 .5–.8 1.5–3.5 8–12 70

Estrus Cycle (days) 4–5 4–5 induced 15–18 daily 21–28 21

Age at Maturity (weeks) 13 7–9 24–35 12–16 22 40–72 15–18 yr.

Gestation Period (days) 21 19–21 29–35 58–70 21 56–58 270

Litter Size 12–16 10–12 5–10 2-4 270/yr. 4–8 1–2

Birth Weight (grams) 4–6 1–2 30–70 80 50 300–500 3200

Eye Opening (days) 12–14 10–12 7 0 0 10 0

Weaning Age (days) 21 21 28 18–24 hatched 42–56 250

Weaning Weight (grams) 40–50 10–12 1800 250 50 1500–2500 8000

Body Temperature (F) 99.5 99.0 103.0 103.0 103.0 101.3 98.6

Heart Rate (beats/minute) 330–480 320–780 205–220 230–380 280 130–150 72

Blood Volume

(percent body weight) 6–7 5 5.5 7–7.5 9 8 8

Respiratory Rate

(breaths/minute) 100 163 35–65 84 12–30 20–25 10–13

Guinea Parameter Rat Mouse Rabbit Pig Hen Dog Human

Comparison of Biological and Physiological Parameters

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• Establish an independent quality assurance unit toinspect and ensure the quality and reliability of all studydata.

Newly purchased animals brought into the testingfacility are quarantined away from all animals alreadyhoused there. They are acclimated to their new quar-ters, food, and environment for two weeks prior totesting. Animals showing signs of illness or diseaseduring the quarantine period are not used for study.The animals’ diet and water are monitored and ana-lyzed for impurities. The sterilized, absorbent beddingplaced in the animals’ cages is changed regularly.Cages, racks, and other equipment are cleanedthoroughly on a regular basis.

Animal Husbandry Procedures

Individual studies are isolated from all other studies.Animals are housed in well ventilated rooms wheretheir environment remains constant; lighting, tempera-ture, and humidity are preset and monitored to preventsignificant fluctuation. All instruments used during eachexperiment are calibrated routinely to ensure accuratemeasurements.

Housing Conditions

Data Collection Procedures and Reviews

GLP regulations require laboratories to accuratelydocument every step of an experiment. All data andobservations are recorded in study notebooks orelectronically recorded by computers interfaced withdata-generating instruments such as weight scales andblood analysis instruments. Changes in procedures orcorrections of errors must be recorded, dated, andinitialed; and reasons for changes in data or text mustbe provided.

EPA’s Laboratory Data Integrity Assurance Divisioninspectors audit the accuracy and integrity of datagenerated by pesticide research facilities. EPA inspec-tors look at employee education and training records,check calibration of analytical equipment, and reviewcompliance with written SOPs. Inspectors also examinethe integrity of the data; the housing, feeding, handling,and care of test animals; the handling of test, control,and reference substances; and the accuracy of thestudy reports. Collectively, the procedures establishedby the manufacturer and the inspections by EPA

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assure that testing is accurate, scientifically sound, andproperly documented, and that the experiments aregenerating meaningful, reliable results.

Typically, the pesticide being tested is administeredin the animals’ food or water, or in the air they breathe;dermal effects are studied by placing the chemical onthe test animals’ skin or in their eyes. The dose iscalculated on each individual animal’s weight and isexpressed in milligrams of administered chemical perkilogram of body weight (mg/kg). As an example, tworabbits weighing 4 and 6 kilograms are to be adminis-tered the same pesticide dose of 5 mg/kg. The smallerrabbit should be administered 20 milligrams, while thelarger should receive 30 milligrams.

Animals are typically administered a pesticide dosevia the predominant route of expected human expo-sure: oral; dermal; inhalation.

Administration of Pesticides to Animals

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The method chosen for administering an oral doseoften depends on the chemical, the animal species,and the duration of the study. In a short-term study, thepesticide might be administered to dogs as a gelatincapsule, or to rodents through a stomach tube; thesemethods place the entire dose directly into the stom-ach. In longer-term studies, the pesticide usually isincorporated into the animals’ feed or water, allowingtheir access to small amounts each time they eat ordrink.

Oral Administration

A syringe with a tube is used to insert a pesticide into the stomach of a rat.

The animals’ fur is clipped prior to placing a pesticidedose directly on the skin. Solid materials are crushed andmixed with a liquid to form a paste, slurry, or solution,then applied to the skin. The site of application is ban-daged to keep the animals from licking the treated areaand ingesting the chemical. Another method employed todeter licking is the placement of a large “collar” aroundthe neck to restrict the animals’ access to the applicationsite.

Dermal Administration

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Animals are confined in air-tight chambers intowhich pesticide vapor, aerosol mists, or dusts areintroduced. It is critical that the test substance beuniformly distributed throughout the chamber for thetime period during which animals are obliged to breathethe treated air. The pesticide concentration and particlesize in the air is monitored regularly. If the particles aretoo large, they are ground to assure accessibility to thelungs. Placing animals in chambers exposes not onlythe respiratory system, but all external body surfaces,as well. Alternative testing systems are available forlimiting exposure to the animals’ nose or face.

Most tests require four groups of animals, eachreceiving a different dose level of the pesticide:

• none (control group—no pesticide whatsoever)• low (an amount of pesticide estimated to produce

no toxic response)• medium (enough pesticide to evoke a moderate

response, between those at low and high doses)• high (in acute studies, enough pesticide to cause

death; in chronic studies, enough to produce significantsigns of toxicity, but not death)

Inhalation Administration

Establishing Dose Levels

An essential part of any toxicological program is theresponse comparison between the animals exposed toa pesticide and those not exposed. The animal groupsexposed to a pesticide are known as “treatmentgroups” and those left untreated are referred to as“controls.” Control groups are handled exactly like thetreatment groups, except that they are not administereda pesticide. Toxicologists use controls to demonstratenormal growth and development and to provide valu-able information on the occurrence, type, and fre-quency of background disease in untreated animals.Data from both groups are evaluated to differentiateabnormal from normal response.

The type of control group used in a toxicologicalstudy may be specified by regulatory protocol, or the

Making Comparisons Between Treatedand Untreated Animals

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manufacturer may choose control groups known toensure levels of performance and reliability of thetesting program.

Untreated Control Group

The most common control is the untreated controlgroup, often called the negative group. Control animalsare housed in the same room with those being treated,and they are fed an identical diet—minus the pesticide.

Some pesticides are incorporated into a capsule ordissolved in a solvent such as corn oil prior to adminis-tration to the test animals. Animals assigned to avehicle control group receive the capsule or the solventwithout the pesticide, which helps differentiate effectscaused by the vehicle.

Positive Control Group

Facilities that supply animals to toxicological testinglaboratories typically maintain historical control records,and information from those records is used to assesschanges in diseases, over time. Testing laboratoriesalso maintain historical control group records of animalparameters and disease rates for the testing facility.The large numbers of animals represented in historicalrecords yield better estimates of normal disease ratesthan do the relatively small numbers of control animalsin individual studies.

Historical Control Group

An animal, under some circumstances, can serve asboth a treated and untreated control. Self controlgroups are especially useful in eye and skin irritationstudies; a response to a pesticide in one eye or on onearea of skin on an albino rabbit, for instance, is com-pared to the untreated (control) eye or skin of the sameanimal.

Self Control Group

Vehicle Control Group

The positive control group also is treated with asubstance known to produce a specific effect; thus,treated control groups ensure that the animal testsystem is appropriately sensitive to the end point ofinterest. For instance, tri-ortho-tolylphosphate (TOTP)is a chemical known to produce certain neurotoxicsymptoms; so neurotoxic effects observed in testgroups treated with TOTP demonstrate those whichmight be expected in groups treated with a similarlyneurotoxic pesticide.

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Many observations in toxicological studies are usedto determine if pesticides impact animal health. Ingeneral, effects can be observed as

• changes in appearance and behavior,

• findings from routine physical examinations,

• changes in body weight,

• shifts in food consumption,

• alterations in blood chemistry,

• physical and chemical changes in urine,

• changes in appearance and weight of internalorgans, and

• microscopic changes of internal organs and

Methods Used for MeasuringToxicological Effects

tissues.

Observations of General BehaviorExperimental animals are typically observed twice

daily for mortality and signs of toxicity. The observerneeds extensive, on-the-job training and experience tobecome proficient in recognizing differences betweennormal and abnormal behavior and symptoms.Examples of observable end points are listed on thefollowing pages.

Animals are handled andtheir reactions and reflexesobserved during detailed physi-cal examinations. Each animalalso is examined for unusualgrowths or lumps; abnormalitiesare recorded each time they areobserved.

Routine PhysicalExaminations

Scientist examining a rat, looking for abnormalitiesand preparing to enter the information into thecomputer.

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Excreta• unusual urine color• blood-like urine color• decreased urination• excessive urination• unusual fecal color• blood-like fecal color• black stool• soft stool• diarrhea• decreased defecation• hard stool• bright yellow urine

Feet and Limbs• focal loss of hair• swelling• abrasions• sores on feet• torn toenails• loss of limb/paw/toes• cysts

General Appearance• emaciation• dehydration• distended abdomen• self-mutilation• intra-abdominal swellings• protrusion of tissue from rectum

Genitalia• urogenital discharge• vaginal discharge• red discharge• yellow/brown discharge• clear discharge• blood-like discharge• swelling• protrusion of tissue• greenish discharge• abnormal penile erection

Observable End PointsObservable End PointsObservable End PointsObservable End PointsObservable End Points ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

Activity• hyperactivity• hypoactivity• nonresponsiveness• prostrate position• aggressiveness

Ears• discharge• tears/lacerations• pallor• redness• swelling• scabs• encrustation

Eyes• red discharge• blood-like discharge• pus-like discharge• lacrimation (tearing)• wetness around the eyes• excessive blinking• partially/completely closed eyelids• opacity (cloudy eyes)• pupil contraction• pupil dilation (expansion)• pallor• yellow/brown conjunctival discoloration• diffuse conjunctival redness• dilation of conjunctival blood vessels• pitted/raised corneal surface• protrusion• necrosis/rupture of globe• loss of eye• periorbital encrustation

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Mouth• emesis (vomiting)• salivation• wetness around the mouth• swollen mouth• bleeding gums• ulceration of lips• ulceration of oral mucosa• blood-like discharge• broken teeth• missing teeth• mismatched teeth• overgrown teeth• pale mucosa

Movement/Posture• head tilt• ataxia (incoordination)• convulsions• twitching• tremors• misuse of limbs• arching of back• circling movements• somersaulting• lateral rolling movements• side-to-side head motion• up-and-down head motion• backward walking

Nose• red/pink discharge• clear discharge• yellow/brown discharge• blood-like discharge• frothy discharge• perinasal encrustation

Respiration• labored breathing• gasping• rapid breathing• slow breathing• shallow breathing• sneezing• high-pitched sounds• rattling sounds• intermittent apnea (stopped breathing)

Skin• hair loss• abrasions• scabs• scars• swelling• flaking/peeling• sloughing• necrosis• ulceration• discoloration• pallor• blood-stained appearance• red/brown encrustation• urine-stained hair• feces-stained hair• anogenital staining• mammary gland staining• mammary gland secretion• cysts

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

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Body weight is an important end point in assessingthe toxicological effects of a pesticide. Animals in short-term studies are weighed at least weekly. Those inlonger studies are weighed weekly for 12 weeks andmonthly, thereafter, as the rate of weight change slows.Decreased body weight may correlate to toxicity, in thatan animal that does not feel well likely will not eat well;another correlation might be that the toxin inhibits theanimal’s ability to utilize the food consumed.

Changes in Body Weight

A measured amount of food is offered to eachanimal. At specified intervals, uneaten food is weighed.The difference between the amount offered and theamount uneaten represents food consumption for thattime interval. Comparisons of what is consumed bycontrols and by treated animals reveal early indicationsof toxicity. Water consumption also may be recorded.

Shifts in Food Consumption

Hematology is the study of cellular components in whole blood. Small amountsof blood are taken from experimental animals for hematological studies. The follow-ing cellular constituents are recognized as key measurements in describing toxicity:hemoglobin concentration and platelet, erythrocyte (red cell) , and leukocyte (whitecell) counts.

Changes in Hematology

Red blood cells—large cell, no nucleus; white blood cells—large

cell, blue nucleus; platelets—small cells.

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When cells are removed from blood, what remainsis the liquid portion known as plasma. Blood chemistrymeasurements are important because cells, tissues,and organs are in close contact with circulating blood.Damaged organs often release enzymes and othersubstances into the bloodstream, which are recogniz-able as indicators of toxicity. The measurement ofenzymes, electrolytes, and biochemical changes inplasma facilitates indirect detection of organ damage.

Alterations in Blood Chemistry

Acid/base balance

Alanineaminotransferase

Albumen

Alkaline phosphatase

Aspartateaminotransferase

Bilirubin

Blood creatinine

Calcium

Chloride

Cholesterol

Cholinesterase activity

Glucose (blood sugar)

Hormones

Lipids

Methemoglobin

Phosphorus

Potassium

Protein

Sodium

Total bilirubin

Urea nitrogen

The following chemicals are typically measured inblood:

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Alterations in Urine

Measurements of hematology, blood chemistry, andurinalysis are similar to analyses performed duringhuman medical examinations.

Appearance

Bilirubin

Cast kidney cells

Glucose

Ketones

Occult blood

pH

Protein

Specific gravity

Urobilinogen

Volume

White blood cells

The following parameters in urine provide clues totoxic effects from pesticides:

Autopsies are performed on animals that have diedor been put to sleep during or at the conclusion ofstudies. Internal body cavities are examined. Thetestes, thyroid, thymus, spleen, heart, lungs, ovaries,brain, liver, and kidneys are individually weighed andexamined for changes in size and color, and for le-sions. Organs are observed to verify presence andproper location within the body cavity. Once all bodytissues have been evaluated and removed, the focusturns to muscles and bones.

Gross Observations of Internal Tissues

As many as fifty tissues from each animal areexamined. Each is sliced into thin sections—a processwhich can take as long as six months—for microscopicobservation to detect the presence of lesions andabnormal growth. Pathologists look at each tissue fromeach animal and examine cell structure, organization,size, and shape; some chronic studies require exami-nation of as many as 20,000 tissue slices. Organs andtissues are subject to routine microscopic evaluation;there is no limitation on the types or numbers of tissuesexamined.

Adrenals

Aorta

Bone marrow

Brain

Caecum

Colon

Duodenum

Esophagus

Eye

Femur (bone)

Gall bladder

Gross lesions

Heart

Ileum

Jejunum

Kidneys

Liver

Lungs

Lymph nodes

Mammary glands

Muscle

Nose

Ovaries

Pancreas

Peripheral nerve

Pituitary

Rectum

Salivary glands

Skin

Spinal cord

Spleen

Sternum

Stomach

Testes

Thymus

Thyroid

Tongue

Trachea

Urinary bladder

Uterus

Microscopic Evaluation of Tissues

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