Lecture 6: Fundamental Principles of Toxicity ... · 50% LD50, ED50, LC50, EC50 Slope NOAEL Figure 3. Sigmoidal distribution of proportion of population responding relative to dose

ES/RP 532 Applied Environmental Toxicology Page 1 of 25

ESRP532 Lecture 6.doc Fall 2004

September 20, 2004

Lecture 6: Fundamental Principles of Toxicity, Toxicokinetics, Risk Assessment,

I. IntroductionA. Toxicity is the inherent capacity of a molecule to cause harm owing to its three-

dimensional structure and the interaction with an appropriate biochemical receptor.1. However, the capacity to cause a response and the actualization of this response may

not be coincident with simple exposure or mere presence of a toxicant.a. For example, mode of action (or the mechanism by which a toxin interacts with a

receptor molecule) does not explain why variation in response exists within apopulation of a single species where all individuals are exposed to the same dose.1. Note that the amount of toxicant can be expressed as dose (absolute mass),

dosage (mass per unit weight), or concentration (mass per unit volume ofmatrix, whether soil, water, or plant).

b. Mode of action may or may not explain why different species may not respondsimilarly to a toxicant.

c. Mode of action does not fully explain why thresholds exist for physiologicalreactions.1. In other words, response tends to increase as dose increases.

d. Mode of action does not explain why degree of toxicity differs for various routesof administration (e.g., whether exposure is via the epidermis [dermal], ingestion[oral or gastrointestinal] the vascular system [intravenous], or the ventilatorysystem (inhalation; lungs).

2. The reason for the incongruity between mode of action (i.e., the ability to react with abiochemical target site) and the likelihood of a physiological response is relatedgenerally to aspects of exposure and dose; i.e., how much is an organism exposed to,how much is absorbed, and how much actually interacts with the target site.a. Thus, if an insect or plant is exposed to a pesticide residing in soil at

concentration of 10 micrograms chemical per gram of soil (10 µg/g = 10 ppm),how much of this chemical actually gets inside of the organism? Is that amountenough to cause harm (by virtue of actual interactions with target sites)?

b. Alternatively, if a child ingests some 2,4-D (a herbicide) straight from the bottle,what is the likelihood that toxicity will result?1. If the ingestion was through residues contained on food, is toxicity likely?

3. More specifically, the generation of in vivo toxic effects requiresa. Absorption from the site of dose administration;

1. In animals, such absorption would include potential to cross the dermis or theintestines. For plants exposed to a chemical via the foliage, the chemical mustdiffuse through the cuticular waxes (thus sharing something in common withArthropods) and through the cutin layer.

2. For exposure of plants to soil-borne chemicals, absorption would beinfluenced by the ability of a chemical to enter the root and cross theendodermis into the vascular bundles.



a. Note, however, that with plants, interaction of the chemical with soil canaffect bioavailability, and thus adds a layer of complexity in predictingtoxicity.1. Similarly, soil dwelling invertebrates may be crawling with highly

contaminated soil, but bioavailability of contaminant residues isaffected by the degree of sorption.

b. Delivery to the target;1. Movement and distribution through the circulatory or vascular system.2. Influenced by—

a. Duration of exposureb. Magnitude of exposurec. Potential for accumulation of chemical at the target tissue

c. Initiation of cellular events at the target organ or tissue; i.e., the mode ormechanism of toxic action.

B. To answer the question of what is the likelihood of toxicity from exposure to a pesticide,and the related question of why do individuals and species differ in their response to agiven amount of toxicant, one relies on two basic types of studies:1. Measurements of toxicity (bioassays)

a. Characterization of dose-response relationship2. Studies of toxicokinetics and toxicodynamics (Figure 1)

a. Toxicokinetics: movement and fate of toxicant in the organism (from initialcontact to final elimination)

b. Toxicodynamics: the actions of a chemical within the target organ.c. Note that the terms toxicokinetics and toxicodynamics are applicable to all

substances, but for pharmaceutical agents, these processes are known aspharmacokinetics and pharmacodynamics. Of course, the physiologicalendpoints of drugs are preferably therapeutic responses, and those ofcontaminants, including pesticides, are toxicity.

Figure 1. Conceptualization of roles for toxicokinetics & toxicodynamics (copiedfrom Heinrich-Hirsch, B. et al. 2001. The use of toxicodynamics in risk assessment.Toxicology Letters 120:131-141).



II. Measuring ToxicityA. Toxicity is measured by determining the relationship between dose or concentration of a

substance and the response of the test organism.1. The response can range from death to subtle changes in enzyme activity or everything

in between.2. The most common parameter to express toxicity is the dose or concentration causing

50% of tested organisms to respond.a. The median population response is expressed as the LD50 or LC50 (if lethality is

the endpoint) or the ED50 or EC50 (if other types of responses are the endpoints).3. In addition to knowing the LD50 or ED50, and more importantly for purposes of

determining “safety”, we also want to know the dose or concentration causing noresponse, known as the NOEL or NOAEL (No Observable Adverse Effect Level) orNOAEC (No Observable Adverse Effect Concentration).

B. How an LD50/LC50 and NOAEL/NOAEC are determined1. Hypothetical response of a population to a stimulus (adverse or favorable) can be

described as a normal distribution (“bell-shaped” curve) if we graph the numbersresponding at each dose or concentration tested (Figure 2).

Figure 2. Normal distribution of responses to increasing dose of toxicant.

a. To generate the required data, organisms are exposed via the diet, skin, orenvironment (i.e., air, water [aquatic organisms], soil [worms, bacteria] to a seriesof increasingly higher doses, starting with zero concentration as a control. Theorganisms are randomly assigned to experimental groups, and each group receivesone dose.

b. The response, i.e., the endpoint, must be strictly specified; the magnitude of thisendpoint is then recorded at each dose; many times we are interested in outrightdeath, but other effects, such as decreased weight or enzyme activity are equallyvalid, just as long as they are specified and measurements can be validated.1. Concerning endpoints, we distinguish between acute toxicity, which is usually

an immediate response to the short term or single dosing of an organism, andchronic toxicity, which is a systemic effect developing over a period of timebeyond the actual dosing;a. We normally think of chronic toxicity as resulting from repeated non-

acutely lethal (i.e., from sublethal) dosing.



2. The cumulative proportion responding (which can be expressed as a percent) toincreasing doses can be depicted as a sigmoidal function; note that the tangent to thefunction would be the slope (Figure 3).a. The slope of the response would indicate the variability in response within the test

population.b. The LD50, ED50, LC50, EC50 represent the dose or concentration corresponding

to the median (or 50%) population response.Cu

mula

tive

Prop

ortio

n Re

spon

ding

Dose, Dosage, Concentration

50% LD50, ED50,LC50, EC50

Slope

NOAEL

Figure 3. Sigmoidal distribution of proportion of population responding relative to dose.

3. Bliss (1935) linearized the sigmoidal function using probability units (i.e., probits, which turnout to be unit standard deviations above and below 50% mortality, or probit 5.0) plottedagainst the logarithmic dose [Bliss, C. I. 1935. The calculation of the dosage-mortality curve. Annals ofApplied Biology 22:134-167]. (Fig. 4).

Figure 4. Transformation of sigmoidal function to probit function

a. The LD50/LC50 is the region having the narrowest confidence intervals, and thusthe most reliable indication of response at a particular dose.



Figure 4a. Transformation of sigmoidal dose-response curve to a linear functionusing probits. FI signifies the fiducial limits, which are similar to aconfidence interval.

4. For any single compound, the slope of the dose-response line helps determine themargin of safety.a. Shallow slope allows greater margin of safety; in other words, comparatively

larger changes in dose result in small changes in response (Figure 5A,B).b. The slope also tells something about the variability in the population (Figure 5A,

B);1. A steep slope indicates little variation in the population response;2. A comparatively shallower slope indicates that the response is much more

variable over a greater dose range.



Figure 5. Relationship between slope and variability (distribution) of response of one or morepopulations to a single chemical, or response of a single population to two differentchemicals; or response of two different species to a chemical.

5. Two different species might respond to a chemical with the same LD50/LC50, but thevariation in susceptibility may differ substantially (Figure 5A,B). Alternatively, theLD50’s may be substantially different, in addition to the variability being different(Figure 5C,D).

6. Note that the slope can also be used to assess the occurrence of resistance in apopulation. Populations naïve to a toxicant are fairly homogeneous in response. Asa toxicant selects for resistant individuals, the variability in response increases(distribution flattens out), and as selection continues, most individuals will eventuallybecome resistant, establishing a new, homogenous distribution but exhibiting asubstantially higher LD50 (Figure 6).

Figure 6. Change in susceptibility after repeated selection for resistant individuals.

7. Bear in mind, however, that by itself, an LD50 is somewhat meaningless; it is usefulonly in the context of comparisons with other chemicals, or alternatively forcomparing the response of two or more different species to a single chemical, orcomparing the responses of two different populations of a single species.a. Nevertheless, a comparison of oral and dermal toxicity can be used to assess the

potential hazard of a compound, especially in occupational settings. (Note thatthe comparison is made for rats).1. Oral and dermal LD50’s close in value and below 50 mg/kg signify a

compound of very high hazard (Figure 7, phosdrin).



DDT phosdrin 2,4-D thifensulfuron1

10

100

1000

10000

LD50

Comparison of Toxicity to Mammals

oral

dermal

Figure 7. Comparative oral and dermal toxicity to rats.

b. Note how one chemical that is toxic to birds may be of low toxicity to fish.(Figure 8 & 9)

DDT diazinon permethrin thifensulfuron1

10

100

1000

10000

100000

LD50

Comparison of Toxicity to Birds

Figure 8. Toxicity of four pesticides to pheasants.

DDT diazinon permethrin(Pounce)

thifensulfuron(Harmony)

0.001

0.01

0.1

1

10

100

LC50 5 0

Comparison of Toxicity to Fish

Figure 9. Toxicity of four pesticides to trout.



C. Toxicokinetics in combination with mode of action studies can explain why fish andbirds respond differently to the same pesticide, and why oral routes of exposure are morehazardous than dermal routes.

III. ToxicokineticsA. Keep in mind that simple exposure of a plant or animal (or any organism) does not

necessarily result in a measurable biological effect owing to the dynamics of the chemicalin the organism. The fate of a chemical within an organism and its interaction with thetarget receptors, tissues, or organs is described by toxicokinetics (or pharmacokinetics ifdrugs are the subject) and toxicodynamics (pharmacodynamics).1. The basic processes of toxicokinetics are absorption, distribution, and elimination.

a. The fate of the chemical can be characterized by the extent and rate of each ofthese basic processes.

B. Absorption (or Penetration)1. Penetration refers to a contaminant crossing the outermost barrier of an organism;

the chemical is transferred from the site of contact into the general circulation.a. The skin of mammals, birds, reptile, etc.;b. The exoskeleton of invertebrates;c. The cell wall of microorganisms;d. The cuticle of plants.

2. For an animal, exposure may be through dermal contact, food, or air; regardless, thechemical must penetrate across an epidermal cell layer, enter the circulatory system,and then be carried to the diversity of tissues.a. The first hurdle following exposure would be absorption by cells.b. The process is essentially controlled by thermodynamic considerations, although

we also measure the kinetics of uptake.1. Consider that cell membranes have been theorized to be lipid bilayers with the

hydrophilic ends of the lipid oriented toward the outside and the hydrophobicends forming the middle of the membrane (Figure 10).

2. The membrane also has proteins extended throughout the bilayer in variousregions; these areas can be described as “aqueous” pores or channels throughwhich ions and water soluble (polar) chemicals cross.

Figure 10. Lipid bilayer nature of cell membrane with channels created by intrusions ofproteins.



c. Diffusion processes are the main mechanisms of entry of most compounds;studies show a positive correlation between hydrophobicity (as represented by themagnitude of the Kow or the ratio of substance in octanol relative to water atequilibrium) and penetration.1. In some cases “carrier” proteins can bind the substrate and move it from one

side of the membrane to the other2. Compounds that are ionized cross membranes very slowly because they

cannot diffuse readily through the lipid bilayers. Thus, pKa of the compound(the pH at which 50% of the compound is ionized and 50% unionized) and pHof the tissue matrix it is crossing influences rate and extent of absorption.

d. In sum, hydrophobicity, pKa, and molar volume (recall its relationship amongphysicochemical properties) control the rate of penetration across membranes.

e. Bioavailability is the fraction of the contacted dose that is transferred from thesite of contact (or administration) into the general circulation (or tissues).1. Recall that bioavailability has an analogous significance when considering

sorbed contaminants and exposure of soil dwelling organisms; only thedesorbed chemical (i.e., the solution phase) is bioavailable, but in the contextof toxicodynamics, not all of the chemical contacting an organism’s externalsurface will penetrate.

3. Some studies have shown that proteins in the blood of vertebrates can act as carriersof certain contaminants; the binding will be dependent on the Km (affinity constant),but nevertheless these interactions facilitate transport;a. On the other hand, protein binding can reduce the amount of chemical that would

interact with a receptor molecule, thereby altering the “toxic” effect.4. Because Arthropods and some other invertebrates (think of them not only as pests but

as fish and bird food!) are either directly and intentionally exposed to insecticides orunintentionally through contamination of water supplies, it is worth considering thepenetration aspects through the cuticle (example for insects follows).a. The cuticle of insects consists of a waxy outer layer (epicuticle, consists mainly of

long-chain hydrophobic hydrocarbons) lying above a proteinaceous-glycoproteininner matrix (exocuticle and endocuticle, cross-linked proteins with chitin, longchain polymers of acetylglucosamine); the exocuticle and endocuticle is polarrelative to the epicuticle. The exocuticle and endocuticle have wax “canals”running through them to the epicuticle; also the tracheae, “breathing tubes’ runthrough them to the surface (the tracheal system is responsible for carryingoxygen and carbon dioxide)

b. An early study showed that the rate of penetration of insecticides into a cockroachwas inversely related to the partition coefficient (olive oil:water); thus, the half-time of penetration for DDT was 1584 minutes and paraoxon (the toxic oxidativemetabolite of parathion was 55 minutes); (Olson and O’Brien, 1963, J. InsectPhysiology 9:777-7786)1. Adsorption to the exocuticle might be directly related to Kow, but penetration

involves a different mechanism where the pesticide must diffuse across layersthat are progressively more polar;



2. Thus, while the insecticide is held up in the cuticular layer due to hydrophobicinteractions, it slowly diffuses into the insect, but the diffusion is controlled byits polarity. Penetration of the polar barrier is the rate-limiting factor. Thosecompounds that have an intermediate hydrophobicity would penetrate thefastest. Parathion, which has a nitro group and is a phosphate ester, is morepolar compared to DDT.a. But the rate of penetration is only one factor that would affect toxicity;

other factors include the rate of metabolism and strength of interaction ofchemical with target receptors (i.e., biomolecules).

C. Distribution1. The process of reversible transfer of a chemical from general circulation (in the case

of animals) into body tissues;2. For a plant, distribution could be thought of as the reversible movement of a chemical

from the xylem and phloem into the foliar cells (or fruit).3. Distribution is usually very rapid, but can be slow under two circumstances:

a. The chemical may have a high affinity for accumulation in a tissue or organwhich is only slowly perfused (i.e., blood circulates slowly through the tissue;e.g., fat or muscle); rate of distribution is thus limited by rate of blood flow;

b. The chemical may be polar, so that its rate of entry into the intracellular fluid ofall the tissues will be limited by its solubility in the lipid of the membrane (i.e.,same problem as encountered in penetration or absorption).

4. Extent of distribution is influenced by:a. A compound’s water solubility;

1. The greater the water solubility, the greater the tendency to partition intoplasma or interstitial and intercellular fluids with limited uptake by fat tissueor the central nervous system.

b. Lipid solubility;1. The greater the lipid solubility (hydrophobicity) the greater the tendency to

concentrate in adipose tissue, central nervous system, or other tissues withhigh lipid content; concentrate in cytoplasmic membranes and endoplasmicreticulum of all cells.

c. Plasma protein binding;1. Causes reduction in tissue distribution and retains compounds longer in

circulation.d. Tissue protein binding;

1. Chemicals with high affinity for tissue protein binding will show acomparatively more extensive distribution.

D. Elimination--Metabolism1. In any one tissue, interaction with enzymes will result in either detoxification

(inactivation) or activation of a chemicala. The reactions involved can be phase I (oxidation, hydrolysis, reduction) or phase

II (conjugations)1. Oxidation of many contaminants occurs by the microsomal oxidase system,

which utilizes the cytochrome known as P450,



a. In vertebrates, the P450 system is located on the rough endoplasmicreticulum; the liver is especially rich in this enzyme system, although itexists in essentially all cells.

b. Oxidations usually results in detoxications, but sometimes the contaminantis “activated” (its toxicity is equal or greater than the parent form becauseit can react with the biochemical receptor more easily);

2. Esterases, which cause hydrolytic type reactions, usually result indetoxifications in vertebrates.a. Figure 11 illustrates the metabolism of an organophosphorus insecticide

by an esterase to inactive products and oxidation to a toxic product.

N

ClClHO

ClC2H5O

C2H5O

S

P- OH

N

ClCl

ClC2H5O

C2H5OP-O

S

N

ClCl

ClC2H5O

C2H5OP-OO

+ +C2H5O

C2H5O

O

P- OHN

ClClHO

Cl

trichloropyridinoldi-ethoxythiophosphate di-ethoxyphosphate trichloropyridinol

chlorpyrifos chlorpyrifos oxon

Figure 11. Metabolism of chlorpyrifos by two pathways, oxidation (leading to activation orincreased toxicity illustrated by chlorpyrifos oxon) and hydrolysis (leading todetoxification, illustrated by trichloropyridinol).

b. Certain herbicide esters are hydrolyzed in plants and soil to the activetoxin. For example, there are many different phenoxy acetic acid esters,but all of them are hydrolyzed in plants and in soil to the phenoxy aceticacid form that is the actual indole acetic acid agonist (Figure 12).

OCH2COHO

Cl

Cl

2,4-D

Cl

Cl

OCH2CO (CH2)nCH3

O

2,4-D ester

Figure 12. Hydrolysis of a 2,4-D ester to 2,4-D (the active toxicant).



b. Enzymatic reductions relevant to toxicokinetics are less common than eitheroxidations or hydrolyses.

c. Phase II reactions involve conjugation of either parent compound or a metabolitewith either glucose (monosaccharide carbohydrate) or glutathione (three aminoacid peptide).1. The group of enzymes catalyzing conjugation with glutathione are called

glutathione transferases. The enzyme is either membrane bound or in thecytoplasm. (Armstrong, R. N. Structure, catalytic mechanism, and evolutionof the glutathione transferases. Chem. Res. Toxicol., 10 (1), 2 -18, 1997)

2. Conjugated contaminants and their transformation products are much morewater soluble than the parent compound and can therefore be more easilyexcreted in animals.

2. When dealing with chemical technology that is used to control pests (for ex.pesticides or disinfectant-containing cleaning solutions), selectivity can provide alarge margin of safety. Selectivity can be considered the differential toxicity of acompound between a pest organism and nontarget organisms. Pests (whether they arebacteria or pathogen or insects or weeds) and nontarget organisms may share similarbiochemical target sites. Metabolism of pesticides is one of the most importantfactors in the basis of selectivity between pests and nontarget organisms (other factorsinclude penetration potential and target site insensitivity).a. A good example of selectivity based on metabolism (i.e., metabolic rate) in

animals is the pyrethroid class of insecticides. Although these compounds havemoderate to very low toxicities in birds and mammals, they are highly toxic tofish and insects. Birds and mammals have very active serum hydrolase (esterase-type enzymes) that hydrolyze pyrethroids very quickly, thus allowing lesschemical to reach the target site (Figure 13). However, in insects, especially,oxidations are more important than esterases in metabolism of pyrethroids, butthese reactions are seemingly much slower.1. Note that the mode of action of pyrethroids is the same regardless of species;

i.e., interaction with nerve membrane proteins responsible for the sodium gaterelated to nerve impulse conduction.

permethrin

OOC

O

Cl

Cl

PBAO

HO

O

+1R trans DCCA 1R cis DCCA

CO

OH

HH

Cl

ClCl

ClH

H CO

OH

Figure 13. Hydrolysis of a pyrethroid to its component dichlorovinyl cyclopropane carboxylicacid and phenoxybenzyl alcohol moieties. Note that metabolism of thephenoxybenzyl alcohol proceeds through oxidation to the corresponding acid.



b. Rate of metabolism can account for large differences in sensitivity betweenspecies (or even within a species); i.e., metabolic differences (rates or qualitativedifferences) can confer selectivity.1. Note that the toxicity of several OPs (organophosphate insecticides) in mice,

cockroaches and flies differs as a result of different levels of the oxonmetabolite and levels of organosoluble metabolites (which could still be toxicproducts as opposed to water soluble metabolites which are likely not toxic)(Krueger et al. 1960 JEE 53:25). In all cases, the mouse is less susceptible tothe insecticides than the housefly or cockroach.

2. In Table 1, the oxidation and further metabolism of three OP insecticides werecompared between houseflies, cockroaches, and mice (i.e., parathion,diazinon, and dimethoate. To manifest toxicity, all three compounds must beoxidized to the oxon form (see Figure 11 for an example of the P=S [thion]and P=O [oxon] form of an OP insecticide).

3. Note the LD50’s were much higher for mice than the insects.a. Correspondingly, there was either more oxon formed and/or more

chloroform (CHCl3) soluble metabolites (which could still represent toxicmetabolites) in the insects than in the mouse (examine the ratios of insectto vertebrate or vice versa).

Table 1. Insect/mammalian ratios of OP oxons, parent OP in chloroform extracts, and LD50s forseveral OP insecticides.Parameters Ratios Parathion Ratios Diazinon Ratios DimethoateP=O house fly/mouse 4.4 -- --CHCl3 extract. Fly/mouse 1.6 1.1 7.7LD50 mouse/fly 6.0 37 325

P=O cockroach/mouse 3.0 11.5 2.3CHCl3 extract roach/mouse 1.5 1.2 11.0LD50 mouse/roach 6.0 20 70

E. Elimination--Excretion1. Metabolic reactions, whether inactivation or activation, ultimately result in a more

water soluble compound that can be more easily excreted.a. In vertebrates, hydrophobic compounds are not readily filtered from the blood by

the kidneys.2. Excretion from the body is the second type of elimination process; either elimination

mechanism, i.e., metabolism or excretion, reduces the amount of parent compoundavailable to the target sites;a. Another concept used by pharmacologists to help describe the amount of

chemical available at the target site and related to the concept of elimination isclearance;1. Clearance refers to the processes that remove a chemical from circulation.2. Defined as the volume of blood (or plasma) cleared of chemical per unit time.

3. Routes of elimination/excretion (vertebrates)



a. Expired airb. Salivac. Biled. Fecese. Urinef. Milkg. Hair

4. Rate of excretion can also be important in insects;a. In one study (Hsin and Coats 1986, Pestic. Biochem.. Physiol. 25:336), rate of

absorption of isofenphos (an OP insecticide) was about three times faster in larvaethan in adults, yet adults were two-fold more susceptible than larvae (Table 2).

b. The difference in toxicity could be explained by both a more rapid metabolism inthe larvae, and a more rapid rate of elimination of the parent isofenphos.1. In Table 1, the external rinse represents the chemical not penetrating the insect

cuticle at the specified time interval of sampling (note that exposure was viadirect application of an LD10 equivalent dose as a droplet on the cuticle)a. The “internal organic extract” represents the whole body extracted with

organic solvent after the external rinse;b. The “container rinse” represents metabolic products present in the excreta

of the insects;c. “Container rinse isofenphos” represents the parent insecticide excreted

Table 2. Toxicokinetics of isophenfos (an OP insecticide) in corn rootworms.% Applied 14C Dose Recovered After Indicated Time (h)

Stage 1 2 4 8 24AdultExternal rinse 16.6 11.3 7.8 4.5 1.1Internal organic extract 71.3 65.4 53.7 44.0 15.8Container rinse metabolites 3.6 3.5 10.2 17.9 35.1Container rinseisofenphos

3.0 2.3 3.3 2.1 0.8

LarvaeExternal rinse 6.9 2.3 1.3 0.4 0.2Internal organic extract 59.4 45.9 37.6 21.4 9.5Container rinse metabolites 9.1 13.3 19.1 32.6 43Container rinseisofenphos

8.6 11.1 13.5 21.0 23.0

5. Note that the extent of excretion (elimination) is less important than the rate. Onehundred percent of a toxicant will eventually be eliminated, but the rate will vary as aresult of absorption, clearance, and metabolism processes.

F. Storage1. The degree of storage is also influenced by the metabolism rate and lipophilicity of

the compound. Obviously, more lipophilic compounds (i.e., hydrophobiccompounds) are not going to be excreted easily until made more water soluble (i.e.,metabolized to more water soluble compounds); if a chemical’s metabolic kinetics are



rather slow (for ex. DDT, dieldrin, PCBs) than there is ample opportunity for storagein adipose tissue

2. A chemical will be released from the storage sites into circulation; indeed there is anequilibrium between the adipose tissue and the blood;a. In the case of DDT and metabolites, DDT in the blood stream probably indicates

a recent exposure, whereas DDE indicates exposure from the past because DDE isthe main storage metabolite.

3. In vertebrates and invertebrates, the bioconcentration factor has been used as anindicator of the potential storage in lipid (adipose) tissue

G. Interaction with Target Site (Toxicodynamics)1. The end result of metabolism is to lower the concentration of the parent chemical on a

whole body basis and ultimately the amount reaching a receptive target site (i.e.,receptor) where mode of action is expresseda. The possibility of interaction will be determined by the kinetics of metabolism,

rate of circulation, as well as the Km (affinity constant) for the target siteb. At the target site, further metabolism of a toxicant may occur, further reducing its

effective concentration.

IV. Case Study—What Do We Know About Human Absorption of Pesticides?A. As a result of human volunteer studies in the past, we actually know quite a bit about

some of the toxicokinetic parameters of pesticides in humans. However, newerchemicals are not being tested in humans (other than worker exposure studies, which donot measure toxicokinetic parameters).

B. One older study with parathion indicates that different areas of the body have differentpotential for absorption of pesticides (Maibach 1971, Arch. Environ. Health 28:203-210)(Figure 20).

Figure 20. Percent of applied dose absorbed through skin specimens taken fromdifferent parts of the body.

C. Studies with human volunteers generally show low absorption potential for OPinsecticides and several herbicides through forearm skin (Figure 21).



1. Carbaryl is a major exception with nearly 80% of the applied dose absorbed throughthe skin.

Figure 21. Percent of applied dose penetrating through human forearm.

D. Studies with human volunteers also show that many herbicides have a low potential forabsorption by human skin. The doses that are absorbed, however, are rapidly eliminated(through excretion), and little metabolism (if any) occurs (Table 3).

Table 3. Absorption, elimination, and metabolism parameters for some vegetation managementherbicides.

Toxicokinetic Parameter clopyralid 2,4-D picloramDermal Absorption Efficiency-Humans (%) no data 6 0.2Half-life for Elimination from Body (h) 3 28 6Metabolites of toxicological importance none none none

E. Studies with rats show that pesticides are efficiently absorbed through the intestine (incontrast to the lower efficiency from the skin) (Figure 22).

Figure 22. Absorption of pesticides (in one hour) by rat gastrointestinal tract



V. Case Study—Influence of Exposure Route on Toxicokinetic Parameters (example fromNolan et al., 1984, Toxicol. Appl. Pharmacol. 73:8-15)A. Human volunteers were dosed orally and dermally with chlorpyrifos.

1. Blood and urine samples taken periodically.2. Trichloropyridinol is the major metabolite of chlorpyrifos and is excreted in the urine.

Thus, its concentration in the urine, along with the volume of urine, can be used toback calculate the amount of absorption and excretion (resulting parameters shown inTable 3).

B. Toxicological endpoints, butyrylcholinesterase (also known as plasma or pseudocholinesterase) and acetylcholinesterase (erythrocyte and central/peripheral nervoussystem are identical enzymes) were measured to determine the relationship betweentoxicokinetics and relevant toxicological endpoints (Table 3).

C. Results1. Why are there differences in plasma cholinesterase inhibition even though oral dose

was ten times less than the dermal dose?2. What conclusions can you make about the extent of chlorpyrifos absorption by human

skin?

Table 4. Toxicokinetic parameters for chlorpyrifos in human volunteers dosed orally anddermally.

Toxicokinetic Parameter Oral Dose (0.5 mg/kg) Dermal Dose (5 mg/kg)Absorption Half-Life (hr) 0.5 22.5Elimination Half-Life (hr) 26.9 Not DeterminedPlasma Distribution Time(µg/mL/hr)

46 6.2

% of Dose Recovered in Urine 70 1.3Plasma Cholinesterase (% ofPredose)

15 70

Erythrocyte Cholinesterase (% ofPredose)

70 80

Signs/Symptoms of Toxicity No NoNote: The half-life refers to a parameter derived from the assumption of first-order kinetics,where the change in concentration of substrate over time (reaction rate) is proportional to theconcentration. The proportional loss of chemical is constant; thus the half-life represents thetime it takes for each successive 50% proportion of chemical to decline from thecompartment in which it is measured.

VI. Plant-Contaminant InteractionsA. All the interactions (processes, mechanisms, etc.) of contaminants with microorganisms

and animals necessarily is applicable to plants;1. However, processes for uptake are somewhat unique in that plants will absorb

chemical through the roots and translocate it to growing parts; but plants also absorbchemicals from the air.a. As with microbes in soil, the rate-limiting factor will still be sorption in soil (i.e.,

desorption rate into the soil water), which is measured by the organic carbon



normalized soil distribution coefficient (Koc; the ratio of concentration of thechemical in soil relative to the concentration in solution).

b. The ability to absorb the chemical will be related to the Kow of the chemical(ratio of chemical concentration in octanol relative to concentration in water) aswell as the Koc(parameters are covariates); translocation within the plant will alsobe a function of Kow.1. For most contaminants, except weak acids and bases, translocation will be a

one-way street in the xylem (i.e., up but not down).a. Movement in the xylem is related to the transpiration stream.

2. Ionizable compounds, especially acids, can move in the phloem, and areconsidered to have true systemic activity.

2. A relatively new area of research concerns the efficiency by which plants absorb air-borne contaminants, whether from the vapor phase or from direct impaction.a. The unique waxy cuticle allows adsorption of hydrophobic compoundsb. These contaminants may not be able to penetrate the cutin and cross into the

epidermis very easily, but their presence on the leaf surface can still act as asource of contaminants to animals feeding on the plant1. Indeed, dioxins are thought to remain in the human food chain because of

atmospheric deposition on plants, which are then eaten by livestock or aredirectly consumed.a. Another source of dioxin in the diet would be from fish consumption

c. It is possible for vapor phase contaminants to enter the leaf through the stomatadirectly, and then sorb to the inner mesophyll layer of cells

d. How much chemical adsorbs to the leaf and where it resides over time willdepend on the length of time between exposure and measurement;1. For example, an experiment with pine trees showed that diazinon insecticide

residues were associated with the pine needles after the surface was washedand the cuticular wax was removed; note in the graph below that only about15 ng/g (ppb) were found in pines sitting in an arboretum (ambient airexposure). (Aston and Seiber, 1996, J. Agric. Food Chem. 44:2726-2735)

2. In pines that were placed in a peach orchard that was then sprayed, 2 dayslater most of the diazinon was associated with a water wash extract of theleaves (Figure 23).a. This indicated that the contaminant residues were mostly dislodgeable (in

other words they could be washed off with rainfall, or fall off with leafmovement [wind action]).

b. After 22 days, however, the residues remaining were much lower (about500 ng/g) but almost all associated with the needles (minus the wax layer)



Ambient(arboretum)

Orchard 2 days

after spray

Orchard22 days

after spray

0

500

1000

1500

2000

2500

3000

3500

ng/g

Concentration of Diazinon in Pine Needle Compartments

Location of Pine Trees

Needle

Cuticular Wax

Water Wash

Surfactant Wash

Figure 23. Recovery of diazinon from pine needles of trees placed remotely from an orchard orplaced within the orchard. Note that most of the chemical is actually sorbed to thesurface of the pines, but a small amount does penetrate to the interior of the needle.

B. Thus, the interaction of contaminants with plants can be described by the model shownbelow (Figure 24), with thermodynamics controlling the uptake processes (whether fromthe soil or the air).1. VP = saturated vapor pressure (solubility of chemical in air at equilibrium);2. Kow = octanol:water partition coefficient (measure of hydrophobicity);3. Cw = water solubility;4. KH = Henry’s Law Constant; tendency to partition from water into air;5. Koc = soil:water partition (or distribution) coefficient normalized for soil organic

carbon content.



Figure 24. Conceptual model of parameters controlling plant-chemical interactions. This modelis a surrogate for toxicokinetic processes discussed for animals, but it includes rate-limiting environmental parameters.

C. Plant metabolism will result in very similar breakdown products and conjugates as animaland microbial metabolism. However, plants do not excrete the products as animals do;1. The roots do excrete various organic acids that serve as bacterial nutrient sources, but

exudation of contaminants has not been studied;a. However, it is unlikely for high Kow compounds to be translocated in the phloem

stream, which is the only way that leaf absorbed contaminants can make it to theroots.

2. Plants do store transformed contaminants in cell walls and vacuoles; this process isknown as compartmentalization.

VII. Overview of Risk AssessmentA. A necessary first step to determining the likelihood (i.e., risk) that exposure to a

contaminant might cause an adverse effect is to review the process of risk assessment.1. Often terms like hazard, exposure, and risk are misunderstood and occasionally used

interchangeably.a. However, each term has a specific meaning in the risk assessment process, which

is applicable to pesticide regulation and eventually to permitted uses.2. Furthermore, risk assessment and risk management are sometimes confused, perhaps

because risk assessment has elements of both science (in the broadest sense of the



process of hypothesis testing) and management (in the sense of policy, which isinfluenced by politics, economics, and sociological factors).

B. Risk assessment as currently practiced by the EPA in its regulation of toxic substances(and recommended by the National Academy of Sciences) consists of four processes:1. Hazard identification (or assessment and characterization)2. Dose-response assessment3. Exposure characterization4. Risk characterization

C. The first three processes in risk assessment are scientific in nature; i.e., experiments areconducted using sound principles of hypothesis testing to determine (characterize)toxicological hazard, the relationship between dosage and response (adverse effects), anddegree of exposure in the environment. Risk characterization, on the other hand, is partlyscientific and partly management.

D. Hazard Assessment: determination of the range of possible biochemical andphysiological responses to a toxicant. The process necessarily involves administeringlow and high doses to achieve a full range of effects.1. Hazard assessment studies are the most common type of experiments found in the

toxicological literature.a. Most of the studies are motivated by the objective of determining the mechanism

of toxicity.1. Thus, these studies are most often characterized by few doses, sometimes only

one, administered to an animal.2. Often to achieve an easily measured effect, the doses are given by injection,

either intraperitoneally or subcutaneously.3. Other variables in dosing include acute (single dosing), short term (perhaps

during gestation only, or several weeks), subchronic (usually 13 weeks or 90days), and chronic (life-time equivalent; the average lifespan of a rat isconsidered about 2 years).

b. Fewer studies are governed by the objective of discovering a dose causing noeffect.1. Ironically, this “negative” data, although less likely to be published by

members of research-oriented institutions like universities, is the informationmost valuable to regulatory agencies for proper risk assessment.

2. Similarly, few investigators ask the question directly, why did this substancecause no effect at this dose but an effect at a higher dose?a. Toxicokinetic studies are valuable in answering such a question.

2. Information about hazards of substances comes from three basic types ofexperiments:a. In Vitro

1. Tests are conducted on purified or gross tissue homogenates of enzymes andreceptors; cell and tissue cultures; perfused organsa. Dosages are difficult to relate to whole body exposures unless

toxicokinetics are well understood.b. In Vivo

1. Live animals are administered doses by various routes, including dermal,inhalational, oral.



2. Subsequent measurements range from biochemical effects to behavioraleffects.

c. Epidemiological1. Epidemiology, which evolved to study incidence of pathogenic diseases and

causes of the disease, relies on Koch’s postulates.2. Post WWII, the traditional role of epidemiology was stretched to include

chemical substances, but in contrast to positive identification of a diseaseorganism and traceability of exposure levels, chemical epidemiology suffersfrom inaccurate (and imprecise, or even non-existent) exposure information.

3. Hazard assessment studies examine the widest possible range of toxicologicalendpoints, includinga. Death

1. Single dose (acute exposures); estimation of LD50b. Systemic effects

1. Pathology of organs2. Weight loss3. Other gross abnormalities4. Blood chemistry

c. Neurotoxicity1. Functional examination of behavior2. Determination of effects on cholinesterase if evidence of Neurotoxicological

effects3. Pathologic examination of nerve tissues if evidence of neurotoxicity

d. Development toxicity1. Maternal exposure during gestation2. Observations of gross morphological effects (teratology)

e. Reproductive toxicity1. Exposure during gestation followed by continuous exposure through next two

generationsf. Immunotoxicity

1. Levels and ratios of various immune system factors2. Very rare to see a study that does pathogen challenge after toxicant exposure

g. Behavior1. Functional observational battery

a. For ex., how long can a rodent pup hang onto a rod, how long does it taketo right itself, ability to learn a maze, and others.

h. Carcinogenicity1. Life time, daily exposure2. Post-mortem examination of multiple tissues

i. Biochemical effects1. Enzyme or receptor function

j. Toxicokinetics1. Extent and rate of toxicant absorption, distribution, and elimination.

4. Note that the FQPA (Food Quality Protection Act) now requires the EPA to make adetermination of whether a pesticide might affect the endocrine system. The Safe



Drinking Water Act also has the same mandates where applicable to regulateddrinking water contaminants.a. However, at this time there are no agreed upon tests that must be submitted for

risk assessment, but the EPA is developing its recommendations.b. Some toxicologists would make the case that developmental and reproductive

toxicity testing has always served as a surrogate for endocrine system effects.5. The EPA’s requirements for hazard characterization studies that are used in risk

assessment of pesticides are published in U.S. EPA (1998), Health Effects TestGuidelines. OPPTS 870.XXX, EPA 712-C-98-XXX, Washington, DC. Updated andavailable online athttp://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/a. Note that the XXX’s refer to specific numbers assigned to each type of test.

E. Dose-Response Characterization1. A series of increasing doses are given to test populations, most often rodents but also

dogs. Of course, one group of animals is given no toxicant.a. Note that for regulatory toxicology studies, males and females are dosed as

independent groups rather than as a mixed group.2. The objective is to determine the lowest dose causing an adverse effect (LOAEL),

and the NOAEL.a. For regulatory toxicology studies, usually three doses are given for the various

required toxicity tests.b. An examination of many of the studies submitted to the EPA shows that the two

highest doses cause effects, and the third dose usually hits upon the NOAEL.c. The separation between the NOAEL and LOAEL is usually approximately 10-

fold or less.1. Note that the NOAEL that the EPA identifies for its use in risk

characterization is an actually observed dosage, not a statistical estimate.Thus, the true NOAEL is somewhat higher than that eventually used inrisk assessments.

3. From the dose-response studies, EPA determines which of the various toxicologicalendpoints (shown under “hazard characterization”) are the most sensitive for bothacute (single, short term) exposure and chronic (lifetime equivalent) exposure.a. Thus, the most sensitive toxicological endpoint is any toxicological response

occurring at the lowest acute and chronic dose.1. For certain compounds, like herbicides and fungicides, toxicity in acute and

short-term tests is essentially nil. In longer term exposures, especially at thehigh doses, weight loss (defines as significant if 10% or more compared tocontrol groups) is often the only toxicologically significant endpoint.

F. Exposure Characterization1. Estimate or direct measure of how much pesticide a person (or nontarget organism)

contacts;a. Does not take into account toxicokinetics unless extrapolations are being made

from one pathway of exposure to another;



1. For example, if an oral exposure toxicity study is used to determine hazardsand potential dose causing toxicity from dermal exposure, than the oral dosageis multiplied by the absorption efficiency.

2. Implicit, therefore, is the assumption that all of an oral dose is absorbed intothe blood and is distributed to the tissues.

2. The FQPA mandates that exposure of pesticides to consumers be aggregated from thediet (i.e., food), water, and residential (indoor or outdoor) use.

3. Food residues are directly measured. If not, than the EPA assumes the tolerancelevel.

4. Water and residential exposure are often measured.a. For some compounds, like atrazine, water residues are well monitored and there is

a large database.b. For some compounds, like chlorpyrifos, there are studies measuring actual

exposure.G. Risk Characterization

1. Risk characterization is partly scientific and partly management (policy).a. For example, from dose-response assessments, one can observe directly a dosage

that causes no adverse effect in the test animal. This dosage is known as theNOAEL (No Observable Adverse Effects Level).1. A level of exposure either estimated or directly measured in the environment

can be compared to the NOAEL, forming a ratio that describes how muchunder or over the NOAEL of a contaminant or drug a person is being exposedto. This ratio is called the margin of exposure (MOE).

€

Margin of Exposure (MOE) = NOAEL (mg/kg/day)Exposure (mg/kg/day)

2. For exposure to contaminants, the risk or likelihood of an adverse effect in theenvironment is characterized after deciding what an acceptable MOE would be. Forconsumer exposure, the EPA considers MOEs greater than 100 to be of no concern(i.e., the Level of Concern or LOC), meaning a reasonable certainty of no harm.

3. Another way to estimate risk is to compare exposure to the Reference Dose (RfD).The RfD is the NOAEL adjusted by a 100-fold safety factor. Any exposure less than100% of the RfD is below EPA levels of concern (LOC).

€

Reference Dose (RfD) = NOAEL100

€

EPA Risk Estimate = Exposure (mg/kg/day)RfD

x 100 , which must be less

than 100% of RfD.

a. Determining an appropriate safety factor is subjective, i.e., it is management.There are no scientific principles that would dictate the use of one safety factorover another.1. Nevertheless the rationale for using a standard 100-fold safety factor in

translating the NOAEL into the risk parameter known as the Reference Dose(RfD) is a 10-fold factor for translation of data from rodents to humans (in



case humans are more sensitive than rodents) and another 10-fold factor forresponse variability in the human population (in case children and seniors aremore sensitive than middle aged men.)

4. Risk characterization when children are deemed more sensitive at a given dose thanadults;a. Note that the FQPA specifically mandates EPA to make a determination of

whether infants and children are more sensitive to a given dose of a pesticide thanadults.

b. If the findings are affirmative, than EPA uses up to a 10-fold additional safetyfactor to estimate the RfD. At this point, the RfD is transformed into thePopulation Adjusted Dose (or PAD).1. Similarly, if the MOE method is used to characterize risk, then the acceptable

MOE will be 1000 rather than 100.

€

Population Adjusted Dose (PAD) = RfD10

, which is substituted into the

EPA Risk Estimate equation above.

2. Note that the acceptable risk will still be below 100% of the PAD.5. Note that risk characterization for compounds deemed genotoxic and carcinogenic are

considered by the EPA to have non-threshold effects. The MOE approach is notused.a. Rather, the EPA estimates the slope factor (as number of tumors per mg toxicant

per kg bw per day) from a modeling of the dose-response data.1. Usually three doses plus an unexposed control are tested over the lifetime of

the test animal (rodent, for example, would be tested for two years).2. The number of tumors relative to dose are fit to a linear model.

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Lecture 6: Fundamental Principles of Toxicity ... · 50% LD50, ED50, LC50, EC50 Slope NOAEL Figure 3. Sigmoidal distribution of proportion of population responding relative to dose