Environmental Epidemiology for Chemists - American Chemical Society

5 Environmental Epidemiology for Chemists Curtis D . Klaassen and William C. Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0241.ch005

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Kersha

w*,2

University of Kansas, School of Medicine, Kansas City, KS 66103 Health Care Technology Division, Preclinical Safety Assessment, Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, O H 45239-8707 1

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The principles of toxicology are utilized to (1) characterize verse effects of chemicals on living organisms,

the ad-

(2) define the condi-

tions of exposure required to produce these pernicious

effects, and

(3) understand the mechanisms by which chemicals produce toxicity. Toxicologists conduct safety studies to determine the intrinsic toxic properties of chemicals and conduct pharmacokinetic

studies to as-

sess the biological disposition of chemicals. The most important tenet of toxicity testing is the dose-response relationship,

because conclu-

sions regarding causality between chemical exposure and the ensuing adverse reaction are based on it. Toxicologists obtain essential information from safety and pharmacokinetic

studies to assess the risks

associated with chemical exposure and, in this manner, protect human health from hazardous materials found in the environment, workplace,

the

and the home.

Introduction to Toxicology Toxicology is the study of the adverse effects of chemicals o n l i v i n g o r g a n isms. T h e toxicologist is an i n d i v i d u a l t r a i n e d to e x a m i n e the nature o f these adverse effects a n d to d e t e r m i n e the p r o b a b i l i t y of t h e i r o c c u r r e n c e . S i n c e the 1970s, several academic institutions have offered s p e c i a l i z e d t r a i n i n g i n this f i e l d , a w a r d i n g b o t h u n d e r g r a d u a t e a n d graduate degrees i n toxicology. I n contrast to this recent t r e n d of s p e c i a l i z e d d i d a c t i c t r a i n i n g , m a n y p r a c t i c i n g toxicologists w i t h graduate degrees i n p h y s i c a l a n d life sciences have a c q u i r e d a w o r k i n g u n d e r s t a n d i n g of toxicology b y a p p l y i n g these sciences to toxicology-related issues i n i n d u s t r y , academia, a n d g o v e r n m e n t .

* C o r r e s p o n d i n g author

0065-2393/94/0241-0039$08.00/0 © 1994 American Chemical Society

Draper; Environmental Epidemiology Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

ENVIRONMENTAL EPIDEMIOLOGY

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0241.ch005

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Different Areas of Toxicology. Toxicologists a p p l y t h e i r skills to three major fields of i n q u i r y : d e s c r i p t i v e , m e c h a n i s t i c , a n d regulatory t o x i cology. D e s c r i p t i v e toxicology is a process w h e r e b y the deleterious effects of chemicals are characterized b y u s i n g laboratory animals a n d various i n v i t r o techniques. T h e purpose of these studies is to define the hazards that occur d u r i n g c i r c u m s c r i b e d exposure conditions. I n the p h a r m a c e u t i c a l i n dustry, one of the goals of the d e s c r i p t i v e toxicologist is to d e t e r m i n e the n o o b s e r v e d adverse effect l e v e l ( N O A E L ) of a d r u g candidate i n o r d e r to set safe dose levels for first-time exposure i n h u m a n s . I n the c h e m i c a l i n d u s t r y , the toxicologist is c o n c e r n e d not o n l y about h u m a n h e a l t h hazards p o s e d b y c o m p a n y products b u t also about the p o t e n t i a l deleterious effects these chemicals may have o n the e n v i r o n m e n t a n d its inhabitants. I n m e c h a n i s t i c toxicology, the mechanisms b y w h i c h chemicals exert t h e i r toxic effects are d e t e r m i n e d i n the laboratory. L e a r n i n g s f r o m this area of study f r e q u e n t l y lead to the d e v e l o p m e n t of m o r e efficient safety tests for the p r e d i c t i o n of h u m a n h e a l t h hazards, the establishment of s t r u c t u r e - a c t i v i t y relationships for the design of molecules w i t h i m p r o v e d safety profiles, the identification of antidotes to treat poisonings, a n d a clearer u n d e r s t a n d i n g of h u m a n p h y s iology a n d disease processes. R e g u l a t o r y toxicology is an activity c o n d u c t e d b y g o v e r n m e n t a l agencies w h e r e b y the hazards posed b y i n d u s t r i a l c h e m i cals or pharmaceuticals are assessed, a n d j u d g m e n t s are m a d e about w h e t h e r the p r o d u c t can b e u t i l i z e d i n the m a n n e r i n d i c a t e d b y the s p o n soring company. R e g u l a t o r y toxicologists base t h e i r h a z a r d assessments o n data p r o v i d e d b y the d e s c r i p t i v e toxicologist e m p l o y e d b y the c o m p a n y that is p e t i t i o n i n g the g o v e r n m e n t a l agency for m a r k e t i n g a p p r o v a l .

Toxicity versus Hazard.

T h e p r i n c i p l e activity of descriptive a n d

regulatory toxicologists is the assessment of the hazards o r risks associated w i t h the use of chemicals a n d not the i n t r i n s i c toxic activity of chemicals p e r se. H a z a r d is the situation i n w h i c h an u n d e s i r a b l e response results f r o m a specified l e v e l of exposure. F o r example, tetrodotoxin is an e x t r e m e l y toxic substance a n d is present i n the l i v e r of puffer fish, whose f l e s h is c o n s i d e r e d a delicacy i n Japan. A l t h o u g h a single dose of tetrodotoxin as l o w as 0.1 m g / k g is l e t h a l to laboratory animals, this substance is not a major h u m a n h e a l t h h a z a r d , because the fish are p r e p a r e d b y e x p e r i e n c e d food handlers w h o are careful not to contaminate the f l e s h w i t h liver. B y c o m p a r i s o n , ethanol is not as intrinsically toxic as tetrodotoxin. A l e t h a l dose of e t h a n o l is approximately 100,000 times greater than that of tetrodotoxin. D e s p i t e the fact that ethanol is t r e m e n d o u s l y less toxic than tetrodotoxin, one c o u l d c o n v i n c i n g l y argue that ethanol is m o r e hazardous, i n v i e w of the n u m b e r of alcohol-related deaths o n o u r n a t i o n s highways each year. B o t h i n t r i n s i c toxicity a n d exposure conditions (i.e., r o u t e , d u r a t i o n , f r e q u e n c y a n d p r o b ability of exposure, a n d dose) d e t e r m i n e w h e t h e r a substance w i l l b e h a z ardous.


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Local versus Systemic Effects. Toxic reactions m a y result f r o m a d i r e c t local action o r from the absorption of the toxicant i n t o systemic c i r culation a n d its subsequent d i s t r i b u t i o n into various tissues. L o c a l effects refer to those that occur at the site of first contact b e t w e e n the toxicant a n d the organism. M o s t substances, except for h i g h l y reactive ones, such as v e r y caustic or i r r i t a t i n g substances, r e q u i r e systemic absorption a n d tissue d i s t r i b u t i o n to p r o d u c e t h e i r toxic effects. T h e s e categories are not m u t u a l l y exclusive, because some substances demonstrate b o t h local a n d systemic effects. F o r instance, t e t r a e t h y l lead damages s k i n o n contact a n d after syst e m i c absorption causes c e n t r a l nervous system intoxication manifested b y restlessness, headaches, loss of m e m o r y , a n d convulsions.

Spectrum of Undesired Effects. T h e s p e c t r u m of u n d e s i r e d effects p r o d u c e d b y systemieally active toxicants is w i d e . Toxicities r u n the gamut f r o m r e l a t i v e l y b e n i g n or b o t h e r s o m e to incapacitating a n d l i f e - t h r e a t e n i n g . D r u g s , for instance, often p r o d u c e a w i d e array of subtle changes i n b o d i l y functions, although the p r e s c r i b i n g p h y s i c i a n often seeks to p r o d u c e a single effect. Results not d i r e c t e d t o w a r d the i n t e n d e d t h e r a p e u t i c objective are c o n s i d e r e d u n d e s i r a b l e effects, or side effects, of the d r u g for a p a r t i c u l a r disease c o n d i t i o n . T h e s e u n t o w a r d effects are u s u a l l y not of major c o n c e r n ; i n fact, an undesirable effect for one t h e r a p e u t i c i n d i c a t i o n m a y be desirable for another c l i n i c a l situation. F o r e x a m p l e , atropine causes dryness o f the m o u t h , w h i c h is c o n s i d e r e d a side effect w h e n the d r u g is p r e s c r i b e d for the treatment of p e p t i c ulcers b u t is the d e s i r e d response w h e n it is g i v e n as a preanesthetic m e d i c a t i o n . M o r e serious toxic reactions that result f r o m systemic exposure can b e characterized b y the onset of toxicity f o l l o w i n g exposure, b y w h e t h e r the deleterious changes are p e r m a n e n t or temporary, b y a genetically r e g u l a t e d a b n o r m a l reactivity of the exposed organism to the toxic agent, a n d b y w h e t h e r the adverse reaction results f r o m an i m m u n e response.

Immediate versus Delayed Toxicity. I m m e d i a t e toxic reactions are those that d e v e l o p w i t h i n hours or days f o l l o w i n g c h e m i c a l exposure, whereas d e l a y e d effects b e c o m e apparent weeks or e v e n years after the exposure i n c i d e n t . C a r b o n t e t r a c h l o r i d e - i n d u c e d hepatotoxicity is a n example of i m m e d i a t e toxicity, because m a r k e d changes i n the b i o c h e m i s t r y a n d structure of rat l i v e r are o b s e r v e d several hours after i n j e c t i o n of toxic doses. I n contrast, tumors often appear 20 years or m o r e after exposure to a c a r c i n ogen. F o r instance, there are n u m e r o u s cases i n w h i c h y o u n g w o m e n have d e v e l o p e d vaginal a n d u t e r i n e tumors as the result of i n utero exposure to d i e t h y l s t i l b e s t r o l w h e n this d r u g was p r e s c r i b e d to t h e i r mothers to p r e v e n t miscarriages. Reversible versus Irreversible Toxic Effects.

D a m a g e to tissues

caused b y c h e m i c a l exposure can b e e i t h e r r e v e r s i b l e or i r r e v e r s i b l e . T h e severity of c h e m i c a l l y m e d i a t e d tissue damage a n d the type of tissue i n


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w h i c h i n j u r y occurs are i m p o r t a n t factors that dictate w h e t h e r toxic effects w i l l b e t e m p o r a r y or b e c o m e p e r m a n e n t . F o r e x a m p l e , l i v e r has a r e m a r k able capacity to regenerate after c h e m i c a l insult. C o n s e q u e n t l y , the c y t o toxic effects of m a n y hepatotoxicants are c o m p l e t e l y r e v e r s i b l e . C h e m i c a l l y m e d i a t e d damage to the c e n t r a l nervous s y s t e m , however, is largely i r r e v e r s i b l e because fully differentiated n e u r a l tissue has r e l a t i v e l y little a b i l i t y to self-repair.

Idiosyncratic Reactions. " I d i o s y n c r a s y " is d e f i n e d as a genetically c o n t r o l l e d , u n e x p e c t e d reaction to a c h e m i c a l agent. T h e response that f o l lows exposure is q u a l i t a t i v e l y s i m i l a r to that o b s e r v e d i n most i n d i v i d u a l s , b u t the intensity of the response is considerably greater or less t h a n exp e c t e d . Idiosyncrasy can b e i l l u s t r a t e d b y the u n a n t i c i p a t e d p r o t r a c t e d d u ration of apnea (i.e., cessation of breathing) that is o b s e r v e d i n 1 i n 2500 patients w h o receive a standard dose of the skeletal m u s c l e relaxant, s u c c i n y l c h o l i n e . I n most p e o p l e , s u c c i n y l c h o l i n e produces transient a p n e a b e cause of its r a p i d degradation a n d inactivation b y p l a s m a cholinesterase. P a tients w h o e x h i b i t this idiosyncratic reaction have an a t y p i c a l f o r m of p l a s m a cholinesterase that has a r e d u c e d a b i l i t y to metabolize the d r u g a n d t h e r e b y to n e u t r a l i z e its activity. T h e familial o c c u r r e n c e of this p h e n o m e n o n s u g gests that h y p e r s u s c e p t i b i l i t y to s u c c i n y l c h o l i n e is u n d e r genetic c o n t r o l . Allergic or Sensitization Reactions. C h e m i c a l allergic reactions are i m m u n o l o g i c a l l y m e d i a t e d a n d r e q u i r e a p r e v i o u s p e r i o d of exposure (i.e., sensitization) to the c h e m i c a l or to a s t r u c t u r a l l y s i m i l a r one. O n e of the hallmarks of c h e m i c a l allergies is a h e i g h t e n e d sensitivity to c h e m i c a l i n t o x ication i n preexposed i n d i v i d u a l s . A p o p u l a t i o n is c o m p o s e d o f " n a i v e " p e o p l e (i.e., nonexposed individuals) as w e l l as p e o p l e w h o have h a d various degrees of p r i o r contact w i t h the c h e m i c a l . C o n s e q u e n t l y , there is m a r k e d person-to-person v a r i a t i o n w i t h respect to the s u s c e p t i b i l i t y to a l l e r g i c chemicals. T h e r e f o r e , it is not s u r p r i s i n g that p o p u l a t i o n - b a s e d , d o s e - r e sponse relationships are often not r e a d i l y apparent. T h e apparent lack of a d o s e - r e s p o n s e relationship suggests that allergic reactions are not t r u e toxic responses. H o w e v e r , a d o s e - r e s p o n s e r e l a t i o n s h i p can b e d e m o n s t r a t e d i n a single allergic i n d i v i d u a l , a n d the allergic responses are often severe a n d occasionally life-threatening. T h e r e f o r e , allergic reactions s h o u l d be r e g a r d e d as a g e n u i n e toxic response. Systemic Effects. I n d e s c r i p t i v e toxicity studies, chemicals are a d m i n i s t e r e d v i a routes a n d dose forms that m a x i m i z e systemic absorption. T h e goal of these studies is to characterize fully a c o m p o u n d s ability to p r o d u c e b i o c h e m i c a l , s t r u c t u r a l , a n d functional changes i n various target o r gans, such as liver, k i d n e y , l u n g , b o n e m a r r o w , a n d b r a i n . C o m p o u n d s are also tested as to t h e i r a b i l i t y to (1) interact w i t h D N A a n d alter the process of h e r e d i t y (i.e., mutagenesis), (2) p r o d u c e t u m o r s (i.e., carcinogenesis,), (3) r e d u c e the fertility o f male a n d female animals (i.e., r e p r o d u c t i v e toxicity),


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a n d (4) cause malformation a n d dysfunction o f offspring (i.e., d e v e l o p m e n t a l toxicity).


The Dose-Response Relationship.

T o establish that the a d m i n i s -

tration o f a c h e m i c a l is causally related to signs o f intoxication, t h e toxicologist must demonstrate a d o s e - r e s p o n s e r e l a t i o n s h i p . T h i s relationship is based o n t h e reasonable p r e s u m p t i o n that the toxic response is a result o f the a d m i n i s t e r e d c h e m i c a l a n d t h e assumption that t h e i n t e n s i t y o f this r e sponse is related p r o p o r t i o n a l l y to the dose. T h e assumption is based o n three others: F i r s t , there is a m o l e c u l a r site w i t h i n t h e organism w i t h w h i c h the c h e m i c a l interacts to p r o d u c e t h e response; second, t h e i n t e n s i t y o f the response is related to t h e concentration o f the c h e m i c a l at that site; a n d t h i r d , the concentration at t h e site is r e l a t e d to t h e dose. D o s e - r e s p o n s e curves can b e e i t h e r " g r a d e d " o r " q u a n t a l " . T h e dose describes t h e increasi n g intensity o f a response o f an i n d i v i d u a l (or a population) o v e r a range o f increasing dosages, whereas t h e response illustrates t h e d i s t r i b u t i o n o f m i n i m a l doses that p r o d u c e a g i v e n " a l l - o r - n o n e " response (e.g., p r e s e n c e o f a tumor) i n a p o p u l a t i o n o f b i o l o g i c a l subjects.

Organizations That Conduct Toxicity Studies. T h e p h a r m a c e u t i cal i n d u s t r y is responsible for c o n d u c t i n g d e s c r i p t i v e a n i m a l toxicity studies that d e t e r m i n e w h i c h organs are t h e p o t e n t i a l targets o f toxicity d u r i n g d r u g therapy as w e l l as the d r u g s mutagenic, carcinogenic, a n d teratogenic p r o p erties. T h e F o o d a n d D r u g A d m i n i s t r a t i o n ( F D A ) is responsible for r e v i e w i n g a n i m a l toxicology reports s u b m i t t e d b y the sponsoring d r u g c o m p a n y a n d for j u d g i n g w h e t h e r the potential benefits o f the d r u g o u t w e i g h its acc o m p a n y i n g hazards. I f a favorable risk-to-benefit p a r a d i g m is c o n c l u d e d , the d r u g candidate is a p p r o v e d for i n i t i a l c l i n i c a l trials that establish safety a n d tolerance i n h u m a n s . T h e E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ( E P A ) r e quires the c h e m i c a l i n d u s t r y to c o n d u c t extensive d e s c r i p t i v e toxicity testing of pesticides a n d i n d u s t r i a l chemicals. T h e r e s u l t i n g data are u s e d b y t h e E P A to assess the possible negative i m p a c t a c h e m i c a l m a y have o n t h e e n v i r o n m e n t a n d to d e t e r m i n e w h e t h e r r e m e d i a l activities against the m a n u facturer s h o u l d b e initiated. Pharmacokinetics and Toxicity Studies T w o f u n d a m e n t a l types o f studies, b r o a d l y r e f e r r e d to as p h a r m a c o k i n e t i c a n d toxicity studies, are c o n d u c t e d to u n d e r s t a n d t h e i n t e r a c t i o n b e t w e e n chemicals a n d l i v i n g organisms. P h a r m a c o k i n e t i c studies d e t e r m i n e t h e body's effects o n the c h e m i c a l , whereas toxicity studies d e t e r m i n e the effects of the c h e m i c a l o n t h e body.

Pharmacokinetic Studies. S t r i c t l y speaking, manifestations o f syst e m i c intoxication d o not d e p e n d d i r e c t l y o n t h e a d m i n i s t e r e d dose o f a toxicant b u t rather o n t h e concentration o f the c h e m i c a l (and its metabolites) i n



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target organs. T h e concentration o f the active c h e m i c a l species i n target o r gans is regulated b y four b i o l o g i c a l processes: a b s o r p t i o n , d i s t r i b u t i o n , m e tabolism, and elimination. Absorption.

T h e process b y w h i c h chemicals cross b i o l o g i c a l m e m -

branes a n d e n t e r b l o o d is c a l l e d absorption. T h e gastrointestinal tract, lungs, a n d s k i n p r o v i d e pathways for chemicals to e n t e r systemic c i r c u l a t i o n . B o t h


the rate a n d the extent w i t h w h i c h toxicants e n t e r b l o o d m a r k e d l y i n f l u e n c e the severity a n d type of r e s u l t i n g l e s i o n . Injection of chemicals into veins or the p e r i t o n e a l cavity (for rodents only) are a d d i t i o n a l routes of exposure that are e m p l o y e d i n the laboratory to evaluate t h e i r p h a r m a c o k i n e t i c a n d t o x i cological p r o p e r t i e s . T h e gastrointestinal tract is an i m p o r t a n t site of absorption t h r o u g h w h i c h foreign chemicals gain access to systemic c i r c u l a t i o n . F o r e x a m p l e , m a n y e n v i r o n m e n t a l pollutants e n t e r the food c h a i n a n d are ingested along w i t h food products. T h i s route of exposure is also e n c o u n t e r e d f r e q u e n t l y i n suicide attempts i n v o l v i n g overdoses of drugs a n d is the most c o m m o n route i n w h i c h c h i l d r e n are p o i s o n e d b y h o u s e h o l d chemicals. A b s o r p t i o n after oral ingestion is i n f l u e n c e d b y p h y s i c o c h e m i c a l p r o p erties of the c h e m i c a l . Factors that favor absorption i n c l u d e h i g h l i p i d - t o water p a r t i t i o n coefficient,

l o w degree of i o n i z a t i o n at various p H levels

along the gastrointestinal tract, l o w m o l e c u l a r w e i g h t a n d r a p i d d i s s o l u t i o n rate for s o l i d materials. P r o p e r t i e s of the exposed i n d i v i d u a l , such as gastric e m p t y i n g t i m e , i n t e s t i n a l m o t i l i t y , s p l a n c h n i c b l o o d flow, b i l e secretion, a n d b i n d i n g to gastrointestinal m u c u s , as w e l l as interactions b e t w e e n chemicals a n d foods also have a significant i n f l u e n c e o n the absorption o f foreign s u b stances that are ingested u n i n t e n t i o n a l l y . Distribution.

F o l l o w i n g absorption, most xenobiotics are c a r r i e d b y

b l o o d i n a n o n u n i f o r m m a n n e r t h r o u g h o u t the body. W h e t h e r a c h e m i c a l remains i n systemic c i r c u l a t i o n , diffuses i n t o extracellular f l u i d , or enters a n d is stored i n various tissue c o m p a r t m e n t s , such as adipose tissue a n d b o n e , d e p e n d s o n its p h y s i c o c h e m i c a l p r o p e r t i e s (i.e., l i p o p h i l i c i t y , d e g r e e of i o n i z a t i o n at p l a s m a p H , m o l e c u l a r size, a n d p e r m e a b i l i t y t h r o u g h p l a s m a a n d tissue membranes) as w e l l as b i o l o g i c a l considerations, such as b i n d i n g to p l a s m a proteins and c e l l u l a r constituents. O f p a r t i c u l a r interest to the toxicologist is the p r o p e n s i t y o f some toxicants to accumulate i n specific tissues i n the body. These storage depots often are the target organs of toxicity; for example, paraquat is a p u l m o n a r y toxicant that accumulates i n l u n g . C o n versely, some toxicants accumulate i n storage depots that are not target tissues; sequestration i n these tissues can b e c o n s i d e r e d a p r o t e c t i v e m e c h anism. F o r example, e n v i r o n m e n t a l contaminants such as l e a d a n d 1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane ( D D T ) d i s t r i b u t e a n d concentrate i n b o n e a n d b o d y fat, respectively. T h e s e c o m p o u n d s have no toxicological i m -


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pact o n t h e i r respective storage sites a n d once sequestered are not available to affect t h e i r target organ, the c e n t r a l nervous system. Metabolism. M o s t chemicals u n d e r g o e n z y m a t i c a l l y catalyzed b i o transformations that p r o d u c e metabolites that are m o r e p o l a r a n d w a t e r - s o l u b l e Than the parent c o m p o u n d . Increasing the water-soluble p r o p e r t i e s of a c o m p o u n d enhances its e l i m i n a t i o n f r o m the b o d y a n d t h e r e b y lowers the b o d y b u r d e n of the material. B i o t r a n s f o r m a t i o n reactions can c o n v e r t a toxic c h e m i c a l to an innocuous metabolite or to a m e t a b o l i t e that has s i m i l a r o r greater toxicologic activity t h a n the parent c o m p o u n d . T h e l i v e r is the p r i n c i p a l site o f xenobiotic m e t a b o l i s m , a l t h o u g h biotransformation also occurs i n k i d n e y , intestinal e p i t h e l i u m , l u n g , b l o o d , a n d i n the l u m e n of the gut b y the action of intestinal flora. Biotransformation reactions are classified as Phase I a n d Phase I I reactions. Phase I reactions u s u a l l y convert the parent c o m p o u n d to m o r e p o l a r metabolites a n d i n v o l v e three types of c h e m i c a l reactions: (1) oxidation (e.g., h y d r o x y l a t i o n , side-chain aliphatic oxidation, d e a m i n a t i o n , sulfoxide f o r m a t i o n , d e a l k y l a t i o n o f O , N, a n d S substitutions, a n d the d e h y d r o g e n a t i o n of alcohols a n d aldehydes), (2) r e d u c t i o n (e.g., azo, n i t r o , k e t o n e , a n d a l d e hyde), a n d (3) the h y d r o l y s i s of esters a n d amides. Phase I I reactions are conjugation o r synthetic reactions. O n e o f the most c o m m o n conjugation reactions is catalyzed b y u r i d i n e d i p h o s p h o g l u curonosyltransferase a n d results i n the formation of g l u c u r o n i c a c i d d e r i v a tives. Various c h e m i c a l moieties are susceptible to g l u c u r o n i d a t i o n , s u c h as aliphatic a n d aromatic amines as w e l l as h y d r o x y l , c a r b o x y l , a n d s u l f h y d r y l groups. O t h e r conjugation reactions i n c l u d e the acetylation of p r i m a r y amines; conjugation w i t h sulphate, g l y c i n e , a n d glutathione; a n d m e t h y l a t i o n of O, N, a n d S atoms i n the parent m o l e c u l e . Phase I a n d Phase I I reactions usually result i n the formation of m e t a b olites that are m o r e r e a d i l y excreted i n the u r i n e . S o m e Phase I I reactions, such as conjugation w i t h g l u c u r o n i c a c i d or g l u t a t h i o n e , f r e q u e n t l y shift the route of excretion from u r i n e to feces, as d e s c r i b e d f u r t h e r o n i n greater detail. Elimination. P a r e n t c o m p o u n d a n d its metabolites are e l i m i n a t e d f r o m the b o d y v i a u r i n e a n d feces a n d , to a lesser extent, b y w a y of r e s p i r a t i o n a n d secretory substances (i.e., m i l k , sweat, saliva, a n d tears). E x c r e t o r y o r gans t y p i c a l l y e l i m i n a t e polar or water-soluble c o m p o u n d s m o r e p r o f i c i e n t l y than they do l i p o p h i l i c c o m p o u n d s . T h u s , x e n o b i o t i c m e t a b o l i s m f r e q u e n t l y enhances the e l i m i n a t i o n o f chemicals a n d so can b e c o n s i d e r e d a detoxification process. K i d n e y , the p r i m a r y organ for c h e m i c a l e l i m i n a t i o n , receives 2 5 % o f the cardiac output, w h i c h allows large v o l u m e s of p l a s m a water to b e filtered. S e v e r a l factors favor g l o m e r u l a r filtration o f c h e m i c a l s , such as l o w m o l e c u l a r



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w e i g h t ; h i g h concentration gradient b e t w e e n p l a s m a w a t e r a n d filtrate; a n d l i m i t e d p l a s m a p r o t e i n b i n d i n g , because o n l y u n b o u n d c h e m i c a l can b e filt e r e d . O n c e chemicals are present i n t u b u l a r filtrate, the extent of i o n i z a t i o n influences r e n a l e x c r e t i o n , because p o l a r c o m p o u n d s a n d ions are not r e a b sorbed i n p r o x i m a l or distal t u b u l e s . N o n i o n i z e d c o m p o u n d s h a v i n g h i g h l i p i d - t o - w a t e r p a r t i t i o n coefficients are passively r e a b s o r b e d t h r o u g h t u b u l a r c e l l m e m b r a n e s . W e a k acids are n o n i o n i z e d i n acidic u r i n e , w h i c h promotes r e c y c l i n g a n d prolongs e l i m i n a t i o n . C o n v e r s e l y , alkalinization o f u r i n e accelerates the excretion o f weak acids. B i o t r a n s f o r m a t i o n reactions f r e q u e n t l y result i n the formation of stronger acids that r e a d i l y i o n i z e i n acidic u r i n e . T h i s effect reduces t u b u l a r r e a b s o r p t i o n , facilitates u r i n a r y excretion, a n d often decreases a toxicants d u r a t i o n o f activity. E n e r g y - d e p e n d e n t , saturable transport of organic acids a n d bases f r o m afferent b l o o d into t u b u l a r u r i n e occurs b y w a y o f p r o x i m a l t u b u l a r cells a n d is m o r e effective than filtration as a means to e l i m i n a t e c o m p o u n d s . T h e fact that o n l y 2 0 % of afferent b l o o d that flows to the k i d n e y is f i l t e r e d , whereas the r e m a i n i n g 8 0 % is d e l i v e r e d to t u b u l a r cells, partially accounts for the effectiveness of secretion. R e v e r s i b l e b i n d i n g o f chemicals to p l a s m a p r o teins does not significantly affect p r o x i m a l t u b u l e secretion, because free a n d b o u n d chemicals are available for transport. T h i s occurs because b o u n d c h e m i c a l dissociates r a p i d l y to m a i n t a i n e q u i l i b r i u m w i t h p l a s m a w a t e r as free c h e m i c a l is r e m o v e d f r o m t u b u l a r cells. C h e m i c a l s can also b e excreted b y hepatocytes across canalicular m e m branes into the b i l i a r y tract a n d pass into intestine. L o w - m o l e c u l a r - w e i g h t molecules are p o o r l y excreted into b i l e , whereas c o m p o u n d s w i t h m o l e c u l a r weights of 350 daltons or m o r e are excreted i n t o b i l e i n appreciable q u a n tities. T h e synthetic nature of Phase I I reactions results i n metabolites w i t h h i g h e r m o l e c u l a r weights than those of the parent c h e m i c a l . T h u s , the p r o d ucts of conjugation reactions that use glutathione a n d g l u c u r o n i c a c i d as l i gands are t y p i c a l l y excreted into b i l e . O n c e i n the i n t e s t i n e , the c h e m i c a l a n d its metabolite can b e e l i m i n a t e d i n the feces. A l t e r n a t i v e l y , i f the p r o p erties of the c o m p o u n d favor intestinal absorption (i.e., l o w degree of i o n i zation, h i g h l i p i d - t o - w a t e r p a r t i t i o n coefficient of the n o n i o n i z e d f o r m , a n d small m o l e c u l a r radius of the w a t e r - s o l u b l e substance); a cycle r e f e r r e d to as " e n t e r o h e p a t i c c i r c u l a t i o n " m a y result, i n w h i c h b i l i a r y secretion a n d i n t e s t i n a l reabsorption c o n t i n u e u n t i l biotransformation a n d u r i n a r y excretion eliminates the c o m p o u n d f r o m the body. G l u c u r o n i d e conjugates are susc e p t i b l e to h y d r o l y t i c reactions c o n d u c t e d b y gut flora. T h e s e reactions f o r m products that have p h y s i c o c h e m i c a l p r o p e r t i e s favorable to intestinal reabsorption. S o m e t i m e s a c h e m i c a l s l o n g persistence i n the b o d y is largely the result of enterohepatic c i r c u l a t i o n , as is the case w i t h cardiac glycosides. I n contrast, b i l i a r y excretion serves as an efficient route o f e l i m i n a t i o n for c e r tain organic acids a n d bases (e.g., tetracyclines a n d streptomycin) that c a n not b e reabsorbed i n the i n t e s t i n e , o w i n g to i o n i z a t i o n at intestinal p H .


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Toxicology Studies. Hazard Evaluation Goals. T h e p u r p o s e of d e scriptive toxicity studies, regardless of the i n t e n d e d use of the test m a t e r i a l , is to d e t e r m i n e the adverse effects p r o d u c e d b y chemicals a n d the dose n e e d e d to p r o d u c e these effects. U l t i m a t e l y , this v i t a l i n f o r m a t i o n is u s e d b y the toxicologist to make h a z a r d assessments to protect h u m a n s against the d e t r i m e n t a l effects of chemicals. Principles of Toxicity Testing. D e s c r i p t i v e toxicity testing is f o u n d e d o n two m a i n p r i n c i p l e s that s u p p o r t its use i n c h e m i c a l h a z a r d assessment. T h e first p r i n c i p l e is that the toxic effects p r o d u c e d b y a c o m p o u n d i n l a b oratory animals, w h e n p r o p e r l y q u a l i f i e d , are applicable to h u m a n s . T h i s tenet is not u n i q u e to toxicology a n d applies to a l l other life sciences. F u r t h e r m o r e , it has b e e n v e r i f i e d n u m e r o u s times for a w i d e variety of c h e m i cals. F o r example, n e a r l y a l l h u m a n carcinogens are also carcinogenic i n at least one species u s e d i n toxicity studies. T h e second p r i n c i p l e is that exposure of e x p e r i m e n t a l animals to h i g h doses of toxic agents is necessary a n d a v a l i d m e t h o d of d i s c o v e r i n g possible hazards to h u m a n h e a l t h . T h i s axiom is f o u n d e d o n the q u a n t a l d o s e - r e sponse concept that the occurrence of a toxic response i n a p o p u l a t i o n i n creases as the dose a n d d u r a t i o n of exposure increases. P r a c t i c a l l i m i t a t i o n s i n the design of toxicological studies dictate that a s m a l l n u m b e r of animals w i l l b e exposed to the c h e m i c a l c o m p a r e d w i t h the size of the h u m a n p o p u l a t i o n p o t e n t i a l l y at risk. C o n s e q u e n t l y , r e l a t i v e l y large doses of chemicals are necessary to increase the frequency w i t h w h i c h treatment effects occur i n s m a l l groups of animals. F o r e x a m p l e , suppose the i n c i d e n c e rate of a particular c h e m i c a l to p r o d u c e a t u m o r i n h u m a n s was as l o w as 0 . 0 1 % . T h i s i n c i d e n c e rate represents 24,000 p e o p l e i n a p o p u l a t i o n of 240 m i l l i o n p e o ple. A toxicological study w o u l d have to use a m i n i m u m of 30,000 animals i n o r d e r to detect a t u m o r i n three animals! It is not feasible to use e x c e e d i n g l y large n u m b e r s of animals to assess the toxic p r o p e r t i e s of chemicals. T h e o n l y available o p t i o n is to collect toxicity data from s m a l l groups of a n imals exposed to large doses o f chemicals a n d to use the p r i n c i p l e s of t o x i cology a n d pharmacokinetics to extrapolate the e x p e r i m e n t a l findings to h u mans. I n this manner, toxicologists can assess h u m a n h e a l t h hazards that result from low-dose exposures to e n v i r o n m e n t a l pollutants a n d o t h e r c h e m icals f o u n d i n the w o r k p l a c e a n d h o m e . Toxicity Studies. Test species, n u m b e r of animals, d u r a t i o n of c h e m i c a l exposure, dose l e v e l , a n d the m e a s u r e d parameters of toxicity are the f u n d a m e n t a l variables that constitute a l l d e s c r i p t i v e toxicological studies. Animals. M o s t often, the selection of species i n toxicity studies u l t i m a t e l y depends o n the regulatory agency that w i l l r e v i e w the data. R e g u latory bodies, i n t u r n , set species r e q u i r e m e n t s based o n m a n y years of as-



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sessing safety data s u b m i t t e d b y i n d u s t r y as w e l l as the advice f r o m r e c o g n i z e d authorities i n the field of c h e m i c a l h a z a r d assessment. T h e most i m p o r t a n t c r i t e r i o n for species selection is that the s p e c t r u m of toxicities o b s e r v e d i n h u m a n s b e r e p r o d u c i b l e i n the test species. T h i s is r e c o g n i z e d retrospectively for a p l e t h o r a o f c o m p o u n d s that e n t e r e d the m a r k e t or e n v i r o n m e n t before the advent of rigorous toxicity testing. T h e s e toxicities preferably are e l i c i t e d at dosages e q u i v a l e n t to, or l o w e r t h a n , those e n c o u n t e r e d b y h u m a n s a n d expressed after r e l a t i v e l y short durations o f exposure. T h e E P A usually r e q u i r e s rats a n d m i c e to be u s e d as test species i n studies that characterize the toxic effects o f pesticides a n d other toxic s u b stances that m a y cause occupational i n j u r y or contaminate the e n v i r o n m e n t . E x c e p t i o n s to this r u l e occur w h e n a specific adverse effect seen i n h u m a n s is not easily r e p r o d u c e d i n rodents. F o r instance, some organophosphorus c o m p o u n d s , such as t r i o r t h o s c r e s y l phosphate, p r o d u c e a neurotoxic effect i n h u m a n s manifested b y a " d y i n g b a c k " of the distal ends of l o n g , l a r g e diameter, p e r i p h e r a l nerves. T h e c h i c k e n is h i g h l y sensitive to this l e s i o n a n d is u s e d r o u t i n e l y to d e t e r m i n e w h e t h e r a n e w p e s t i c i d e has this toxicological property. T h e F D A t y p i c a l l y r e q u i r e s a rodent a n d a n o n r o d e n t species for the assessment of d r u g safety i n s u b c h r o n i c a n d c h r o n i c studies that d e t e r m i n e w h i c h organs are affected b y high-dose d r u g treatment. T h e rat is the r o d e n t species used most often, whereas the n o n r o d e n t species is e i t h e r the beagle d o g or the cynomolgus monkey. A c u t e toxicity tests (i.e., single-dose a d m i n istration) evaluate the possible consequences of d r u g overdose a n d e m p l o y t h r e e test species, usually the mouse, rat, a n d rabbit. Rats a n d rabbits are u s e d to evaluate the effects o f chemicals o n m a l e a n d female f e c u n d i t y as w e l l as fetal d e v e l o p m e n t . M i c e a n d rats are u s e d to d e t e r m i n e w h e t h e r a c o m p o u n d produces tumors (i.e., carcinogenicity study) w h e n a d m i n i s t e r e d t h r o u g h o u t an a n i m a l s e n t i r e life span (e.g., 1.5-2 years for m i c e ; 2 - 2 . 5 years for rats). I n a d d i t i o n , most studies that evaluate the systemic toxicity of chemicals contain b o t h male a n d female animals. Numbers of Animals. T h e n u m b e r of animals e m p l o y e d i n r e g u l a t e d toxicity studies that support the m a r k e t i n g a p p r o v a l of a d r u g , food a d d i t i v e , or agricultural c h e m i c a l is d e f i n e d b y the regulatory agency that has j u r i s d i c t i o n o v e r the i n d i c a t e d usage of the c h e m i c a l . T h e m i n i m u m n u m b e r can b e as l o w as a single a n i m a l as i n the case w i t h acute toxicity tests that e m p l o y dogs as the test species. F o r e x a m p l e , the lethal dose i n dogs is a p p r o x i m a t e d b y i n c r e a s i n g the dose g i v e n to the same a n i m a l each day u n t i l serious side effects of the d r u g are o b s e r v e d . T h e d o g is m o n i t o r e d for signs of recovery, r e m o v e d f r o m the study, a n d r e t u r n e d to the d o g colony. W h e n rodents are u s e d i n acute toxicity tests, as m a n y as 10 animals p e r sex are assigned to a dose l e v e l .



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I n subacute studies, the test m a t e r i a l is u s u a l l y a d m i n i s t e r e d for 14 days. T h e s e studies p r o v i d e an i n i t i a l identification of target organs a n d are u s e d to set dose levels for s u b c h r o n i c studies, that is, studies of 1 a n d 3 months i n d u r a t i o n . Subacute studies usually are not r e g u l a t e d , because they are not u s e d d i r e c t l y for h u m a n h a z a r d assessment. A c c o r d i n g l y , the d e s c r i p t i v e toxicologist a n d not a g o v e r n m e n t a l g u i d e l i n e selects the n u m b e r of rats a n d dogs to be u s e d i n subacute testing. F r e q u e n t l y , sample sizes for rats a n d dogs are 5 - 1 0 p e r sex a n d 2 - 4 p e r sex, respectively. I n contrast, repeat-dose, target organ studies s u b m i t t e d to regulatory bodies (i.e., s u b c h r o n i c a n d c h r o n i c studies) are r e q u i r e d to have 1 0 - 2 5 rodents p e r sex p e r dose l e v e l a n d 3 - 6 dogs or m o n k e y s p e r sex p e r dose l e v e l . I n carcinogenicity studies, as m a n y as 5 0 - 7 5 rodents p e r sex p e r dose l e v e l are p l a c e d o n diets that contain the test substance. S t u d y designs i n c l u d e at least three treatment groups o n diets that have i n c r e a s i n g c o n c e n trations of test m a t e r i a l a n d often call for two groups to b e p l a c e d o n c o n t r o l diets. T h u s , these lifetime studies f r e q u e n t l y b e g i n w i t h 500 or m o r e r o dents. L a r g e n u m b e r s of animals are n e e d e d to d i s c r i m i n a t e b e t w e e n treatm e n t - r e l a t e d increases i n t u m o r i n c i d e n c e a n d those that occur spontaneously i n senescent animals. A c c o r d i n g l y , regulatory agencies m a y request that a m i n i m u m of 30 animals p e r sex p e r dose l e v e l s u r v i v e to the e n d of the study. F o r reasons that r e m a i n obscure, the 2 4 - m o n t h s u r v i v a l rate of naive rats e m p l o y e d i n toxicity studies has decreased somewhat over the years. C o n s e q u e n t l y , large n u m b e r s of animals are p l a c e d o n study to ensure that the 3 0 - a n i m a l r e q u i r e m e n t is met. Route of Administration and Dose Levels. T h e route of a d m i n i s t r a t i o n e m p l o y e d i n toxicity studies is the same as that expected for h u m a n exposure a n d is often oral. O r a l a d m i n i s t r a t i o n is a c h i e v e d b y d i r e c t p l a c e m e n t of the test m a t e r i a l i n the stomach (i.e., oral gavage) or b y m i x i n g the c o m p o u n d into the diet, w h i c h is t h e n fed to the a n i m a l . T o x i c i t y studies e m p l o y a m i n i m u m of three dose levels a n d a single c o n t r o l g r o u p . Ideally, the h i g h dose s h o u l d clearly demonstrate some f o r m of a noxious response. T h e l o w est dose l e v e l s h o u l d identify the dose that produces no observable adverse effects ( N O A E L ) , whereas the m i d - d o s e l e v e l is i n c l u d e d to establish a d o s e - r e s p o n s e relationship a n d d e t e r m i n e the lowest dose that produces adverse effects ( L O A E L ) . Parameters of Toxicity. T h e parameters r e c o r d e d i n subacute a n d s u b c h r o n i c studies i n c l u d e c l i n i c a l observations (i.e., b o d y w e i g h t g a i n , food c o n s u m p t i o n , p h y s i c a l appearance, c l i n i c a l behavior, cardiovascular a n d resp i r a t o r y distress, loose stools, a n d macroscopic appearance of the eyes), c l i n ical c h e m i s t r y (i.e., s e r u m levels or activities of s o d i u m , p o t a s s i u m , c a l c i u m , c h l o r i d e , glucose, b l o o d u r e a n i t r o g e n , c r e a t i n i n e , b i l i r u b i n , alanine a m i notransaminase, aspartate aminotransaminase, s o r b i t o l dehydrogenase, a l -



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fcaline phosphatase, 7-glutamyl transpeptidase, t r i g l y c e r i d e s , a l b u m i n , a n d total protein), hematology (i.e., assessment o f b l o o d for h e m o g l o b i n , h e m a tocrit, r e d b l o o d c e l l count, w h i t e b l o o d c e l l count a n d t h e i r subtypes, p l a t e let count, a n d c l o t t i n g time), urinalysis (i.e., e x a m i n a t i o n of u r i n e for color, v o l u m e , specific gravity, p H , b l o o d , glucose, ketones, p r o t e i n , b i l i r u b i n , a n d u r o b i l i n o g e n a n d the presence of crystals, casts, e p i t h e l i a l cells, r e d b l o o d cells, w h i t e b l o o d cells, a n d microorganisms), gross a n d l i g h t m i c r o scopic appearance o f a l l major tissues (i.e., b r a i n , heart, liver, k i d n e y , s p l e e n , testes, prostate, e p i d i d y m u s , s e m i n a l vesicles, ovaries, u t e r u s , t h y r o i d , p a r a t h y r o i d , adrenals, t h y m u s , eyes, l u n g s , l y m p h nodes, m a m m a r y glands, s p i n a l c o r d , sciatic n e r v e , p i t u i t a r y g l a n d , pancreas, b o n e , b o n e m a r row, s k i n , skeletal m u s c l e , a n d various portions o f the u r i n a r y a n d gastrointestinal tracts), a n d the w e i g h t of the first 15 a f o r e m e n t i o n e d tissues. Chronic and Lifetime Carcinogenicity Studies. C h r o n i c studies a n d lifetime carcinogenicity studies are u s e d to assess adverse reactions that r e q u i r e p r o t r a c t e d exposure times. T h e study d e s i g n a n d objectives of the c h r o n i c study are s i m i l a r to those of the s u b c h r o n i c study. T h e d u r a t i o n o f the c h r o n i c studies depends o n the l e n g t h o f h u m a n exposure e n v i s i o n e d for the test m a t e r i a l as w e l l as the test species. F o r example, a 6 - m o n t h c h r o n i c study is appropriate to assess the c h r o n i c target organ toxicity of a n a n t i m i c r o b i a l agent h a v i n g an anticipated c l i n i c a l treatment r e g i m e n of 10 days i n d u r a t i o n . I n general, the p e r i o d of exposure i n c h r o n i c r o d e n t studies is b e t w e e n 6 a n d 12 m o n t h s . D o g s a n d monkeys t y p i c a l l y receive test materials for 1 year. H o w e v e r , m u c h l o n g e r exposures are occasionally e m p l o y e d , as was the case w i t h o r a l contraceptives that w e r e a d m i n i s t e r e d to m o n k e y s for several years. C h r o n i c studies f r e q u e n t l y incorporate r e c o v e r y groups that are m o n i t o r e d for several m o n t h s or m o r e after the cessation of c o m p o u n d a d m i n i s t r a t i o n to d e t e r m i n e w h e t h e r the toxicities n o t e d d u r i n g t r e a t m e n t are r e v e r s i b l e . T h e l i f e t i m e carcinogenicity study is c o l l o q u i a l l y r e f e r r e d to as a "bioassay" a n d is u t i l i z e d to d e t e r m i n e w h e t h e r a c o m p o u n d has the ability to p r o d u c e t u m o r s . D o s e selection is c r i t i c a l for carcinogenicity studies, b e cause m a r k e d c h r o n i c intoxication, m o r b i d i t y , a n d m o r t a l i t y w i l l r e d u c e the n u m b e r o f animals that s u r v i v e to the t e r m o f t h e study, w h i c h has a serious i m p a c t o n the acceptability o f the study b y foreign a n d domestic regulatory agencies. T h u s , a m a x i m u m tolerated dose ( M T D ) is u s e d as the highest dose l e v e l i n carcinogenicity studies, a n d fractions t h e r e o f are u s e d as m i d - a n d low-dose levels (e.g., o n e - h a l f a n d o n e - q u a r t e r of the M T D ) . T h e M T D is frequently d e f i n e d i n the p h a r m a c e u t i c a l i n d u s t r y as a dose that suppresses b o d y w e i g h t gain b y a m a x i m u m of 1 0 % a n d is often i d e n t i f i e d i n the 3 - m o n t h s u b c h r o n i c study. A strong c o m m i t m e n t to b r i n g i n g a p r o d u c t to m a r k e t is m a d e w h e n a p h a r m a c e u t i c a l , food, o r a g r i c u l t u r a l c h e m i c a l c o m p a n y decides to conduct carcinogenicity studies, because t h e y are a n i m a l - ,


5.

KLAASSEN & KERSHAW

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test m a t e r i a l - , t i m e - , a n d labor-intensive a n d are b y far the most expensive toxicity tests to conduct. T h e c u r r e n t cost of c o n d u c t i n g carcinogenicity studies i n rats a n d m i c e is approximately $ 2 m i l l i o n .


Summary I n summary, the p r i n c i p l e s of toxicology are e m p l o y e d to design sensitive and p r e d i c t i v e a n i m a l safety studies w i t h the u l t i m a t e goal of extrapolating the data to h u m a n a n d e n v i r o n m e n t a l c h e m i c a l exposure situations. I n this manner, the hazards that chemicals pose to the h e a l t h of humans a n d the w e l l - b e i n g of the e n v i r o n m e n t can be assessed a n d , m o r e i m p o r t a n t , averted. RECEIVED for r e v i e w February 11, 1993.

September

3,

1992.

ACCEPTED

revised manuscript


Environmental Epidemiology for Chemists - American Chemical Society

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