In Vitro and In Vivo Assays to Screen for Reproductive Toxicants in

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7 In Vitro and In Vivo Assays to Screen for Reproductive Toxicants in Animals and Humans Barbara S. Shane Institute for Environmental Studies, Louisiana State University, Baton Rouge, L A 70803

Although more than 800 compounds that have been tested in animals have been shown to be embryotoxic or teratogenic, little is known of the reproductive toxicity of these and other chemicals in humans. Because the compounds that have been tested account for only 2.5% of the potentially embryotoxic or teratogenic compounds to which humans are exposed, emphasis has been placed on the development of short-term reproductive assays to screen these compounds more rapidly. In some cases, the results obtained in the in vivo assays in intact animals correlate well with those obtained in the short-term in vitro assays; however, the usefulness of these assays will be evident only after extensive validation. Also, epidemiological studies in humans are difficult to interpret when the cohorts are small and the adverse reproductive outcomes very subtle. In this chapter, the most commonly used in vitro and in vivo assays are described in association with the outcomes elicited by certain classes of chemicals. Additionally, epidemiological studies in humans in which adverse reproductive outcomes have been elicited by industrial chemicals are also discussed.

THE HUMAN POPULATION HAS BEEN EXPOSED (I)

to a p p r o x i m a t e l y 6 0 , 0 0 0

c h e m i c a l s , a n d this n u m b e r is i n c r e a s i n g b y a few t h o u s a n d a n n u a l l y b e c a u s e of t h e synthesis o f n e w c o m p o u n d s , i n a d v e r t e n t p r o d u c t i o n i n i n d u s t r i a l p r o cesses, a n d c h e m i c a l i n t e r a c t i o n s i n t h e e n v i r o n m e n t (2). O f these c h e m i cals, o n l y 1600 have b e e n e v a l u a t e d for t h e i r fetotoxic a n d teratogenic

po-

t e n t i a l i n a n i m a l s . C l o s e to o n e - h a l f o f these c o m p o u n d s have b e e n s h o w n

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to b e teratogenic i n animals, whereas less t h a n 50 c o m p o u n d s have b e e n s h o w n to b e teratogenic ( i ) or cause spontaneous a b o r t i o n i n h u m a n s (3-5). T h e n u m b e r o f categories into w h i c h these chemicals fall is n u m e r o u s a n d includes pesticides, i n d u s t r i a l c o m p o u n d s , metals, organic solvents, l a b o ratory chemicals, drugs, feed a d d i t i v e s , e n v i r o n m e n t a l c h e m i c a l s , a n d naturally o c c u r r i n g substances such as p l a n t , fungal, a n d b a c t e r i a l b y p r o d u c t s . T h e majority of these chemicals exist i n the e n v i r o n m e n t i n n e g l i g i b l e amounts; however, a small percentage have b e e n s h o w n to persist at toxic levels (2). T h e toxic p o t e n t i a l of c h e m i c a l c o n t a m i n a t i o n o f the e n v i r o n m e n t p r e sents hazards not o n l y to the c u r r e n t p o p u l a t i o n b u t also to future g e n e r a tions b y adverse effects to the genetic m a t e r i a l a n d r e p r o d u c t i o n . E x p o s u r e of b o t h male a n d female organisms to chemicals d u r i n g the r e p r o d u c t i v e cycle can have deleterious effects o n the d e v e l o p m e n t of the fetus. T h e toxic action of e n v i r o n m e n t a l chemicals o n r e p r o d u c t i o n occurs b y a d i r e c t effect on the conceptus or b y effects o n m a t e r n a l organs that can result i n a l t e r e d h o r m o n e secretion a n d hence a l t e r e d r e p r o d u c t i o n (6). E s t i m a t i n g the i n c i d e n c e o f these effects is difficult, a n d d e t e r m i n i n g t h e i r causes is e v e n more c o m p l e x because o f the large n u m b e r o f c o n f o u n d i n g factors. T h e s e factors i n c l u d e b u t are not l i m i t e d to the f o l l o w i n g : i n c o m p l e t e i n f o r m a t i o n c o n c e r n i n g dose, t i m i n g a n d d u r a t i o n of exposure; u n k n o w n interactions a m o n g causes; difficulty i n o b t a i n i n g s p e c i m e n s ; difficulty i n d e t e r m i n i n g e n v i r o n m e n t a l concentrations; the large n u m b e r of p o t e n t i a l causes of abort i o n ; a n d variations i n i n d i v i d u a l s u s c e p t i b i l i t y d u e to differences i n g e n o type (7). T o u n d e r s t a n d these effects o n r e p r o d u c t i o n , this r e v i e w addresses the physiology of p r e g n a n c y a n d fetal d e v e l o p m e n t , the etiology a n d m e c h anisms of r e p r o d u c t i v e failure a n d fetal malformations (teratogenesis), the evaluation of e n v i r o n m e n t a l pollutants for t h e i r d e v e l o p m e n t a l toxicity i n i n v i t r o a n d i n v i v o assays, a n d the assessment o f the toxicity of these c o m pounds i n e p i d e m i o l o g i c a l studies.

Physiology of Pregnancy and Fetal Development G e s t a t i o n is the p e r i o d o f fetal d e v e l o p m e n t f r o m the t i m e o f c o n c e p t i o n to the t i m e of b i r t h . T h e average l e n g t h of gestation i n m a n is 266 days, or approximately 9 m o n t h s (8). G e s t a t i o n is c o m m o n l y d i v i d e d into three p e riods each o f 3 months d u r a t i o n , r e f e r r e d to as the first, second, a n d t h i r d trimesters. T h e greatest damage to the d e v e l o p i n g e m b r y o f o l l o w i n g exposure to toxic c o m p o u n d s occurs d u r i n g the first trimester, w h e n the e m b r y o differentiates. T h e blastocyst, w h i c h is f o r m e d d u r i n g the first w e e k of gestation, consists o f cells that have not u n d e r g o n e differentiation. I n j u r y to a s m a l l n u m b e r o f these cells does not result i n a specific d e v e l o p m e n t a l defect b u t i n an o v e r a l l delay i n the d e v e l o p m e n t of the fetus or i n death i f the dose is

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h i g h (9). E m b r y o n i c differentiation begins d u r i n g the t h i r d w e e k of gestat i o n , w h e n the cells of the blastocyst are segregated into three e m b r y o n i c g e r m layers, r e f e r r e d to as e c t o d e r m , m e s o d e r m , a n d e n d o d e r m . D u r i n g weeks 4 t h r o u g h 8, each of the three g e r m layers gives rise to a n u m b e r of specific groups o f cells, k n o w n as p r i m o r d i a . T h e differentiated cells have m o r e s p e c i a l i z e d metabolic r e q u i r e m e n t s a n d are, therefore, m o r e v u l n e r able to damage b y adverse influences. D a m a g e to a p a r t i c u l a r p r i m o r d i u m at this t i m e m a y later result i n specific structural defects i n the fetus ( F i g u r e 1). O n c o m p l e t i o n of organ formation (organogenesis) at the e n d of the twelfth w e e k , the i n d u c t i o n of major structural defects is no l o n g e r a factor of c o n c e r n (9). T h e p e r i o d f r o m the b e g i n n i n g of the t h i r d m o n t h to b i r t h is k n o w n as the fetal p e r i o d . It is c h a r a c t e r i z e d b y r a p i d g r o w t h o f the b o d y a n d c o n t i n u e d tissue differentiation or histogenesis (10). T h e progress of histogenesis is closely c o r r e l a t e d w i t h the d e v e l o p m e n t o f the f u n c t i o n a l activity o f the fetal organs. A d v e r s e influences d u r i n g this stage of d e v e l o p m e n t result i n microscopic structural defects a n d possibly i n functional abnormalities (9).

- — F e t a l Period (in weeks) 1

2

Prenatal death

3

4

5

6

Major morphological abnormalities Spontaneous abortion

7

8

12

16

- • Full Term

20-36

38

Physiological defects and minor morphological abnormalities

Figure 1. Human developmental stages and susceptibility of organ systems to reproductive toxicants. Dark hatched areas are time periods at which tissue is highly susceptible to environmental toxicants.

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Because structural a n d functional m a t u r a t i o n continues after b i r t h i n m a n y organ systems such as the i m m u n o l o g i c system, nervous system, liver, k i d neys, a n d other e n d o c r i n e organs, c o n c e r n of the possible adverse effects of e n v i r o n m e n t a l factors o n l a t e - m a t u r i n g functions d u r i n g infancy a n d c h i l d h o o d is g r o w i n g (9).

Reproductive Failure and Teratogenesis Interference of n o r m a l d e v e l o p m e n t t h r o u g h exposure to various c o m p o u n d s may have one of four possible outcomes: d e a t h , malformation, g r o w t h retardation, o r organ dysfunction of the fetus (9). D e a t h of the e m b r y o d u r i n g the early stages of gestation (weeks 1 t h r o u g h 4) results i n r e sorption of the conceptus b y the m a t e r n a l system, whereas death of the e m b r y o d u r i n g weeks 4 t h r o u g h 8 results i n heavy b l e e d i n g that is freq u e n t l y u n d e t e c t e d as a fetal death b y the mother. M a n y pregnancies are not diagnosed u n t i l the w o m a n seeks m e d i c a l attention, 8 - 1 0 weeks after conception. D e a t h of the fetus d u r i n g weeks 8 t h r o u g h 36 results i n e x p u l sion of the u t e r i n e contents, r e f e r r e d to as a spontaneous abortion. T h e rate (percentage) of spontaneous abortions is d e t e r m i n e d b y m u l t i p l y i n g the n u m b e r of spontaneous abortions b y 100 a n d d i v i d i n g b y the s u m of the n u m b e r of spontaneous abortions a n d the n u m b e r of b i r t h s i n a specific p o p ulation (II). T h e estimated rate of abortion r e p o r t e d i n the l i t e r a t u r e is v a r i able. T h e reason for these discrepancies is the i n c l u s i o n or exclusion of the e m b r y o n i c p e r i o d (weeks 1-8) i n the estimation of spontaneous a b o r t i o n . O n e source estimates that 7 5 - 7 8 % of all conceptions are r e s o r b e d or a b o r t e d (12), whereas another estimates the rate as b e i n g b e t w e e n 30 a n d 5 0 % (13). A n o t h e r study indicates the i n c i d e n c e of spontaneous a b o r t i o n of r e c o g n i z e d pregnancies to be 1 5 - 2 0 % (3). U n t i l recently, it was i m p o s s i b l e to detect pregnancy i n its early stages, a n d thus the i n c i d e n c e of p r e g n a n c y wastage was frequently u n d e r e s t i m a t e d because of the inadequacies of the methods of analysis of pregnancy loss (13). T h e d e v e l o p m e n t of a n e w a n d i n n o v a t i v e immunoassay to detect the presence of u r i n a r y @ c h o r i o n i c gonadotropin, a h o r m o n e p r o d u c e d b y the placenta 9 days after c o n c e p t i o n (14), has made it possible to d e t e r m i n e m o r e accurately the rate of spontaneous a b o r t i o n i n the first 2 months of pregnancy. T h e outcome of pregnancy f o l l o w i n g exposure to a c h e m i c a l substance depends o n the l e n g t h of exposure, the stage of fetal d e v e l o p m e n t at the t i m e of exposure, the m a g n i t u d e of exposure, a n d the nature of the c h e m i c a l substance. N o t a l l exposures result i n fetal death a n d spontaneous a b o r t i o n . M a n y exposures result i n teratogenesis, w h i c h is d e f i n e d as the p r o d u c t i o n of any significant change i n structure or f u n c t i o n of the fetus that can be detected d u r i n g the postnatal p e r i o d (I). T h e possible consequences of t e r atogenesis are death of the fetus (spontaneous abortion), congenital malform a t i o n , g r o w t h retardation, a n d functional d i s o r d e r of an organ system (15).

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T h e final manifestation i n the fetus depends p r i m a r i l y o n the s u s c e p t i b i l i t y of the e m b r y o at the t i m e of exposure a n d the m a g n i t u d e of the dose (9). T h e dose of the c h e m i c a l that reaches the e m b r y o or fetus d e p e n d s o n several factors: the m a g n i t u d e of the dose, the p h y s i c a l f o r m of the agent, the route of exposure, the rate of absorption b y the m a t e r n a l system, a n d the effectiveness of m a t e r n a l metabolic processes that function to protect the fetus. T h e p r i m a r y f u n c t i o n of m a t e r n a l m e t a b o l i s m is to r e d u c e the b l o o d concentration of the toxicant so that the n u m b e r of molecules free to cross the placenta is m i n i m i z e d . T h i s is a c c o m p l i s h e d b y detoxification, exc r e t i o n , or storage of the toxicant b y the m a t e r n a l system. T h e e m b r y o or fetus is thought to have a t h r e s h o l d dose for most toxic substances. B e l o w this dose, no effect occurs; above i t , p e r m a n e n t changes i n the fetus may b e i n d u c e d (9). A l t h o u g h the e m b r y o or fetus m a y o n l y receive a s m a l l fraction of the m a t e r n a l dose, it is f r e q u e n t l y sufficient to p r o d u c e an e m b r y o t o x i c or teratogenic response. Because of the extraordinary sensitivity of the c o n ceptus, it is possible for e m b r y o t o x i c i t y to occur i n the absence of m a t e r n a l toxicity f o l l o w i n g exposure (16). Teratogens may alter n o r m a l d e v e l o p m e n t i n a n u m b e r of ways. T h e s e i n c l u d e gene mutations, c h r o m o s o m e breaks, interference w i t h mitosis of cells, alteration i n the n o r m a l f u n c t i o n of the genes, lack of substrates r e q u i r e d for g r o w t h t h r o u g h alteration i n the source of energy, i n h i b i t i o n of certain e n z y m e s , an osmotic f l u i d imbalance w i t h i n the d e v e l o p i n g fetus, a n d changes i n m e m b r a n e characteristics. A l l of these c e l l u l a r processes, w h i c h are f u n d a m e n t a l to the d e v e l o p m e n t of an e m b r y o , can be m e a s u r e d i n v i t r o a n d thus can be s t u d i e d separately. A l t h o u g h m a n y teratogenic assays are r e l a t i v e l y cheap a n d can be p e r f o r m e d r a p i d l y a n d cheaply, they m o d e l o n l y a single d e v e l o p m e n t a l process. A n alternative strategy b e i n g evaluated is the d e v e l o p m e n t of m o r e c o m p l i c a t e d assays i n w h i c h a n u m b e r of d e v e l o p m e n t a l processes, i n c l u d i n g c e l l g r o w t h a n d d i v i s i o n , c e l l - t o - c e l l interaction a n d c o m m u n i c a t i o n , a n d differentiation are m e a s u r e d s i m u l t a neously.

In Vitro and In Vivo Assays To Monitor for Reproductive Toxicants R e p r o d u c t i v e toxicity assays have b e e n d e s i g n e d to detect embryotoxicity, embryolethality, a n d teratogenicity. S o m e assays may detect a l l three e n d points, b u t most detect o n l y one. T h r e e levels of s c r e e n i n g are u t i l i z e d i n the evaluation of a c o m p o u n d for its effects o n the d e v e l o p i n g fetus. 1. P r e s c r e e n i n g e m p l o y s s i m p l e , i n e x p e n s i v e , s h o r t - t e r m testi n g a n d is most c o m m o n l y p e r f o r m e d i n v i t r o (17,18). 2. A n i m a l testing involves the testing of c h e m i c a l agents for t e r atogenicity i n s m a l l laboratory animals.

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ENVIRONMENTAL EPIDEMIOLOGY 3. E p i d e m i o l o g i c a l studies i n h u m a n s attempt to show an association b e t w e e n a c h e m i c a l a n d its r e p r o d u c t i v e effect (I).

In Vitro Short-Term Assays. I n v i t r o assays can b e p e r f o r m e d b y u s i n g cells, tissues, or intact organisms of various levels of complexity. E s tablished c e l l lines assess the effect of chemicals o n a single d e v e l o p m e n t a l l y relevant e n d p o i n t of toxicity; p r i m a r y cultures d e r i v e d f r o m e m b r y o s assess one or m o r e of the d e v e l o p m e n t a l processes; cultures of organ p r i m o r d i a d e r i v e d f r o m embryos assess the d e v e l o p m e n t of a p a r t i c u l a r organ; a n d intact e m b r y o s , i n c l u d i n g rodent e m b r y o s i n c u l t u r e or f r e e - l i v i n g e m b r y o s of s u b m a m m a l i a n species, assess the toxicity of a c o m p o u n d s o n a n u m b e r of d e v e l o p m e n t a l processes. M a n y c o m p o u n d s e x a m i n e d i n these assays have b e e n s h o w n to be e m b r y o l e t h a l or to r e t a r d g r o w t h b u t not to b e t e r atogenic. T h e r e is some confusion, however, as to w h e t h e r the degree of damage is related to dose or w h e t h e r some of the assays are biased t o w a r d d e t e c t i n g o n l y e m b r y o l e t h a l e n d points o r growth-retardation effects. I n fact, it has b e e n stated that m a n y of the i n v i t r o tests are d e s i g n e d to detect e m b r y o l e t h a l i t y a n d not teratogenicity. S o m e i n v i t r o assays can be u s e d to study the m e c h a n i s m b y w h i c h a c o m p o u n d elicits d e v e l o p m e n t a l toxicity. I n assays u s i n g r o d e n t e m b r y o c u l ture, the m a t e r n a l factors that m a y c o n t r i b u t e or alleviate toxicity are e l i m i n a t e d , as the embryos are r e m o v e d from the m o t h e r a n d c u l t u r e d i n v i t r o . Because these embryos are u n a b l e to metabolize chemicals to active i n t e r mediates, the role of m a m m a l i a n e n z y m e s i n the m e t a b o l i s m of the test c o m p o u n d can b e evaluated. F o r example, it has b e e n s h o w n that a teratogenic effect is e l i c i t e d b y c y c l o p h o s p h a m i d e i n rodent e m b r y o s i n c u l t u r e w h e n adult m e t a b o l i z i n g enzymes are i n c l u d e d i n the assay b u t not w h e n these enzymes are o m i t t e d . Interestingly, o n l y two metabolites of c y c l o p h o s p h a m i d e have b e e n s h o w n to be teratogenic (19, 20). F o r any i n v i t r o screen to be useful, it must have four attributes: p r e dictivity, sensitivity, specificity, a n d elicitation of a quantitative response. It must be p r e d i c t i v e of the toxicity of a c o m p o u n d i n h u m a n s , a n d it must be quantitative, that is, there must be a d o s e - r e s p o n s e relationship so that w i t h increasing concentrations of the c h e m i c a l , an increase i n an effect is o b served. C h e m i c a l s have a t h r e s h o l d at w h i c h they cause d e v e l o p m e n t a l toxicity. Because v e r y few h u m a n e p i d e m i o l o g i c a l studies of the teratogenicity of chemicals have b e e n u n d e r t a k e n , these screens have b e e n u s e d to p r e d i c t teratogenicity i n h u m a n s indirectly. Difficulties i n extrapolation occur, b e cause the dose levels r e q u i r e d to e l i c i t d e v e l o p m e n t a l toxicity i n h u m a n s are largely u n k n o w n , except i n the case of a few drugs such as t h a l i d o m i d e a n d methotrexate. A sensitive test is one that successfully identifies d e v e l o p m e n t a l toxicants w i t h few false-negative results. A specific screen successfully classifies nontoxicants w i t h v e r y few false-positive results. F a l s e - n e g a tive or false-positive findings can be m i n i m i z e d b y m a n i p u l a t i o n . F o r

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e x a m p l e , Steele et a l . (21) a n d t h e N a t i o n a l Toxicology P r o g r a m (22) f o u n d that m o r e false-negative results are generated w h e n data f r o m o n l y t h e l o w est concentrations of 44 substances, tested at concentrations o f 0 . 5 - 4 0 m M , w e r e evaluated. A t a concentration o f 0.05 m M , a l l the nonteratogens w e r e c o r r e c t l y classified, g i v i n g a n o v e r a l l accuracy o f 5 4 % . A s t h e c o n c e n t r a t i o n was increased, t h e n u m b e r o f false-negative findings decreased a n d t h e n u m b e r of false-positive results increased. O v e r a l l , t h e accuracy was highest (71%) w h e n t h e concentration was 1 0 - 2 0 m M . A t t h e highest c o n c e n t r a t i o n , accuracy was l o w e r a n d the n u m b e r o f false-positive results b e c a m e ext r e m e l y h i g h , close to 9 0 % . T h e accuracy o f t h e b a t t e r y was lowest at t h e concentrations at w h i c h false-positive a n d false-negative results w e r e o p t i mized. N o n e of t h e i n v i t r o assays has b e e n sufficiently v a l i d a t e d o r s h o w n to b e sufficiently p r e d i c t i v e to b e accepted as a n all-purpose screen for d e v e l o p m e n t a l toxicants (Table I). H o w e v e r , o n e i m p o r t a n t a p p l i c a t i o n o f r a p i d i n v i t r o screens is i n r a n k i n g chemicals w i t h i n a f a m i l y for d e v e l o p m e n t a l toxi c i t y so that c o m p o u n d s w i t h t h e highest p o t e n c y can b e s t u d i e d i n m o r e d e p t h i n other systems. F o u r such studies w e r e r e c e n t l y r e p o r t e d : o n e b y O g l e s b y et a l . (23) o n t h e e m b r y o t o x i c i t y o f p a r a s u b s t i t u t e d p h e n o l s i n t h e p o s t i m p l a n t a t i o n assay, a second b y M a y u r a et a l . (24) o n c h l o r i n a t e d p h e nols u s i n g t h e Hydra attenuata a n d p o s t i m p l a n t a t i o n rat e m b r y o assays, a t h i r d b y K i s t l e r (25) o n t h e teratogenic p o t e n t i a l o f a n u m b e r o f synthetic retinoids i n the i n v i t r o l i m b b u d c e l l c u l t u r e screen, a n d a f o u r t h b y R a w l ings et a l . (26), w h o r a n k e d alkoxy acids (metabolites o f g l y c o l ethers) i n t h e rodent p o s t i m p l a n t a t i o n assay. A l t h o u g h t h e r a n k i n g system is i n f o r m a t i v e , it is difficult to extrapolate f r o m these data to t h e c o n c e n t r a t i o n that m i g h t b e expected to e l i c i t a toxic effect i n e m b r y o s i n v i v o o r i n h u m a n s . Table I.

Predictability of Short-Term Tests

Assay

Predictability

Cell culture Mouse ovarian tumor H u m a n embryonic palatal mesenchyme Neuroblastoma Organ Culture Mouse limb b u d Whole Embryo Culture A . Invertebrates Hydra B. Non-mammalian Frog Chick C . Mammalian Rats—postimplantation S O U R C E : Adapted from Daston and D'Amato (18).

No. of Compounds

79 64 86

178 99 57

89

27

100

24

93

—

43 130

97

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A second approach, u s e d b y F a b r o a n d B r o w n (27) a n d J o h n s o n (28), has b e e n the d e t e r m i n a t i o n o f the ratio b e t w e e n the concentration o f a c o m p o u n d that causes toxicity i n an adult to one that causes d e v e l o p m e n t a l toxicity i n an e m b r y o or fetus. F a b r o a n d B r o w n (27) advocated u s i n g a relative teratogenic i n d e x (RTI), w h i c h t h e y calculated b y d i v i d i n g the adult dose L D j (the concentration that kills 1 % of adults) b y the teratogenic dose T D (the concentration that causes teratogenesis i n 5 % of embryos). J o h n s o n (28) p r o p o s e d an A / D ratio, w h i c h h e d e f i n e d as the lowest o b s e r v e d adverse effect l e v e l ( L O A E L ) of the adult toxic dose (A) d i v i d e d b y the L O A E L for d e v e l o p m e n t a l toxicity (D). Recently, this was m o d i f i e d b y d i v i d i n g the adult no adverse effect l e v e l ( N O A E L ) b y the d e v e l o p m e n t a l N O A E L (29). T h e R T I a n d A / D ratio are f u n d a m e n t a l l y similar, b u t the R T I is a statistical value calculated f r o m a dose—response c u r v e , whereas the A / D ratio is a ratio of L O A E L s i n w h i c h no consideration is g i v e n to the slope of the d o s e response c u r v e . B o t h ratios are based o n the concept that most teratogens are specifically toxic to the e m b r y o a n d m u c h less toxic to adults. J o h n s o n stated that a c r i t i c a l A / D ratio of 3 or h i g h e r is i n d i c a t i v e of a r e p r o d u c t i v e h a z a r d (28). Teratogens, therefore, w o u l d have a h i g h R T I o r A / D ratio. I n practice, as far as h u m a n teratology is c o n c e r n e d , this m a y not b e t r u e , because c e r t a i n c h e m o t h e r a p e u t i c agents, anticonvulsants, a n d e t h a n o l are o n l y teratogenic at o r near levels that cause m a t e r n a l toxicity. 5

Tissue Culture. C e l l lines have b e e n established f r o m mouse ovarian t u m o r ( M O T ) cells (30), h u m a n e m b r y o n i c palatal m e s e n c h y m e cells (31), a n d h u m a n n e u r o b l a s t o m a cells (32). C e l l attachment, g r o w t h i n h i b i t i o n , a n d i n h i b i t i o n or s t i m u l a t i o n of differentiation are the respective parameters m e a s u r e d after exposure to nontoxic doses of the test c h e m i c a l . T h e rationale of the M O T assay is based o n the g e n e r i c p r o p e r t y of e m b r y o n a l cells to adhere to surfaces or lectins. T h e assay is p e r f o r m e d b y a d d i n g the test c h e m i c a l to a suspension of M O T cells p r e t r e a t e d w i t h r a d i o l a b e l e d t h y m i d i n e . A plastic sheet coated w i t h l e c t i n is p l a c e d i n the c u l ture bottle so that the M O T cells can adhere to it. I n h i b i t i o n of adherence is m e a s u r e d b y d e t e r m i n i n g the radioactive counts associated w i t h the plastic sheet a n d the m e d i u m a n d b y c o m p a r i n g these values w i t h the c o n t r o l . I n a study of m o r e than 100 teratogens a n d nonteratogens, a 7 9 % accuracy c o m p a r e d w i t h i n v i v o results was o b t a i n e d . T h e assay i n w h i c h h u m a n e m b r y o n i c palatal m e s e n c h y m e cells are u s e d is based o n the p r e m i s e that teratogens w i l l i n h i b i t the g r o w t h of these r a p i d l y d i v i d i n g cells. G r o w t h i n h i b i t i o n is m e a s u r e d b y c e l l c o u n t i n g . A c r i t i c a l concentration of 1 m M was chosen for the u p p e r m o s t concentration i n the assay. A n evaluation o f k n o w n teratogens a n d nonteratogens s h o w e d that 6 6 % of the teratogens w e r e correctly i d e n t i f i e d as positives a n d that 6 0 % of the nonteratogens w e r e i d e n t i f i e d as true negatives (33). T h e t h i r d assay uses h u m a n n e u r o b l a s t o m a cells d e r i v e d f r o m c h i l d h o o d malignant tumors that i n c u l t u r e can differentiate into neurites. O f 42 te-

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ratogens tested, 7 6 % s t i m u l a t e d differentiation a n d 7% i n h i b i t e d differentiat i o n , a n d of the nonteratogens tested, 7 4 % d i d not alter differentiation (34). Organ Culture. Mouse Embryo Limb Bud Cell Culture Assay. I n this assay, 11- to 12-day-old m i c e e m b r y o s are r e m o v e d from the dams a n d the l i m b b u d dissected away from the e m b r y o u n d e r a stereomicroscope (35). T h e b u d s are i n c u b a t e d for several days i n a n u t r i e n t m e d i u m i n the p r e s ence or absence o f the test c h e m i c a l . T h e c u l t u r e d l i m b s can t h e n b e scored qualitatively for the presence or absence of malformations a n d quantitatively for the amount of cartilage f o r m e d i n the l i m b b y staining w i t h t o l u i d i n e b l u e . A quantitative approach to the m e a s u r e m e n t of the formation of cartilage is the d e t e r m i n a t i o n of the uptake of H - p r o l i n e or ^S-sulfate into c h o n d r o i t i n sulfate, the major c o m p o n e n t of cartilage. A modification of this m e t h o d , d e v e l o p e d b y G u n t a k a t t a et a l . (36), d e p e n d s o n the disassociation of the l i m b b u d into i n d i v i d u a l cells, w h i c h are t h e n exposed to ^ - t h y m i dine and -sulfate i n c u l t u r e . A n o v e r a l l accuracy of 8 9 % was r e p o r t e d b y G u n t a k a t t a et a l . (36) after the testing of b o t h teratogens a n d nonteratogens that do not r e q u i r e metabolic activation. A n advantage of this assay is the fact that the l i m b b u d s can be c u l t u r e d for 9 days, d u r i n g w h i c h t i m e c a r t i lagenous b o n e structures d e v e l o p . T h i s p e r m i t s the evaluation of a n u m b e r of e n d points, i n c l u d i n g chondrogenesis a n d collagen biosynthesis (35). M e t a b o l i z i n g e n z y m e s a d d e d e i t h e r as cell-free extracts or intact cells have successfully b e e n used i n the assay. 3

Whole Embryo Systems. Invertebrate Embryos. H y d r a , a test syst e m , w h i c h was d e v e l o p e d b y Johnson a n d co-workers (28, 37, 38), utilizes the freshwater coelenterate H. attenuata a n d is based o n the reaggregation a n d differentiation of the coelenterate. A d u l t h y d r a polyps are exposed to l o g concentrations of the test substance (1 X 1 0 " to 1 X 10 ( x L / m L ) a n d the m i n i m u m concentration r e q u i r e d to p r o d u c e a toxic response is determ i n e d . T h e dose of a test c o m p o u n d that results i n toxicity i n the adult (A) is t h e n c o m p a r e d w i t h that w h i c h causes d e v e l o p m e n t a l toxicity (D). T h e e m b r y o is p r e p a r e d b y disassociating 7 0 0 - 1 0 0 0 h y d r a into t h e i r c o m p o n e n t cells to form an "artificial e m b r y o " . These preparations consist of two c e l l types, the major c o m p o n e n t of w h i c h consists of groups of cells that give rise to a n e w p o p u l a t i o n of adult polyps i n about 90 h . T h e second g r o u p consists of a small n u m b e r of undifferentiated interstitial cells capable of r a p i d p r o liferation a n d subsequent differentiation into a t y p i c a l adult h y d r a . T h e c o n centration that prevents the formation of the adults f r o m the u n d i f f e r e n tiated cells is the dose that causes d e v e l o p m e n t a l toxicity. A n A / D ratio can t h e n b e calculated. I f this ratio is greater t h a n 3, it has b e e n suggested that the c o m p o u n d is a r e p r o d u c t i v e toxicant. M o r e than 30 c o m p o u n d s that have b e e n tested i n this assay h a d i n v i t r o A / D ratios that c o m p a r e d w e l l w i t h i n v i v o A / D ratios (38, 39). T h u s , this assay seems to b e e x t r e m e l y p r o m i s i n g i n p r e d i c t i n g the r e p r o d u c t i v e toxicity of c e r t a i n c o m p o u n d s . 3

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Planaria, a system d e v i s e d b y Best a n d M o r i t a (40) u s i n g the freshwater planarian Dugesia dortocephala, has b e e n u s e d to assess the effects of t o x i cants o n r e p r o d u c t i o n a n d teratology. P l a n a r i a e x h i b i t b i l a t e r a l s y m m e t r y , are h e r m a p h r o d i t i c , can r e p r o d u c e sexually o r asexually, a n d have a t r u e b r a i n i n d i c a t i v e of c o m p l e x behavior. P l a n a r i a r e p r o d u c e b y fission i n the transverse plane w i t h regeneration of the anterior or posterior section. R e generation involves the m i g r a t i o n of undifferentiated stem cells or neoblasts. Because this process resembles embryogenesis, it is thought to be sensitive to teratogenic agents. T w o e x p e r i m e n t a l approaches have b e e n d e v e l o p e d : (1) S u r g i c a l fragments of planaria can be exposed to the test m a t e r i a l d u r i n g regeneration to assess the effect of the c o m p o u n d o n the r a p i d m u l t i p l i c a t i o n of cells, or (2) intact p l a n a r i a can be exposed to the test m a t e r i a l a n d e x a m i n e d for m o r p h o l o g i c o r b e h a v i o r a l abnormalities. T h i s assay has b e e n s h o w n to be less sensitive to three k n o w n teratogens than the h y d r a assay (41). Lower Vertebrate Embryos. F i s h , (Japanese m e d a k a , r a i n b o w trout, fathead m i n n o w , a n d z e b r a fish), have b e e n u s e d to test for adverse r e p r o d u c t i v e outcomes, b u t o n l y a few c o m p o u n d s have b e e n evaluated i n fish. T h e m e d a k a seems to be the most p r o m i s i n g species, because it is s m a l l , oviparous (producing eggs that hatch outside the body), a n d easy to c u l t u r e i n the laboratory; m u c h of its biology is k n o w n ; a n d it has a clear c h o r i o n (outer e m b r y o n i c membrane) so that adverse changes can easily be seen at various stages of d e v e l o p m e n t . A n u m b e r of e n d points can be m o n i t o r e d , i n c l u d i n g d e a t h , specific malformations, d e l a y e d h a t c h i n g , a n d functional ones, such as i m p a i r e d s w i m m i n g m o v e m e n t s . E x p o s u r e can be d o n e d i rectly into the water (42) or b y m i c r o i n j e c t i o n of the e m b r y o at various stages of d e v e l o p m e n t (43). T h e most extensive studies have b e e n u n d e r t a k e n b y B i r g e et a l . (42), w h o found r a i n b o w trout to b e the most sensitive to t e r a togenic agents, followed b y goldfish, sunfish, a n d l a r g e m o u t h bass. I r r e spective of w h e t h e r exposure was to inorganic or organic c o m p o u n d s , the majority of the malformations i n v o l v e d the skeletal system. I n frogs, the frog e m b r y o teratogenesis assay (known as F E T A X ) , uses the S o u t h A f r i c a n c l a w e d toad, Xenopus laevis. F e r t i l i z e d eggs are exposed to the toxicant i n question for the first 96 h of d e v e l o p m e n t , a n d the m e d i u m is r e p l a c e d e v e r y 24 h . A f t e r 4 days, the s u r v i v i n g e m b r y o s are fixed i n f o r m a l i n , malformations n o t e d , a n d the d e v e l o p m e n t a l stage d e t e r m i n e d (44). T h e n u m b e r of dead e m b r y o s are r e c o r d e d daily. A 9 6 - h L C (mortality) a n d E C (concentration i n d u c i n g terata i n 5 0 % of the s u r v i v i n g e m bryos) are d e t e r m i n e d . T h e teratogenic i n d e x ( L C / E C 5 o ) is calculated. B o t h methotrexate a n d 5-azacytidine, w h i c h are h i g h l y teratogenic i n F E T A X , have also b e e n s h o w n to be teratogenic i n m a m m a l i a n a n d i n h u m a n studies. A s p a r t a m e a n d amaranth, w h i c h are b o t h w e a k teratogens or negative i n F E T A X , have not b e e n s h o w n to be teratogenic i n h u m a n s (45). E y e defects a n d anencephaly (lack of c e r e b r a l hemispheres) have b e e n o b s e r v e d i n b o t h 5 0

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m a m m a l i a n assays a n d F E T A X (46, 47) f o l l o w i n g exposure to methotrexate. I n h u m a n s , absence of frontal bones a n d p r e m a t u r e c l o s i n g o f the b r a i n s u tures have b e e n d o c u m e n t e d i n c h i l d r e n whose mothers w e r e exposed to methotrexate (48). It has b e e n shown that c y t o c h r o m e P-450 e n z y m e s can be a d d e d to the F E T A X assay to metabolize teratogens such as d i p h e n y l h y d a n t o i n to active i n t e r m e d i a t e s . C r a n i o f a c i a l defects have b e e n f o u n d i n c h i l d r e n b o r n to mothers treated w i t h h y d a n t o i n for e p i l e p s y d u r i n g p r e g nancy (49). Mammalian Embryos. I n the p r e i m p l a n t a t i o n assay early m a m m a l i a n e m b r y o s are collected t h r o u g h the blastocyst stage a n d c u l t u r e d i n i n v i t r o i n s e r u m - a m e n d e d m e d i a (50, 51). E x p o s u r e of the e m b r y o can b e u n d e r taken d u r i n g c u l t u r i n g or b y exposure o f the m o t h e r before the e m b r y o is f l u s h e d from the r e p r o d u c t i v e tract. M o r p h o l o g i c a l alterations i n d e v e l o p m e n t to the blastula stage, h a t c h i n g from the zona p e l l u c i d a , attachment to a c u l t u r e d i s h , a n d o u t g r o w t h of the t h r e e types o f cells, are the e n d points m e a s u r e d i n the assay. To date, o n l y a few c o m p o u n d s (cyclophosphamide, c a d m i u m c h l o r i d e , i r r a d i a t i o n , m e t h y l m e r c u r y , m e r c u r i c c h l o r i d e , potassium dichromate, nitroquinoline N-oxide, mitomycin C , bleomycin, diethylstilbestrol, a n d ochratoxin A) have b e e n evaluated for r e p r o d u c t i v e toxicity u s i n g this assay (52). C y t o l o g i c a l e n d p o i n t s , such as sister c h r o m a t i d exchanges a n d c h r o m o s o m a l aberrations, have b e e n d e t e c t e d i n the e m b r y o s that have shown adverse effects. E x t r a p o l a t i o n of these findings to h u m a n s is difficult, because u n d e r n o r m a l c o n d i t i o n s , o n l y 1 0 % of e m b r y o s i n the mouse are lost d u r i n g the first few days after fertilization, whereas i n h u mans, this may be as h i g h as 7 5 % (53). T h e p o s t i m p l a n t a t i o n assay, w h i c h was o r i g i n a l l y d e v e l o p e d b y N e w (54), involves the r e m o v a l of 9- or 10-day-old (equivalent to 4 - 1 2 somites) m i c e or rat e m b r y o s from the uterus f o l l o w e d b y i n v i t r o c u l t u r i n g i n rat a n d h u m a n s e r u m for a p e r i o d r a n g i n g from 1 to 48 h (55-57). D u r i n g i n c u b a t i o n , test chemicals are a d d e d to the m e d i u m . A f t e r exposure, e m b r y o s are exa m i n e d for changes i n heartbeat, y o l k sac c i r c u l a t i o n , c r o w n r u m p l e n g t h , somite n u m b e r s , closed or o p e n n e u r a l tubes, otic a n d optic vesicles, a n d d e v e l o p m e n t of l i m b s . A m o r p h o l o g i c a l s c o r i n g system for teratogenic changes i n rats has b e e n d e v e l o p e d b y B r o w n a n d F a b r o (58). I n a d d i t i o n to m o r p h o l o g i c a l characteristics, various b i o c h e m i c a l parameters, i n c l u d i n g p r o t e i n content, D N A content, uptake of H - t h y m i d i n e a n d C - a m i n o acids i n t o D N A a n d p r o t e i n s , respectively, can b e m e a s u r e d . 3

1 4

A study b y S c h m i d (59), i n w h i c h a c t i n o m y c i n D , a z a t h i o p r i n e , c o l c h i c i n e , c o u m a r i n , d o x o r u b i c i n , h y c a n t h o n e , ketoconazole, methotrexate, a n d t r y p a n b l u e w e r e u s e d , d e m o n s t r a t e d that the n u m b e r of somites after exposure was as r e l i a b l e an i n d e x of teratogenicity as any other m o r p h o l o g i c a l change. H o w e v e r , the author also d e t e r m i n e d that each c o m p o u n d e l i c i t e d a specific response r e s u l t i n g i n a defect at a p a r t i c u l a r e m b r y o n i c sites. T h e

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concentrations that e l i c i t e d a teratogenic response r a n g e d f r o m 3 n g / m L (act i n o m y c i n D ) to 150 j x g / m L (trypan blue). Results of this study (59) w e r e similar to those o b t a i n e d i n i n v i v o studies w i t h a c t i n o m y c i n D (60), h y c a n thone (61), a n d methotrexate (62). A l t h o u g h c o u m a r i n was s h o w n to cause c e n t r a l nervous system malformations i n this assay (89) a n d has b e e n i m p l i cated i n congenital defects of the c e n t r a l nervous system i n h u m a n s (63, 64), it was negative i n i n v i v o studies i n rabbits (65) a n d m i c e (66). Advantages and Disadvantages of In Vitro Assays. I n v i t r o assays have m a n y advantages as w e l l as a n u m b e r of i n h e r e n t p r o b l e m s . Advantages are the r e l a t i v e l y l o w costs, the s i m p l i c i t y a n d the r a p i d i t y w i t h w h i c h results are o b t a i n e d , the m a n y p e r m u t a t i o n s that can b e u s e d for the scientific p r o tocol, the precise c o n t r o l o v e r exposure to a specific d e v e l o p m e n t a l stage of the e m b r y o , the c o m p o s i t i o n of the c u l t u r e m e d i u m , the concentration of the test c o m p o u n d , a n d the d u r a t i o n of exposure. T h e major disadvantages i n c l u d e the isolation of the e m b r y o from the mother, thus e l i m i n a t i n g the m o d u l a t i o n o f the m a t e r n a l m e t a b o l i c system a n d the difficulty i n assessing the l o n g - t e r m consequences o f b r i e f e m b r y o n i c exposures u s e d i n these assays. A d d i t i o n a l p r o b l e m s i n c l u d e the lack of standardization of the methods a n d quantitation of n o r m a l a n d a b n o r m a l e m b r y o n i c d e v e l o p m e n t . T h e i n fluence of c h r o n i c exposure cannot be a n a l y z e d b y any of these m e t h o d o l o gies, a n d the extrapolation of the results f o u n d i n laboratory animals to that of h u m a n s is difficult. H o w e v e r , i n v i t r o assays do have p o t e n t i a l value for the f u t u r e , as a good c o r r e l a t i o n b e t w e e n i n v i t r o a n d i n v i v o studies for a few c o m p o u n d s has b e e n d o c u m e n t e d .

In Vivo M a m m a l i a n Assays. Mammalian Three-Generation Studies. I n these r e p r o d u c t i v e toxicity tests, t h r e e generations of animals are exposed to the toxic agent i n q u e s t i o n at various dose levels. T h e test s u b stance is a d m i n i s t e r e d to b o t h parents t h r o u g h o u t the study; the highest dose l e v e l is toxic to the test a n i m a l a n d the lowest is i n n o c u o u s (67). F o l l o w i n g exposure of the animals d u r i n g gestation, various d e v e l o p m e n t a l p a rameters are m e a s u r e d i n the p u p s . O f f s p r i n g of the m a t i n g are c o n t i n u o u s l y exposed to the c o m p o u n d , w e a n e d , a l l o w e d to m a t u r e , a n d m a t e d a m o n g themselves. T h i s p r o c e d u r e is f o l l o w e d for t h r e e s u c c e e d i n g generations. T h e advantage of this t e c h n i q u e is the d e t e r m i n a t i o n of the effect d u r i n g i n utero exposure a n d subsequent r e p r o d u c t i v e p e r f o r m a n c e . A c o m p l i c a t i o n that does arise is the selection of the animals for m a t i n g for the s u c c e e d i n g generations. F e t a l s u r v i v a l , l i t t e r size, a n d w e i g h t of the p u p s may affect the selection process. A t the e n d of each r e p r o d u c t i v e c y c l e , a l l the p u p s are e x a m i n e d for p h y s i c a l abnormalities. T h e n u m b e r of v i a b l e , s t i l l b o r n , a n d dead p u p s i n each l i t t e r is r e c o r d e d , a n d the n u m b e r s of survivors o n days 1, 4, 7, 14, a n d 21 are r e c o r d e d . B o d y w e i g h t of the p u p s is r e c o r d e d at w e a n i n g . T w o i n d i c e s , the gestation i n d e x a n d the v i a b i l i t y i n d e x (VI), can

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be calculated. T h e gestation i n d e x , w h i c h is a measure of the percentage o f pregnancies r e s u l t i n g i n l i v e litters, is not a m e a n i n g f u l measure, because i t does n o t take into account s t i l l b o r n p u p s unless t h e w h o l e l i t t e r succumbs. T h e V I refers to t h e percentage of p u p s that s u r v i v e for a specific p e r i o d . T h i s i n d e x is e x t r e m e l y i m p o r t a n t , because early s u r v i v a l o f t h e p u p s m a y b e d e p e n d e n t o n the excretion o f a toxic c o m p o u n d into the m i l k o f t h e lactating d a m a n d subsequent ingestion b y t h e p u p s . I n these three-generation studies, a n u m b e r o f parameters cannot b e m e a s u r e d . F o r example, t h e actual litter size is s e l d o m k n o w n , because the mothers are not sacrificed after p a r t u r i t i o n . N o i n f o r m a t i o n is c o l l e c t e d o n s p e r m m o t i l i t y o r viability. G r o u p s o f u n t r e a t e d c o n t r o l animals m u s t b e i n c l u d e d i n these studies so that comparisons w i t h t h e exposed g r o u p c a n be made. T h e r e p r o d u c t i v e organs of o n l y pups not b e i n g u s e d for b r e e d i n g can b e evaluated b y w e i g h i n g or b y e x a m i n a t i o n for histological changes. I n most studies, o n l y the male r e p r o d u c t i v e organ, that is, t h e testicle, is s t u d i e d , b u t i n some situations, the ovary m a y be e x a m i n e d . Mammalian

Single-Generation

Studies.

In single-generation

repro-

d u c t i v e studies, b o t h male a n d female animals receive the c o m p o u n d for 60 days p r i o r to c o n c e p t i o n a n d t h e n t h r o u g h gestation. T h e effect o n l i b i d o , estrus cycle, r e p r o d u c t i v e capability, toxicity of t h e c o m p o u n d to b o t h m o t h e r a n d fetus, postnatal p u p d e v e l o p m e n t , a n d adequacy of lactation i n the m o t h e r c a n b e evaluated. I n these c o n v e n t i o n a l r e p r o d u c t i o n studies, the animals are m a t e d once o r twice f o l l o w i n g exposure to t h e test c h e m i c a l . A n e w protocol, k n o w n as t h e F e r t i l i t y Assessment b y C o n t i n u o u s B r e e d i n g , w h i c h has b e e n d e v e l o p e d b y t h e N a t i o n a l Toxicological P r o g r a m ( N T P ) , allows m u l t i p l e matings for each p a i r o f animals d u r i n g t h e 14 weeks o f cohabitation a n d exposure to the toxicant. I n these studies, females generally d e l i v e r four to five litters d u r i n g t h e exposure p e r i o d ; thus, a m e a n i n g f u l fertility index can be calculated (68). A n o t h e r approach, o n e that is used m o r e frequently, is t h e a d m i n i s t r a t i o n of the toxicant to t h e m o t h e r f o l l o w i n g m a t i n g . S e v e r a l dose levels of the c h e m i c a l are a d m i n i s t e r e d to different groups o f females. T h e highest dose is based o n t h e m a x i m u m tolerated dose ( M T D ) a n d f r e q u e n t l y results i n m a t e r n a l toxicity, w h i l e t h e l o w e r doses are set at specific fractions o f the M T D . T o p r e v e n t c a n n i b a l i s m of the p u p s , t h e fetuses are usually d e l i v e r e d b y caeserean section p r i o r to t h e i r expected date of b i r t h . T h e n e w b o r n p u p s are e x a m i n e d for external a n d i n t e r n a l abnormalities. A major p r o b l e m associated w i t h this approach is that teratogenic changes that w i l l o c c u r later i n life are not detected. A m o d i f i e d e x p e r i m e n t a l p r o t o c o l that has b e e n u s e d b y m a n y investigators is exposure o f the rodents o n days 1 t h r o u g h 19 o f gestation to t h e toxicant f o l l o w e d b y sacrifice o f the dams a n d r e m o v a l o f the fetuses for m o r p h o l o g i c a l a n d sometimes histological evaluation. F r e quently, the route o f exposure is similar to that w h i c h h u m a n s w o u l d e n counter, b u t the outcomes are different.

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Effects of Chemicals on Reproductive Outcomes in Laboratory Animals. Single-generation assays have f o u n d m a n y c o m m o n solvents to b e teratogenic or e m b r y o t o x i c i n rodents. T h e s e i n c l u d e b e n z e n e (69), xylene (70), cyclohexanone (71), p r o p y l e n e g l y c o l (72), alkane sulfones (73), glycol ethers (74), acetamides, a n d formamides (75). I n general, insecticides appear to have l o w e m b r y o t o x i c p o t e n t i a l i n m a m m a l s , b u t a n u m b e r of teratogenic effects have b e e n r e c o r d e d w i t h organochlorine insecticides, i n c l u d i n g a l d r i n , d i e l d r i n , a n d e n d r i n i n the mouse a n d hamster (76). A few carbamates a n d organophosphates, s u c h as carbaryl i n the g u i n e a p i g (77) a n d dog (78), d e m e t o n (79), a n d p a r a t h i o n (80) i n the mouse, a n d dichlorvos i n the rabbit (81), are k n o w n to b e t e r a togenic. E v i d e n c e that the fungicides mancozeb, dinocap, a n d nitrofen are potent teratogens has b e e n p r e s e n t e d (82). D D E , a metabolite of 1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane ( D D T ) is k n o w n to have d e t r i m e n t a l effects o n r e p r o d u c t i o n i n b i r d s b y i n t e r f e r i n g w i t h the h o r m o n a l activity of estrogen, w h i c h results i n decreased d e p o s i t i o n of c a l c i u m i n the eggshell. T h e h o r m o n e methyltestosterone has b e e n s h o w n to be teratogenic i n dogs, r e s u l t i n g i n p s e u d o h e r m a p h r o d i t i s m i n female offspring (83). A study b y N e l s o n et a l . (84) that assessed the d e v e l o p m e n t a l toxicity of 13 alcohols s h o w e d that teratogenic effects w e r e not e l i c i t e d by l o w - m o l e c u l a r - w e i g h t alcohols (methanol a n d ethanol), as h a d b e e n o r i g i n a l l y h y p o t h e s i z e d . A l s o , the concentrations that e l i c i t e d effects e x c e e d e d 5000 p p m a n d caused m a ternal toxicity.

Epidemiological Studies in Humans E p i d e m i o l o g i c a l studies for r e p r o d u c t i v e loss are usually u n d e r t a k e n u s i n g retrospective approaches, i n c l u d i n g personal i n t e r v i e w s a n d m e d i c a l r e cords. I f o n l y personal i n t e r v i e w data are u s e d , m a n y c o n f o u n d i n g factors can interfere w i t h an accurate assessment of a spontaneous a b o r t i o n . P a tients are f r e q u e n t l y confused about d e l a y e d m e n s t r u a t i o n a n d cannot r e c a l l events. T h e confirmation of a miscarriage t h r o u g h m e d i c a l d o c u m e n t a t i o n is thus necessary to obtain relevant data (67, 85). Selection bias of exposed a n d nonexposed w o m e n m u s t also be a v o i d e d i n these studies. A n u m b e r of factors not i n v o l v i n g exposure have also b e e n associated w i t h spontaneous abortion. T h e s e i n c l u d e m a t e r n a l age, previous spontaneous abortions, c e r v i c a l i n c o m p e t e n c e , m a t e r n a l fever (78, 85, 86), d i e t , h e a l t h status, a n d w e i g h t (87). I n an e p i d e m i o l o g i c a l study, evaluation o f t i m i n g a n d d u r a t i o n of exposure is c r u c i a l . E x p o s u r e of the father for 4 months p r i o r to c o n c e p t i o n m u s t be m o n i t o r e d , because this is the i n t e r v a l r e q u i r e d for a c o m p l e t e cycle of spermatogenesis. Possible fetal exposure d u r i n g the first t r i m e s t e r is the most i m p o r t a n t , although exposure d u r i n g the second a n d t h i r d trimesters can result i n adverse effects. Q u a n t i f i c a t i o n of exposure is f r e q u e n t l y the

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most difficult p a r a m e t e r to measure. A n i n n o v a t i v e approach that has b e e n a p p l i e d i n assessing exposure has b e e n b i o l o g i c a l m a r k e r s , w h i c h are n o n invasive measures of dose that can b e r e l a t e d to p o t e n t i a l adverse r e p r o d u c tive outcomes (88). U r i n a r y mutagens, thioethers, or D - g l u c a r i c are f r e q u e n t l y m e a s u r e d b i o m a r k e r s . H o w e v e r , use o f these b i o m a r k e r s r e q u i r e s information about t h e i r n o r m a l values, i n t e r - a n d intravariability a m o n g i n d i v i d u a l s , a n d possible c o n f o u n d i n g effects. T o assess the effect of exposure o n r e p r o d u c t i v e outcomes, two s e q u e n tial e p i d e m i o l o g i c a l t e c h n i q u e s , d e s c r i p t i v e a n d analytical have b e e n u s e d (89, 90). I n d e s c r i p t i v e studies, i n f o r m a t i o n r e g a r d i n g the d i s t r i b u t i o n a n d f r e q u e n c y of an o u t c o m e is o b t a i n e d . T h e i m p e t u s for these studies is usually based o n an increased n u m b e r o f case reports o n spontaneous a b o r t i o n o r teratology i n a particular geographical area. T h e second stage is an analytical study d e s i g n e d to test a hypothesis or to generate a n e w hypothesis about an association b e t w e e n exposure a n d r e p r o d u c t i v e outcomes. E i t h e r casec o n t r o l or cohort studies are u s e d . I n the case-control study, a retrospective assessment of the exposure factors of the cases a n d controls is p e r f o r m e d . I n a cohort study, b o t h i n d i v i d u a l s exposed a n d not exposed to a p a r t i c u l a r factor are f o l l o w e d over t i m e ; e i t h e r retrospective or prospective t e c h n i q u e s are u s e d to observe the o u t c o m e of interest.

Chemicals Causing Spontaneous Abortion.

A n u m b e r of c h e m i -

cals have b e e n i m p l i c a t e d as b e i n g abortifacient i n h u m a n s , a l t h o u g h d e f i n itive e p i d e m i o l o g i c a l data o n m a n y c o m p o u n d s are still l a c k i n g (91). H o w ever, there is some e v i d e n c e that arsenic, l e a d , a n d anesthetic gases can cause spontaneous a b o r t i o n , p a r t i c u l a r l y d u r i n g the early weeks of p r e g nancy. E x t e n s i v e e p i d e m i o l o g i c a l studies i n S w e d e n b y N o r s t r o m a n d cow o r k e r s (92-94),

have r e p o r t e d an i n c r e a s e d p r e v a l e n c e of

spontaneous

a b o r t i o n i n w o m e n l i v i n g close to or w o r k i n g i n a s m e l t e r that discharges arsenic and lead. D e c r e a s e d b i r t h w e i g h t of offspring not associated w i t h cigarette s m o k i n g was s h o w n (94). H e a l t h p r o v i d e r s seem to be at greater risk of adverse r e p r o d u c t i v e o u t comes than m a n y o t h e r occupationally exposed groups. F o r e x a m p l e , e p i d e m i o l o g i c a l studies have s h o w n that exposure of o p e r a t i n g r o o m nurses a n d female anesthesiologists (95, 96) to anesthetic gases r e s u l t e d i n an i n c r e a s e d rate of spontaneous a b o r t i o n , c o m p a r e d w i t h u n e x p o s e d c o n t r o l w o m e n . A l s o , exposure of nurses d u r i n g the first t r i m e s t e r to antineoplastic d r u g s , i n c l u d i n g c y c l o p h o s p h a m i d e , d o x o r u b i c i n , a n d v i n c r i s t i n e , has r e s u l t e d i n an increased i n c i d e n c e of fetal loss (97). T h e i n d e p e n d e n t effect of each d r u g c o u l d not be p r o v e d , because m o r e t h a n one d r u g was h a n d l e d b y each nurse d u r i n g the exposure p e r i o d . In F i n l a n d , an increase i n spontaneous a b o r t i o n was also o b s e r v e d i n w o m e n i n v o l v e d i n the sterilization of h o s p i t a l i n s t r u m e n t s u s i n g e t h y l e n e oxide (98). E x p o s u r e of p e r s o n n e l ranged f r o m 5 to 10 p p m , t h e r e w e r e

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occasional acute concentrations of 250 p p m . N e i t h e r teratogenic effects n o r decreased b i r t h weights w e r e o b s e r v e d . A l t h o u g h e t h y l e n e oxide ranks 2 6 t h i n the v o l u m e of organic chemicals p r o d u c e d i n the U n i t e d States, less t h a n 1% is u s e d i n hospitals (5). A second cohort that m a y have a h i g h e r risk of spontaneous abortions are w o r k e r s exposed to pesticides. A significant increase i n abortions a m o n g occupationally exposed couples i n I n d i a has b e e n r e p o r t e d (99-101). T h e s e w o r k e r s w e r e exposed to n i n e pesticides, i n c l u d i n g D D T , d i e l d r i n , d i c h l o r vos, p a r a t h i o n , metasystox, a n d c o p p e r sulfate. A n increase i n c h r o m o s o m a l aberrations i n l y m p h o c y t e was also d o c u m e n t e d i n these sprayers (102). I n S w e d e n , a statistically h i g h e r rate of miscarriage was f o u n d a m o n g w o m e n w o r k i n g at a p e t r o c h e m i c a l p l a n t , b u t no differences w e r e o b s e r v e d i n w o m e n l i v i n g close to the plant (103). A s i m i l a r nonsignificant increase i n r e p r o d u c t i v e outcomes was n o t e d i n a group of w o m e n l i v i n g close to a p h e n oxy h e r b i c i d e plant (104).

Chemicals Causing Teratology. Teratogenic effects are u s u a l l y manifest at b i r t h , b u t w i t h some c o m p o u n d s an adverse response is n o t e d o n l y d u r i n g the teens or early twenties. F o r e x a m p l e , p r e n a t a l exposure of males to d i e t h y l s t i l b e s t r o l , a d r u g u s e d to p r e v e n t miscarriage, r e s u l t e d i n abnormalities i n the male r e p r o d u c t i v e tract, manifested as e p i d i d y m a l cysts, s e m i n a l vesicle e n l a r g e m e n t , prostatic i n f l a m m a t i o n , u n d e s c e n d e d testes, a n d the p r o d u c t i o n of a b n o r m a l s p e r m (105). B y i n t e r f e r i n g w i t h the male hormones necessary for d e v e l o p m e n t and differentiation of the m a l e r e p r o d u c t i v e tract, p r e n a t a l d i e t h y l s t i l b e s t r o l exposure was responsible for m o r p h o l o g i c a l abnormalities expressed i n later life. I n the case of the female fetus exposed i n utero to d i e t h y l s t i l b e s t r o l , adenocarcinoma, a malignant cancer of the r e p r o d u c t i v e tract, has b e e n diagnosed i n a n u m b e r of y o u n g women. M e r c u r y is the m e t a l for w h i c h the greatest d o c u m e n t a t i o n of teratology has b e e n r e c o r d e d . D u r i n g the p e r i o d of 1954-1960, the p o p u l a t i o n of M i n amata B a y i n Japan was exposed to m e t h y l m e r c u r y v i a the food c h a i n . A chlor-alkali plant, i n w h i c h m e r c u r y was u s e d as a catalyst, discharged its wastes into the bay. S u b s e q u e n t m e t h y l a t i o n of the m e r c u r y b y bacteria a n d uptake of the l i p o p h i l i c m e t h y l m e r c u r y b y fish r e s u l t e d i n b i o c o n c e n t r a t i o n of the c o m p o u n d into fat a n d m u s c l e . Because fish was the staple source of p r o t e i n for the inhabitants of the area, a n u m b e r of p e o p l e w e r e exposed to the c o m p o u n d . Infants exposed i n utero v i a the c o n s u m p t i o n of c o n t a m i nated fish b y t h e i r mothers d e m o n s t r a t e d v a r y i n g degrees of n e u r o l o g i c a l symptoms r e s e m b l i n g c e r e b r a l palsy (106, 107). S i m i l a r effects of m e r c u r y o n d e v e l o p m e n t has also b e e n d o c u m e n t e d i n the U n i t e d States (108) a n d I r a q (109) f o l l o w i n g accidental i n c l u s i o n of m e r c u r o u s fungicides into grains. O t h e r metals for w h i c h e p i d e m i o l o g i c a l e v i d e n c e exists r e g a r d i n g t h e i r teratogenic effects are a l u m i n u m a n d l i t h i u m . I n a study i n S o u t h W a l e s , a

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significant positive correlation b e t w e e n c e n t r a l nervous system m a l f o r m a tions a n d a l u m i n u m was n o t e d (110). L i t h i u m i n the f o r m of l i t h i u m c a r b o n ate, w h i c h is used therapeutically to treat p s y c h i a t r i c disorders, has b e e n associated w i t h teratogenicity i n infants b o r n to mothers exposed d u r i n g the first t r i m e s t e r of pregnancy (111, 112). A c c i d e n t a l exposure of m o r e than 2000 p e o p l e to c o o k i n g o i l c o n t a m i nated w i t h p o l y c h l o r i n a t e d b i p h e n y l s ( P C B ) a n d dibenzofurans d u r i n g a 6m o n t h p e r i o d i n T a i w a n r e s u l t e d i n teratogenicity. M a l f o r m e d c h i l d r e n w e r e still b e i n g b o r n to mothers exposed m o r e t h a n 6 years p r e v i o u s l y to the c o o k i n g o i l (113). T h i s long-lasting p h e n o m e n o n may be related to the bioacc u m u l a t i o n i n fatty tissue of the ingested P C B , w h i c h i n h u m a n s has an estimated half-life of 7 years. A b n o r m a l i t i e s of the g i n g i v a , s k i n , nails, t e e t h , a n d lungs w e r e o b s e r v e d i n a statistically larger percentage of offspring of exposed mothers. Neurologically, the exposed infants d i d not differ f r o m the unexposed controls, b u t a delay i n the performance of c e r t a i n tasks r e q u i r i n g motor coordination was observed. A l s o , the exposed c h i l d r e n scored l o w e r o n three I Q tests. A n earlier study i n Japan w i t h a smaller cohort of c h i l d r e n whose mothers h a d b e e n exposed to t h e r m a l l y d e g r a d e d c o o k i n g o i l s h o w e d similar abnormalities (114). A t 7 years of age, 7 0 % of these Japanese c h i l d r e n w e r e apathetic a n d h a d I Q s i n the 70s. C o n f l i c t i n g reports o n the teratogenic p o t e n t i a l of 2 , 4 , 5 - t r i c h l o r o p h e n oxyacetic acid (2,4,5-T) a n d its major contaminant 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o d i o x i n has appeared i n the literature (5). A n extensive study, b y the U . S . D e p a r t m e n t of Defense o n 0.5 m i l l i o n exposed V i e t n a m e s e , r e p o r t e d no increased i n c i d e n c e of e i t h e r miscarriages or teratogenic outcomes (115), whereas an A m e r i c a n Association for the A d v a n c e m e n t of Science ( A A A S ) report s h o w e d an increase i n cleft palate a n d s p i n a b i f i d a i n c h i l d r e n i n V i e t n a m from 1964 to 1968 (116). A study b y L a P o r t e (117) substantiated the results of the A A A S , whereas a study b y K u n d s t a d t e r (J 18) f o u n d no i n creased i n c i d e n c e from the retrospective examination of hospital records. L i f e - s t y l e factors, such as s m o k i n g a n d c o n s u m p t i o n of ethanol, have b e e n clearly i m p l i c a t e d as teratogenic agents. S m o k i n g d u r i n g pregnancy has b e e n associated w i t h r e d u c e d b i r t h w e i g h t of infants (J 19) a n d an i n creased risk of spontaneous a b o r t i o n (85,120). T h i s effect, w h i c h has b e e n c o u p l e d to g r o w t h retardation, has b e e n f o u n d to be more p r o n o u n c e d i n blacks than whites (121). A prevalent s y n d r o m e , k n o w n as fetal alcohol s y n d r o m e , has b e e n diagnosed i n infants whose mothers c o n s u m e d m o r e t h a n two d r i n k s p e r day d u r i n g pregnancy (122). T h e s e c h i l d r e n have v a r y i n g craniofacial malformations i n c l u d i n g midfacial a n d m a x i l l a r y hypoplasia (inc o m p l e t e development), m i c r o e n c e p h a l y (small brain), a b n o r m a l joints, a n d heart abnormalities. T h e y also have severe g r o w t h deficiencies a n d i n t e l l e c tual handicaps (123). D r u g s that are k n o w n h u m a n teratogens i n c l u d e t h a l i d o m i d e ; a n d r o gens; a m i n o p t e r i n (2); c o u m a r i n derivatives; t r i m e t h a d i o n e , an a n t i c o n v u l -

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sant; a n d i s o t r e t i n o i n , u s e d i n aene treatment (124). Isotretinoin contains an aromatic r e t i n o i d , etretinate, u s e d to treat s k i n diseases. M a l f o r m a t i o n s i n n e w b o r n s manifested as craniofacial a n d l i m b defects w e r e r e p o r t e d to be associated w i t h intake of etretinate b y pregnant mothers (125). T h r e e aborted fetuses e x a m i n e d b y these investigators w e r e f o u n d to have severe central nervous system anomalies.

Chemicals Affecting the Normal Production of Spermatozoa. M a l e - m e d i a t e d adverse r e p r o d u c t i v e outcomes result e i t h e r f r o m d i r e c t c h e m i c a l effects o n the testes a n d g e r m cells o r t h r o u g h interference w i t h the steroidogenic function of the testes. D i b r o m o c h l o r o p r o p a n e ( D B C P ) , a potent alkylating agent that was d e v e l o p e d as a pesticide, is k n o w n to i n h i b i t (azoospermia) or decrease (oligospermia) the d e v e l o p m e n t of s p e r m . R e d u c e d s p e r m counts, testicular atrophy, a n d decreased fertility w e r e d o c u m e n t e d i n male employees i n a plant m a n u f a c t u r i n g D B C P (126,127). S i m ilar r e p r o d u c t i v e effects w e r e n o t e d i n occupationally exposed w o r k e r s a p p l y i n g the nematocide (128). S u b c h r o n i c exposure of laboratory animals to D B C P p r o d u c e d similar changes to those o b s e r v e d i n h u m a n s (129). A second c o m p o u n d that alters s p e r m p r o d u c t i o n is l e a d , w h i c h p r o duces m a l f o r m e d s p e r m (teratospermia) i n the ejaculate (130). H o w e v e r , n o relationship b e t w e e n the a b n o r m a l l y shaped s p e r m a n d adverse r e p r o d u c tive outcomes have b e e n established (131). D e l e t e r i o u s changes i n l y m p h o cytes a n d s p e r m have b e e n d o c u m e n t e d a m o n g male w o r k e r s exposed to arsenic a n d lead at a smelter, there w e r e respective increases i n c h r o m o somal aberrations i n c u l t u r e d l y m p h o c y t e s a n d i m p a i r e d m o r p h o l o g y a n d m o t i l i t y of s p e r m (132). Because these w o r k e r s w e r e c o n c o m i t a n t l y exposed to c a d m i u m a n d other s m e l t e r fumes, the effects m a y not be d u e to a p a r ticular m e t a l alone b u t rather d u e to a synergistic i n t e r a c t i o n b e t w e e n m o r e than one toxin simultaneously.

Extrapolation from Animal Studies to Humans. I n extrapolating f r o m a n i m a l studies to h u m a n s , a n u m b e r of factors must be taken i n t o c o n sideration: T h e appropriate laboratory a n i m a l must be u t i l i z e d , the n u m b e r of animals o n each treatment m u s t a l l o w for statistical evaluation, a n d the dose levels s h o u l d be relevant to h u m a n exposure. F a r m o r e a n i m a l than h u m a n teratogens have b e e n i d e n t i f i e d because of the way i n w h i c h w e are able to test animals. E x p e r i m e n t a l animals have a short r e p r o d u c t i v e cycle a n d m u l t i p l e offspring. T h e r e f o r e , a large n u m b e r of fetuses can be e x a m i n e d i n a short t i m e a n d at r e l a t i v e l y l o w cost. A n e q u i v a l e n t h u m a n e p i d e m i o l o g i c a l study w o u l d cost m i l l i o n s of dollars a n d r e q u i r e a n u m b e r of years. T h e dosage a d m i n i s t e r e d to animals can be a d j u s t e d so that it is m a n y times greater than w o u l d be expected i n a n o r m a l h u m a n exposure situation. T h e e x p e r i m e n t m a y also be d e s i g n e d to c o r r e late exposure of the e m b r y o or fetus w i t h its most sensitive t i m e i n d e v e l -

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o p m e n t (I). A l l of these factors i m p r o v e the p o s s i b i l i t y of i d e n t i f y i n g a c h e m ical c o m p o u n d as a teratogen i n animals. A n i m a l studies are, however, not w i t h o u t fault. T h e v a r i a t i o n i n genot y p e a m o n g different i n d i v i d u a l s influences t h e i r response to a teratogen a n d h e n c e t h e i r s u s c e p t i b i l i t y to a p a r t i c u l a r toxicant. E v e n greater variations i n genotype exist a m o n g different species. T h e r e f o r e , a c h e m i c a l p r o v e d to be nonteratogenic i n a c e r t a i n laboratory species may w e l l b e teratogenic i n a different laboratory species or e v e n i n h u m a n s . A classic example is that of t h a l i d o m i d e . T h e laboratory animals most w i d e l y u s e d i n teratogenicity testi n g (mouse, rat, a n d rabbit) are 5 - 5 0 0 times less sensitive t h a n h u m a n s to this sedative (5), w h i c h is one o f the most potent h u m a n teratogens k n o w n . F r o m a risk p e r s p e c t i v e , however, a n i m a l studies have generally b e e n good predictors of teratogenicity. Studies b y S c h a r d e i n (133), F a b r o (134), a n d Jelovsek et a l . (135) have i n d i c a t e d that h u m a n teratogens y i e l d positive results i n a n i m a l a n d i n v i t r o studies m o r e often t h a n do nonteratogens. It has b e e n estimated that 7 5 % of h u m a n d e v e l o p m e n t a l toxicants can b e p r e d i c t e d o n the basis of a n i m a l studies o n l y (136), b u t unfortunately false-negative results are also h i g h (25%). O t h e r factors for consideration are the combinations a n d p e r m u t a t i o n s of chemicals after they e n t e r the e n v i r o n m e n t . E v e n i f a c h e m i c a l is f o u n d to be nontoxic i n a n i m a l studies, the safety of the c h e m i c a l can not b e e n sured. T h e possible potentiation of a c h e m i c a l s effect b y its i n t e r a c t i o n w i t h other c h e m i c a l agents or o t h e r toxic factors once it enters the e n v i r o n m e n t cannot be d i s m i s s e d .

Conclusion It is e v i d e n t that m a n y possible adverse h e a l t h effects m a y result f r o m exposure to the n u m e r o u s chemicals i n the e n v i r o n m e n t . E x p o s u r e of b o t h male a n d female organisms to chemicals d u r i n g the r e p r o d u c t i v e cycle can have deleterious effects o n the d e v e l o p m e n t of the fetus. T h e s e effects are manifested as fetal death, m a l f o r m a t i o n , r e t a r d e d g r o w t h , a n d organ d y s function. O u r k n o w l e d g e of the adverse h e a l t h effects that are p r o d u c e d as the result of a c o m p l e x n e t w o r k of factors is l i m i t e d . T h i s is the k e y p o i n t : M u c h is u n k n o w n about the chemicals to w h i c h h u m a n s are exposed a n d h o w they interact w i t h biological systems. T h e r e is m u c h progress to b e made i n d e f i n i n g the i n c i d e n c e of such effects, the m a g n i t u d e of dose i n specific exposure situations, a n d the p o s s i b i l i t y of interactions a m o n g causes. To a i d i n assessing w h e t h e r a c o m p o u n d is a teratogen, a n u m b e r of shortt e r m i n v i t r o assays have b e e n d e v e l o p e d a n d are p r e s e n t l y b e i n g v a l i d a t e d . T h e progress m a d e so far has i n d i c a t e d the i m p o r t a n c e of l i m i t i n g exposure to chemicals to the most p r a c t i c a l a n d feasible extent. F u t u r e research efforts s h o u l d b e d i r e c t e d t o w a r d establishing the m e c h a n i s m s of toxicological effects a n d d e v e l o p i n g m o r e accurate means of i d e n t i f y i n g hazardous c o m pounds.

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