11 Toxicology: A Summary LEON
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GOLBERG
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Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709
There has never been a more exciting time in Toxicology. Never before have we had such a wealth of new ideas and concepts pumped in by the basic sciences, such a multiplicity of new methods and approaches, such sophisticated and sensitive analytical procedures. Our problem is to assimilate and apply all these opportunities, which bid fair to revolutionize the classical approaches to safety evaluation. Hence an even greater source of concern may be expressed thus: will we be afforded a breathing-space to develop the new tests to a satisfactory point, before they become a part of government regulation? In my introductory address, I referred to a different kind of regulation, namely the basic biological regulatory and defensive mechanisms that exist within each cell and between cells, making possible the integrated harmonious functioning of the whole organism that we call "homeostasis". Superimposed on this basic concept of the capacity of the organism to adapt to change is the clear evidence of limits to the capacity of these defensive mechanisms to cope with endogenous changes (for instance, caused by disease processes) or exogenous environmental changes. Equally, the body's defenses can be overwhelmed by the action of toxicants - both physical and chemical agents.
Five f a c t o r s help to determine the impact of a t o x i c agent on any population of experimental animals or people. These are as f o l l o w s : the p o t e n t i a l of the compound t o b r i n g about s p e c i f i c t o x i c e f f e c t s ; i t s potency under defined experimental c o n d i t i o n s ; the degree and circumstances of exposure of the p o p u l a t i o n ; the range of i n d i v i d u a l s u s c e p t i b i l i t i e s w i t h i n t h a t p o p u l a t i o n ; and the s y n e r g i s t i c or a n t a g o n i s t i c i n t e r a c t i o n s occasioned by simultaneous exposure t o a m u l t i p l i c i t y of t o x i c agents. S e l e c t i o n of a compound f o r t o x i c i t y t e s t i n g o f t e n r a i s e s many d i f f e r e n t questions that need to be addressed i n advance of b i o l o g i c a l experimentation. These i s s u e s i n c l u d e the d e c i s i o n on s p e c i f i c a t i o n of the t e s t m a t e r i a l , the nature and c o n c e n t r a t i o n s of t r a c e i m p u r i t i e s , as w e l l as the changes t h a t occur on storage o r admixture w i t h animal d i e t s . 1
Current address: 2109 Nancy Nanam Drive, Raleigh, NC 27607. 0097-615 6/81/0160-0187$05.00/0 © 1981 American Chemical Society Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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In the h i e r a r c h y of t o x i c i t y t e s t i n g , the use of s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s i s becoming an i n c r e a s i n g l y important p r e d i c t i v e t o o l . The r e l i a b i l i t y of t h i s t o o l i s , of course, dependent on the accuracy of the data bases which i t i n c o r p o r a t e s . E f f o r t s are under way to make these bases more r e l i a b l e . Further steps i n s a f e t y e v a l u a t i o n may f o l l o w a " d e c i s i o n t r e e ' approach; any scheme adopted should i n v o l v e a v a r i e t y of screening procedures, i n c l u d i n g t e s t s f o r genetic t o x i c i t y , as w e l l as e a r l y s t u d i e s of metabolism and pharmacokinetics. F i n a l l y , the importance of human s t u d i e s was emphasized not only s t u d i e s i n human v o l u n t e e r s , or e p i d e m i o l o g i c a l r e s e a r c h , but a l s o the use of breath a n a l y s i s , human lymphocytes, hemoglob i n , e t c . as i n d i c e s of exposure and of e f f e c t s of such exposure. The suggestion of a human l i v e r bank was a l s o d i s c u s s e d . Consideration was next given (by Dr. B. Schwetz) to the widening concepts of t o x i c o l o g y , s p e c i f i c a l l y i n r e l a t i o n to changes i n t i m e , space, s p e c i e s , t o x i n s , c o n c e n t r a t i o n s and parameters of concern. Time a t which t o x i c e f f e c t s might be manifested had extended beyond the immediate f u t u r e to subsequent generations as y e t unborn, and t o x i c o l o g i c a l t e s t procedures had been developed to cope w i t h these concerns. The l o c a l i z a t i o n and d i s t r i b u t i o n of t o x i n s to remote recesses of the environment and ecosystems now i n v o l v e d a huge range of species and, most immediately, p e t s , w i l d a n i m a l s , beasts of burden and food sources. Among the compounds being s t u d i e d , there was i n c r e a s i n g emphasis on environmental t o x i c a n t s l i k e P C B ' s , P B B ' s , TCDD and other d i o x i n s and dibenzofurans. In p a r a l l e l w i t h developments i n a n a l y t i c a l c h e m i s t r y , t o x i c o l o g y was now concerning i t s e l f w i t h amounts as low as a few molec u l e s . S o c i e t a l concern now embraced the q u a l i t y of l i f e and, o f t e n , a misplaced i n s i s t e n c e on zero r i s k . The trends i n t o x i c o l o g y r e f l e c t e d these developments. More reproductive s t u d i e s of a more s o p h i s t i c a t e d kind were f o l l o w i n g e f f e c t s on sperm and ova, as w e l l as on the complete reproductive process. Studies of the developing embryo and f e t u s r e f l e c t e d a more c r i t i c a l and reasonable a t t i t u d e to thresholds and dose-response aspects of t e r a t o g e n i c i t y . Greater involvement i n behavioral s t u d i e s had l e d to combined approaches t h a t permitted a n a l y s i s of the postnatal consequences of i n utero exposure. F i n a l l y , the c o n t r i b u t i o n of immunotoxicology was i n c r e a s i n g ; not i n f r e q u e n t l y , compounds s t i m u l a t e d the immune system a t low l e v e l s of exposure and i n h i b i t e d i t a t high l e v e l s . A n a l y s i s of the biochemical aspects of organ s p e c i f i c i t y i n t o x i c a c t i o n (by Dr. J . S. Dutcher) began w i t h a survey of the f a c t o r s t h a t i n f l u e n c e patterns of o r g a n - s p e c i f i c t o x i c i t y . Notable among these are the mechanisms of d e t o x i c a t i o n and meta b o l i c a c t i v a t i o n , l e a d i n g to covalent i n t e r a c t i o n w i t h c e l l u l a r macromolecules and consequent t o x i c i t y . A superb example of the s h i f t i n g t a r g e t of t o x i c a c t i o n
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Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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11.
GOLBERG
Toxicology:
A
Summary
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had been found i n the furanoterpene, 4-ipomeanol. Organ-specific t o x i c i t y r e f l e c t e d t i s s u e l e v e l s o f a l k y l a t i o n consequent upon metabolic a c t i v a t i o n . In the r a t , b r o n c h i o l a r c e l l n e c r o s i s r e s u l t e d from a c t i v a t i o n of the compound i n C l a r a c e l l s . In the mouse, beside the l u n g , renal t u b u l a r n e c r o s i s occurred. The l i v e r was the t a r g e t organ a t a l l doses i n the Japanese q u a i l , whereas i n the Syrian golden hamster both lung and l i v e r n e c r o s i s was observed. The e x p l a n a t i o n f o r these t a r g e t s hinged upon l o c a l s i t e s o f metabolic a c t i v a t i o n . P o s s i b l e s t a b i l i t y of the r e s u l t i n g m e t a b o l i t e s formed, and subsequent t r a n s p o r t from the s i t e o f a c t i v a t i o n , had been r u l e d out as a mechanism f o r t o x i c i t y i n other organs. Studies i n v o l v i n g microsomal enzyme i n d u c t i o n or i n h i b i t i o n had revealed changes i n s e v e r i t y or s i t e of t o x i c a c t i o n . E q u a l l y , the p r o t e c t i v e e f f e c t of g l u t a t h i o n e had been e s t a b l i s h e d i n various organs and s p e c i e s . As a s t a r t i n g - p o i n t i n h i s d i s c u s s i o n of g e n o t o x i c i t y , Dr. G. M. W i l l i a m s analyzed animal c a r c i n o g e n i c i t y from the standpoint of the d e f i n i t i o n of a c a r c i n o g e n , based upon an o p e r a t i o n a l d e s c r i p t i o n , and the c l a s s i f i c a t i o n o f carcinogens. Two broad c a t e g o r i e s of carcinogens are recognized: those t h a t are genotoxic and e l i c i t DNA damage, and e p i g e n e t i c carcinogens t h a t i n v o l v e no DNA damage. This dichotomy excludes r e v e r s i b l e b i n d i n g t o r e c e p t o r s i t e s and a c t i o n of i n t e r c a l a t i n g agents, which are mutagenic but not c a r c i n o g e n i c . The c o r r e l a t i o n between evidence o f g e n o t o x i c i t y and f i n d i n g of c a r c i n o g e n i c i t y i s g r e a t e s t f o r DNA r e p a i r , r e s u l t i n g from covalent damage t o DNA and a m p l i f i c a t i o n of DNA r e p a i r s y n t h e s i s i n response t o the l e s i o n i n DNA. Good c o r r e l a t i o n e x i s t s f o r mutagenicity observed i n b a c t e r i a l and mammalian c e l l systems. A c o r r e l a t i o n has not been e s t a b l i s h e d f o r s i s t e r chromatid exchange and n e o p l a s t i c t r a n s f o r m a t i o n of c e l l s i n v i t r o . Thus the u l t i m a t e purpose of i n v i t r o t e s t s i s t o l i m i t f u r t h e r t e s t i n g , p a r t i c u l a r l y t o e l i m i n a t e long-term s t u d i e s and t o provide an understanding of the mechanism of a c t i o n of the t e s t compound. A f t e r reviewing the various a v a i l a b l e t e s t s , and emphasizing the shortcomings o f the S9 f r a c t i o n (which e n t a i l s a s e l e c t i v e l o s s of d e t o x i c a t i o n p o t e n t i a l ) , the make-up of a b a t t e r y of a p p r o p r i a t e short-term t e s t s was d i s c u s s e d . A b a t t e r y comprising the f o l l o w i n g f i v e t e s t s had shown a high degree o f s e n s i t i v i t y : a b a c t e r i a l t e s t ; DNA r e p a i r w i t h primary c u l t u r e s o f hepatocytes; mammalian c e l l mutagenesis using various c e l l l i n e s , i n c l u d i n g r e p l i c a t i n g l i v e r e p i t h e l i a l c e l l s ; s i s t e r chromatid exchange, the most o b j e c t i v e evidence of chromosomal damage; and c e l l transformation i n v i t r o . I f a t e s t m a t e r i a l i s p o s i t i v e i n the f i r s t two of these t e s t s , there i s a d e f i n i t e presumption of c a r c i n o g e n i c i t y . A b r i e f account was presented o f abbreviated i n vivo b i o assays f o r c a r c i n o g e n i c i t y . These i n c l u d e d s k i n p a i n t i n g , w i t h or without a promoting agent (TPA); the production o f pulmonary tumors i n s t r a i n A mice; the development of breast cancer i n
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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female Sprague-Dawley r a t s by day 55; and a l t e r e d f o c i produced i n rodent l i v e r , which c o r r e l a t e w e l l w i t h subsequent development of h e p a t o c e l l u l a r carcinoma. A p o s i t i v e r e s u l t i n any i n v i t r o bioassay, coupled w i t h a p o s i t i v e r e s u l t i n one of the l i m i t e d i n vivo bioassays probably r e f l e c t s c a r c i n o g e n i c p o t e n t i a l . Discussing the mode of a c t i o n of c a r c i n o g e n i c p e s t i c i d e s , Dr. Williams dwelt on the p o l y c h l o r i n a t e d compounds t h a t e l i c i t tumors i n rodents, mouse l i v e r being the p a r t i c u l a r t a r g e t and, o c c a s i o n a l l y , the t h y r o i d . I t seemed c l e a r t h a t these were e p i g e n e t i c carcinogens t h a t d i d not form covalent adducts w i t h DNA nor damage DNA. Various t e s t s f o r unscheduled DNA s y n t h e s i s , point mutations and n e o p l a s t i c transformation were a l l n e g a t i v e . Tests f o r promotional e f f e c t , i n systems t h a t revealed phénobarb i t a l to be a prototype promoter, served to e s t a b l i s h t h a t the c h l o r i n a t e d p e s t i c i d e s acted i n the same way. P o s s i b l e mechanisms of promoting a c t i o n were reviewed. P r e d i c t i o n of c a r c i n o g e n i c p o t e n t i a l of p e s t i c i d e s was taken a step f u r t h e r by Dr. S. Nesnow, who described EPA's "phased approach" f o r the a p p l i c a t i o n of short-term t e s t s to these compounds. Phase 1 i n v o l v e d d e t e c t i o n of p o i n t mutations, DNA damage and chromosomal e f f e c t s i n a p p r o p r i a t e microorganisms. Phase 2 aimed a t v e r i f i c a t i o n of any p o s i t i v e f i n d i n g s by use of higher-order t e s t systems (human lung f i b r o b l a s t s , r e c e s s i v e l e t h a l i t y i n D r o s o p h i l a , dominant l e t h a l i t y i n the mouse and n e o p l a s t i c t r a n s f o r m a t i o n i n c e l l c u l t u r e systems). The f i n a l stage c a l l e d f o r q u a n t i t a t i v e r i s k assessment through the use of rodents to study gene mutations and chromosomal e f f e c t s , as w e l l as long-term c a r c i n o g e n e s i s bioassay. A p p l i c a t i o n of these approaches to 38 p e s t i c i d e s was des c r i b e d i n d e t a i l . T e s t i n g was f a r from complete as y e t - as an example, f o r only 13/38 compounds were c a r c i n o g e n i c i t y data a v a i l a b l e i n evaluated form. The fundamentals of t e s t i n g f o r r e p r o d u c t i v e and t e r a t o g e n i c e f f e c t s of chemical agents were described by Dr. R. T y l . She l a i d emphasis on t e r a t o g e n i c phenomena i n man, o n e - f i f t h of which were a t t r i b u t a b l e to germinal mutations. Developmental c r i t e r i a and landmarks i n rodents were t a b u l a t e d to i l l u s t r a t e the important a p p l i c a t i o n s of such data i n s a f e t y t e s t i n g . The r o l e of Epidemiology was presented by Ms. M. W. Palshaw, w i t h a c l e a r a n a l y s i s of occupational s t u d i e s i n t h i s f i e l d . She s t r e s s e d t h a t , w h i l e an a s s o c i a t i o n between exposure and i l l - h e a l t h may be e s t a b l i s h e d by such means, cause and e f f e c t r e l a t i o n s h i p s cannot be proved. The i n t e r f a c e between epidemiol o g i s t s and experts i n other s c i e n t i f i c d i s c i p l i n e s (chemists, chemical engineers, i n d u s t r i a l h y g i e n i s t s , t o x i c o l o g i s t s , p h y s i cians and b i o s t a t i s t i c i a n s ) was d i s c u s s e d , emphasizing the c r u c i a l importance of exposure d a t a . C o n s i d e r a t i o n of the OSHA c a t e g o r i e s f o r occupational i l l n e s s was f o l l o w e d by i l l u s t r a t i o n s drawn from b i o l o g i c a l monitoring of c h o l i n e s t e r a s e l e v e l s
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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11.
GOLBERG
Toxicology:
A
Summary
191
i n red c e l l s and plasma, as w e l l as n e u r o l o g i c a l and r e p r o d u c t i v e e f f e c t s . The c o n t r i b u t i o n o f Epidemiology t o worker s a f e t y puts i t i n the f r o n t l i n e of h e a l t h p r o t e c t i v e measures. In c o n s i d e r i n g the e v a l u a t i o n of r i s k s to human h e a l t h , Dr. A. C. Kolbye reviewed the mathematical approaches to r i s k asssessment but concluded that such mathematical models ignore b i o l o g i c a l v a r i a b l e s . Damage and r e p a i r , as w e l l as c e l l r e p l i c a t i o n a c t i n g as a f i x a t i v e of DNA damage i n the r e p l i c a t i n g genome, need to be weighed i n r e l a t i o n to the no-threshold hypothesis which was developed by analogy w i t h p e n e t r a t i n g r a d i a t i o n . Dose-response c o n s i d e r a t i o n s should loom large i n r i s k assessment. Where a compound acts as a promoter, i t s e f f e c t i v e ness a t lower doses i s l i k e l y to be decreased. By using "Maximum T o l e r a t e d Doses", c e l l and t i s s u e damage i s brought about t h a t s t i m u l a t e s h y p e r p l a s t i c a c t i v i t y t h a t not i n f r e q u e n t l y causes the a c t i o n o f endogenous i n i t i a t o r s t o be promoted t o cancer. A c c o r d i n g l y an a p p r o p r i a t e s t r a t e g y f o r cancer prevention c a l l s f o r c l a s s i f i c a t i o n i n t o two c a t e g o r i e s : f i r s t order carcinogens or i n i t i a t o r s t h a t are e f f e c t i v e a t subtoxic doses; and second order promoters t h a t b r i n g about h y p e r p l a s t i c t o x i c i t y and a s s o c i a t e d phenomena. Such second-order compounds should not be regarded as carcinogens. They may i n f l u e n c e f i r s t - o r d e r compounds, depending on the b i o l o g i c a l defense mechanisms present and the mode and amount of exposure. Experimental procedures are a v a i l a b l e t h a t permit a c l e a r d i s t i n c t i o n t o be drawn between f i r s t - and second-order compounds. RECEIVED February 12, 1981.
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
