Systematic Toxicity Testing for Xenobiotics in Foods - ACS Symposium

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Systematic Toxicity Testing for Xenobiotics in Foods MORRIS M. JOSELOW University of Medicine of New Jersey, Newark, NJ 07103

Substances that are not natural to a food, xenobiotics, may become a part of the food by several routes. They may be deliberately added as food or color additives; they may result indirectly from migration of additives from wrappings into the food; they may also result from the incorporation of environmental pollutants into the food while that food is growing or maturing. All such xenobiotics inevitably raise questions of safety. This, of course, does not infer that a l l natural foods are safe (1). We know this to be untrue--but it does recognize that anything added to foods will be regarded with suspicion until adequate toxicity testing has established its safety--a concept that now applies to almost a l l foods and drugs introduced into commerce. As might be expected, there i s a large interface between toxicity testing and legal requirements. Some formal definitions, originally proposed by the National Academy of Sciences, are in order. Toxicity is defined as the capacity of a substance to cause injury, in a very broad sense, and includes injury to any mechanism or tissue of the body, as well as i r r i t a t i o n , behavioral changes, or mental disturbances. Hazard, as used, for example, in defining a hazardous substance, involves a probability concept, like so much else in toxicology. It is an estimate of the probability or likelihood that a substance may cause injury. The converse of this i s the meaning reserved for the term safety, namely, the probability (or practical certainty) that injury will not result when a substance i s used in a particular manner and quantity (2). The use aspect is a major qualification that inheres in these definitions. Any substance can cause injury i f high enough concentrations are administered. The evaluation of safety or hazard must therefore take into account the conditions of use or possible predictable misuse of the substance. Toxicity testing has become a major concern nationally because of the extensive legislation that now mandates such testing (Food, Drug, and Cosmetic Act; Hazardous Substances Act; Occupational Safety and Health Act; Toxic Substances Control Act) as well as i n ternationally, because of the need for mutually acceptable products as a prerequisite to economic cooperation in the marketing of foods and chemicals. 0097 -6156/83/0234-0001 $06.00/0 © 1983 American Chemical Society

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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X E N O B I O T I C S IN F O O D S A N D

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In no o t h e r a r e a has t o x i c i t y t e s t i n g assumed g r e a t e r i m p o r t a n c e t h a n w i t h f o o d s , f o r t h e o b v i o u s r e a s o n t h a t f o o d s r e p r e s e n t pot e n t i a l l y t h e g r e a t e s t p o p u l a t i o n i m p a c t , and t h e r e f o r e t h e need for greatest surveillance. B e f o r e p r o c e e d i n g w i t h c o n s i d e r a t i o n o f some o f t h e f o r m a l t e s t p r o c e d u r e s t h a t have been d e v e l o p e d and a r e now i n u s e , we s h o u l d recognize a fundamental flaw i n the s c i e n t i f i c b a s i s f o r a l l safety e v a l u a t i o n s . A l l t e s t s t o evaluate safety are designed to demonstrate a negative, the p r o b a b i l i t y t h a t i n j u r y w i l l not r e s u l t , w h i c h , by d e f i n i t i o n and l o g i c , c a n n o t be p r o v e n . We may sometimes f e e l , a f t e r h a v i n g p e r f o r m e d a s u f f i c i e n t number of t e s t s o f a s u f f i c i e n t v a r i e t y over a s u f f i c i e n t length o f time, t h a t s a f e t y has been f u l l y d e m o n s t r a t e d . But i t o n l y t a k e s a t h a i i d o m i d e - t y p e d i s a s t e r , w i t h t h e a p p e a r a n c e o f a new u n e x p e c t ed e f f e c t , t o r e m i n d u s t h a t a b s o l u t e s a f e t y -- t h e g u a r a n t e e d a b s e n c e o f any harm -- i s an i l l u s i o n t h a t can n o t be u n e q u i v o c a l l y e s t a b l i s h e d , u n l e s s we c o u l d t e s t a l l human b e i n g s o v e r an i n f i n i t e p e r i o d o f time, an o b v i o u s l y impossible task. Faced with t h i s c o n f l i c t between w i s h and r e a l i t y , we have come t o r e l y on " s c i e n t i f i c j u d g m e n t " -- a much o v e r u s e d and a b u s e d p h r a s e t h a t s h o u l d a l w a y s be s u s p e c t -- t o make p r e d i c t i o n s a b o u t s a f e t y t h a t we t h e n c l a i m a r e beyond r e a s o n a b l e d o u b t , o r t h a t t h e r e i s no unr e a s o n a b l e r i s k " , a s r e q u i r e d by t h e T o x i c S u b s t a n c e s C o n t r o l A c t . Of n e c e s s i t y , heavy r e l i a n c e i n t o x i c i t y t e s t i n g must be p l a c e d on t h e use o f a n i m a l s a s s u r r o g a t e s o r s u b s t i t u t e s f o r humans. T h i s r e q u i r e m e n t has imposed some q u a l i f i c a t i o n s -- a t t i m e s s e v e r e -- i n t h e i n t e r p r e t a t i o n o f r e s u l t s . Choice o f S p e c i e s o f T e s t Animal In d e c i d i n g o n t h e animal t o be u s e d , t o x i c o l o g i s t s a t t e m p t t o f o l l o w a b a s i c g u i d e l i n e : w h e r e v e r p o s s i b l e , use a s p e c i e s t h a t b i o l o g i c a l l y handles the m a t e r i a l under study a s s i m i l a r l y a s p o s s i b l e t o man; and, i n d e c i d i n g s i m i l a r i t y t o man, c o n s i d e r a t i o n s o f m e t a b o l i s m , a b s o r p t i o n , e x c r e t i o n , and o t h e r p h y s i o l o g i c a l p a r a m e t e r s s h o u l d be t a k e n i n t o a c c o u n t . T h i s g u i d e l i n e , w h i c h w o u l d seem t o be s o f u n d a m e n t a l , n e v e r t h e l e s s , p o s e s a dilemma. The t o x i c o l o g i s t would l i k e t o use s p e c i e s t h a t i s a s s e n s i t i v e t o t h e i n j u r y e x p e c t e d f o r t h e substance under study, a s p e c i e s t h a t would r e a c t p h y s i o l o g i c a l l y l i k e humans, o r be even more s e n s i t i v e . But t h e s t a t e o f t h e a r t i n t o x i c o l o g y i s n o t s u f f i c i e n t l y developed t o permit p r e d i c t i o n s as to which species of animals are most s i m i l a r t o man i n t h e i r r e s p o n s e s t o c h e m i c a l c h a l l e n g e s . In p r a c t i c e , t h e c h o i c e o f a s p e c i e s i s o f t e n d i c t a t e d more by p r a c t i c a l c o n s i d e r a t i o n s t h a n by s i m i l a r i t y o f r e s p o n s e . C o n -


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JOSELOW

Systematic

Toxicity

Testing in Foods

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venience, ease o f h a n d l i n g , a v a i l a b i l i t y o f stock, housing f a c i l i t i e s , c o s t o f m a i n t e n a n c e , and p r e c e d e n c e o f use -- a l l o f t h e s e e n t e r i n t o a judgment o f t h e c h o i c e o f s p e c i e s . A l b i n o r a t s and dogs have been most f r e q u e n t l y u s e d , b e c a u s e t h e y a r e t h e most e a s i l y a v a i l a b l e a n i m a l s and have a l o n g h i s t o r y o f u s e . The f a c t t h a t r a t s and dogs a r e most w i d e l y u s e d i s a l s o s e l f perpetuating. I n any s t u d y o f a new c h e m i c a l , t h e o b v i o u s f i r s t c h o i c e w o u l d be an animal s p e c i e s whose r e a c t i o n s a r e w e l l - k n o w n and documented. A l s o , t h e p r a c t i c a l p r o b l e m o f o b t a i n i n g s u i t a b l e a n i m a l s i s m i n i m i z e d by t h e use o f t h o s e f o r w h i c h p r e v i o u s demands have c r e a t e d a d e q u a t e , d e p e n d a b l e , a n d i n e x p e n s i v e s o u r c e s of supply. F u r t h e r m o r e , some f e d e r a l r e g u l a t i o n s have recommended and t h e r e by a l m o s t mandated t h e use o f r a t s and d o g s , p a r t i c u l a r l y , f o r l o n g - t e r m t o x i c i t y s t u d i e s o f f o o d a d d i t i v e s and p e s t i c i d e s , e v e n t h o u g h t h e r e may be i m p o r t a n t d i f f e r e n c e s i n m e t a b o l i c c a p a b i l i t i e s and p h y s i o l o g i c a l r e s p o n s e s between t h e s e s p e c i e s and man. The r a t i s a p o o r c h o i c e t o e v a l u a t e t h e l i v e r i n j u r y p o t e n t i a l o f a s u b s t a n c e f o r man, b e c a u s e t h e r a t l i v e r i s r e s i s t a n t , a n d r e g e n e r a t e s r a p i d l y . The g u i n e a p i g o r t h e r a b b i t w o u l d be a b e t t e r c h o i c e o f t h i s . The dog does n o t a c e t y l a t e o r d e t o x i f y aroma t i c a m i n e s . The monkey and t h e g u i n e a p i g r e q u i r e exogenous a s c o r b i c a c i d , w h i l e t h e r a t does n o t , w h i c h makes t h e r a t u n s u i t a b l e f o r t h e d e m o n s t r a t i o n o f a n a s c o r b i c and d e f i c i e n c y . For long-term s t u d i e s , a s p e c i e s having a r e l a t i v e l y s h o r t l i f e span p e r m i t s d e t e r m i n i n g t h e e f f e c t s o f a c h e m i c a l o v e r c o u r s e o f a l i f e t i m e . The l a t t e r r e q u i r e m e n t , i n a p r a c t i c a l s e n s e , l i m i t s l i f e - t i m e s t u d i e s t o r a t s or mice o r hamsters. But h e r e t o o , some b a s i c d i f f i c u l t i e s can p r e s e n t t h e m s e l v e s . F o r a s s e s s i n g t h e p o s s i b i l i t y o f c a n c e r -- t h e main o b j e c t i v e i n l o n g t e r m t e s t s -- we r e l y o n t h e use o f h e a l t h y , d i s e a s e - f r e e i n b r e d mice, which represent c h a r a c t e r i s t i c s t h a t are h a r d l y r e p r e s e n t a t i v e o f a h e t e r o g e n e o u s human p o p u l a t i o n a t r i s k . T h i s d i f f e r e n c e o n l y c o m p l i c a t e s known d i f f e r e n c e s i n m e t a b o l i c r e s p o n s e s ; e.g., m i c e can n o t d e c a r b o x y l a t e and e l i m i n a t e some c a r c i n o g e n i c h y d r o c a r b o n s t h a t humans can r e a d i l y " d e t o x i f y " . F o r p r e l i m i n a r y s t u d i e s and s c r e e n i n g , r a t s , m i c e , r a b b i t s , o r g u i n e a p i g s a r e s e l e c t e d f o r economy, e a s e o f t r e a t m e n t , and, t o a l a r g e e x t e n t , a c c o r d i n g t o t h e r e p o r t e d work o f o t h e r s who have done s i m i l a r s t u d i e s . F o r t h e c h r o n i c s t u d i e s r e q u i r e d by some F e d e r a l r e g u l a t i o n s , t h e s p e c i e s s p e c i f i e d by t h e a g e n c i e s a r e u s e d , u s u a l l y r a t s and b e a g l e d o g s . U n d e r t h e F e d e r a l H a z a r d o u s S u b s t a n c e A c t ( 3_ ) f o r e x a m p l e , t h e d e f i n i t i o n s o f t o x i c , h i g h l y t o x i c , and n o n - t o x i c


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a r e b a s e d i n p a r t on r e s p o n s e s o f r a t s t o o r a l o r i n h a l a t i o n exp o s u r e s . T h a t recommendation has made i t a l m o s t m a n d a t o r y t o use r a t s and f o l l o w t h e p r e s c r i b e d p r o t o c o l s . In summary, t h o u g h we o u g h t t o use a s p e c i e s r e a c t i n g q u a l i t a t i v e l y and q u a n t i t a t i v e l y most l i k e man, what we a c t u a l l y use most o f t e n i s the s p e c i e s f o r which h e a l t h y commerical s t o c k s , reasona b l y p r i c e d and r e a s o n a b l y c o n s t a n t , a r e a v a i l a b l e . A d m i n i s t r a t i o n o f Dose The r o u t e c h o s e n f o r a d m i n i s t r a t i o n o f t h e s u b s t a n c e t o t h e t e s t animal and t h e manner o f i t s a d m i n i s t r a t i o n s h o u l d be t h e same a s t h a t by w h i c h man w i l l be e x p o s e d . I n a c u t e t o x i c i t y t e s t i n g , however, t h i s i s a p r a c t i c e t h a t i s i n t e n t i o n a l l y v i o l a t e d , s i n c e a s i n g l e m a s s i v e dose i s u s u a l l y a d m i n i s t e r e d by i n t u b a t i o n o r gavage. T h e r e w i l l a l w a y s be some d i f f e r e n c e s between t h e e f f e c t s o b s e r v ed i n a n i m a l t e s t s , and a c t u a l human e x p e r i e n c e . No i n t u b a t e d o r a l d o s e can be r e l i e d upon t o be a sound model o f t h e e f f e c t o f a c h e m i c a l i n t h e d i e t , even i f i t i s r e p e a t e d day a f t e r day. T h e s i n g l e d o s e w i l l o f n e c e s s i t y y i e l d a peak c o n c e n t r a t i o n i n body f l u i d s h i g h e r t h a n what w o u l d r e s u l t from t h e s l o w a b s o r p t i o n o f a c h e m i c a l d u r i n g s l o w d i g e s t i o n o f f o o d . I n t u b a t i o n a l s o has a g r e a t e r c h a n c e o f b e i n g i n j u r i o u s , e i t h e r by t h e g r e a t e r m a g n i tude o f t h e peak b l o o d c o n c e n t r a t i o n o r by o v e r w h e l m i n g a metab o l i c pathway t h a t c o u l d i n normal c i r c u m s t a n c e s h a n d l e a l o w e r c o n c e n t r a t i o n o f the t o x i c substances. Dose L e v e l s and S a f e t y F a c t o r s A b a s i c assumption underlying t o x i c i t y t e s t i n g i s t h a t responses a r e d o s e - r e l a t e d , and t h a t , i n t e s t i n g , s e v e r a l d o s e s s h o u l d be administered to e l i c i t graded responses. F u r t h e r m o r e , t h e r e must a l s o be some dose below w h i c h no r e s p o n s e w i l l be shown ( o r , more p r e c i s e l y , be i n c a p a b l e o f d e t e c t i o n ) . T h i s dose has been v a r i o u s l y t e r m e d t h e " b i o l o g i c a l l y i n s i g n i f i c a n t d o s e " o r t h e "NOEL" (No O b s e r v e d E f f e c t L e v e l ) , and i t s d e t e r m i n a t i o n i s one o f t h e primary o b j e c t i v e s o f t o x i c i t y t e s t i n g . The term " a d v e r s e e f f e c t " i s n o t e a s i l y i n t e r p r é t a b l e . A p h y s i o l o g i c a l r e s p o n s e t o a s t r e s s t h a t i s r e a d i l y r e v e r s i b l e , such a s a change i n enzyme c o n c e n t r a t i o n s , m i g h t n o t n e c e s s a r i l y be a n a d v e r s e e f f e c t , p a r t i c u l a r l y i f no damage i s d e t e c t a b l e . The a b i l i t y o f t o x i c i t y t e s t i n g t o s e t s a f e l e v e l s i s i t s most i m p o r t a n t p r a c t i c a l f u n c t i o n . The p r o c e d u r e has been d e v e l o p e d most r i g o r o u s l y i n t h e f i e l d o f f o o d and c o l o r a d d i t i v e t e s t i n g -- c h e m i c a l s added t o f o o d s -- where a s e r i e s o f s p e c i a l s t i p u l a -


1.

JOSELOW

Systematic

Toxicity

Testing in Foods

5

tions that permit estimates o f " t o x i c o l o g i c a l l y i n s i g n i f i c a n t l e v e l s " o f c h e m i c a l s i n f o o d s , have been p r o p o s e d . I n g e n e r a l , a " s a f e " l e v e l o f a s u b s t a n c e i s a r b i t r a r i l y s e t by a p p l y i n g a s a f e t y f a c t o r t o the h i g h e s t intake t h a t i s found not t o i n j u r e experimental animals exposed f o r extended p e r i o d s ; i . e . , over a l i f e t i m e . The "safe" level i s f r e q u e n t l y expressed as l/100th o f t h e e x p e r i m e n t a l l y d e t e r m i n e d h i g h e s t No O b s e r v e d E f f e c t L e v e l . T h i s r a t i o , 1:100, i s d e r i v e d by a p p l y i n g a f a c t o r o f 10 f o r ext r a p o l a t i o n o f t h e f i n d i n g s o f a n i m a l s t o man, a n d a n o t h e r f a c t o r o f 10 t o a c c o u n t f o r v a r i a t i o n s i n s u s c e p t i b i l i t i e s among p e o p l e . A r a t i o o f 100 i s t h o u g h t t o p r o v i d e a c o n s e r v a t i v e e s t i m a t e o f t h e s a f e t y f a c t o r needed t o a f f o r d a d e q u a t e p r o t e c t i o n , even t o p e r s o n s whose d i e t a r y p a t t e r n s o r 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 m i g h t be u n u s u a l . A n d t h i s p r o c e d u r e s has been s o w i d e l y a d o p t e d by n a t i o n a l a n d i n t e r n a t i o n a l b o d i e s c o n c e r n e d w i t h s a f e t y t h a t the term " s a f e l e v e l " , a s a p p l i e d t o chemicals i n f o o d , has a l s o become a l a r g e e x t e n t a p p l i c a b l e t o c h e m i c a l s a n d h a z a r d o u s s u b s t a n c e s t h a t impinge on man f r o m w h a t e v e r s o u r c e . The c o n c e p t t h a t t h e r e must be some dose l e v e l below which an a d v e r s e e f f e c t w i l l n o t a p p e a r has n o t met w i t h u n i v e r s a l a c c e p t a n c e , p a r t i c u l a r l y w i t h r e g a r d t o t o x i c a g e n t s t h a t have l o n g t e r m , i r r e v e r s i b l e e f f e c t s , such a s i o n i z i n g r a d i a t i o n o r c a r c i n o g e n i c s u b s t a n c e s . Some t o x i c o l o g i s t s have p r o p o s e d t h a t a d e q u a t e t e s t i n g c a n s e t s a f e l i m i t s even f o r c a r c i n o g e n i c a g e n t s ; o t h e r s c h a l l e n g e t h i s b e l i e f , and a r g u e t h a t i t i s i m p o s s i b l e t o p r e d i c t s a f e l e v e l s f o r c a r c i n o g e n s by t a k i n g an a r b i t r a r y f r a c t i o n o f t h e l o w e s t No O b s e r v e d E f f e c t a n i m a l dose i n any p a r t i c u l a r e x p e r i m e n t a l s i t u a t i o n . S h a r p d i s t i n c t i o n s have a l s o been made between r e v e r s i b l e and i r r e v e r s i b l e e f f e c t s . F o r a d d i t i v e s t h a t can i n d u c e r e v e r s i b l e t o x i c e f f e c t s , t h r e s h o l d l e v e l s below which human e x p o s u r e w o u l d be s a f e c a n be r e a s o n a b l y d e t e r m i n e d . Howe v e r , f o r c h e m i c a l s i n d u c i n g i r r e v e r s i b l e and p o s s i b l y c u m u l a t i v e e f f e c t s , such t h r e s h o l d s c a n n o t be d e t e r m i n e d ; and a z e r o t o l e r a n c e has t o be s e t . I f a z e r o t o l e r a n c e i s m a n d a t e d , t h e n t h e r e i s no need f o r t o x i c i t y t e s t i n g . O n l y t h e t e c h n i q u e s o f a n a l y t i c a l c h e m i s t r y need be a p p l i e d t o d e t e r m i n e w h e t h e r a s u b s t a n c e i s p r e s e n t o r n o t , and t h e r e f o r e , w h e t h e r i t s u s e i s p e r m i s s i b l e o r n o t . Such an a p p r o a c h however, r u n s t h e r i s k o f becoming a r e d u c t i o a d a b s u r dum, a s a n a l y t i c a l t e c h n i q u e s become more r e f i n e d and s e n s i t i v e . D e s i g n s and O b j e c t i v e s f o r Formal T e s t i n g The movement toward s y s t e m a t i c f o r m a l i z e d t o x i c i t y t e s t i n g b e g a n , as m i g h t be e x p e c t e d , w i t h t h e p r o b l e m o f c h e m i c a l s i n f o o d s i n t h e 1940's. A Food and Drug A d m i n i s t r a t i o n r e p o r t p u b l i s h e d i n 1943 ( 4 ) o f f e r e d some g e n e r a l p r o t o c o l s . As a d d i t i o n a l t o x i c e f f e c t s ' - - u n s u s p e c t e d a t t h a t t i m e -- became known, t h e s e r e c -


6


FEEDS

ommendations were a m p l i f i e d i n d e t a i l and expanded i n s c o p e . T h e r e c e n t l y p u b l i s h e d monograph ( ) o f t h e Bureau o f Goods, Food and Drug A d m i n i s t r a t i o n , e n t i t l e d , " T o x i c o l o g i c a l P r i n c i p l e s " can be r e g a r d e d a s t h e c u l m i n a t i o n o f t h e s e e f f o r t s . I t i s a c o n s i d e r a b l y updated v e r s i o n o f the o r i g i n a l recommendations, and i s s i n e qua non f o r t h o s e c o n c e r n e d w i t h t o x i c i t y t e s t i n g . I t p r o v i d e s d e t a i l s r e g a r d i n g t e s t r e q u i r e m e n t s and p r o t o c o l s , and r e p r e s e n t s t h e c o n s e n s u s o f j u d g m e n t s o f t h e a g e n c y most c o n cerned with the s a f e t y o f x e n o b i o t i c s i n foods. The o r i g i n a l g u i d e l i n e s recommended o n l y t h r e e b a s i c t y p e s o f t e s t s t o be p e r f o r m e d on l a b o r a t o r y a n i m a l s t o e v a l u a t e s a f e t y . W h i l e o t h e r t e s t s f o r s p e c i f i c t o x i c e f f e c t s have s i n c e been a d d e d , t h e s e b a s i c t e s t s s t i l l r e m a i n t h e backbone o f c u r r e n t t o x i c i t y t e s t i n g procedures. These t e s t s d i f f e r p r i m a r i l y i n t h e i r d u r a t i o n s and o b j e c t i v e s . T e s t s t h a t use o n l y s i n g l e d o s e s o f t h e c h e m i c a l , a d m i n i s t e r e d on one o c c a s i o n , a r e r e f e r r e d t o a s "acute t e s t s " . Longer t e s t s , i n which the chemical i s given a t l e a s t o n c e d a i l y , f o r p e r i o d s o f up t o t h r e e months, a r e commonly r e f e r r e d t o as "sub-acute" o r "sub-chronic" o r "prolonged" t e s t s . S t i l l l e n g t h i e r t e s t s , i n v o l v i n g the a d m i n i s t r a t i o n o f a chemical t o a n i m a l s d a i l y f o r p e r i o d s o f one t o two y e a r s t o s i m u l a t e l i f e time exposure, are r e f e r r e d t o as " c h r o n i c " o r "long term" o r "extended" t o x i c i t y t e s t s . Acute T o x i c i t y Tests The s i n g l e t e s t t h a t i s c o n d u c t e d on p r a c t i c a l l y a l l s u b s t a n c e s of b i o l o g i c i n t e r e s t i s the acute t o x i c i t y t e s t . I t i s undoubte d l y r e s p o n s i b l e f o r t h e g r e a t e s t r i t u a l mass s l a u g h t e r o f a n i mals i n t h i s country. The t e s t r e q u i r e s t h a t t h e a n i m a l s be e x p o s e d t o a r e l a t i v e l y l a r g e d o s e , o n a t l e a s t one o c c a s i o n , t o t h e c h e m i c a l o f i n t e r e s t . The p r i n c i p a l p u r p o s e i s t o d e t e r m i n e t h e l e t h a l i t y o r LD50 f o r t h e c h e m i c a l ( t h e d o s e t h a t w i l l be f a t a l t o 50% o f t h e t e s t population.) A l m o s t a l l a c u t e t o x i c i t y t e s t s a r e done w i t h r a t s o r m i c e , l a r g e ly b e c a u s e o f t h e low c o s t , e a s y a v a i l a b i l i t y , e x p e n d a b i l i t y o f t h e s e a n i m a l s , and t h e f a c t t h a t an a b u n d a n t l i t e r a t u r e e x i s t s f o r t h e s e two s p e c i e s . I n i t i a l l y , t h e c h e m i c a l may be g i v e n t o a s i n g l e s p e c i e s ; a t s e v e r a l dosage l e v e l s , t o p r e - c o n d i t i o n e d g r o u p s o f a n i m a l s , a s p r e s c r i b e d by F e d e r a l r e c o m m e n d a t i o n s . F o l l o w i n g a d m i n i s t r a t i o n o f t h e d o s e s , o b s e r v a t i o n s a r e made o f t h e a n i m a l s f o r p e i o d s r a n g i n g f r o m a few m i n u t e s t o two weeks, t o c o m p l y w i t h t h e r e q u i r e m e n t s o f some r e g u l a t i o n s e.g.; t h e F e d e r a l H a z a r d o u s S u b s t a n c e s A c t . T h e l e t h a l i t y i s d e t e r m i n e d on t h e b a s i s o f d e a t h o c c u r i n g w i t h i n t h e o b s e r v a t i o n p e r i o d (14 days)-


1.

JOSELOW

Systematic

Toxicity

Testing in Foods

7

Subchronic o r Subacute T o x i c i t y T e s t i n g The a c u t e t e s t i s p r i m a r i l y a f a c t - f i n d i n g , e x p l o r a t o r y e x p e r i m e n t t o o b t a i n some i n d i c a t i o n o f t o x i c i t y . Such i n f o r m a t i o n i s u s e d n o t o n l y t o p e r m i t c o m p a r i s o n s among d i f f e r e n t a g e n t s (by compari n g t h e i r L D s q ' S ) , b u t a l s o t o p r o v i d e a more r e f i n e d b a s i s f o r t h e s e l e c t i o n o f dose l e v e l s f o r t h e more p r o l o n g e d t y p e o f s t u d i e s , e x t e n d i n g f o r 3-4 months, t h e s o - c a l l e d " s u b c h r o n i c " o r "subacute animal s t u d i e s . These more p r o l o n g e d s t u d i e s a r e d e s i g n e d t o p r o v i d e a d d i t i o n a l i n f o r m a t i o n on t o x i c i t y . They have t h r e e main p u r p o s e s : 1)

T o e s t a b l i s h more n a r r o w l y a maximum t o l e r a t e d d o s e ; i . e . , a dose t h a t w i l l p r o d u c e o v e r t a d v e r s e symptoms b u t n o t death.

2)

T o p r o v i d e an e s t i m a t e o f t h e h i g h e s t dose t h a t w i l l show any e f f e c t ; i . e . , t h e "no e f f e c t " l e v e l .

3)

T o e s t a b l i s h t h e b i o l o g i c a l n a t u r e o f t h e damage p r o d u c e d as r e v e a l e d by c l i n i c a l and p a t h o l o g i c a l e x a m i n a t i o n o f the s a c r i f i c e d animals.

not

Chronic Toxicity I f t h e s u b s t a n c e has been f o u n d t o be s a t i s f a c t o r y , i . e . , nont o x i c , i n t h e s u b c h r o n i c t e s t s , i t may t h e n be s u b j e c t e d t o c h r o n i c , l o n g - t e r m , o r e x t e n d e d t o x i c i t y t e s t s . These t e s t s a r e meant t o s i m u l a t e l i f e t i m e human e x p e r i e n c e , and t h e e x p e r i m e n t a l p r o t o c o l s , t h e r e f o r e , c a l l f o r dosing the animal with the substance, o v e r t h e c o u r s e o f i t s l i f e - t i m e , which means, f o r p r a c t i c a l p u r p o s e s , t h a t an a n i m a l w i t h a s h o r t l i f e span must be u s e d ; i . e . , r a t s or mice. The r a t i o n a l e f o r a c c e p t i n g t h i s i s t h a t i n a s h o r t l i v e d s p e c i e s , p r o g r e s s i v e i n j u r y p r o c e e d s more r a p i d l y , and can be d e t e c t e d more e a s i l y t h a n i n a l o n g - l i v e d s p e c i e s . L i f e t i m e d o s i n g o f a r a t ; i . e . , f o r 30 months, i s c o n s i d e r e d e q u i v a l e n t t o 70 y e a r s e x p o s u r e i n man. The F . D . A . t h u s s p e c i f i e s and a c c e p t s 30 month e x p o s u r e i n r a t s f o r a l i f e t i m e s t u d y . But t h i s r a t i o n a l e does n o t h o l d f o r dogs o r o t h e r a n i m a l s . Two y e a r s f o r a dog i s s t i l l o n l y a f r a c t i o n (20%) o f i t s l i f e t i m e . By t h e t i m e a s u b s t a n c e i s c o n s i d e r e d f o r c h r o n i c t o x i c i t y s t u d i e s , i n f o r m a t i o n w i l l have a l r e a d y been o b t a i n e d r e g a r d i n g t h e n a t u r e o f i t s t o x i c i t y and i t s t o l e r a b l e a s w e l l a s l e t h a l d o s e s . The main p u r p o s e s o f l o n g - t e r m t e s t i n g t h e n a r e t w o f o l d : 1)

To f i n d o r c o n f i r m t h e " N o - E f f e c t " l i f e t i m e dosage


level

X E N O B I O T I C S IN F O O D S A N D F E E D S

8

f o r the a d d i t i v e , i . e . , t h e maximum t h a t can be t a k e n t h a t w i l l n o t p r o d u c e any o b s e r v a b l e a d v e r s e e f f e c t o v e r a 1 i f e time. 2)

T o d e t e c t any more s i g n i f i c a n t a b n o r m a l i t i e s t h a t may become a p p a r e n t o n l y o v e r t h e c o u r s e o f a l i f e t i m e ; a n d t h e change g e n e r a l l y s o u g h t most commonly i s t h e d e v e l o p ment o f a c a n c e r o f some k i n d .

T h i s i s u n d o u b t e d l y t h e most i m p o r t a n t o b j e c t i v e o f t h e c h r o n i c t e s t -- t o s e a r c h f o r t h e d e v e l o p m e n t o f a c a r c i n o g e n i c e n d r e s u l t . I n f a c t , some t o x i c o l o s i t s b e l i e v e t h a t i f e f f e c t s o t h e r than c a n c e r a r e s o u g h t , t h e s e s h o u l d have become a p p a r e n t i n t h e s h o r t e r s u b c h r o n i c s t u d i e s -- a n d t h e r e would t h e n u s u a l l y be no need t o c a r r y the t e s t beyond t h e s u b c h r o n i c p e r i o d . As m i g h t be e x p e c t e d when c o n d u c t i n g t e s t s w i t h a n i m a l s t h a t w i l l u n a v o i d a b l y v a r y t o some e x t e n t i n t h e i r g e n e t i c make-up, v a r i a t i o n s i n r e s u l t s may be e n c o u n t e r e d , p a r t i c u l a r l y i f t e s t c o n d i t i o n s a r e not c a r e f u l l y c o n t r o l l e d . R e c o g n i t i o n o f t h i s p r o b l e m w h i c h has a c c o u n t e d f o r w i d e d i s p a r i t i e s r e p o r t e d i n t h e l i t e r a t u r e i n t o x i c i t y t e s t f i n d i n g s , h a s l e d F.D.A. and o t h e r s (6) t o s t a n d a r d i z e the r e l e v a n t f a c t o r s i n v o l v e d i n conducting t o x i c i t y t e s t s . T e s t c o n d i t i o n s , i f n o t c o n t r o l l e d , can l e a d t o s p u r i o u s r e s u l t s among d i f f e r e n t l a b o r a t o r i e s , and even w i t h i n t h e same laboratory (7). The s t a n d a r d i z e d f a c t o r s i n c l u d e n o t o n l y t h e c o n d i t i o n s u n d e r w h i c h t h e t e s t s a r e c o n d u c t e d -- and good l a b o r a t o r y p r a c t i c e s a r e now a n added minimum r e q u i r e m e n t -- but a l s o p r e s c r i b e t h e c l i n i c a l t e s t i n g and o b s e r v a t i o n s t h a t must be made on t h e t e s t a n i m a l s d u r i n g and a f t e r the t e s t p e r i o d ( T a b l e s I - I V ) . R e c e n t a d d i t i o n s t o t h e t o x i c i t y t e s t p r o t o c o l s may now r e q u i r e formulized examinations f o r genetic e f f e c t s ( i . e . , teratogenesis, m u t a g e n e s i s , r e p r o d u c t i o n ) a s w e l l a s d e t e r m i n a t i o n o f t h e metabo l i c a l t e r a t i o n s o f t h e a d d i t i v e a n d t h e i r d i s p o s i t i o n . Each o f t h e s e a c t i v i t i e s has become an i m p o r t a n t s u b - b r a n c h o f t o x i c i t y testing. A t y p i c a l manner i n w h i c h a d e c i s i o n on s a f e t y may be made, b a s e d on s e q u e n t i a l t e s t i n g , i s shown i n F i g u r e 1. Such t e s t i n g o b v i o u s l y r e p r e s e n t s an e x t e n s i v e and e x p e n s i v e u n d e r t a k i n g . The s e r v i c e s o f v a r i e d p r o f e s s i o n a l s w i l l be r e q u i r e d : t o x i c o l o g i s t s , p a t h o l o g i s t s , chemists, biochemists, v e t e r i n a r i a n s , s t a t i s t i c i a n s , and a t t o r n e y s ( t o p r e p a r e t h e p e t i t i o n s ) and a h o s t o f o t h e r s u p p o r t ing p e r s o n n e l . From a l l t h e t o x i c i t y d a t a , i f t h e s t u d y works o u t , w i l l emerge a r e a s o n a b l e f i g u r e f o r a "No E f f e c t L e v e l " a s w e l l a s an i n d i c a t i o n


1.

JOSELOW

Systematic

Toxicity

Testing in Foods

TABLE 1 OUTLINE OF GENERAL PROCEDURES*

Animals

No. o f Dose L e v e l s

No. o f A n i m a l s (minimum/group)

ACUTE

WTrs) r a t s , mice, guinea pigs

Dermal

4 -5

5-10 e a c h , e q u a l n o s . o f e a c h sex

dogs

u s u a l l y t o t a l o f 12

rabbits

2 male & 2 f e m a l e e a c h

INTUBATION 1 - 8 hrs SUBACUTE (90 d a y s )

Dermal

rats


dogs


rabbits


CHRONIC (2 y r s ) rats


dogs


* From A p p r a i s a l o f S a f e t y o f C h e m i c a l s i n F o o d s , D r u g s , and C o s m e t i c s . A s s o c i a t i o n o f Food and Drug O f f i c i a l s o f U.S., 1959.


10


TABLE I I SUBCHRONIC ORAL TOXICITY TESTING * STANDARDIZED FACTORS* 1.

Test Duration

2.

Species

3.

Age o f Animals

4.

Number o f A n i m a l s

5.

C o n t r o l Groups

6.

Dose G r o u p s

7.

Diet

8.

Route o f A d m i n i s t r a t i o n

9.

Clinical Testing (a) (b) (c)

10.

Ophthalmological Examination Hematology Clinical

Chemistry

Observation o f Animals

11.

Gross

Necropsy

12.

Organ

Weights

13.

H i s t o p a t h o l o g i c a l E x a m i n a t i o n on a t l e a s t 32 p r i n c i p a l

*

tissues

Toxicological Principles. A d m i n i s t r a t i o n , 1982.

U.S. Food a n d Drug


FEEDS

JOSELOW

Systematic

Toxicity Testing in Foods

TABLE I I I SIGNS AND SYMPTOMS FOUND IN ANIMALS UNDERGOING TOXICOLOGIC TESTS Signs Aggressiveness toward experimenter A l t e r e d muscle tone A l t e r a t i o n s i n cardiac rate and rhythm Catatonia (phases of stupor or excitement) Coma Convulsions to touch Paralysis Change i n p u p i l l a r y s i z e S e n s i t i v i t y to pain Skin l e s i o n s Corneal o p a c i t i e s Placing reflexes Righting r e f l e x e s Grasping r e f l e x e s Death Symptoms

Abnormal excreta Exploratory behavior Inactivity Convulsions , spontaneous Dyspnea (shortness of breath) Sedation Nystagmus (involuntary r a p i d movements of eyeballs) Cyanosis Salivation Nasal Discharge P i l o e r e c t i o n (erection of hair) Phonation (utterance of vocal sounds) Unusual p h y s i c a l p o s i t i o n s Unusual t a i l p o s i t i o n s


XENOBIOTICS IN FOODS

A N D FEEDS

TABLE IV CLINICAL PROCEDURES COMMONLY EMPLOYED IN ANIMAL TOXICOLOGICAL TESTS

Blood chemistry studies Sodium Potassium Blood urea nitrogen Glucose Urinalysis pH and s p e c i f i c g r a v i t y Protein Glucose Ketones Crystals Blood c e l l s Bacteria Hematology Hematocrit T o t a l red blood c e l l counts T o t a l and d i f f e r e n t i a l white blood c e l l counts Organ f u n c t i o n t e s t s Bromsulfophthalein r e t e n t i o n ( l i v e r function) Thymol t u r b i d i t y ( l i v e r function) Serum a l k a l i n e phosphatase ( l i v e r function) Blood urea nitrogen (kidney function)



Figure 1. Safety decision tree. Key: + , presents socially unacceptable risk; - , does not present a socially unacceptable risk; S, metabolites known and safe; U, metabolites unknown or of doubtful safety; and ?, decision requires more evidence. (Reproduced with permission jrom Kef. 8. Copyright 1978, F o o d C o s m e t . T o x i c o l . )

Exposure

Assessment

Defined

Test Material


14

FEEDS

of the carcinogenic potential o f the substance. Thes a f e t y o f the s u b s t a n c e f o r humans may t h e n be e s t i m a t e d . The word " e s t i m a t e " i s u s e d a d v i s e d l y . A f t e r c o m p l e t i o n o f a l l t h e a n i m a l t e s t s , o n l y a c t u a l e x p o s u r e i n humans w i l l r e v e a l any s u b t l e o r u n f o r e s e e n t o x i c i t y . T o x i c i t y t e s t i n g c a n n o t be c o n s i d e r e d c o m p l e t e u n t i l t h e s u b s t a n c e has gone t h r o u g h e x t e n s i v e u s e i n humans. A n d even t h e n , i t may n o t be c o m p l e t e , u n l e s s ade q u a t e r e c o r d s , f o l l o w - u p s , e t c . , have been m a i n t a i n e d and p r o s p e c t i v e e p i d e m i o l o g i c a l s t u d i e s a r e made.

Literature Cited 1. Toxicants Occurring Naturally i n Foods. Report, Committee on Food Protection, National Academy of Sciences, Washington, DC, 1973. 2. Principles and Procedures f o r Evaluating the Safety of Food Additives. Food Protection Committee, Food and Nutrition Board, National Academy of Sciences, Publ. No. 750, December 1959. 3. Hazardous Substances Act. Consumer Product Safety Commission, Washington, DC, 1959. 4. Appraisal of Safety of Chemicals i n Foods, Drugs, and Cosmet i c s . Association of Food & Drug O f f i c i a l s of the United States, Washington, DC, 1959. 5. Toxicological Principles f o r the Safety Assessment of Direct Food Additives and Color Additives Used i n Food. U.S. Food and Drug Administration, 1982. 6. O.E.C.D. Guidelines f o r Testing of Chemicals. O.E.C.D. P u b l i cations, Washington, DC, 1982. 7. Weil, C.S. and Scala, R. A. Tox. Appl. Pharmacol., 1971, 19, 276. 8. Food and Cosmetics Toxicology, 16, 9 (1978), Pergamon Press, Oxford. General Bibliography Doull, J.; Klaasen, C.; Amdur, Μ., Eds. Toxicology, The Basic Science of Poisons, Macmillan Publishing C o . , New York, 1980. Hayes, A. W. Principles and Methods of Toxicology, Raven Press, New York, 1982. Loomis, T . A. Essentials of Toxicology, 3rd ed., Lea & Febiger, New York, 1978. Mehlman, Μ. Α.; Blumenthal, H.; Shapiro, R . , Eds. Advances i n Mod ern Toxicology, New Concepts i n Safety Evaluation, V o l . 1, Part 1, 2. John Wiley & Son, New York, 1979. Paget, G. E., Ed. Methods i n Toxicology. F. A. Davis & Co., P h i l a delphia, 1970. RECEIVED July 6, 1983


Systematic Toxicity Testing for Xenobiotics in Foods - ACS Symposium

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