Problems and Pitfalls in Analytical Studies in ... - ACS Publications

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Problems and Pitfalls in Analytical Studies in Toxicology J. D. McKINNEY, P. W. ALBRO, R. H. COX, J. R. HASS, and D. B. WALTERS Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 The major interrelated divisions of environmental health sciences necessary to define a human health hazard are epidemiology, toxicology and chemistry. If one or more of these areas is lacking or incomplete in studies of environmental agents such as pesticides, then health hazard potential can not be fully assessed. Chemistry is essential in these relationships in order to provide timely and effective solutions to environmental health problems. Environmental Health Chemistry has been presented (1) as a new subdiscipline which emphasizes the chemistry needed to establish these relationships and permit assessment of the potential human health hazards associated with chemical contamination of our environment. Fundamental to these various programs is assurance that good, reliable analytical data is generated especially in those cases which most directly affect the public's health and well being. How one generates good, reliable analytical data is the primary focus of the papers in this session. Our position, which is similar to that taken by others (2), is one of adopting good analytical practices as an alternative to standard methods, licensing and certification. Our position is presented in brief in the following outline of criteria for analytical protocols. Good A n a l y t i c a l P r a c t i c e s General C o n s i d e r a t i o n s . I t i s o f t e n more d i f f i c u l t to judge the v a l i d i t y of an a n a l y t i c a l study than to perform the analyses. Since the d i f f i c u l t y most commonly a r i s e s because of f a i l u r e to s p e c i f y the f u l l d e t a i l s of procedures i n advance or r e p o r t them adequately a t the c o n c l u s i o n of a study, the present o u t l i n e d e s c r i b e s the types of information considered e s s e n t i a l f o r both preparing p r o t o c o l s f o r p r i o r approval and submitting f i n a l a n a l y t i c a l r e s u l t s i n r e p o r t form. Planning and r e p o r t i n g c a t e g o r i e s f o r the f i n a l processing steps, the a c t u a l determinat i o n procedures, w i l l be the subject of f u t u r e documents. The present g u i d e l i n e s p e r t a i n to a l l of those steps p r e l i m i n a r y to

This chapter not subject to U.S. copyright. Published 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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determination; that i s , from sample c o l l e c t i o n to p r e s e n t a t i o n of the f i n a l t e s t p r e p a r a t i o n . The emphasis i s upon v a l i d a t i o n of procedures, and upon r e p o r t i n g i n s u f f i c i e n t d e t a i l the procedures that can i n f a c t be evaluated subsequent to the submission of analytical results. Sample S e l e c t i o n . Assuming that the samples to be analyzed are to be s e l e c t e d from a l a r g e r population or populations, the manner i n which samples w i l l be s e l e c t e d must be d e s c r i b e d . I f s e l e c t i o n i s intended to be random, enough d e t a i l s must be given to enable one to d i s t i n g u i s h random from haphazard. Since some s t a t i s t i c a l a n a l y s i s may e v e n t u a l l y be a p p l i e d to the a n a l y t i c a l r e s u l t s , one must be able to determine whether the sample groups are f u l l y independent, or the observations a r e n a t u r a l l y p a i r e d . F i n a l l y , evidence f o r c o n s i d e r i n g the samples as r e p r e s e n t a t i v e of the l a r g e r populations must be d e s c r i b e d . Sample C o l l e c t i o n and Storage. This step i n an a n a l y t i c a l study o f f e r s many o p p o r t u n i t i e s f o r l o s s of i n t e g r i t y of samples, and must be described i n f u l l d e t a i l . P r e c i s e l y what t o o l s w i l l be used to a c q u i r e the samples? Disposable s c a l p e l s , f o r example, are coated with an o i l that can contaminate t i s s u e s . Metal t o o l s obviously should not be used to take samples f o r t r a c e metal determination. Good judgement can not be assumed; d e t a i l s must be provided. The sample containers must be f u l l y d e s c r i b e d . What m a t e r i a l i s used? Aluminum f o i l i s coated with drawing o i l . B o t t l e cap l i n e r s a r e a common source of contamination. How a r e the cont a i n e r s cleaned? How w i l l samples be stored p r i o r to a n a l y s i s ? At what temperatures and f o r how long? W i l l they be exposed to l i g h t ? A i r ? D e t a i l s must be g i v e n . Sample Workup. Most t r a c e l e v e l analyses s t a r t with some sort of e x t r a c t i o n step. This should be described i n d e t a i l ("the samples were extracted with chloroform" i s not enough). How long does each step take? For how long a r e e x t r a c t i o n mixtures s t i r r e d or shaken? How many c y c l e s of a Sohxlet extractor? Enough information must be given to permit someone to d u p l i c a t e the procedure. I s the sample f i n e l y d i v i d e d p r i o r to or during the e x t r a c t i o n ? What i s the solvent to sample r a t i o ? Vague terms such as "warm s o l v e n t " , "extracted e x h a u s t i v e l y " , or the l i k e must be avoided. I f temperature, time, e t c . a r e important, they should be p r e c i s e l y s p e c i f i e d . Sources of s o l vents and reagents, and s p e c i a l means used to p u r i f y or dry them must be given. Terms such as "reagent grade" provide no assurance of freedom from r e l e v a n t , i n t e r f e r i n g i m p u r i t i e s . Cleanup Procedures. Most t r a c e l e v e l analyses i n v o l v e some sort of cleanup of the crude e x t r a c t . D e t a i l s of these procedures


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must i n c l u d e sources and means of a c t i v a t i o n of chromatographic media, sources and some d i s c u s s i o n of p u r i t y of s o l v e n t s used, and s u f f i c i e n t d e s c r i p t i o n of the procedures themselves to permit their duplication.


V a l i d a t i o n S t u d i e s . P r i o r to the a n a l y s i s of unknown samples, a l l procedures must be v a l i d a t e d i n terms of recovery, r e p r o d u c i b i l i t y , s e n s i t i v i t y , freedom from i n t e r f e r e n c e , and accuracy. Recovery S t u d i e s . Since these s t u d i e s w i l l g e n e r a l l y i n v o l v e the use of " s p i k e s " , the f i r s t requirement i s f o r the s p i k i n g or f o r t i f i c a t i o n procedure i t s e l f to be v a l i d a t e d . E i t h e r i t must be shown that the "spiked" chemicals e q u i l i b r a t e with the c o r r e sponding endogenous ones, or i t must be e m p i r i c a l l y demonstrated that the recovery of exogenous " s p i k e " i s the same as the recovery of endogenous compound, over the f u l l range of c o n c e n t r a t i o n l e v e l s to be sought i n the a n a l y s i s of unknowns. The dependence of both percentage recovery and i t s standard d e v i a t i o n on concent r a t i o n must be determined and r e p o r t e d . The a c t u a l f o r t i f i c a t i o n procedure must be described i n s u f f i c i e n t d e t a i l to permit d u p l i cation. L i m i t s of D e t e c t i o n . Since the clean-up and recovery achieved i n the o v e r a l l sample workup e f f e c t s the d e t e c t i o n l i m i t of the measurement technique, the a c t u a l l i m i t of d e t e c t i o n i s , r e l a t i v e to the amount of sample a v a i l a b l e f o r a n a l y s i s , always l e s s than the l i m i t of the measurement technique. Thus, i f a measurement technique can respond to one microgram of m a t e r i a l , but the recovery i n the p r e l i m i n a r y sample workup i s 50%, then the t r u e e f f e c t i v e d e t e c t i o n l i m i t i s two micrograms, and i t i s t h i s l a t t e r v a l u e that must be r e p o r t e d . A d d i t i o n a l l y , the l i m i t of d e t e c t i o n of the measurement technique must be taken as not the smallest amount causing a response, but r a t h e r the smallest amount to which the a n a l y t i c a l c r i t e r i a employed f o r q u a l i t a t i v e i d e n t i f i c a t i o n can be a p p l i e d . C r i t e r i a f o r Q u a l i t a t i v e I d e n t i f i c a t i o n . The c r i t e r i a to be a p p l i e d f o r q u a l i t a t i v e i d e n t i f i c a t i o n must be d e s c r i b e d . I d e a l l y , they should i n c l u d e a t l e a s t one c r i t e r i o n unique to the compound of i n t e r e s t ; f a i l i n g t h i s , they must i n c l u d e a combinat i o n of c r i t e r i a , which combination JLS unique to the compound sought. In a d d i t i o n , the c r i t e r i a by which i n t e r f e r i n g substances may be recognized must be d e s c r i b e d . C r i t e r i a f o r Q u a n t i t a t i v e Determination. Not a l l assays provide l i n e a r c a l i b r a t i o n curves. C a l i b r a t i o n curves must be constructed from the a n a l y s i s of spiked samples, to v i s u a l i z e any n o n l i n e a r i t y i n recovery. I f i n t e r p o l a t i o n i s u t i l i z e d , the i n t e r p o l a t i o n procedure must be f u l l y d e s c r i b e d ; e x t r a p o l a t i o n should not be used. Standard curves must span the complete range


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of v a l u e s found i n the set of unknown samples. The samples used f o r s p i k i n g should i d e a l l y c o n t a i n no d e t e c t a b l e endogenous compounds responding to the assay, but i f zero background can not be achieved, the endogenous (background) l e v e l i n the spiked samples must be a c c u r a t e l y determined and compensated f o r . Blanks. "Procedure blanks", samples of the same matrix m a t e r i a l that w i l l subsequently be analyzed i n the study but known to l a c k d e t e c t a b l e l e v e l s of the compounds of i n t e r e s t , must be put through the e n t i r e procedure i n c l u d i n g storage f o r the same (mean) l e n g t h of time i n the same environment as the t e s t samples. E x t r a c t i o n and cleanup procedures a p p l i e d to these "blanks" must i n v o l v e the same amounts of s o l v e n t s and chromatographic media as w i l l be used on the r e a l samples. In no other way can the l i k e l i h o o d of i n t e r f e r e n c e s ( f a l s e p o s i t i v e s ) be c o n v i n c i n g l y assessed. This paper i d e n t i f i e s some unique problems and p i t f a l l s i n providing a n a l y t i c a l support f o r t o x i c o l o g i c a l r e s e a r c h c o n s i s t e n t with our p o s i t i o n on good a n a l y t i c a l p r a c t i c e s . Major areas r e l e v a n t to t o x i c o l o g i c a l r e s e a r c h r e c e i v i n g some a t t e n t i o n include environmental a n a l y s i s , mechanism e l u c i d a t i o n , t e s t i n g programs, t o x i c i t y p r e d i c t i o n , chemical epidemiology, and s a f e t y monitoring. Environmental A n a l y s i s Assessment of the human h e a l t h hazard p o t e n t i a l of e n v i r o n mental chemicals r e q u i r e s study of the environmental transformat i o n products. The products can i n c l u d e a v a r i e t y of m e t a b o l i t e s generated i n p l a n t s , s o i l s and animal t i s s u e as w e l l as nonb i o l o g i c a l products such as those d e r i v e d from h y d r o l y s i s or p h o t o l y s i s . Therefore, environmental a n a l y s i s i s of b a s i c importance i n determining the substances reaching the human e n v i r o n mental i n t e r f a c e . The commercial uses of p o l y c h l o r i n a t e d b i p h e n y l mixtures (PCBs) as i n s u l a t i n g and d i e l e c t r i c f l u i d s i s an example of t h i s problem. The commercial mixture c o n s i s t s of a complex mixture of c h l o r o b i p h e n y l s v a r y i n g i n degrees and p o s i t i o n s of c h l o r i n e s u b s t i t u t i o n along w i t h a few p a r t - p e r - m i l l i o n (ppm) c h l o r i n a t e d dibenzofuran contaminants. We now know that the PCBs can be transformed c h e m i c a l l y during use ( 3 ) , b i o l o g i c a l l y i n c u l t u r e s and s o i l ( 5 ) , and animal t i s s u e s (4) and under p h y s i c a l i n f l u e n c e such as u l t r a v i o l e t l i g h t (5) and i n c i n e r a t i o n c o n d i t i o n s (6) (Table 1) . Residues i n humans (7) l a r g e l y c o n s i s t of those PCB isomers with high c h l o r i n e content which a r e g e n e r a l l y more r e s i s t a n t to chemical and b i o l o g i c a l transformation. The exact nature of these r e s i d u e s i s s t i l l being i n v e s t i g a t e d i n an attempt to assess the r e a l human h e a l t h hazard a s s o c i a t e d with the PCBs ( 1 ) .


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Table I C l a s s i f i c a t i o n and Occurrence of Compounds Associated with the PCB Problem In Commercial Product of Use Mixtures or T r a n s p o r t a t i o n

Compounds


PCBs and Related D e r i v a t i v e s : Non-ortho s u b s t i t u t e d with high meta-para s u b s t i t u t i o n (Type I)

X

Ortho s u b s t i t u t e d V i c i n a l unsubstituted carbon atoms absent

(Type I I )

V i c i n a l unsubstituted carbon atoms present (Type I I I )

Χ X a

Biphenyl Dimers and Trimers Oxygenated C h l o r i n a t e d Aromatic

Χ Compounds:

C h l o r i n a t e d Dibenzofurans

Χ

X a

C h l o r i n a t e d Diphenylethers

X

Hydroxychlorinated Biphenyls and Related Oxygen Containing Compounds

X

^

a

T h i s i s based on p r e l i m i n a r y data ( 4 ) from a n a l y s i s of the used f l u i d s as w e l l as a n a l y s i s of model chemical r e a c t i o n s under simulated use c o n d i t i o n s . Biological

Transformation.

I t has been only r e c e n t l y (8) that the c h l o r i n a t e d dibenzo furans have been t e n t a t i v e l y i d e n t i f i e d along with PCBs i n a higher animal. A r e l a t i v e l y new technique using negative chemical i o n i z a t i o n mass spectrometry provides a r a p i d and s e n s i t i v e q u a l i t a t i v e screen f o r these compounds. T h i s technique has had l i m i t e d a p p l i c a t i o n thus f a r . However, i t i s not c l e a r whether or not these furans a r e the o r i g i n a l contaminants of the PCB mixtures and/or transformation products of PCBs or other compounds i n the environment. Confirmation i s needed to r u l e out p o s s i b l e i n t e r f e r i n g compounds such as c h l o r i n a t e d diphenyl e t h e r s . The important p o i n t to make i s that the t o x i c i t y of the r e l e a s e d products can be c o n s i d e r a b l y d i f f e r e n t from the modified product which u l t i m a t e l y reaches man or any other higher monogastric animal.


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Mechanism E l u c i d a t i o n One of the more e x t e n s i v e l y studied areas i n drug metabolism has been the o x i d a t i v e metabolism of alkenes and p o l y c y c l i c aromatic hydrocarbons (PAHs). The evidence i s convincing that t h i s proceeds by formation of epoxides and arene oxides i n a r e a c t i o n mediated by the cytochrome P-450 dependent monoxygenase system. The f a t e of these r e a c t i v e oxides i n b i o l o g i c a l systems and the r e l a t i o n s h i p s to b i o l o g i c a l endpoints and t o x i c i t y are r e c e i v i n g c o n s i d e r a b l e a t t e n t i o n ( 9 ) . Improved and more s o p h i s t i cated methodology f o r e l u c i d a t i n g these mechanisms of a c t i o n a r e c o n t i n u a l l y being developed. Two examples w i l l be d e s c r i b e d , one considered to be a major d e t o x i c a t i o n pathway and the other a major a c t i v a t i n g pathway, i l l u s t r a t i n g some of the problems and some of the newer a n a l y t i c a l approaches to overcoming these problems. Conjugation of oxides with g l u t a t h i o n e (GSH) c a t a l y z e d by g l u t a t h i o n e t r a n s f e r a s e s i s a major d e t o x i c a t i o n process f o r removal of these r e a c t i v e molecules (10). However, s t u d i e s of t h i s metabolic process o f t e n do not f u l l y recognize the p o t e n t i a l complexity of the metabolic p r o f i l e . For example, i n our s t u d i e s (11) of the metabolism of styrene oxide, i t has been demonstrated that both p o s i t i o n a l and d i a s t e r e o i s o m e r i c m e t a b o l i t e conjugates of GSH and mercapturic a c i d s a r e formed. The t o t a l c h a r a c t e r i z a t i o n of t h i s process has r e q u i r e d the s y n t h e s i s of styrene oxide conjugates of GSH, c y s t e i n y l g l y c i n e , c y s t e i n e and N - a c e t y l c y s t e i n e (mercapturic a c i d ) , both p o s i t i o n a l and d i a s t e r e o i s o m e r i c using o p t i c a l l y a c t i v e isomers of styrene oxide. C h a r a c t e r i z a t i o n and measurement of these isomers i m m i x t u r e s f u r t h e r r e q u i r e d the development of methodology i n C nuclear magnetic resonance (NMR) spectroscopy and high pressure l i q u i d chromatography (HPLC). The GSH conjugates have a l s o been prepared f o r α-methylstyrene oxide, trans-β-methylstyrene oxide, 1,2,3,4-tetrahydronaphthalene1,2-oxide, phenanthrene-9,10-oxide, pyrene-4,5-oxide and benzo[a] pyrene-4,5-oxide (BP-4,5-oxide). Using standards a v a i l a b l e from our s y n t h e t i c s t u d i e s , we have compared the enzymatic and chemical conjugation of g l u t a t h i o n e with epoxides and found some i n t e r e s t ing d i f f e r e n c e s i n the r eg i o s p e c i f i c i t y and stereo s p e c i f i c i t y of the conjugation r e a c t i o n . For example, the chemical conjugation of GSH with benzo[a]pyrene-4,5-oxide produces equal amounts of the 4- and 5-thioether isomers as a mixture of d i a s t e r e o i s o m e r s . The enzymatic conjugation using r a t l i v e r c y t o s o l produces a mixture of the 4- and 5-thioether conjugates. However, the diastereoisomers of the conjugates a r e not formed i n equal amounts. With a p u r i f i e d enzyme from the L i t t l e River Skate, an equal mixture of the 4- and 5-thioether conjugates are produced. But i n t h i s case, only one of the diastereoisomers of each p o s i t i o n a l isomer i s produced. Product a n a l y s i s of the^jGSH conjugates obtained from C-labeled BP-4,5-oxide (4,5- C) e s t a b l i s h e d some d e f i n i t e stereochemical requirements f o r the c a t a l y t i c step (12).



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The data demonstrate that the skate l i v e r enzyme has h i g h r e g i o s e l e c t i v i t y and s t e r e o s p e c i f i c i t y f o r each BP-4,5-oxide enantiomer. The mechanistic i m p l i c a t i o n s of these r e s u l t s concerning the r e g i o s e l e c t i v i t y and s t e r e o s p e c i f i c i t y of enzymatic c o n j u g a t i o n are being evaluated f u r t h e r . The importance and potent b i o l o g i c a l a c t i v i t y of c e r t a i n g l u t a t h i o n e conjugates i s being i n c r e a s i n g l y recognized (13) . Most chemical carcinogens r e q u i r e metabolic a c t i v a t i o n to h i g h l y r e a c t i v e e l e c t r o p h i l i c intermediates to be c a r c i n o g e n i c . Such intermediates can bind c o v a l e n t l y to c e l l u l a r c o n s t i t u e n t s such as RNA, DNA, and p r o t e i n s . Therefore, one approach to the study of chemical c a r c i n o g e n e s i s i s to determine the nature and degree of covalent binding processes i n b i o l o g i c a l systems. The r o l e of arene oxides as r e a c t i v e metabolic intermediates has been i n v e s t i g a t e d e x t e n s i v e l y , and the subject of r e a c t i v e metabol i t e s i s addressed i n a separate paper i n t h i s conference. Evidence i s i n c r e a s i n g to support the formation of arene oxides during the metabolism of c e r t a i n PCBs (14) . I n a d d i t i o n , chemical synthesis of c e r t a i n PCB arene oxides has been completed and these oxides show the expected chemical p r o p e r t i e s c o n s i s t e n t with t h e i r p o t e n t i a l f o r rearrangement and covalent b i n d i n g to various nucleophiles. However, r e p o r t s of PCB binding to biopolymers i n v i v o and i n v i t r o g e n e r a l l y do not d i f f e r e n t i a t e between bound and simply adsorbed r e s i d u e s . In order to prove that a chemical binds c o v a l e n t l y to a biopolymer, i t i s necessary to i s o l a t e and c h a r a c t e r i z e the modified polymers and monomers. Simple f a i l u r e s to e x t r a c t PCBs from t i s s u e s with organic s o l v e n t s or p h y s i c a l methods of f r a c t i o n a t i o n alone, do not c o n s t i t u t e evidence of covalent b i n d i n g . Such s t u d i e s a r e g r e a t l y f a c i l i t a t e d by the use of r a d i o i s o t o p e s s i n c e the amount of covalent b i n d i n g i s usually quite small. Recent work (15) i n mouse l i v e r ( i n vivo) w i t h a slowly metabolized PCB (2,4,5,2 ,4 ,5 -hexachlorobiphenyl) and a r a p i d l y metabolized PCB (2,3,6,2',3 ,6 -hexachlorobiphenyl) along with the a p p r o p r i a t e c o n t r o l s has c l e a r l y demonstrated increased binding of the more r a p i d l y metabolized isomer to biopolymers. This was determined through i s o l a t i o n and c h a r a c t e r i z a t i o n of PCB bound biopolymers and monomers. The g r e a t e s t b i n d i n g was observed i n RNA followed by p r o t e i n and DNA, r e s p e c t i v e l y , and binding occurs i n t i s s u e s other than l i v e r as w e l l . T h i s b i n d i n g i s l i k e l y to be covalent and the r e s u l t o f metabolic a c t i v a t i o n , but proof of t h i s awaits f u r t h e r chemical c h a r a c t e r i z a t i o n of the i s o l a t e d m a t e r i a l s . F i e l d d e s o r p t i o n mass spectrometry (MS) and other s p e c i a l i z e d MS techniques should be u s e f u l i n c h a r a c t e r i z i n g such adducts (16). The a n a l y t i c a l c a p a b i l i t y now e x i s t s to determine the nature and extent of c o v a l e n t l y bound chemicals i n t i s s u e s . Further work should provide a data base upon which to draw some g e n e r a l f

1

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f

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i z a t i o n s concerning l i k e l y t a r g e t s f o r a t t a c k by r e a c t i v e metabo lites. However, the q u a n t i t a t i v e aspects of the problem have received l i t t l e a t t e n t i o n so f a r . In e l u c i d a t i n g the mechanism of chemical c a r c i n o g e n e s i s , one must determine the nature and degree of " e f f e c t i v e b i n d i n g " to biopolymers, i . e . what s p e c i f i c perturbations of the macromolecules a r e s u f f i c i e n t to induce n e o p l a s t i c transformation. Progress i n t h i s area i s l i k e l y to be dependent upon the development of good ±a v i t r o models f o r studying n e o p l a s t i c transformation i n c e l l s (17). S t r u c t u r e - A c t i v i t y and T o x i c i t y P r e d i c t i o n The t o x i c p r o p e n s i t y of a molecule r e s i d e s i n the chemical makeup of the molecule. The a v a i l a b i l i t y of m u l t i p l e measures of p h y s i c a l / c h e m i c a l , t o x i c o l o g i c a l and pharmacokinetic p r o p e r t i e s allows us to understand the t o x i c i t y of a molecule. One can measure or c a l c u l a t e molecular p r o p e r t i e s and compare the magni tude of these p r o p e r t i e s with the magnitudes of observed b i o l o g i c a l responses of animals exposed to the molecule. I t i s e s s e n t i a l to have r e l i a b l e and accurate b i o l o g i c a l measures of t o x i c i t y f o r c o r r e l a t i o n purposes. We have used the e x q u i s i t e s e n s i t i v i t y (LD^Q) of the guinea p i g to the t o x i c e f f e c t s of halogenated aromatic hydrocarbons as a b i o l o g i c a l response f o r comparison with some measured molecular p r o p e r t i e s considered important i n a s p e c i f i c receptor i n t e r a c t i o n (18) . Measured molecular f e a t u r e s include s i z e , shape, symmetry and p o l a r i z a b i l i t y . A v a r i e t y of techniques have been used to make these measurements i n c l u d i n g Xray c r y s t a l l o g r a p h y , nuclear magnetic resonance and mass spec troscopy and gas chromatography. A number of isomers and homologs i n the halogenated dibenzop - d i o x i n , dibenzofuran, b i p h e n y l and naphthalene c l a s s e s have been tested i n the guinea p i g . Based on these and other r e l e v a n t s t u d i e s , some g e n e r a l i z a t i o n s about s t r u c t u r a l p r o p e r t i e s important i n t h e i r t o x i c i t y can be made. The c r i t i c a l halogenation p a t t e r n takes the approximate form of a 3 Χ 10 A box f o r c h l o r i n e s and can be somewhat smaller and t r i a n g u l a r f o r bromines. P l a n a r i t y or c o p l a n a r i t y of r i n g s i s necessary only to e f f e c t j u x t a p o s i t i o n of four l a t e r a l c h l o r i n e s . T h i s imparts a c e r t a i n degree of symmetry to the molecule, but symmetry per se i s not a requirement. Studies (19) of v a r i o u s isomers and homologs i n the biphenyl s e r i e s suggest that the u n d e r l y i n g f a c t o r r e s p o n s i b l e f o r b i n d i n g i s net molecular p o l a r i z a b i l i t y which has a p r e f e r r e d d i s t r i b u t i o n along the 10 A receptor d i s t a n c e i n the molecule. We are c u r r e n t l y i n v e s t i g a t i n g ways to measure t h i s property d i r e c t l y . Some v a r i a t i o n i n these p r o p e r t i e s through molecular conforma t i o n a l preferences i n the biphenyls i s thought to e x p l a i n the apparent "mixed i n d u c t i o n " a c t i v i t y seen f o r c e r t a i n PCB isomers. Pharmacokinetic s t u d i e s a r e o b v i o u s l y important f o r d e t e r mining the f a t e and d i s p o s i t i o n of chemicals i n b i o l o g i c a l systems. The cost e f f e c t i v e n e s s of such work i s g r e a t l y increased by the



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use of r a d i o l a b e l e d compounds to f a c i l i t a t e the monitoring of a b s o r p t i o n , d i s t r i b u t i o n and e x c r e t i o n of the parent compound and i t s m e t a b o l i t e s . A few examples w i l l be described that i l l u s t r a t e some of the problems i n using both r a d i o a c t i v e and s t a b l e l a b e l s as b i o l o g i c a l t r a c e r s . The a v a i l a b i l i t y of l a b e l e d m a t e r i a l s with the d e s i r e d p r o p e r t i e s can be a problem. I n t e r e s t i n c a r r y i n g out pharmacok i n e t i c s t u d i e s on the t o x i c environmental contaminant 2,3,7,8t e t r a c h l o r o d i b e n z o f u r a n (2^,7,8-TCDF) engendered a need f o r s y n t h e s i s of the compound C l a b e l e d a t a high s p e c i f i c a c t i v i t y (greater than 50 mCi/mmol). A f t e r c o n s i d e r a b l e s y n t h e t i c work (20), the l a b e l e d 2,3,7,8-TCDF was obtained i n low y i e l d Jjjt a t high p u r i t y v i a Pschorr c y c l i z a t i o n of o^-phenoxyaniline-U C, c h l o r i n a t i o n of the r e s u l t a n t dibenzofuran and s e p a r a t i o n of the t e t r a c h l o r o isomers by high pressure l i q u i d chromatography. Completely anomalous r e s u l t s were obtained when more convenient s y n t h e t i c routes were t r i e d that had been p r e v i o u s l y reported f o r " c o l d " m a t e r i a l . These r e s u l t s were i n t e r p r e t e d i n terms of the i n t e r v e n t i o n of "hot" f r e e r a d i c a l intermediates. The e f f e c t s observed i n the promotion of r e a c t i v e f r e e r a d i c a l formation are perhaps not widely known. S i m i l a r e j e c t s could p o s s i b l y o b t a i n i n the metabolic degradation of the C-2,3,7,8-TCDF a f f o r d i n g again r e a c t i v e r a d i c a l intermediates which could a l t e r the "normal" course of metabolism. There i s a t l e a s t one r e p o r t (21) describing^£he s i g n i f i c a n t l y d i f f e r e n t gas chromatographic behavior of C-labeled 2,3,7,8-tetrachlorodibenzo-rj-dioxin (2,3 ,7,8-TCDD) , s p e c i f i c a c t i v i t y greater than 150 mCi/mmole. T h i s r e s u l t suggests that at s u f f i c i e n t l y high s p e c i f i c a c t i v i t i e s there can be an effect on p h y s i c a l / c h e m i c a l p r o p e r t i e s . Whether the h i g h l y l a b e l e d C2,3,7,8-TCDD behaves i n b i o l o g i c a l systems as unlabeled TCDD can not be answered d e f i n i t i v e l y , but i t i s important to be aware of t h i s p o t e n t i a l problem. The use of s t a b l e isotopes i s becoming i n c r e a s i n g l y pc-gular i n b i o l o g i c a l work (22). Progress has been made i n using Cl a b e l e d compounds to f a c i l i t a t e m e t a b o l i t e i s o l a t i o n and i d e n t i f i c a t i o n by MS techniques. Other work has been ^ n e using Cl a b e l s to help e l u c i d a t e biomechanism. Several C-labeled benzo[a]j^rene metabolites were prepared with s p e c i f i c i n c o r p o r a t i o n of C-labels. J ^ l a b e l e d compounds proved to be u s e f u l i n the assignment of C-NMR s p e c ^ a to the BP m e t a b o l i t e s . The (+) benzo[a]pyrene-4,5-oxide-4,5 C a l s o proved to be u s e f u l i n determining the p o s i t i o n a l and d i a s t e r e o i s o m e r i c g l u t a t h i o n e conjugates as a r e s u l t of i t s r e a c t i o n wj^h g l u t a t h i o n e t r a n s f e r a s e i n b i o l o g i c a l systems (12). The C enriched compound s i g n i f i c a n t l y s i m p l i f i e d the a n a l y t i c a l problem by enabling measurements to be made on much smaller amounts of m e t a b o l i t e s . The double l a b e l a t C-4 and C-5 i n the BP-oxide was intended to f a c i l i t a t e the determination of r e g i o s p e c i f i c i t y of i t s r e a c t i o n s , but a s i n g l e l a b e l a t e i t h e r C-4 or C-5 would have been j u s t as e

s

e

American Chemical Society Library 1155 16th St., N.W.

Bandal et al.; The Pesticide Chemist and Modern Toxicology Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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u s e f u l f o r t h i s purpose and would have f u r t h e r improY^d J^e a n a l y t i c a l problem s i n c e the r e d u c t i o n i n s i g n a l by CC coupling would have been e l i m i n a t e d . The a v a i l a b i l i t y of the s y n t h e t i c conjugates as standard r e f e r e n c e compounds was e s s e n t i a l i n t h i s work.


Chemical Epidemiology The importance of chemical epidemiology i n assessing the health hazard p o t e n t i a l of environmental chemicals i s obvious. The v a r i e t y of demands that are made on the a n a l y t i c a l chemist i n support of epidemiology s t u d i e s has r e c e n t l y been discussed (23) and major f a c t o r s i d e n t i f i e d which impact on the chemist. Two areas of concern c u r r e n t l y r e c e i v i n g a t t e n t i o n by the epidemiologist w i l l be d e s c r i b e d . The f i r s t i s the problem of PCBs i n mother's m i l k and the p o t e n t i a l t r a n s f e r of these chemic a l s to the baby during breast feeding; the second i s the potent i a l problem of d i o x i n (2,3,7,8-TCDD) residues i n humans (milk and f a t ) a s s o c i a t e d with exposure to 2,4,5-T and r e l a t e d h e r b i c i d e s and t h e i r p r e c u r s o r s . A study (24) i s i n progress that w i l l attempt to c o r r e l a t e h e a l t h e f f e c t s i n the developing i n f a n t with l e v e l s of PCBs and r e l a t e d compounds measured i n mother's m i l k . Figure 1 i l l u s t r a t e s the s p e c i f i c problem of analyzing f o r PCB i n human m i l k by e l e c tron capture-gas chromatography. Although s e v e r a l peaks appear to c o i n c i d e with the A r o c l o r standard, the p a t t e r n i n m i l k i s c l e a r l y d i f f e r e n t from the standard p a t t e r n . One r e a l l y can not compare these peaks s i n c e each one l i k e l y contains more than one compound with varying r e l a t i v e detector response. Therefore, there i s no true standard f o r q u a n t i t a t i o n purposes and absolute q u a n t i t a t i o n by t h i s method i s not p o s s i b l e . The best that can be hoped f o r i s a r e p r o d u c i b l y q u a n t i t a t i v e method that can be used f o r r e l a t i v e comparisons. In developing such r e p r o d u c i b l e methods, our experience has shown that extensive method v a l i d a t i o n i s r e q u i r e d f o r each sample matrix of i n t e r e s t . L i t e r a t u r e methods have been of l i t t l e v a l u e i n f a c i l i t a t i n g the v a l i d a t i o n work. R e p r o d u c i b i l i t y of methods i s c l e a r l y a f u n c t i o n of both method technology and o p e r a t i o n a l techniques. However, the a n a l y t i c a l problems of PCBs i n human t i s s u e s i s f u r t h e r complicated by the uniqueness of the p a t t e r n found i n the general population ( 7 ) . Residues i n humans appear to be l a r g e l y the o r t h o - s u b s t i t u t e d type PCBs with high c h l o r i n e content which are not r e a d i l y metabolized and eliminated from the body. These isomers are a l s o not p a r t i c u l a r l y t o x i c on a short term b a s i s . Therefore, i t i s the PCBs which can not be measured by an e x t r a c t i o n method that may be of greater b i o l o g i c a l consequence. As a part of t h i s e p i d e m i o l o g i c a l study, a method r e l y i n g on neutron a c t i v a t i o n a n a l y s i s has been developed (25) that w i l l allow determination of t o t a l organic c h l o r i n e r e s i d u e s i n body f l u i d s and t i s s u e s which i n c l u d e s both bound and unbound m a t e r i a l s .


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Figure 1. PCB pattern in human milk compared with Arochlor 1254 Standard. Peak off-scale is DDE. GC parameters: 3% OV-1, 220°C, 6 ft X 4 mm column.


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The a n a l y t i c a l problems a s s o c i a t e d with d i o x i n r e s i d u e s i n humans are s i m i l a r i n many ways but there are some key d i f f e r e n c e s which should be pointed out (26) (Table 2). Absolute q u a n t i t a t i o n i s a c h i e v a b l e s i n c e standard r e f e r e n c e compounds are a v a i l a b l e or can be made a v a i l a b l e . However, because of the high t o x i c i t y of these compounds, the d e s i r e d d e t e c t i o n l i m i t i s i n the low p a r t p e r - t r i l l i o n (ppt) range instead of the ppb to ppm range r e q u i r e d for PCBs. Dioxins are a l s o not g e n e r a l l y considered to have ubiquitous d i s t r i b u t i o n i n the environment although t h i s has r e c e n t l y been challenged by the "chemistry of f i r e " proposal of Dow Chemical Company (27). As a r e s u l t of the low concentrations being sought and the non-ubiquity of these r e s i d u e s , a c o n s i d e r a b l y more complex and s o p h i s t i c a t e d a n a l y t i c a l method i s r e q u i r e d to achieve the d e s i r e d s e n s i t i v i t y and s p e c i f i c i t y needed. The method p r e s e n t l y i n use g e n e r a l l y c o n s i s t of some form of low or high r e s o l u t i o n chromatography and high or low r e s o l u t i o n mass spectrometry. Table 2 Problems i n Development of TCDD A n a l y s i s i n Human Fat 1)

Sample s i z e u s u a l l y small ( f a t biopsy - 0.5

g or l e s s ) .

2)

Low p a r t - p e r - t r i l l i o n (ppt) s e n s i t i v i t y d e s i r e d because of e x q u i s i t e t o x i c i t y of the compound (1 ppt on 0.5 g sample r e q u i r e s 0.5 picogram i n t o t a l sample).

3)

The a n a l y s i s must not only be h i g h l y s e n s i t i v e but h i g h l y s p e c i f i c f o r the 2,3,7,8-tetra isomer.

4)

Requires s y n t h e t i c work to provide a n a l y t i c a l standards and h i g h l y compatible s e p a r a t i o n s c i e n c e and s p e c i f i c measurement c a p a b i l i t i e s under high r e s o l u t i o n c o n d i t i o n s (must e l i m i n a t e interferences).

5)

Requires s t r i n g e n t a n a l y t i c a l p r o t o c o l s and safe handling procedures and f a c i l i t i e s to maintain sample i n t e g r i t y and avoid contamination of f a c i l i t i e s and exposure of personnel.

Although e x i s t i n g methods f o r TCDD have already been put i n t o use f o r supporting l i m i t e d e p i d e m i o l o g i c a l s t u d i e s , they can not be defended as unequivocal f o r determination of 2,3,7,8-TCDD. The methods a v a i l a b l e l a c k complete v a l i d a t i o n i n the a p p r o p r i a t e sample matrices and s i z e s . T h i s i n t u r n i s due to the l a c k of s u f f i c i e n t a n a l y t i c a l standards i n c l u d i n g other t e t r a d i o x i n s , i n t e r n a l standards and p o s s i b l e i n t e r f e r i n g compounds. These standards are needed to v a l i d a t e a l l aspects of the method i n c l u d i n g s p i k i n g , e x t r a c t i o n , cleanup, and measurement f o r the sample type, s i z e s and concentrations of i n t e r e s t . Such extensive method v a l i d a t i o n and e l a b o r a t i o n are needed f o r unequivo c a l determinations of 2,3,7,8-TCDD r e s i d u e s i n human samples.


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Of p a r t i c u l a r concern i s the problem of p o t e n t i a l l y u b i q u i tous PCB m e t a b o l i t e s that are exact mass e q u i v a l e n t s of d i o x i n s and can lead to f a l s e p o s i t i v e s (28). Such t e t r a c h l o r o b e n z o q u i none m e t a b o l i t e s a r e known to occur i n the metabolism of c e r t a i n hexachlorobiphenyl isomers. At l e a s t one m e t a b o l i t e of t h i s type can be drawn which shows a remarkable resemblance to the d i o x i n molecule and a t the same time can p o s s i b l y d e p o l a r i z e i t s e l f through e l e c t r o n i c i n t e r a c t i o n s between the two r i n g systems.

H--C

Our present technique involves h i g h r e s o l u t i o n mass spectrometry u s i n g an instrument with reversed geometry capable of doing mass analyzed i o n k i n e t i c energy mass spectrometry (MIKES) experiments. Sample i n t r o d u c t i o n i s by c a p i l l a r y gas chromatography. The information generated i s the elemental composition of the molecular i o n , the elemental composition of the most unique fragment i o n (M-COCl), the i n t e n s i t y r a t i o of these two ions, and the r e t e n t i o n time of the presumptive TCDD. The a v a i l a b i l i t y of radioimmunoassay (RIA) procedures f o r environmental agents holds some promise i n minimizing the need f o r the more s o p h i s t i c a t e d and expensive instrumental methods of a n a l y s i s by e l i m i n a t i n g "negative" samples and f o r r o u t i n e monit o r i n g of exposure i n environments known to be contaminated by c e r t a i n c l a s s e s of compounds. There are a number of fundamental problems involved i n development of such RIA procedures and i n t h e i r use (Table 3 ) . Double-antibody RIA's have been developed (29) f o r q u a n t i t a t i n g a number of c h l o r i n a t e d aromatic hydrocarbons of c u r r e n t concern from environmental samples i n c l u d i n g animal t i s s u e s . These c h l o r i n a t e d hydrocarbons i n c l u d e members of the dibenzo-£-dioxin, dibenzofuran, and b i p h e n y l c l a s s e s of compounds. The use of RIA procedures f o r t r a c e r e s i d u e a n a l y s i s i s discussed f u r t h e r i n another paper i n t h i s conference. These examples h o p e f u l l y i l l u s t r a t e the complexity and c o n s i d e r a t i o n s necessary f o r developing and a p p l y i n g a n a l y t i c a l methodology to support r e g u l a t o r y d e c i s i o n s made i n the i n t e r e s t of p r o t e c t i n g human h e a l t h .


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Table 3 Problems i n Development of Radioimmunoassay f o r TCDD 1)

Choice of Dibenzodioxin D e r i v a t i v e

2)

Choice of Coupling Method

3)

Choice of C a r r i e r P r o t e i n

4)

C h a r a c t e r i z a t i o n of Antigen

5)

Immunization

Schedule

6)

S o l u b i l i z a t i o n of TCDD

7)

S e l e c t i o n of A n t i s e r a

8)

P a r t i a l P u r i f i c a t i o n of A n t i b o d i e s to Improve S p e c i f i c i t y . 125

9)

S e l e c t i o n of Appropriate

10)

I - l a b e l e d Reagent.

Prevention of N o n s p e c i f i c A d s o r p t i o n .

Toxicity Testing The N a t i o n a l Toxicology Program (NTP) was formed i n 1978 to coordinate t o x i c o l o g i c a l t e s t i n g e f f o r t s of the Department of Health and Human S e r v i c e s ( p r e v i o u s l y DHEW) and amounted to $42 m i l l i o n f o r FY-79. Components of the N a t i o n a l Toxicology Program include ^ n v i t r o and in v i v o bioassay t e s t i n g programs a t the N a t i o n a l I n s t i t u t e of Environmental Health Sciences (NIEHS), the N a t i o n a l Cancer I n s t i t u t e (NCI), the N a t i o n a l Center f o r T o x i c o l o g i c a l Research (NCTR) and the N a t i o n a l I n s t i t u t e f o r Occupational Safety and Health (NIOSH) ( 3 0 ) . Sound a n a l y t i c a l chemistry support i s e s s e n t i a l f o r the performance of these tasks. Chemistry a s s i s t a n c e f o r the NTP i n v i t r o genetic t o x i c o l o g y and c e r t a i n i n v i v o programs i s c u r r e n t l y provided by c o n t r a c t l a b o r a t o r i e s . Chemistry problems a r e comp l i c a t e d by the sheer numbers of chemical r e q u i r i n g t e s t i n g i n c l u d i n g storage, d i s t r i b u t i o n , a n a l y s i s , d i s p o s a l , computer inventory, e t c . A n a l y t i c a l c a p a b i l i t i e s i n c l u d e : 1. Chemical assay to determine the p u r i t y of the p r i n c i p a l chemical component(s) of commercial chemicals and major i m p u r i t i e s (% l e v e l ) which may be present. 2. S t a b i l i t y and s o l u b i l i t y determinations performed as necessary to a s c e r t a i n f a t e and d i s t r i b u t i o n of a chemical under s p e c i f i c c o n d i t i o n s (time d u r a t i o n , temperature, l i g h t , s o l u t i o n media, etc.) as r e q u i r e d f o r the p a r t i c u l a r bioassay experiment. 3. Comprehensive a n a l y s i s to determine a l l p o s s i b l e chemical components with i d e n t i f i c a t i o n and q u a n t i t a t i o n of t r a c e i m p u r i t i e s a t the r e s i d u e l e v e l .



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The f i r s t two c a p a b i l i t i e s a r e s t r a i g h t forward; however, an explanation of the need f o r comprehensive a n a l y s i s i s u s e f u l . The f i r s t step i n the t i e r system of genetic t o x i c o l o g y b l i n d bioassay t e s t i n g i s the Salmonella, m i c r o b i a l (Ames) t e s t . Because of the low cost of t h i s bioassay t e s t ($500-1,000/chemical) r e l a t i v e to the o f t e n u n p r e d i c t a b l e and always more c o s t l y demands of chemical a n a l y s i s , most chemicals a r e bioassayed by Salmonella before they a r e c h e m i c a l l y analyzed. Plans c a l l f o r 300 chemicals to be tested i n the m i c r o b i a l system i n FY-80 w i t h a eventual goal of 1,000 bioassays of new chemicals a year by 1983. Test chemicals producing p o s i t i v e , ambiguous and s e l e c t e d negative bioassay r e s u l t s a r e then subjected to comprehensive chemical a n a l y s i s . An important aspect i s that the s e p a r a t i o n technique used i n the chemical a n a l y s i s must i n s u r e : 1.

Composition altered.

of b i o l o g i c a l l y a c t i v e compounds i s not

2.

Maximum c o n s e r v a t i o n of a l l components.

3.

Maximum separation of chemical

4.

Minimum i n t r o d u c t i o n of i m p u r i t i e s .

groups.

A f t e r s e p a r a t i o n , the r e s u l t a n t f r a c t i o n s a r e r e t e s t e d and the p o s i t i v e f r a c t i o n s a r e f u r t h e r f r a c t i o n a t e d or subjected to complete component i d e n t i f i c a t i o n and q u a n t i t a t i o n using s o p h i s t i c a t e d chromatographic and s p e c t r o s c o p i c techniques, GC/MS (EI and C I ) , HPLC, NMR, IR, e t c . Each component of the f i n a l a c t i v e f r a c t i o n i s then bioassayed s e p a r a t e l y to determine the chemical(s) r e s p o n s i b l e f o r the o r i g i n a l observed b i o l o g i c a l a c t i v i t y . I t may a l s o become necessary to synthesize the i s o l a t e d a c t i v e chemical f o r c o n f i r m a t i o n purposes. The i n v i v o NTP e f f o r t s i n c l u d e the NCI l i f e t i m e rodent bioassay f o r c a r c i n o g e n e s i s . Approximately 75-100 new chemicals are s t a r t e d on t e s t each year a t a t o t a l bioassay cost of about $500,000 per chemical. Chemical support f o r the bioassay t e s t i n g , as shown i n F i g u r e 2, i s a l s o provided by c o n t r a c t l a b o r a t o r i e s and i n c l u d e s : 1. Chemical p u r i t y and s t a b i l i t y a n a l y s i s of bulk chemicals before t e s t i n g and development of p r o t o c o l s f o r r e a n a l y s i s of bulk chemicals. 2. Development of p r o t o c o l s f o r assay and s t a b i l i t y d e t e r minations of c h e m i c a l / v e h i c l e mixes and dosage a n a l y s i s . 3. Development of procedures f o r a n a l y s i s and s t a b i l i t y of reprocured as w e l l as r e s i d u a l bulk chemicals. 4. Development of procedures and p r o t o c o l s f o r a chemical/ v e h i c l e q u a l i t y c o n t r o l a n a l y s i s program. Such programs a r e designed to insure that r e l i a b l e procedures a r e used by the bioassay and chemistry l a b o r a t o r i e s f o r the a n a l y s i s of bulk chemicals and chemicals i n the dosage/feed mixtures.


THE

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CHEMICAL "SELECTION


PREPARE TOXICITY & SAFETY DOCUMENTS

PROCUREMENT

RECEIVE BULK CHEMICALS

REPROCURE TEST CHEMICALS

HOMOGENIZE

(PACKAGE,

REPOSITORY INVENTORY, DISPOSAL)

ANALYTICAL

,

LABORATORY

PURITY ( % ) , STABILITY

RESIDUAL CHEMICALS FOR ANALYSIS & DISPOSAL

STORAGE RECOMMENDATIONS SPECIAL CHEMISTRY: COMPREHENSIVE A N A L Y S I S , QUANTITATTON-TISSUE & FLUID ANALYSIS, SYNTHESIS DEVELOP CHEMICAL VEHICLE QUALITY ASSURANCE PROGRAM FOR EACH TEST CHEMICAL

DISTRIBUTE CHEMICALS — FOR BIOASSAY

Figure 2.

Flow chart of chemistry support to in vivo bioassay testing


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S p e c i a l analyses as needed f o r : a.


Analytical

I d e n t i f i c a t i o n and q u a n t i t a t i o n of i m p u r i t i e s i n bulk chemicals.

b.

A n a l y s i s of v e h i c l e s f o r t o x i c components.

c.

A n a l y s i s of chemicals i n body f l u i d s and t i s s u e s .

d.

Development of chemical monitoring techniques to be used f o r s a f e t y and p o l l u t i o n concerns.

A n a l y s i s to Insure Safe Working Environment Chemistry operations to support the NTP in v i t r o e f f o r t s a r e performed i n a s p e c i a l l y designed Hazardous M a t e r i a l s Laboratory. The Laboratory (31,32,33) i s under negative atmospheric pressure and a l l a i r and water e f f l u e n t s a r e f i l t e r e d through p a r t i c u l a t e , c h a r c o a l and other a p p r o p r i a t e media before being discharged. The design of the Laboratory i s based on the p r i n c i p l e s of containment and the e f f e c t i v e use of engineering c o n t r o l s r a t h e r than r e l i a n c e on personnel p r o t e c t i o n (34,35). O v e r r e l i a n c e on personnel p r o t e c t i o n can lead to a f a l s e sense of s e c u r i t y r e s u l t i n g i n an overt exposure to hazardous chemicals. Only through an understanding of the substances' chemical, p h y s i c a l and t o x i c o l o g i c a l p r o p e r t i e s can adequate f a c i l i t i e s be designed and monitored f o r the safe use of these chemicals. Inherent with the use of containment f a c i l i t i e s i s a r o u t i n e monitoring program which should i n c l u d e l a b o r a t o r y a i r , t r e a t e d waste water and s u i t a b l e s u r f a c e areas. Because of the wide range of compound types and c l a s s e s used i n the Hazardous M a t e r i a l s Laboratory, a general monitoring procedure i s necessary. Laboratory a i r i s r o u t i n e l y monitored q u a r t e r l y by the NIOSH c h a r c o a l tube sampling procedure. Laboratory a i r i s drawn through the tube f o r an 8 hour p e r i o d and the c h a r c o a l adsorbant i s extracted with carbon d i s u l f i d e or other s u i t a b l e s o l v e n t s . The e x t r a c t i s analyzed by gas chromatography u s i n g both flame i o n i z a t i o n and e l e c t r o n capture d e t e c t o r s . Chromatograms from each sample are compared to those of blank samples c o l l e c t e d p r i o r to i n i t i a t i o n of Hazardous M a t e r i a l s Laboratory o p e r a t i o n s . Standard a n a l y t i c a l techniques (HPLC, GC/MS, etc.) are used, as r e q u i r e d , f o r i d e n t i f i c a t i o n , c o n f i r m a t i o n and q u a n t i t a t i o n . S i m i l a r l y , s u r f a c e samples are c o l l e c t e d q u a r t e r l y . Cotton swabs saturated with acetone are used to c o l l e c t samples from s i x 100 cm s u r f a c e areas i n the Laboratory. The swabs a r e extracted with acetone and analyzed by methods analogous to the c h a r c o a l e x t r a c t above. Samples from the waste water e f f l u e n t p u r i f i c a t i o n system are c o l l e c t e d and analyzed q u a r t e r l y . The samples a r e extracted according to the EPA's P r i o r i t y P o l l u t a n t P r o t o c o l and analyzed analogously to the above method.



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Coupled with these f a c i l i t i e s and a n a l y t i c a l procedures are programs f o r r o u t i n e weekly decontamination; waste d i s p o s a l by i n c i n e r a t i o n and b u r i a l ; personnel p r o t e c t i o n and s a f e t y t r a i n i n g . Chemical a n a l y s i s i s used i n the i n v i v o program to i n s u r e that a safe working environment e x i s t s i n which to do t o x i c o l o g i c a l research. In a d d i t i o n to a l l the problems faced by the i n v i t r o support l a b o r a t o r y the jm v i v o f a c i l i t y i s p a r t i c u l a r l y concerned about weighing and p r e p a r a t i o n of feed mixtures cont a i n i n g l a r g e q u a n t i t i e s ( s e v e r a l kg) of hazardous chemical. A s i m i l a r negative pressure type mixing and feed p r e p a r a t i o n area i s used f o r t h i s work. S e v e r a l papers have appeared d e s c r i b i n g p o t e n t i a l hazards of such operations (36,37,38,39). Only through e f f e c t i v e chemical monitoring and p e r i o d i c use of marker compounds such as f l u o r e s c e i n can the extent of the problem be r e a l i z e d and assurance f o r s a f e working environments be given. The NIEHS has two h i g h hazard containment l a b o r a t o r i e s ; one for chemical r e s e a r c h (34) and the other f o r b i o l o g i c a l r e s e a r c h i n v o l v i n g hazardous chemicals (35). Programs are c u r r e n t l y being developed f o r monitoring these rooms. The Chemical Containment Laboratory i s designed f o r three purposes: (1) organic s y n t h e s i s of hazardous m a t e r i a l s ; (2) a n a l y s i s by GC and HPLC of hazardous r e a c t i o n mixtures and products; and (3) r o u t i n e weighing

of mg

q u a n t i t i e s of hazardous compounds.

Monitoring procedures and f r e q u e n c i e s f o r a i r , water and surfaces are s i m p l i f i e d s i n c e the room i s used almost e x c l u s i v e l y f o r aromatic h a l i d e compounds such as 2,3,7,8-tetrachlorodibenzo-£d i o x i n (TCDD) and s i m i l a r compounds. Samples are prepared from a i r sampling through a s u i t a b l e sorbate (polyurethane foam, PUF) followed by e x t r a c t i o n or chromatography with an a p p r o p r i a t e s o l v e n t ( s ) and a n a l y s i s by GC/MS. Water samples are extracted and concentrated before a n a l y s i s using a Kuderna-Danish r e c e i v e r . Surface wipe samples are g e n e r a l l y extracted overnight on a Soxhlet apparatus using a s o l v e n t chosen on the b a s i s of compound c l a s s i f i c a t i o n ; methanol, toluene or methylene c h l o r i d e . The High Hazard Laboratory f o r L i f e S c i e n t i s t s i n v o l v e s a wide assortment of compounds. For t h i s reason, a broader general method f o r chemical monitoring was needed which could be followed up when necessary with a s p e c i f i c technique. Such a scheme i s c u r r e n t l y being developed and i n v o l v e s monitoring of s u r f a c e samples by the researcher using an inexpensive spectrophotof l u o r i m e t e r . I f a reading i s recorded above the p r e v i o u s l y determined background l e v e l of f l u o r e s c e n c e the sample i s submitted to the Laboratory of Environmental Chemistry f o r i d e n t i f i c a t i o n and c o n f i r m a t i o n . The only requirement f o r t h i s technique i s a short t r a i n i n g p e r i o d f o r a l l users of the High Hazard Laboratory; submission of a small amount (mg) of a l l r e s e a r c h chemicals used i n the f a c i l i t y as r e f e r e n c e m a t e r i a l and back-up



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by more s o p h i s t i c a t e d in-house c a p a b i l i t i e s . The b e n e f i t s a r e speed, s e l f - m o n i t o r i n g , low cost and minimal l o s s of research time. In c o n c l u s i o n , a n a l y t i c a l chemistry i s an underlying f a c t o r i n e s s e n t i a l l y a l l aspects of t o x i c o l o g i c a l work. I t i s evident from these examples that the s o p h i s t i c a t i o n of the a n a l y t i c a l methods a v a i l a b l e f o r use can to a l a r g e extent determine the complexity of the t o x i c o l o g i c a l problem that can be approached and solved. The best a n a l y t i c a l approach i s designed to meet the s p e c i f i c needs and emphasis of the t o x i c o l o g i c a l r e s e a r c h and i s c o n s i s t e n t with good a n a l y t i c a l p r a c t i c e s .

Literature Cited 1. McKinney, J.D., Environmental Health Chemistry. Definitions and Interrelationships. A Case in Point. Environmental Health Chemistry. Chemistry of Environmental Agents as Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 1. 2. Amore, F., Good Analytical Practices as an Alternative to Standard Methods, Licensing and Certification. In Quality Assurance of Environmental Measurements, Information Transfer, Inc., Silver Spring, MD, 1979, pp. 183-190. 3. Albro, P. and C. Parker, General Approach to the Fractionation and Class Determination of Complex Mixtures of Chlorinated Aromatic Compounds, J. Chromatog., In Press, 1980. 4. Hutzinger, O., S. Safe and V. Zitko, Metabolism of Chlorobiphenyl. In The Chemistry of PCB' s, Hutzinger, Safe and Zitko (eds.), CRC Press, Cleveland, Ohio, 1974, Chapter 7 for general review. 5. Hutzinger, O., S. Safe and V. Zitko, Photodegradation of Chlorobiphenyls, In The Chemistry of PCB's, Hutzinger, Safe and Zitko (eds.), CRC Press, Cleveland, Ohio, 1974, Chapter 6. 6. Buser, H.R. and C. Rappe, Formation of Polychlorinated Dibenzofurans (PCDFs) from the Pyrolysis of Individual PCB Isomers, Chemosphere, 8(3), 115-124 (1979). 7. Jensen, S. and G. Sundstrom, Structure and Levels of Most Chlorobiphenyls in Two Technical PCB Products and in Human Adipose Tissue, Ambio 3, 70-76 (1974). 8. Kuehl, D.W., R.C. Dougherty, Y. Tondeur, D.L. Stalling and C. Rappe, Negative Chemical Ionization Studies of Polychlorodibenzodioxins and Dibenzofurans in Environmental Samples, In Environmental Health Chemistry. The Chemistry of Environmental Agents As Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 12. 9. Miller, J.A., Carcinogenesis by Chemicals: An OverviewG.H.A. Clones Memorial Lecture, Cancer Res., 30, 559 (1970). 10. Jerina, D.M. and J.R. Bend, Glutathione S-Transferases. In Biological Reactive Intermediates, D.J. Jallow, J.J. Koesis, R. Snyder and H. Vainio (eds.), Plenum Press, New York, 1977, pp. 207-236.


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11. Cox, R.H., O. Hernandez, B. Yagen, B. Smith, J.D. McKinney, and J.R. Bend, C NMR Studies of the Structure and Stereochemistry of Products Derived from the Conjugation of Glutathione with Alkene and Arene Oxides. In Environmental Health Chemistry. The Chemistry of Environmental Agents as Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 20. 12. Hernandez, O., M. Walker, R. Cox, G.L. Foureman, B. Smith, and J.R. Bend, Regioselectivity and Stereospecificity in the Enzymatic Conjugation of Glutathione with (+) Benzo(a)pyrene 4,5-oxide, Biochem. Biophys. Res. Comm. in press (1980). 13. Corey, E . J . , D.A. Clark, G. Goto, A. Marfat, C. Mioskowski, B. Samuelsson and S. Hammerstrom, Stereospecific Total Synthesis of a "Slow Reacting Substance" of Anaphylaxis, Leukstriene C-l, J. Am. Chem. Soc. 102(4), 1436-1439 (1980). 14. Forgue, S.T., B.D. Preston, W.A. Hargraves, I.L. Reich, and J.R. Allen, Direct Evidence that an Arene Oxide is a Metabolic Intermediate of 2,2',5,5'-Tetrachlorobiphenyl, Biophys. Res. Commun. 91(2), 475-483 (1979) and references therein. 15. Morales, N.M. and H.B. Matthews, In vivo Binding of 2,3,6,2',3',6'-Hexachlorobiphenyl and 2,4,5,2',4',5'-Hexachlorobiphenyl to Mouse Liver Macromolecules, Chem.-Biol. Interact., 27, 99-110 (1979). 16. Harvan, D., J.R. Hass, and M. Lieberman, Interaction of N-acetyl-N-acetoxyaminofluorene with Synthetic DNA. Chem.-Biol. Interact., 17, 203-210 (1977). 17. Barrett, J.C., Neoplastic Transformation Induced by a Direct Perturbation of DNA, Nature, 274, 229 (1978). 18. McKinney, J.D., P. Singh, L. Levy, and M. Walker, High Toxicity and Cocarcinogenic Potential of Certain Halogenated Aromatic Hydrocarbons. In Safe Handling of Chemical Carcinogens, Mutagens and Teratogens: Chemist's Viewpoint. Vol. II, D. Walters (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 22. 19. McKinney, J.D. and P. Singh, Structure-Activity Relationships in Halogenated Biphenyls: Molecular Polarizability in the Most Toxic Halogenated Biphenyls, Chem.-Biol. Interact., submitted, 1980. 20. Gray, A.P., W.J. McClellan and V.M. Dipinto, Carbon-14Labeled 2,3,7,8-and 1,2,7,8-Tetrachlorodibenzofuran, J . Labelled Compounds and Radiopharmaceuticals, in press, 1980. 21. Oswald, E.O., P.W. Albro, and J.D. McKinney, Utilization of Gas Liquid Chromatography Coupled with Chemical Ionization and Electron Impact Mass Spectrometry for the Analysis and Characterization of Potentially Hazardous Environmental Agents and their Metabolites. J . Chromatog. 98, 363 (1974). 22. Tolbert, B.M. and N.A. Matwiyoff, Medical and Biological Applications of Stable Carbon, Nitrogen and Oxygen Isotopes, Radiopharm. Label. Compounds, Proc. Symp. New Develop. Radiopharm. Label. Compounds, Vienna, IAEA, 2, 241-253 (1973). 13


TOXICOLOGY



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23. Rogan, W., Analytical Chemistry Needs for Environmental Epidemiology. In Environmental Health Chemistry. Chemistry of Environmental Agents as Potential Human Hazards. J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 6. 24. Rogan, W.J. and B. Gladen, The Breast Milk and Formula Project - Current Findings. Environ. Hlth. Perspect., in press (abst.). 25. Fawkes, J., D.B. Walters and J.D. McKinney, Neutron Activation Analysis of Organically Bound Chlorine. Precautions to Minimize Extraneous Halide Contamination in Collection, Storage, and Analysis of Human Milk and Formula, 178th ACS National Meeting, Washington, DC, September, 1979, abst. No. 83. 26. McKinney, J.D., Analysis of TCDD in Environmental Samples. Proceedings of the Conference on Chlorinated Phenoxy Acids and Their Dioxins. In Ecol. Bull. (Stockholm), C. Ramel (ed.), 53-66 (1978). 27. Report by Rebecca L. Rawls in Chemical Engineering News, February 12, 1979, Dow Finds support, doubt for dioxin ideas. 28. McKinney, J.D., Toxicology of Selected Symmetrical Hexachlorobiphenyls. Correlating Biological Effects with Chemical Structure. Proceedings of the National Conference on PCB's, Chicago, Illinois, November, 1975. In EPA Publication-560/6-75004, pp. 73-76. 29. Luster, M.I., P.W. Albro, K. Chae, S.K. Chaudhary, and J.D. McKinney, Development of Radioimmunoassays for Chlorinated Aromatic Hydrocarbons. In Environmental Health Chemistry. Chemistry of Environmental Agents as Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 14. 30. National Toxicology Program, Annual Plan for Fiscal Year 1980 (NTP-79-8), November, 1979. 31. Walters, D.B., L.H. Keith and J.M. Harless, Chemical Selection and Handling Aspects of the National Toxicology Program, In Environmental Health Chemistry: The Chemistry of Environmental Agents As Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1981 (in Press). 32. Keith, L.H., J.M. Harless and D.B. Walters, Analysis and Storage of Hazardous Environmental Chemicals for Toxicological Testing, In Environmental Health Chemistry: The Chemistry of Environmental Agents as Potential Human Hazards, J.D. McKinney (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1981 (in press). 33. Harless, J.M., K.E. Baxter, L.H. Keith, D.B. Walters, Design and Operation of a Hazardous Materials Laboratory, In Safe Handling of Chemical Carcinogens, Mutagens, Teratogens and Other Hazardous Materials, Vol. I, D.B. Walters (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 4.



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34. Walters, D.B. J.D. McKinney, A. Norstrom and D. DeWitt, Control of Potential Carcinogenic, Mutagenic and Toxic Chemicals via a Protocol Review Concept and Chemistry Containment Laboratory, In Safe Handling of Chemical Carcinogens, Mutagens, Teratogens and Other Hazardous Materials, Vol. I, D.B. Walters (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 1. 35. Hunt, C.L., Jr., D.B. Walters and E. Zeiger, Approaches for Safe Handling Procedures and Design of a High Hazard Laboratory for Life Scientists, In Safe Handling of Chemical Carcinogens, Mutagens, Teratogens and Other Hazardous Materials, Vol. I, D.B. Walters (ed.), Ann Arbor Science Publishing Co., Ann Arbor, Michigan, 1980, Chapter 2. 36. Sansone, E.B. and M.W. Slein, Application of the Microbiological Safety Experience to Work with Chemical Carcinogens, Am. Ind. Hyg. Assoc. J., 37, 711-720 (1976). 37. Sansone, E.B. A.M. Losikoff, R.A. Pendleton, Sources and Dissemination of Contamination in Material Handling Operations, Am. Ind. Hyg. Assoc. J., 38, 433-442 (1977). 38. Sansone, E.B. A.M. Losikoff, Contamination from Feeding Volatile Test Chemicals, Toxicol. Appl. Pharmacol. 46, 703-708 (1978). 39. Sansone, E.B. and A.M. Losikoff, Chemical Contamination Resulting from the Transfer of Solid and Liquid Materials in Hoods, Am. Ind. Hyg. Assoc. J., 38, 489-491 (1977). RECEIVED

February 2, 1981.


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