red world of
edited by
W. C. FERNELIUS Kent State University Kent. OH 44242
HAROLDWITTCOFF Kaor Chemicals Ltd. Beer-Sheva. Israel P.O.B. 60
The Role of Structural Chemists in Consumer-Products Safety Research Richard P. Oertel The Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, OH 45247
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its longstanding feature column on "Safety in the he mi& Laboratory" and through articles dealing with, for exapple, chemical toxicology. This article considers a further aspect of the chemical-safety story: how scientists in industry ensure the safety of their nroducts. I will focus on the consumer-vroducts industrv, and. in particular, on one of its larger members, The Procter & Gamble Comvanv. Product-safetv research has long been an :mpurtant activity u,ithin I'ructcr b. (;iiitilrle;scientist: r'r u m many disritilines are in\.,rl\.ed. Thv -ma1 oi their rcswn h is 11, provide comprehensive assurance that, under intended, and sometimes unintended, conditions of exposure, our products are safe for people and the environment. For illustration, I will highlight how one particular group of scientists, namely structural chemists-molecular spectroscopists and crystallographers-fits into Procter & Gamble's overall productsafety program and contributes toward its goal. The reader will be given a glimpse of how a large consumer-products company musters its technical resources, both people and equipment, to meet its social and ethical obligations regarding product safety.
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Safety-Testing Scheme Among consumer-products companies, there is no single, uniformly-adopted approach to product safety assessment; the approach that a particular company uses is determined bv such factors as the vroduction volume and chemical compiexity of the productsit sells in the marketplace. The process that Procter & Gamble has developed to make and review product-safety decisions is described helow. Good scientific judgment and many decisions are required throughout the process, including the decision whether or not to proceed with each step. Knowledge of a material's chemical and physical properties and of its patterns of handling and usage allows us to determine the likely routes and estimate the amounts of both human and environmental exposure. These estimates must cover such diverse situations as accidental exposure of factory workers, intentional exposure of consumers, and incidental
46
Journal of Chemical Education
exuosure of humans from drinkine water. Usine this exnosure information, we design appropriate tests: tests to determine what happens to the material when it is released into the en vironment, which information helps to refine the estimates of environmental concentration; tests to find out if the anticipated concentrations harm the environment; and, most important, tests to determine the possible effects of the material on human health. These health tests, most of which are actually carried out in laboratory animals, can range from short-term oral-toxicity tests to long-term tests for carcinogenicity. From information about the kinds of effects produced in animals and the doses of material required to produce them, i t is possible to estimate both the no-effect levels for humans and the safety factors associated with the expected human exposures. To asseis the risk to humans and the environment, we need to compare the quantities predicted to cause harm with the exposure levels dredicted to result from handling and use of the material. If this risk is iudged unacceptable, it may be possible to devise restrictions on the use of the material that would diminish the anticipated exposure and lower the risk (e.g., use of warning labels). If the material ultimately is used in a product, monitoring may be desirable to ensure that the resulting concentrations and effects are those that were expected. Such monitoring very likely will become more common in the future. The Participants Sound product-safety decisions require the coordinated efforts of many groups of persons. The close partnership of toxicologists and analytical chemists is essential. Analytical chemists determine the purity, . . stability, and dosage strength of a test substance, determine environmeptal concintrations, and isolate test-substance metabolites. Toxicoloaists plan and oversee the safety testing and evaluate the resulting safety data. Supporting this partnership are synthetic chemists, who are often asked to make test compounds and reference materials, sometimes in radiolaheled form; advisory personnel from inside and outside Procter & Gamble; pathologists, who Based on a talk presented at the ACS National Meeting. March, 1980, as part of the Analytical Division's Symposium on Industrial Problem Solving-A Multitechnique Approach.
assess the biological response of organisms t o test materials; chemists and engineers who have direct responsibility for developing safe products; scientists in contract laboratories, who sometimes are called on to provide any of the required research functions; chemists who monitor radiolabeled materials; clinical chemists, who analyze samples of animal origin (e.g., blood and urine); and structural chemists, that is, molecular spectroscopists and crystallographers. Structural chemists contribute to tbe safety-testing scheme in two principal ways. First, they provide probably the most important piece of information related to chemical and physical properties-proof of identity of test substances. And second, they contribute to health testing by identifying any testsubstance metabolites that analytical chemists isolate. Since I have spent most of my career as a structural chemist, it is on this aspect that I will direct attention in this article.
Table 1. Major BDAB Urinary Metabolites from Rabbits (Oral)
Metabolite
Abundance ( % of Urin.
Methods to
Radioact.)
identify
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IR, 'H NMR, ElMS
ldentification of Test Substances Two test materials will be discussed: benzyl2,5-diacetoxybenzoate, BDAB, a potential anti-inflammatory agent coordinate, the coordination geometry approximating trigonal bipyramidal.
and bis(N-oxopyridine-2-thionato)zinc(II),ZPT, an antimicrobial agent used as the active ingredient in some antidandruff shampoos.
Throughout most of the safety testing of these materials, nonradiolaheled samples sufficed, but for the metabolism studies radiolabeled materials were required to simplify the recovery and chromatographic characterization of metabolites. I t was the nonlabeled materials that were structurally characterized in detail. As with all compounds for which structural reference data are lacking in the literature, we used fur each pruof ut ~tructureat least twtr .~r~.tro;n,pi:nlrfhudi or, it apl)rupri,tte,sinnlt-crvstill X-ray diiir3ctim. 'I'hc. It(:laheled coumrroarta were then s\iitheai;.cd i t d o w i ~1111. ~r i m e routes used to make the respective nonlabeled compounds, and their structures were confirmed through use of one or two spectroscopic or chromatographic comparisons. For themetabohsm studies. BDAB was 14C-labeled on the benzoate &bonyl and ZPT on the 2 and 6 positions of both rings. We used a combination of infrared (IR), proton and carbon-13 nuclear magnetic resonance ( N M R ~and , accuratemass electron-im~actmass s~ectrometw(EIMS) to Drove the BDAB structure:^^ provided evidence for two typ& of ester (acetyl and conjugated) and for the tri and mono ring substitutions. Proton NMR chemical shifts and spin-spin coupling patterns established the precise isomeric form of the 1,2,5 trisubstitution; in addition, all carbon-13 NMR resonances were assignable to the structure drawn. The ions in the mass p w t r u n were i n agreement u,itlt 113thrhe mt,lecuIar \\eight ;ual predicted fraymcntali~mpathway. Indeed, I hc. ; q q r m n w ~f the C-H-' cdritm at rn 2 91 w.,> the ntrmyeit evidence thar the t a ring, ~ are cunnevted ;IS 6hou.n. The ;ecwld te,t marrrial. %lJ'l'.ha.; \.PI\. l i m i t 4 airter s o . l l d i t y 31ld r ~ i s t a~ ) r t ~ ~ I c ~ t n i ;IS ~ i ~A ~dispersed t~tly ting,t h t , ~a~; ~, sthe imu ur rhc ?in,. ~)\.rlill~tt>tl~ic>tte t m w t t t i ~ wlid t %l"l'. \Vt, it4 111.11 the l e s t a1ii11i:uuua nirthud to characterize this complex was single-crystal X-ray diffraction. In the unusual dimeric structure that we determined (I),the two zinc atoms are oxygen-bridged and each is five-
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ldentification of Metabolites Metabolism studies are not carried out with all test suhstances, but for some materials, including the two under discussion, they can he a very useful component of the overall health-testing scheme. Such studies are designed to answer questions like: Is the material absorbed from the gastrointestinal tract? Is it altered within the body? If so, to what? By what route is the material or its metabolites excreted? By ex~loriuehow the animal affects the chemical (usuallv raderstood and provide additional insight for the toxicologist who must make sound safety judgments. Table 1 lists the three major BDAB metabolites found in the urine of orally-dosed rabbits (2). Listed also are the percent total urinary radioactivity for each metabolite, and the snectrosco~icmethods used together to Drove each structure shown. It is quite common forproductsbf metabolism to be excreted mainlv in the urine; this was true for both of the test substances under discussion. For example, 99% of the administered dose of BDAB was accounted for in the urine within 48 br, although BDAB itself was not detected. The three major metabolites of BDAB accounted for 90% of the urinary radioactivity; several other minor metaholites were Table 2. Major ZPT Urinary Metabolltes from Dogs (Oral) Abundance (Sb of
Metabolite
Volume 59
Urin. Radioact)
Methods to identity
Number 1 January 1982
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observed, but no attempt was made to identify them. BDAB readily undergoes enzymatic hydrolysis, total hydrolysis leading to metabolites A and B but only partial hydrolysis leading to metabolite C. This is followed by a process known as conjugation to form a sulfate conjugate (B) and glucuronide conjugate (C). Conjugates are end products of reactions with natural body chemicals; they are products of the body's attempt to make foreign materials more watersoluble to facilitate excretion. Since the oral dose of BDAB was relatively high in this study, a large fraction of the gentisic acid metabolite (A) remained unconjugated. The animal's supply of available sulfate must simply have been exhausted. None of these findings came as a surprise; BDAB's metabolic route is as expected. It is important to know the metabolic
Research Triangle Institute (RTI) in North Carolina. (The IR analyses, however, were done at Procter & Gamble). We simply do not have time to undertake all product-safety studies using only Procter & Gamble resources. Here, RTI chemists isolated the metaholites, determined tentative structures spectroscopically, synthesized and characterized compounds having the proposed structures, and, finally, showed agreement between the spectral and chromatographic properties of the isolated and synthesized materials. As in the BDAB study, carbon-13 NMR was very useful: in addition to providing stereochemical information, i t showed that the saccharide is attached a t the sulfur and not the oxygen or nitrogen, and that the oxidation states of sulfur and nitrogen are as shown for each structure. Further, these carbon-13 NMR data were obtainable under mild conditions, so that the possibility of sulfur-to-oxygen or sulfur-to-nitrogen migration of the saccharide ort ti on was minimized. With metabolite C. however, for IR and EIMS.
again identified fragmentacon pattern. For metabolite B ~ I R the rine substituents. while the ~ r e c i s ecarbon-13 NMR NMR was of prime imp&tance. By comparing the carbon-13 snectrum of this comolex material with spectra of reference c&npounds, we were able to make detail& assignments that accounted for all the carbon atoms in this structure. Carbon-13 NMR has proven to be an exceedingly valuable tool in our product-safety research, and, indeed, in all of our structural work. The three major ZPT urinary metabolites from orally-dosed dogs are listed in Table 2 (3).Similar resultswere found using rats, rabbits, and monkeys. Apparently the ZPT complex dissociates in the body, with the zinc presumably entering the body's zinc pool. Conjugation a t the sulfur atom is the predominant metabolic pathway for the organic ligand, with two thioglucuronides (A and C ) and a thioglucoside (B) being formed. In one of the metaholites (C) the N-oxide is reduced; there was no evidence of substantial sulfur oxidation. Three additional minor metabolites were tentatively identified, but will not be discussed here. With ZPT, much of the metabolite isolation and identification work was done for us by a contract laboratory, the
48
Journal of Chemical Education
Concluding Remarks
In focusing on the contribution of structural chemists, I have dealt with only one of several important aspects of consumer-products safety research. For this resarch to be successful, structural chemists must have access to the latest instrumentation and must coordinate their efforts with those workers must share is to pro;ide an informed answer to the question: Is this material safe for its intended use? Acknowledgment
In addition to the researchers cited in references (1-3).the following persons are acknowledged for their contri'buti& to the work described here: A. J. Fehl, S. A. Goldman, D. M. Herbers, L. R. Isbrandt, D. L. McKean, R. L. Neal, C. D. Sazavsky, L. C. Strickland, J. D. Wendel, and W. I. Zorh. Literature Cited (1) Bsrnett,B. L.,Kretschmar,H. C.,andHaitman, F. A.,Inorg. Chem., 16,1834(1977). (21 Domeyer. B. E., Ridder, G. M., and Buinlin, P. M., presented at 19th Annual Meeting
