Document 1gJzz9MYDGj6DzZREqdyqwRgm

Seminar in Occupational Medicine PLAINTIFF'S EXHISIT Toxicology: Assessing the Hazard Robert E. Eckardt, M.D., and Robert A. Scala, P h . 0 At the start of any discussion of the fundamentals of toxicol- plication? Finally, were the effects acute or chronic? Thus, high ogy, it is necessary to make a distinction between toxicity and concentrations of sulfur dioxide produce an acute inflammation of , hazard. Toxicity is an inherent property of a compound, like its the trachea or bronchi from which you either die or recover fully. melting point or boiling point. Hazard, on the other hand, is the Repeated low concentrations may cause emphysema which is a likelihood that a compound may cause harm under its intended debilitating long-lasting condition. use. The function of the toxicologist is ct simply to determine the ! toxicity of a compound but rather t o perform the infinitely more ~ Dose-Response Curve &fficult-t assessing its hazard. How does he go about this .t k k . and what are the things ne nas to take cognizance of in / cassessing toxicity? One of the first things a toxicologist does is determine a dose- response curve. A typical dose-response curve is shown in Fig I . If deaths are the response being determined. the results determine -3iShown i n m are some of the factors that a toxicologist has the LDso o i the compound, that is the dose that kills 50h of the to consider. Was the dose large, or was it small? Was the route of animals. Generally the toxicologist will plot these results on log- administration appropriate? After all. you can easily drown in probability paper which tends to convert the data into a straight water, but that route of administration is not appropriate for line as shown in Fig 2. This is really just a convenience to make it. assessing the hazard o i drinking water or washing your hands with easier to determine what the 50% response point is. But the it. What was the frequency and duration of the administered dose toxicologist is not satisfied in knowing only the L050. He is in- or doses? What species of animals was used? In the car- terested in knowing the slope of the dose-response line. Thus in cinogenesis experiments on trichloroethylene reported by the U.S. Fig 3, Compound 1 would be more hazardous than Compound 2. National Cancer institute, liver tumors were produced in mice but even though they both have the same LDso. The reason for this is not in rats. This raises the question of whether man is more like the that to go from an LOOto an LDim with Compound 1 requires a rat, the mouse, the guinea pig, the hamster, or the pig. In terms of much smaller increase in dose than with Compound 2. Perhaps Vitamin C requirements, he is more like the guinea pig, which doubling the dose with Compound 1 may kill all the animals, but ' _ can't synthesize Vitamin C, while both the rat and the mouse syn- doubling it with Compound 2 may cause only a 5 or 10% increase thesize Vitamin C in their intestinal tracts. O n the other hand, in deaths. In assessing hazard the toxicologist wants to know the many people believe the skin of the pig is histologically more like slope of his dose-response or Lo50 line. that of the human than other species, and have, therefore, recom- Genetic factors must be taken into consideration by the mended the pig for skin toxicity studies. What was the age of the toxicologist as shown in Table 2. In the case of isoniazid, a well animals used? This can be important because very young animals known antituberculosis drug, the Oriental acetylates it much more may not yet be producing certain detoxifying enzymes and, as a result, may be sensitive to some materials that do not affect adults who have these enzymes in place. What was the sex o f the animals? Returning to the trichloroethylene experiment mentioned above, the male mice developed liver tumors in a statistically significant increased number of animals (about 45%) but in the female mice (6% liver tumors) the results were bare!y I Dose Route Frequency Duration -Table 1. Toxicity and Hazard. SDeCles Local Sea I 1 significant, if at all. Were the toxic effects local (such as an ulcer produced at the site of application) or were they systemic, damaging some organ (liver or kidney) distant from the site of ap- From the Exxon Corporaton. PO Box 45, Linden, NI 07036. Presented at the Fourth Esso Eastern Medlcal Conterence. November 15. 1977. Singapore 490 -Table 5. Classification of Toxicity Influencing Factors. FMOK Related to the ExDosure Situation Dose concentration and wlume of administralion Route. rate and site of administration Duration and frequency of exmure Time of administration ltim of day season of the year etc -Table 6. Classification of Toxicity Influencing Factors. inherent Factors Related to the SubMt Species and strain (laxonomic classilicatim) Genetic stalus (littermate siblings mulrigmration etfects etc I lmmumlogic status Nutritional stitus ( d e l faclors state of hyiratitm etc 1 Hormonal status (pregnancy etc Age sex body weight and maturity Central ~ ~ N U JsySstem status (activity crowding handling presem of other species e l c ) Presence of disease gr specific wgan pathology I I dose -Fig 1. Typical dose-response curve rapidly than the Occidental Since acetvlation is a detoxifying mechanism, the Oriental is much less prone to get toxic eftects, such as liver damage or neuropathy than the Occidental Other examples of genetic factors that may influence toxicitv are shown in'Table 2 In Fig 4, an attempt i s made to show the influence of genetic effects on an experiment In the upper left-hand corner, numbers of deaths are plotted against dose for a genetically homogeneous population When this is plotted as cumulative deaths versus dose the usual dose-response curve shown in the upper right-hand corner is obtained If a genetically nonomogeneous population is being dealt with. it is as if there were o separate populations each responding in its own way A plot numbers of deaths versus dose would give a curve like the one the lower left-hand corner The initial small bump represents the genetically susceptible group, while the second large bump represents the rest of the population If this i s plotted as cuinuiative deaths versus dose, a skewed dose-response curve as shown in the lower right-hand corner is obtained The toxicologist must know not only what the LD5o is, but also what the slope and shape of the dose-response curve look like. Influences on Toxicity The termination of a toxic effect can occur through several mechanisms as shown in Table 3. ,A material may be excrete$ either unchanged, as much of the inhaled- b the expired air, or changed, as urinary phenol. The compound may u)dergo metabolic transformation in the body which may serve either to increase or decrease its toxicity. Vinyl chloride is not carcinogenic itself, but after it is metabolized in the body, the chloroethylene oxide metabolite is carcinogenic. Finally. materials may be removed from the circulation and stored in relatively unreactive regions of the body. DDT is stored in the fat and lead in the bones. As long as they remain in the fat or bones, they produce no harm, but if mobilized out of these storage areas at Excretion (permanent1 Metabolic transfwmation (inneases or decreases toxicity1 Storage (depasitim) -Table 4. Classification of Toxicity lnfluencing Factors. F+Ion Related to the Toric & a t Chemical composition IpH choice of anim etc I Physical characteristics (particle size method of fcfmulation elc) Presence of impurities a contaminants Stability and staage characteristics of the ~ O K K agent Sol~bilityOf the tOXiC Sgml in biologic fluids Choiee of the vehicle Presence of excipients adiuvants emulsifiers surlactants binding agents mating agents colaing agents L*.*" flavoring agents peservalives antioxidants and other intentional and nonintentional additiws 'I . Journal of Occupational MediclneNol 20 No 7/July 1978 1 .% - log dose -Fig 2. Dore-response curve converted to straight line. 491 100 % deaths 50 0 log of dose two different dose-responses -Fig 3. Comparison of dose-response curves for 2 different compounds. -Table 7. Classification of Toxicity Influencing Factors. Environmental F K l o n Related to the S u b k t ~- ~ ~~Temperature and hunidity B a r m t r i c pressure (hyper. and hypbaric effects) Ambient 3tmaspheric cmpositlon Light and other forms of radiation HWSing 3rd caging effects Noise and other geographic influences Social factors Chemical factors some future time, they may then again create trouble. in removing stored lead from bone with EDTA, caution must be exercised or acute lead intoxication can be produced. Acute lead encephalitis has been produced or exacerbated in children treated with EDTA. In Fig 5 (adapted from reference 1 ,p. 271, a schematic of various body components is shown and glancing at this can indicate the importance of the route of administration. Ingested materials pass through the liver and may undergo metabolic transformation. O n the other hand. inhaled materials may enter the blood stream directly, bypassing the liver. and be excreted by the kidney in the urine unchanged. The effects produced by the same compound may be quite different depending on the route ot administration. In Table 4 are listed a series ot additional factors that may influence toxicity. The pH may affect toxicity. Large particles perhaps cannot be inhaled. Impurities or contaminants may be most important. The highly toxic dioxin, present as an impurity, was largely responsible for certain of the toxic effects ot 2.4.5-T. f i rs -Fig 4. Influence of genetic facton on an experiment. *s Y 5s "a '3+- Sometimes the compound mav break down in storage to form either more toxic or less toxic compounds It is essential to know what happens to a compound in storage Carcinogenic oils are kept in brown bottles and stored in dark cabinets because we know that ultraviolet light mav destroy polynuclear aromatics. Other factors that may intluence results are shown in Table 5. Most of these have already been discussed. The time of day when a material is administered may intluence results because of diurnal variations in the animals. S t i l l further factors related to the animal which may atfect results are shown in Table 6 Some of these have been discussed already. it is well known that mice put on a restricted caloric intake do not grow tumors very well. Further, if animals are too crowded, they may become belligerent, and in some cases become lonely if they i are housed alone Thus, in our carcinogenesis experiments, we usually house several animals in a cage because they groom each other and keep themselves more free of parasites If housed alone. ~~ 1 I N -fig 5. Schematic I the ger an I unt i Pr( 7 trc Ot ter av me for in\ th; mt tir. VI( th, be CA trr T de Ta Ot to aiI la1 W tir T,. IC la1 TOXICANT PATHWAYS 492 Toxicology: Assessing the IC ~ I . -x* - -Table 8. Interlabontory Variability Oral Dosing. American Cyanamid Atlas Dow duPOnt Laboratories lnwolvsd ~ ~~ Kodak Bio-lest Shell Carbide I - -Table 10. Interlabontory Variability Oral Dosing. I Male Wistar rats l M a m Farms 150-200 g) Overnight fast dosed 8 10 am 14 d observation Croups of 5 2X dosing fxtor lundiluted) Purina Lab Chow der 1 R. "* I Dlethanolamine I 1 I 2 4 Pentanedione they sometimes become so covered with parasites that their general health is affected and the results become less clear-cut If animals are handled too much, or too roughly, they become unhappy and may not give consistent results Finally. other factors in the environment of the animals may profoundly affect the results of an experiment as shown in Table 7. Animals are usually housed in temperature and humiditv controlled rooms If their quarters get too hot or too cold, the results of an experiment may be invalidated In fact, in manv animal quarters, back-up systems for controlling temperature and humidity are available so that if the main system breaks down, a whole experiment will not be lost When animals have been on an experiment for a year or more, several hundred thousands ot dollars have been - -Table 11. lnterlaboratory Variability Oral Dosing. Ratio of Highat to Lowest LD50 Sample Ratio Sample Ratio 1 1 60 6 2 07 ~~ Absolute value 0 2 to 8 0 milkg 2 2 35 7 I30 3 2 52 8 4 98 4 167 9 283 5 209 10 192 pretty good agreement. O n the other hand, with Compound 8 , one laboratory found it five times less toxic than another laboratory. Thus. under the very best of circumstances, the variability between laboratories may be as much as five-fold. With this in mind, quality control checks should be done periodically with toxicological laboratories. The same compound under two different code numbers can be sent to the laboratory. In this way, the laboratory unknowingly tests the same compound twice and a check can be made on how well they are able to repeat themselves. At other times, a compound already tested in one laboratory i s sent to a different laboratory and the same test is performed. If the results are fairly comparable, one can have greater confidence, but if a wide difference shows up, then a quandary develops to try to understand which was, in truth, the real result. (Tables 8 , 9.10 and 11 have been adapted from Reference 2.) Z F carrying out his experiments. (Tables 4, 5, 6 and 7 have been taken from Reference 1, p 134.) Test Repeatability Finally, let us examine how reproducible toxicity testing is under the best of circumstances A series of laboratories. shown in ,Tabie 8, did an LD50 determination on a series of compounds Each of these laboratories is a very good and highly respected tojcicological laboratory The compounds selected for test, ten in all, are shown in Table 9 It was agreed in advance that each tory would conduct the test as shown in Table 10 In other , methods were standardized insofar as possible, even to the time of day the animals would be dosed The results are shown in Table 11 With Compound 7,the laboratory reporting the highest LDso he., the least toxic) had a value 1.3 times that of the laboratory reportiwg the lowest LD5o (most toxic) This was a Summary Some of the fundamentals of toxicology that are used by the toxicologist in assessing the toxic properties of compounds have been discussed. It is easy to see that a large amount of keen professional judgment must enter into the toxicologist's decisions. It is not a job that can be done by an amateur, even though some well meaning lay and political associates may believe they are capable of doing so. The toxicologist's job is not finished when he has determined the toxicological properties of a compound. He then has t o assess the hazard of the compound, an infinitely more complex and complicated task. In the end, as stated by Dr. Mrak, "There are no harmless substances, there are only harmless ways of using substances" (quoted in Reference 1. p. 11). References 1 Toxicology The Basic Science of Poisons L J Casarett and J Doull IEds i New York Macmillan Publishing Company Inc 1975 768 p 2 Well CS and Wright GJ Intra- and interlaboratory comparative evaluation of single oral test Toxicol Appl Pharmacol 11 378-388 1967 f 2'O: Y ,%f Journal of Occupational MedicineNol 20 No. 7/July 1978 493