Document vN77k2gOqjRq2qj9LEEvwbyE

1 1 IN THE CIRCUIT COURT TWENTIETH JUDICIAL CIRCUIT OP ILLINOIS 2 ST. CLAIR COUNTY 3 FRANCES E. KEMNER, et al. 4 Plaintiff, 5 VS. 6 MONSANTOCOMPANY, 7 Defendant. ) ) ) ) ) ) ) ) ) No.80-L-970 8 Before the HON. RICHARD P 0 GOLDENHERSH, Judge 9 10 11 REPORT OF PROCEEDINGS 12 JURY TRIAL 13 November 4, 1985 14 15 16 APPEARANCES: 17 MR. REX CARR & MR. JERRY SEIGFREID, Attorneys at Law Appeared on Behalf of the Plaintiff. 18 19 MR. KENNETH R. HEINEMAN & MR. JOSEPH NASSIF, Attorneys at Law 20 Appeared on Behalf of the Defendant. 21 22 23 MARSHA SCHNIPPER 24 Official Court Reporter 1 INDEX 2 DR. FRANK DOST .................. DIRECT EXAMINATION BY MR. HEINEMAN 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 PAGE 3 1 1 EXHIBITS Page Page 2 Identified Admitted 3 EXHIBITS SUBMITTED ON BEHALF OF THE DEFENDANT: 4- Defendant's Exhibit No.: 5 1270 6 1271 1272 7 1273- 1277 1278 8 1279 1280 9 6 41 41 62 64 131 131 42 42 42 131 131 131 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2 1 BE IT REMEMBERED AND CERTIFIED that heretofore, on 2 to^wit: Monday, November 4, 1985, being one of the regular 3 judicial days of this Court, the matter as hereinbefore set 4 forth came on for hearing before the Honorable Richard P. 5 Goldenhersh, a Judge in and for the Twentieth Judicial 6 Circuit of the State of Illinois, Belleville, St. Clair 7 County, Illinois, and the following was had of record, 8 to-wit: 9 10 * * * * * * * 11 12 (The following proceedings were had in open Court.) 13 MR. HEINEMAN: Your Honor, if the Court please, I 14 think the last thing we did was the Court admitted Exhibit 15 No. 1269A. 16 THE COURT: Right. 17 MR. HEINEMAN: And I'd like to pass copies to the 18 jury. 19 THE COURT: Fine, go right ahead. 20 21 22 23 24 3 1 PRANK DOST, 2 resuming the witness stand, having been previously sworn, 3 testified further as follows: 4 DIRECT EXAMINATION (Continued) 5 BY MR* HEINEMAN: 6 Q* Dr. Dost, when we left off Friday afternoon, we 7 were talking about this Exhibit 1269A and the dose response 8 curves* Is this a test in the intact animal? 9 A* This is a study in the intact animal, yes* 10 Q. All right* And what is the significance of the 11 upper and lower levels of response in connection with the 12 frequency or the intensity of the effect. How do those -- 13 how are those reflected there? 14 A. In biological systems there will be an upper limit 15 that in this case is expressed at the top where the effect no 16 longer is increasing with increasing dose* At the bottom 17 there is a point at which no effect occurs. In other words, 18 we find a point whereas the dose decreases, we reach a point 19 where there is no response, there is a no effect dose or no 20 effect level. 21 Q. And did I understand you to say that -- well what 22 was the figure on this curve that you attributed as the no 23 effect level? 24 A. In t he c a s e of r e l a t i v e l y -- the m o use strain of 4 1 limited responsivity, the no effect level is clearly at about 2 -- this is a logarithmic scale, keep in mind, and this is a 3 value of about 2.3, so that the no effect level for this 4 strain of mice is at about 3 tenths of a microgram per 5 kilogram for this The nature of the curve, this is very 6 nearly down at zero effect, and the no effect level here is 7 at about tenfold lower, at about.03 micrograms per kilogram* 8 Q. Now, the no effect level that you've stated there 9 is expressed in terms of an effect or impact on the body, is 10 it not? 11 A. Well, it's expressed in terms of whichever 12 parameter we're -- we're examining, because if we're looking 13 at enzyme induction, that does not tell us about other 14 parameters. We have to examine a large number of different 15 parameters to get some idea of what's going in the rest of 16 body, because some effect would be perhaps less sensitive, 17 some more sensitive, just as we have two strains of animals, 18 two separate strains, one of which is sensitive and one of 19 which is less sensitive. 20 Q. What I'm trying to get at, and X guess I expressed 21 my question pretty inarticulately* Is this dose down here or 22 exposure? 23 A. This is dose. In other words$ you recall in the -- 24 well, here. I drew a dotted line that bypassed the 5 1 interaction with the environment when we were talking about 2 some earlier principles. 3 Q. Would you like to put that up here? 4 A. Well, perhaps. And what we have done is administer 5 this directly to the animal, and actually we could have drawn 6 that line down here. The exposure and the dose, in effect, 7 are the same in a situation where we inject the material into 8 the animal or if we put it into the digestive tract so what 9 we have done here is administer the dose directly to the 10 animal without the-- 11 MR. CARR: Your Honor, I will object. I think the 12 witness is saying that we have done that, and I thought this 13 was a study by somebody else? 14 A. I beg your pardon. Dr. Kouri's group has injected 15 it directly into the animal, and the environment, so to 16 speak, has not intervened and done anything to the chemical. 17 Q. All right, sir. Mow, so this does this figure in 18 this particular type of experiment, does the dose figure 19 equate with the exposure? 20 A. In this situation, yes, because we have -- the 21 nature of the exposure is injection of the material, and it 22 is all -- it all goes into the animal, whereas in -- if the 23 material was on the skin, only a very, very small fraction of 24 it would be -- would go into the animal and so the exposure 6 1 would be much, much larger than the dose. 2 Q. All right. Now# are there experiments in which this 3 type of phenomenon has been observed in an in vitro 4 situation? 5 A. Oh/ yes. 6 Q. This is what you would call what? 7 A. This is in vivo# in the animal/ in the animal. In 8 vitro is the other term. It means literally in glass. 9 Q. Okay. Have you had a chart prepared of an in vitro 10 experiment? 11 A. I do exactly/ yes. 12 Q. Would this assist you in your explanation of the 13 matter to the jury? 14 A. Y6S. 15 Q. Let me have this marked/ if I may. 16 (At this time Defendant's Exhibit 1270 was marked 17 for identification.) 18 .Q. Dr. Dost/ let me show you what's been marked as 19 Defendant's Exhibit No. 1270/ and ask you to examine that and 20 identify it for me please. 21 MR. CARR: Counsel/ may I see it please? 22 MR. HEINEMAN: Certainly. \ 23 MR. CARR: I'm through with it/ Counsel. 24 MR. HEINEMAN: Okay. 7 1 Q. Again, sir, this is Defendant's Exhibit 1270. 2 Would you examine that and identify it for me please? 3 A. This is a graph that has been drawn from numerical 4 data in an article by Niwa, who was at that time working in 5 the laboratory of Dr. Daniel Nieburg at the National 6 Institutes of Health, and it's published in Molecular 7 Pharmacology, Volume 11 in 1975. I'd say it's taken from 8 Table 1. 9 Q. All right. This is a graph prepared by whom? 10 A. I prepared the draft of the graph. 11 Q. All right. Now would you explain what this 12 demonstrates to the jury, please? 13 A. Again, this is a dose response curve that is -- 14 that is information quite similar to what was shown in the 15 last graph. It is an experiment in which TCDD, 2,3,7,8-TCDD, 16 was put into an-- into a culture medium. These are cultured 17 cells taken from -- from rat, and what we see here rather 18 than a dose is a concentration, because we have a homogenous 19 system here, and what we are interested in is the 20 concentration of the chemical in the medium in micrograms per 21 liter. 22 Very crudely you might relate that to an intact 23 animal, because one liter weighs one kilogram, and the animal 24 -- the animal is not homogenous, the material doesn't .8 1 distribute uniformly in the animal as it is in this-- in this 2 culture, in this solution, but a microgram per liter is very, 3 very crudely equivalent to a microgram per kilogram if we 4 tried to compare it. It's very difficult to compare a culture 5 system with an intact animal, but the biochemistry is the 6 same. 7 Q. By a culture system what do you mean, how do they 8 do this? 9 A. Well, these cells were derived from a rat tumor. 10 This is a very well established tumor or cell line, and cells 11 in culture after they have been changed, can by either 12 entering into the tumor process or other biochemical changes 13 can be cultured. They will reproduce, and we can -- in the 14 laboratory cell biologists can grow cells, mammalian cells in 15 the same fashion that we would grow bacteria, for example, 16 not quite the same process, because it requires different 17 kinds of culturing conditions, but it is possible to maintain 18 a cell line like this indefinitely, and the practice around 19 the country is that people who have established these lines 20 generally maintain the culture and make them available to 21 other researchers. 22 Q. Hence a name of some sort, they'll be identified as 23 something? 24 A. It's the id e n t i t y of t h a t p a r t i c u l a r line. In this 9 1 particular case, we have a typical dose response 2 relationship. The concentration down here where the activity 3 is at zero, and I should point out that in a culture system 4 many biochemical systems are actually resting at zero rather 5 than at some level. In the -- in the last graph you will 6 notice that these level out at some activity; in other words, 7 where there is no induction by TCDD, there is a base line of 8 activity. And the reason for this is that in an intact 9 animal that system is under at least some demand all the 10 time, and almost all kinds of activity in a normal cell 11 inside the animal, when the animal is all intact, they have 12 some resting level. It's very much like, for example, your 13 resting respiratory rate when you're asleep, which is quite 14 low relative to the rate when you're awake and around doing a 15 lot of things. That's the way it is in an intact animal. 16 However, in the cell, the isolated cell, there is 17 not all of the other activity in the animal going on, and so 18 at rest oftentimes that enzyme level is essentially zero, 19 because there is no need for it. The cells are in a totally 20 protected environment, they aren't coming in contact with 21 agents that will induce them. We're in contact with things 22 that will induce enzyme induction all of the time. 23 What we see here is a number of points, and then 24 interestingly, here is a point. One of the reasons that I 10 1 chose this particular example is because we can rely so 2 heavily on the dose response relationship and the orderliness 3 of this system that when we see a piece of information like 4 this, we report it* When I find something in my laboratory 5 that is like that, it is reported just as they reported it, 6 but finding that it does not fit into an orderly dose 7 response indicates very strongly that in that particular 8 group of cells something went wrong, something went wrong, 9 whether it was an error in recording, in the process of 10 bringing the data together, whether it was an error in 11 actually conducting an experiment would be impossible to tell 12 from here, but it is clearly an erroneous data point, and I 13 put that in there deliberately, because it tells us the level 14 of confidence that we have in the dose response in general* 15 In any case there is a no effect level here at*003 16 micrograms per liter of medium. Now in this system there 17 isn't much metabolism* The material goes in, there's no 18 circulation around the body, there's no excretion so when 19 that material is put in, it stays there* 20 Q. Now, what is -- what is the figure when you created 21 this graph, what was the figure that caused you to put the. 22 "X" there. 23 A* My recollection is that it was -- was an activity 24 of 94 units 11 1 Q. All right* If you inverted those numbers,, what 2 would the figure be? 3 A* It happens. You recall Ijjust mentioned that it's 4 possible that an error in recording data could imaginably be 5 the case. If you were to make 49 out of that 94, it would 'L 6 fall on the curve. I would not presume to judge that that's 7 what happened. That may just be a coincidence* 8 Q. All right. Now would you talk to us about the no 9 observable effect level. The jury has heard the expression 10 NOEL, N-O-E-L. What is that and what's its significance? 11 A. No observed effect level and the "0", the observed 12 is very important, because it depends on what we can 13 observe. We cannot take this prior graph and say we have a 14 no effect level and assume that that represents everything 15 else that's happening in the animal. It is what we are 16 observing here. Now the idea of the no observed effect level 17 is that it is the -- it is the highest dose that does not 18 produce an effect. 19 Q. All right. Now, what if you have a situation in 20 which there are a number of effects going on in the animal. 21 How do you select an N-O-E-L, how do you find one? 22 A. Well, -it depends on how much information we have, 23 but if we are seeking a no effect level or a no observed 24 effect level that we would like to relate to the whole 12 1 animal, we would find the lowest. We wouldn't take in the 2 case of -- in this situation here, let's say we wanted a no 3 effect level for the mouse, we certainly would not take the 4 highest of these two numbers, we would take the lowest, the 5 most conservative. We would not -- we require a substantial 6 amount of information to represent a no effect or a no 7 observed effect level for a whole animal. A single 8 experiment is not going to give us that much information. 9 Q. How does one go about determining what'a no effect 10 level is? Does it require a series of doses, one dose, how 11 do you go about doing that? 12 A. It requires -- it requires a series of doses. If we 13 have -- if we had only these two doses, we would know that 14 somewhere down in here there is probably a no effect level. 15 I won't say that there is going to be one. I certainly won't 16 say that it's going to be here or here or some other place. 17 We need a sufficient number of doses. There has to be a full 18 range of dosages to show particularly the no effect level. 19 Sometimes we can't find the point where the effect 20 tops off, because we may be reaching a dose so high.that the 21 animal fails before that particular function fails, and then, 22 of course, we don't know anything, but down at the low level 23 we have to have enough doses so that we can tell, a couple of 24 doses or one dose, and many studies are done for the purpose 13 1 of identifying the mechanism by which the chemical is 2 working, and they are only interested in one dose, a dose 3 that will cause the effect. That tells us nothing about the 4 dose response. r 5 Q. All right. Now I'm not sure I'm understanding this. You say there may be a test where they only use one 7 dose? 8 , A. Well, many experiments only use one dose. They 9 want to produce the effect and haying done that,'they want to 10 evaluate the characteristics of the system and see how it has 11 been changed from the normal. 12 Q. All right. 13 A. That's a perfectly valid kind of experiment for the 14 kind of determination that they are making. 15 Q. But that doesn't tell you anything about a no 16 effect level? 17 A. That's correct. 18 Q. Because the dose was selected to produce an effect? 19 A. That's right. 20 Q. So a one shot deal like that, will that tell you 21 anything about a no effect level? 22 A. No, it wouldn't tell us anything about it even if 23 it were down here at a point where it caused no effect, 24 because we don't know for any given chemical, it maybe that 14 1 the no effect dose is done here, and that's, of course, a 2 useless experiment, but the no effect dose may be here or 3 here or here. There would be no information. 4 Q. All right. So how many -- for example, the Kociba 5 experiments of Dow Chemical that the jury has heard a lot 6 about. In the Kociba experiments have they attempted to find 7 a no effect level? 8 A 7ss 9 Q. All right. How did they go about doing" that? 10 A. Well, that was -- Those where long terra * 11 experiments in which the material was administered, in this 12 case TCDD, 2,3,7,8-TCDD, in the diet over a lifetime, and so 13 it was a chronic dose, a chronic experiment, and they used 14 three doses in their two-year experiment. They used four 15 doses in their shortrange finding experiment. Those doses 16 were.001 micrograms per kilogram per day,.01 and.l in the 17 long-term experiment, and in the shorter one they started at 18 one microgram per kilogram per day, and that was-- caused the 19 animals, they carried it through the experiment, but it 20 caused a lot of damage, so quite a bit of toxicity. 21 Q. What did the Kociba experiments find to be the no 22 effect level? 23 A. They did find a no effect level at a daily dose of 24 .001 micrograms per kilogram. 15 1 Q, Which would correspond to what/ what's ,001 2 micrograms? 3 A. That's one nanogram per kilogram. 4 Q. Now# why is it that AAH activity is studied in so 5 many of these experiments? 6 A. AHH activity is one of the -- one of the enzyme -- 7 one of the enzymes that is induced in animals that are 8 responsive to the -- with the AH locus* In other words, there 9 are -- there is a process in the cell that responds to a wide 10 variety of polycyclic hydrocarbons 3-methylcholanthrene, 11 benzpyrene, dimethylbenzanthrozene, TCDD, and other, other 12 generally related compounds. And this particular enzyme is 13 often used as an index of -- that an effect has taken place 14 when researchers are looking for other effects. That's the i 15 reason why -- why in Kouri's experiments they did this, 16 because they were working on a general problem, and they 17 wanted an idea of how much of an induction response these 18 animals had experienced with the doses they were using. 19 Q. Well, does AHH induction have anything to do with 20 toxicity? 21 A. It has to do with the fact that a foreign chemical 22 has come into the body. It is not a toxic response as such. 23 It is something that we are doing all of the time, you and I, 24 everyone here, we have that capacity, all mammals have that ___________________ :____________________________________________________________________________________________________ _____________________ 16 1 capacity. In fact, lots of non-mammals have similar 2 capacity/ and when we encounter chemicals of this class, we 3 go into -- we make a biochemical change that the purpose of 4 which is to reduce, detoxify, and reduce the amount of that 5 chemical that is in the body, 6 Q. Okay. What is it that AHH does? 7 A. AHH is one of the enzymes that detoxifies, if you 8 will, aromatic hydrocarbons. In fact, that's the name of it, 9 aromatic hyrdocarbon hydroxylase. By that I mean~that it 10 attaches -- I believe that on Friday I talked about the 11 hydroxyl group, and this enzyme attaches a hydroxyl group to 12 foreign compounds, relatively non-soluble, relatively fat 13 soluble compounds, makes them more water soluble, and makes 14 them less fat soluble, and much more easily excreted. 15 Q. What is the difference between a threshhold and a 16 no observed effect level? 17 A. Well it's partly a semantics problem, just what 18 words mean. It's also somewhat of a philosophical problem. I 19 mentioned that a no effect level is the highest dose that 20 doesn't produce a effect, and a threshhold often is defined 21 as the lowest dose that does produce a effect. It's very 22 difficult to sort those two out, particularly since there is 23 always statistical variation in any experiment, and so 24 there's a sort of blurry area here, and generally in -- just 17 1 in the common discussion the two. are used interchangeably 2 even though they don't exactly mean the same thing. 3 Q. Now, how is a no observed effect level used in 4 determining a safe level for a human exposure? 5 A. Well, experimentally, after we have gathered a 6 great deal of information about a chemical, any chemical,,and 7 we can relate -- we find a number of no effect levels, we 8 gathered a great deal of toxicological information, 9 identified no effect levels not only for specific functions 10 like this, but for much more general functions. In the 11 Kociba study that you mentioned a little while ago these 12 animals were observed throughout their lifetime, a lot of 13 clinical chemistry, the same kind of tests that are run if 14 you go into the hospital for lab tests, the same kind of 15 information obtained, pathology, microscopic examination of 16 tissues, blood values, every bit of information that can be 17 extracted from the animal, so that there is a good index of 18 the general condition of the animal, or a test may be a whole 19 animal test on some kind of reproductive function again, 20 which is a general test. That coupled with specific kinds of 21 studies that look for certain biochemical changes, eventually 22 give us a number that we can consider is, in fact, a no 23 effect level for -- for the animal. 24 Now your question was how does this relate to 18 1 humans? And when we are trying to identify what might be a 2 safe level of a chemical for the human, we would take a small 3 fraction of the no effect level in animals and make the 4 assumption, and this is a very common practice in toxicology 5 that we would drop, depending on how much information we 6 have, tenfold lower, a hundred fold lower, a thousand fold 7 lower. Generally, we would want to have a human exposure at 8 least a hundred fold lower than the no effect level. 9 Q. All right. How does that relate to the amount of 10 data that's available in the human population? 11 A. Well, the amount of data in the human population 12 and the animal population are both of importance. If we have 13 lots and lots of data in the animal population, it's possible 14 to make -- to work with a narrower margin, because the 15 biochemistry of the animal and the human are, of course, are 16 alike. If there is a lot of data in the human population, 17 epidemiological studies, actual clinical studies of people 18 known to have been exposed to some chemical. The more data 19 of that sort that is available obviously the more data there 20 is that of direct applicability to the human for making a 21 judgment about what levels will have no effect. 22 Q* All right. Now, when you say the biochemistry is 23 alike, isn't it a fact -- well, with respect to TCDD, are 24 there species variances? 19 1 A. Yes, there are species variances for every 2 chemical. 3 Q. So what kind of problems, if there are any 4 problems, do you have in extrapolating animal data to humans? 5 A. We have in the case of -- in the case of TCDD or 6 any substance? In the case of TCDD. We have to look at the 7 reasons why there are variabilities among the animals species 8 and in terms of -- there are two problems. One is predicting 9 effects one is judging effects that might take place, that 10 might have -- on the basis of an, existing exposure as 11 differentiated from a general, potential situation. 12 In the CDC study a whole group of calculations were 13 made based on exposure, absorption, and using a no effect 14 level of.001 raicrograms per kilogram and extending that into 15 a-- into a calculation of acceptable, what is presumed to be 16 acceptable exposure in humans. So the process is rather 17 involved, and it really is a matter of judging risks. 18 Q. All right. Now, do all symptoms have no observed 19 effect levels? 20 A. With respect to TCDD? 21 Q. Yes. 22 A. In every experiment that I have been able to find, 23 in every experiment in which there has been an adequate range 24 of doses, a dose, a no effect dose has emerged, and there is 20 1 no -- in many of them/ of course/ perhaps the curve may stop 2 here. They may have not used sufficient number of doses. 3 The curve is certainly aiming toward a no effect dose, but I 4 can't say where it is. But in every case where there is a 5 sufficient amount of datar where the points come down to or 6 even below the zero effect level/ it is very clear that a no 7 effect level has emerged with TCDD in every parameter that 8 has been studied. 9 Q. Now, Doctor, you're, familiar -- well, have you read 10 the testimony of Dr. Ellen Silbergeld? 11 A. Yes. 12 Q. You have? Do you recall Dr. Silbergeld referring 13 to the work by a Dr. Kaminsky? 14 A. Yes. 15 Q. All right. Do you recall, sir, that she testified 16 that based upon th experiments that Dr. Kaminsky has done 17 there is no threshhold, there is no dose that does not cause 18 an effect on the liver. Do you recall that in her testimony? 19 A. Yes, I do. 20 Q. Okay. Now, are you familiar with the tests that 21 she's referring to, the studies that she's referring to? 22 A. Yes, I am. 23 Q. Okay. Is Dr. Kaminsky is he the first named author 24 in those tests? 21 i A. Dr. Kaminsky is in a -- works with a rather large 2 group of investigators in New York, and I believe he is 3 senior author of one of those papers. I believe J. Silkworth 4 is senior author of one of those papers. There are three 5 actually that he has published on this subject and and though 6 DeCaprio -- yes, he is the senior author of the third paper. 7 Q. Now, what incident were those papers studying? 8 A. These papers -- that research was the outgrowth of 9 the -- there was a fire in an office, of a state~office 10 building in Binghamton, New York, in which an FCB transformer 11 blew up and contaminated, X believe, pretty much the entire 12 building with soot that as it turned out had a substantial 13 amount dibenzofurans and chlorinated dibenzofurans, and 14 dibenzodioxins, including apparently TCDD, 2,3,7,8-TCDD. 15 Q* All right. Now, based upon your examination of 16 these studies, do you agree with Dr. Silbergeld's assessment 17 of their result? 18 A. No,, no. 19 Q. Why not? 20 A. Well, each of those studies did show clearly a no 21 effect dose. Two of them involved parallel studies of the 22 soot, and they were well-done experiments. Parallel studies 23 of soot and TCDD, and in one case they were studying guinea 24 pigs, which is-a-vsry sensitive species, and they a U 6 - "sed a 22 1 model, a chick embryo model, in which the material is 2 actually injected into a fertile egg and following through 3 the incubation period* This is a sensitive system, because 4 TCDD cannot escape. That experiment showed a threshhold* 5 They were looking at mortality in that case* 6 The guinea pig study showed a threshhold and did 7 not show appreciable liver effects, which is not surprising, 8 because the guinea pig is not very sensitive to liver 9 damage* Oftentimes guinea pigs will succumb to TCDD without 10 a great deal of liver damage. They are peculiar. 11 Q. Now, let me just-- may I interrupt you a minute? 12 We have heard that the sensitive measure of TCDD exposure is 13 measured in a guinea pig, that it's a very, very sensitive 14 animal. 15 A. The thymus weight is very sensitive* 16 Q. All right. And its ability to live? 17 A. Excuse me* 18 Q. And its ability to live? 19 A. Well, yes, the guinea pig -- the guinea pig is very 20 sensitive to the lethal effects of TCDD, and the weight of 21 the thymus is also a very sensitive indicator that -- it's / 22 also more sensitive in the guinea pig than in the -- in other 23 species* And in all of the indexes that they looked at they 24 clearly showed a no effect dose* In the third paper, which 23 * 1 was the one authored by DeCaprio their objective, their 2 stated objective in the paper was to find a no effect dose, 3 and they did a chronic, a subchronic experiment so-called. 4 They fed various concentrations of the soot over a 90-day 5 period to guinea pigs, and they found a no effect level. 6 They stated that as their objective, and they stated it in 7 their conclusions. 8 Q. All right. Now, is there -- is there other evidence 9 of the fact that there is a no observed effect level in -- in 10 liver experiments or other experiments? 11 A. Every experiment, as I said earlier, every 12 experiment that I Know of in which a sufficient number of 13 doses has been used, there has been no effect. We talked 14 about -- we talked about Kociba's studies a moment ago, and 15 they clearly showed no effect, both with respect to all of 16 the biochemistry and with respect to liver changes, 17 pathology, and so forth. 18 Q. Now when were these DeCaprio, Silkworth and 19 Kaminsky papers published, sir? 20 A. They were published in, I think, 1983, 1984, 1983, 21 I believe. 22 Q# All right* Now, let me ask you a bit about species 23 variability. What is the variability with respect to TCDD 24 among species, how does it appear to vary? 24 1 A. Well, one index is the lethality, how much does it 2 take to kill an animal, and the guinea pig is very 3 sensitive. Something around a microgram per kilogram, some 4 sex difference, some strain difference, but a microgram per 5 kilogram, the lowest figure that has been found is 6 tenths 6 of a raicrograra per kilogram, which is really not very much. 7 The hamster the median lethal dose, that is, the 8 dose that is required to kill half of the animals in the 9 experiment -- That's a kind of experiment we don't do very 10 often, believe me, but it is necessary in some circumstances, 11 and that, the LD-50 for the hamster is about 5,000 micrograms 12 per kilogram. 13 Q. So, how many times more than the guinea pig? 14 A. Roughly five thousand. 15 Q. All right. Are there any studies which have looked 16 into this variability of species susceptibility to dioxin? 17 A. A great number, because one of -- this enormous 18 difference causes a good deal of confusion when trying to 19 identify the mechanism by which TCDD actually exerts its 20 lethal effect, which we still do not understand, so efforts 21 have been made to find out whether the biochemical 22 sensitivities follow the whole animal sensitivity, and 23 whether the effects on reproductive tracts or reproductive 24 systems or other systems follow the lethality, and they tend \ 1 not to i. i i 2 We don't see the enormous different / 3 hamster and guinea pig* for example, in the L, Vx.-.' >-- \ S. 4 metabolism of TCDD or in the sensitivity to induction. There" 5 are also differences -- I mentioned that the guinea pig does 6 not# is not highly susceptible to the liver injury that is so 7 common with TCDD while the rat is, so there are a lot of 8 mysteries there, but where similar functions take place the 9 range is nowhere nearly as narrow as the lethali-ty. 10 Q, Now with respect to the -- with respect to the 11 human being, where does the human seem to fit into this 12 spectrum? 13 A* Noone has ever known to have died as a result of 14 TCDD intoxication There have been events that have taken 15 place in which mahy animals of various kinds have died. In 16 Seveso, Italy when that accident occurred, there was a 17 substantial mortality in particularly small animals, rabbits, 18 dogs, cats, and so on. The impact on the human population, 19 there was chloracne, there were some effects observed, but 20 the overall impact was apparently not that severe, and noone 21 died of the exposure certainly. 22 The Missouri horse arena, episode that has been so 23 well-known, where the waste oil containing TCDD was mixed in 24 with the arena soil, and the children playing in that and so 26 1 forth, there were a great number of deaths of horses, because 2 horses will pull the material up apparently through their 3 hooves, songbirds that are around the area, small rodents, 4 apparently cats and dogs, a substantial number of animals 5 died, and there was human intoxication, there was human 6 toxicity, but those individuals, according to the information 7 I have, have recovered. None died. 8 Q. The -- one of the things that you mentioned Friday 9 was in relation to metabolism of chemicals. Would you tell 10 us about the metabolism of chemicals within the animal's body 11 and what is the effect of that metabolism on toxicity? 12 A. Well, the whole idea Of metabolism of foreign 13 chemicals, and I will use the term metabolism very narrowly 14 to refer only to what happens to foreign substances once in 15 the body rather than all of the other multitude of metabolic 16 activities that are taking place, animals have to find a way 17 of disposing of foreign chemicals if they aren't easily 18 removed 19 I think I mentioned earlier that if a substantial 20 is water soluble, obviously it's going to stay in the body 21 water, and it will be processed by the kidney, and it will.be 22 removed, and that's a relatively easy process, provided that 23 the chemical is not causing some kind of damage while it is 24 there, and of course, any chemical in sufficient dose is 27 1 going to produce toxicity, but the idea here is to find a way 2 of getting it out of the body. 3 So in the case of substances like -- that are 4 insoluble or relatively insoluble in water, it's very 5 difficult to move them. The amount that goes out in through 6 the kidneys is very, very small, because that kind of 7 material, not only is it insoluble, but it would tend to bind 8 to the proteins into the plasma, and that would keep it from 9 going into the urine, and it would -- a number of things 10 would keep those kind of substances in the body. Their fat 11 solubility would help to keep them in the body. So the whole 12 idea is to make it more soluble and make it less toxic, and 13 often that can be accomplished in the same operation, which 14 is fortunate. 15 In other words, we don't have to undertake two 16 processes, one of which makes it less toxic and other of 17 which makes it more soluble. Most of the time it is a 18 relative simply process, and it would have to be because of 19 the thousands and thousands of different kinds of chemicals 20 that we are continually exposed to. If you walk through a 21 forest, there are enormous number of aromatic hydrocarbons 22 that volatilize from trees. In fact, in Oregon on a hot 23 summer day if you look across the valley ---- if anyone's been 24 in the Smoky Mountains in the east, you can see a haze out 28 1 over these valleys. There isn't a bit of industrial 2 activity, hardly any vehicles, and yet there is this haze out 3 there, and in large measure that is material, organic 4 material that is volatilized from the vegatation". So not 5 only do we have synthetic substances, but there's lots of 6 natural materials out there that we've got to do something 7 with, because they are going to come into the body. 8 Q. In terms of actual exposure to human beings, what 9 part does -- and those things that have to be metabolized, 10 what part does synthetic material play in connection with the 11 overall numbers of naturally occurring things that the body 12 is exposed to? 13 A. Probably pretty small, pretty small Bruce Ames 14 published a paper in Science a couple of years ago that 15 described very vividly the enormous number of natural-- 16 MR. CARRs Object to this line of testimony, 17 clearly hearsay, objectionable, counsel knows it. 18 THE COURTS Objection sustained. It is hearsay. 19 Q. Well, Doctor, are there -- what is the relationship 20 between the exposure levels that thebody -- not levels, the 21 numbers of items that the body is exposed to that occur 22 naturally versus the number of itemsthat are exposed to that 23 occur as a result of synthesis? 24 A. Probably far, far more natural substances of 29 1 toxicological significance, 2 Q. Now, when you were talking about the forests, for 3 example, what specifically are the materials that trees give 4 off? 5 A, Turpenes, particularly, and this is a very wide 6 range of compounds, but a number of what we have called 7 polyaromatic hydrocarbons, organic materials with a number of 8 aromatic rings. 9 Q. Is that where the name turpentine comes from? 10 A* Yeah, yes, it is. 11 Q. Okay. Now, what is it that the body does with 12 these materials when they -- when they come into body through 13 inhalation or dermal exposure or whatever? 14 A. As 1 just said, obviously we can't devise a whole 15 separate system for every single kind of chemical that's 16 going to come into the body. Fortunately, in spite of the 17 enormous number of combinations that can that can and -- 18 and the enormous number of different kinds of organic 19 molecules, they have a lot of common characteristics, and so 20 we don't necessarily have to devise a separate system. The 21 AHH will attack a vast variety of compounds that have some 22 common moiety, namely this benxene ring that has-- that has-- 23 that is part of the molecule, and so the whole idea is to 24 make it more soluble, as I've said many times, by attaching a 30 1 hydroxyl group to it, and then quite possibly on that 2 hydroxyl group attaching another even more soluble substance, 3 and then the material is excreted very rapidly. 4 Q. Now, in order for the body to attach this hydroxyl 5 group biochemically, does it have to do anything to the 6 chemical itself, may it necessarily knock some atom off? 7 A. Two general processes, one of which involves an 8 intermediate in which -- that is very reactive, and then the 9 hydroxyl group goes on. What has to happen is we have two 10 chlorines, for example, side by side. One of them will be-- 11 may be driven off and -- or even both, and an oxygen, a very 12 active oxygen attached, and sometimes that can get us into 13 trouble. That's what is known as a reactive intermediate, 14 and that's the kind of -- that's what happens with oh, 15 benzanthracene, for example, and that reactive intermediate 16 if it isn't changed quickly enough to put another hydrogen on 17 and make a hydroxyl out of it could very well react with 18 DNA. Other organic rings simply have an exchange of the 19 chlorine for a hydrogen, but usually in a biological system 20 they exchange for a hydroxyl group, and there is no reactive 21 intermediate in the process. So there's two general steps 22 that can take place there. 23 Q. How does the body metabolize TCDD? 24 A. It clearly metabolizes it, and it metabolizes it 31 1 apparently by the latter process, one in which a chlorine is 2 removed and a hydroxyl group is substituted. 3 Q. Would it be helpful for you to use an easel and 4 show the jury at this point. 5 A. It would be much easier, I think. 6 Q. I guess the best thing to do is to start with the 7 TCDD molecule that I suspect you have seen on a number of 8 occasions. There are also, by the way, hydrogens in each of 9 these corners. We never -- organic chemists never show the 10 hydrogens, and they never show the carbons. Bach of these 11 intersections there's a carbon molecule, so one of these 12 corners would look something like this. 13 What we're interested in doing with this substance, 14 which is really much, much more fat soluble than it is water 15 soluble, is to find a way of getting it out of the body, and 16 it doesn't react very well, it's hard to get at because of 17 shape of the molecule, but what happens is essentially this. 18 A chlorine will be taken off, and a hydroxyl group will be 19 put on. Now, that could happen at any of these corners. 20 There is also some evidence that a hydroxyl group might be 21 attached here either after removing a chlorine or even 22 leaving it intact, but in general it is very clear that there 23 is a hydroxyl and sometimes two hydroxyls attached. 24 Now, it's i n t e r e s t i n g that b y m a k i n g that very, 32 1 very minor change that that'molecule, number one, will lose 2 its toxicity, a large part of its toxicity. The other thing 3 is that it will become quite soluble. Then the other thing 4 that can happen in the next step if this molecule as it 5 stands is not -- is not carried out of the body, that there 6 may be attached, the hydrogen will be taken off, and there 7 may be attached a relatively large molecule -- I think that 8 Friday I mentioned a molecule that's something like glucose, 9 and it's a 6 carbon glucuronide that attaches, and that's got 10 a lot of hydroxyls on it, and it makes it extremely soluble. 11 So this is the i process in general that would be 12 undertaken. So all we have done is really-- all we have 13 done is change a corner of that molecule. That's the only 14 change that's been made, and the material then becomes 15 soluble enough to be removed, and that takes place almost 16 entirely in the liver. 17 Q. Now, Dr. Dost, are there -- when TCDD comes into 18 the body, does it tend to store in certain places? 19 A. Well, because of its fat solubility, it tends to 20 store in adipose tissue. It also tends to store in the fat 21 deposits that are in every organ, so it would -- and then it 22 also tends to store very vigorously in the liver. Early on 23 more of it will store in the liver or a higher concentration 24 will store in the liver than in fat and at higher 33 1 concentrations there is greater amounts in the liver. The 2 reason for this is because the proteins of the liver have a 3 nonspecific affinity for TCDD, they tend to bind to TCDD, and 4 as I think I've mentionedr this is different from the binding 5 to the receptor that we've talked about a number of times. 6 This is a nonspecific binding, and it stores a lot of TCDD in 7 that form. Relatively small amounts in the rest of the 8 tissues. 9 Now for a normal human about ten percent of the 10 body weight is fat and maybe two and a half percent of the 11 body weight is liver, so the total amount stored would be 12 factored by the size of the organ or the storage site. 13 Q. Now, how does that storage affect the ability of 14 the body to metabolize the compound? 15 A. . It doesn't affect it appreciably. In other words, 16 it's not locked into those places. There is -- there is a 17 equilibrium. TCDD or any other substance in the body is 18 continually moving back and forth even from a site like fat 19 where~itvs'Stored in substantial amounts, it's moving back 20 and forth. I guess a good -- a good analogy would be the 21 movement of -- you could think of water, a vessel of water, 22 where there are molecules of water moving into the vapor 23 phase in atmosphere. This cup, even though it's cold water, 24 there is a,little water coming into the atmosphere, and there 34 1 is also water going back in, but in spite of the fact that 2 the tendency is to stay in the water phase, there is always 3 some coining out, and the same thing is true in fat with 4 TCDD. Even though it is soluble in fat, has an affinity for 5 fat, there's always movement out as well as movement in. 6 Q* So that does its storage in fat affect metabolism? 7 Is the body still able to metabolize the material? 8 A. The body is still able to metabolize the material. 9 Q. Now, if the body metabolizes some of the material, 10 and it is excreted, for example, in the urine, what then 11 happens to that which remains in the fat and has not yet been 12 metabolized? 13 A. Well, let me make another illustration. Let's -- 14 we'll just draw some compartments. Biologists love to talk 15 about compartments, we make compartments into everything. 16 But we're going to have, we have a fat compartment here, and 17 and we have connecting the fat compartment with the liver, 18 and for the moment we'll ignore the TCDD that is just stored 19 in the liver, we'll only think about the TCDD that is being 20 altered in the liver for excretion, and we have the liver 21 here, and the liver is removing now some of this material. 22 Now, TCDD does not metabolize as rapidly as many other 23 chemicals. It's faster than DDT perhaps, but it's not as 24 fast as a number of other substances, so we have -- going to 35 1 the kidney, and we'll ignore bile for the moment, because 2 it's the same idea, we've got ultimately some TCDD coming 3 out* This is a slow process here. What happens in the liver 4 is not going on really very fast, at least when we relate it 5 to the ability of the kidney to get rid of the product. The 6 liver is making this material. The kidney has got lots and 7 lots of capacity to get rid of it, so anything that comes out 8 of here is gone. 9 If we have in the fat a lot of material, and it's 10 bound there pretty tightly, and the amount that can be in the 11 blood at any time is very slight, because it doesn't really 12 bind to very much in the blood, and it's not soluble in water 13 to any great extent. So there is a -- there is what we would 14 describe as an equilibrium here. There is -- if the material 15 has just come into the body, we could draw a large arrow here 16 pointing into the fat showing that it's moving into the fat 17 rather rapidly. At the same time there is always a little 18 coming back out. It may turn around and go right back in, but 19 there's always an exchange here, and that's carried by the 20 blood, and even though the blood can't hold very much it's 21 being removed so fast, and the blood is moving so fast that 22 it can actually transmit, it can transport quite a bit over 23 time. 24 Well let's get to the point where it is in the body 36 1 and it is -- we've come to a steady state so to speak. The 2 liver is continually receiving some/ because there's a little 3 in the blood/ always/ because of this exchange here,, there's 4 always a littlef and we'll get rid of this arrow, and we'll 5 make these two-- almost these two arrows almost the same so 6 it's coming in about the same rate as it's going out, except 7 since there's no more coming in it's slightly faster coming 8 out, because the liver is taking it out of the blood, and in 9 its slow fashion is getting rid of it. So there- is a 10 continual drain on the body burden of TCDD. 11 So what happens is that over time, even though the 12 material is sort of stuck in the fat, it's not stuck 13 permanently, and it moves out slowly, eventually gets to the 14 liver, and once the liver has it, it is disposed of so in 15 time the levels in fat will slowly come down, the levels in 16 all the other and tissues will come down similarly, and 17 sooner or later the material will be down to some level that 18 would probably depend on how much we're just taking in 19 adventitiously in our daily everyday life. 20 Q. What do you mean by adventitiously in our everyday 21 life? 22 A.' Well, we know that a lot of people, roost everyone 23 who has been evaluated has some TCDD in their fat, and it -- 24 so most of us must have some kind of an -- of a low level 37 1 exposure out in prairies, anywhere. 2 Q. Now, you mentioned something about a reactive 3 intermediate. You've read the testimony of Dr. Zahalsky? 4 A. Yes. 5 Q. Who testified before here? 6 A. Yes. 7 Q. Do you recall, sir, that Dr. Zahalsky testified 8 that dioxin when it's metabolized is changed into a highly 9 reactive metabolite? 10 A. I remember reading that. 11 Q. You remember that, sir? Do you agree with that? 12 A. No, I do not. 13 Q. All right. Have there been any studies to -- that 14 demonstrate whether or not it's -- the dioxin is turned into 15 a highly reactive metabolite? 16 A. Yes, there have. 17 Q. What are those studies? 18 A. Well, they have shown just the opposite, and the 19 evidence, there's a variety of evidence. To begin with if 20 dioxin was made more reactive and more toxic by -- by the 21 intermediate, through a reactive intermediate or if it was 22 made more toxic in its metabolism, animals with a high AHH 23 level and a high level of all of the P-450 enzymes would 24 suffer greater toxicity, and some very early studies that 38 1 were done in Robert Neal's lab by Patrick Beatty showed that 2 in animals with normally higher AHH levels, higher P-450 3 levels as a function of sex, had lower, were less responsive 4 to TCDD. 5 Later work that's been done has shown the nature of 6 the metabolite that I just illustrated. That's been very 7 clearly shown. The Swiss workers, Poiger and Schlatter, have 8 done experiments in dogs, in which they have taken the TCDD 9 metabolites from dogs. The dog is a big animal, and it's 10 easier to work with, and have found that those metabolites of 11 TCDD are at least a hundred fold less toxic in the guinea 12 pig. So they have taken the metabolites from one species and 13 introduced them to a species that is much more sensitive and 14 shown that they are, in fact, less toxic. 15 Q. If -- what would be the effect, if any, on DNA if 16 dioxin were metabolized into a reactive intermediate. 17 A. If it were metabolized into a reactive 18 intermediate, particularly where this is taking place in the 19 liver, it would be possible to find on DNA, it would be 20 possible to identify DNA damage, and it would be possible to 21 find the binding of TCDD or its metabolite to DNA, and 22 efforts to identify such binding have simply disclosed that 23 it doesn't happen. Allen Poland has done this and Gunther in 24 the National Institute of Health has also shown that the 39 1 binding of TCDD either with or without induction of 2 microsomal drug metabolizing enzyme just is not there. The 3 level are so low that they cannot imaginably account for any 4 binding. 5 There is also a wealth of mutagenesis studies in 6 which the -- in these test systems it is possible to identify 7 biological changes that result from DNA damage in the cellr 8 and they have all shown no response that could possibly 9 account for any kind of interaction with DNA. 10 Q. Has -- has your position on this matter and what 11 you have described found any support in the Environmental 12 Protection Agency? 13 A. Environmental Protection Agency states very clearly 14 that the metabolites of TCDD are less toxic than TCDD 15 itself 16 Q. Now, if I can deal briefly again with the storage 17 question, how is it that the dioxin or other chemicals that 18 enter the body get distributed around to various organs? 19 A. When a material comes into the body by whatever 20 route, by whatever -- whether it's taken in by mouth or 21 whether it's absorbed through the the skin, by whatever 22 route, it's going to get into the bloodstream sooner or 23 later, and once it's in the bloodstream -- 1 mentioned in the 24 case of TCDD that it's really not very soluble, and there's 40 1 been some speculation that some of the lipids in the blood 2 may actually have some influence in transporting. This is 3 not clearly shown, but in ahy case the TCDD will distribute 4 to all organs. It has to because it's in the blood, and 5 blood goes to all of the organs, and it goes to a number of 6 organs in rather large amounts because of their great 7 activity, the heart muscle, the kidney, because it processes 8 all of the blood, and the liver particularly have a very high 9 rate of supply of blood, and so there would be presumably a 10 opportunity for a lot of contact of those organs. 11 Then the other factor is what organs and tissues 12 have an affinity for the substance and would tend to draw it 13 out of the blood, and that would include the fat. Fat has a 14 substantial circulation. All of the fat cells are supplied 15 by blood vessels ultimately, so there is an opportunity and 16 once it's in the fat it tends, has a much lesser tendency to 17 come out* again, whereas in many cells it would have just as 18 great a tendency to come back into the circulation as it did 19 to go into the cell. That's the reason, for example, that 20 the brain has very, very little TCDD in -- in distribution 21 studies. So what happens is that the material moves through 22 the the body and incites -- where it has some affinity, it 23 will tend to store. 24 THE COURT: Mr. Heineman, is this a good point for a 41 1 short break? 2 MR. HEINEMAN: Yes, it is. 3 THE COURT: Ladies and gentlemen, we'll take a short 4 break at this time. We'll resume testimony after that. I 5 would remind you that you're not to discuss this matter among 6 yourselves or with anyone outside the jury panel or as of yet 7 form any opinions or conclusions about the matters on trial. < 8 Court's in a short recess. 9 (At this time a short recess was taken.) 10 Q. Dr. Dost, I have had the first drawing that you 11 made today marked Defendant's Exhibit 1271. Is that -- what 12 is that drawing again? 13 A. That drawing is a general description of the 14 metabolism of TCDD. 15 Q. All right. 16 THE COURT: What was that number? I'm sorry. 17 MR. HEINEMAN: 1271, Your Honor. 18 THE COURT: Thank you. 19 Q. And the second drawing I have had labeled 20 Defendant's Exhibit 1272. Would you tell us briefly that is 21 again? 22 A. It's a very simple representation of the 23 equilibrium between TCDD stored in fat and moving to the 24 liver for metabolism and out through the kidney. 42 1 HR. HEINEMAN: Your Honor, at this time defendants 2 would move the admission of Exhibits 1271 and 1272. 3 MR. CARR: We have no objection, Your Honor. 4 THE COURT: They're both admitted without objection. 5 MR. HEINEMAN: And I'm told that I neglected to 6 offer Defendant's Exhibit 1270, which is the Niv/a chart as 7 well. 8 THE COURT: Any' objection to that?. 9 MR. CARR: I thought the witness said that he made 10 that chart. 11 MR.HEINEMAN: Yes. 12 HR. CARR: If the witness made the chart, I don't 13 object to it. If it's a Niwa chart I do object to it, it's 14 obviously hearsay. 15 MR.HEINEMAN: The witness made the chart based upon 16 data from the Niwa-- 17 HR. CARR; I would object to it if it's based upon 18 some data that's not in evidence and can't be put in 19 evidence. I thought this witness did this work. 20 THE COURT; Do you have that article? 21 MR. HEINEMAN: Yes, I do, Your Honor. 22 THE COURT: Could you approach the bench with it 23 please. 24 (At this t i m e a c o n f e r e n c e w a s h a d at the b e n c h out 43 1 of the hearing of the jury.) 2 THE COURT: What data was that graph made from? You 3 indicated it wasr that the graph was drawn from data in this 4 article, 5 MR. HEINEMAN: Table 1. 6 THE COURT: Table 1? Have you seen this? 7 MR. CARR: Just now. I object to it. Your Honor. 8 It's clearly based upon data that is not in evidence and 9 can't be put into evidence. I understood the man was 10 testifying he made the chart, but he's apparently made it 11 from some data from some other study. We have no absolutely 12 way of knowing whether this data is correct or not. 13 THE COURT: I think we went through this the other 14 day, the arguments on it. 15 MR. HEINEMAN: Yes, Your Honor, the question was at 16 that time whether or not the witness was going to testify as 17 to the data itself. 18 THE COURT: Urahm. 19 MR. HEINEMAN: Whether he was going to sit there 20 and talk about everything that the study showed, and the -- 21 and at that time what we did was the Court permitted in a 22 table which was taken from the paper itself. 23 THE COURT: Right. 24 MR. HEINEMAN: Which is Kouri exhibit, which he is 44 1 referring to here. This is very little different from that. 2 THE COURT: I think it's different enough. The \ 3 objection is sustained, it's not admitted. 4 MR. CARR: I did not object to the other one# 5 because it Was for illustrative purposes. It was not for the 6 purpose of giving any dater simply to illustrate his 7 testimony. 8 MR. HEINEMAN: That's all this has been used for. 9 MR. CARR: This is clearly using data from an 10 inadmissible document. 11 THE COURT: It is. Objection is sustained. 12 13 (The following proceedings were had in open Court.) 14 THE COURT: The objection to the admission of 15 Defendant's Exhibit 1270 is sustained. 16 MR. CARR: Your Honor/ I know it's a little latef 17 but could the jury be instructed to disregard the information 16 that's apparently on this exhibit that they have seen? 19 MR. HEINEMAN: Your Honor/ the witness used this 20 chart to illustrate his opinion and testimony regarding the 21 dose response relationship/ and it's exactly the same kind of 22 thing that Dr. Ellen Silbergeld was permitted to do. 23 THE COURT: No objection -- that is not an accurate 24 statement of what happened/ and in no part in Dr. 45 1 Silbergeld's testimony was data transformed in the manner in 2 which it is in 1270. The jury is so instructed to disregard 3 the testimony by Dr. Dost that pertains to and uses 4 Defendant's Exhibit 1270. The objection is sustained. 5 MR. HEINEMAN: Your Honor, may I address it further 6 at this moment. 7 THE COURT: No, not at this moment. 8 Q. Dr. Dost/ with respect to the fate of TCDD in the 9 body, now, you talked about what happens to TCDD when it gets 10 actually into the bloodstream, but let's assume that the -- 11 that food which has TCDD put in it for experimental purposes 12 is consumed by the rat or the mouse, all right? 13 A . Yes. 14 Q. Mow, what happens to that as it goes down the 15 alimentary canal? 16 A. Some of the TCDD would be absorbed, some of the 17 TCDD would remain bound to the particulate material in the 18 food, and there will also -- that's the -- those are the two 19 things that will happen first obviously. Those are the only 20 things that can happen. It will either be absorbed or not 21 absorbed. 22 THE COURT: Excuse me just a minute, Doctor. Could 23 the two of you approach the bench for a minute, please. 24 (At this time a c o n f e r e n c e was h ad at the b e n c h o u t 46 1 of the hearing of the jury.) 2 THE COURT: Is that 1270 that's up on the thing? 3 Turn it around or put it against the rail or something. It's 4 standing up. 5 MR. CARR: The chart. 6 THE COURT: The chart that I've just objected, that 7 I've just sustained. 8 MR. HEINEMAN: All right. 9 (The following proceedings were had in open Court.) 10 THE COURT: I'm sorry. Doctor, go right ahead. 11 A. The -- as I said, obviously only two things can 12 happen, and one is that it is absorbed and the other is that 13 it is not absorbed. The material that's in the digestive 14 tract will have a significant effect on the amount of 15 material that is absorbed. In other words, if in the 16 experimental context, and this has been demonstrated, I must 17 say, if the TCDD is introduced into the digestive tract in a 18 solution of methanol, in which it is reasonably soluble, 19 anywhere from perhaps 60 to 80 percent of it will be 20 absorbed, because -- and this is in an animal with an empty 21 digestive tract. If it is introduced in some other medium, 22 there will be enough binding that this amount will be 23 reduced. If it were on soil, the data indicate that that 24 absorption rate will be -- 47 1 HR. CARR: Your Honor, I would object to this unless 2 the witness is testifying from his own experiments, and it's 3 my understanding that he has never done any work, any 4 research work with TCDD whatsoever. 5 MR. HEINEMAN: Your Honor, may I address that 6 point. 7 THE COURT: Sure. 8 MR. HEINEMAN: Your Honor, witnesses who have 9 testified for the plaintiffs in this case, notably Dr. 10 Silbergeld and Dr. Zahalsky, made a extremely large amount of 11 reference to the work of others. Dr. Silbergeld testified 12 with respect to the Eisen chart, the Eisen study. Dr. 13 Zahalsky testified with respect to the data from the Borman 14 study, which was used in his testimony, and there's a 15 Plantiff's Exhibit on the point, and this witness is doing 16 nothing more than giving his own opinion and then basing it 17 upon literature which he's read, and Mr. Carr previously took 18 the position with his experts that, and the Court ruled that 19 they were permitted to rely upon literature that they had 20 read, and that's what this witness is doing. 21 MR. CARR; Your Honor, if I might respond to the 22 argument counsel has made in front of the Court, and he knows 23 it was improperly made. The Eisen chart that was referred to 24 by Dr. Silbergeld was referred to without any objection by 48 1 counsel at the time. Any other data that was referred to 2 that could be considered hearsay was done so without an 3 objection by counsel* They are the ones that cross-examined 4 had Dr. Silbergeld and Dr. Zahalsky and Dr. Carnow about the 5 great body of literature dealing with this. There is 30 or 6 40 articles made Monsanto exhibits, none of which were made 7 plaintiff1s exhibits dealing with the literature. All of the 8 literature that counsel refers to was literature that 9 Monsanto introduced into this case. Counsel knows that the 10 -- all experts may testify to opinions that they've arrived 11 at based upon the scientific studies done by others. Counsel 12 knows that, and that's the way science progresses. What 13 counsel is doing with this witness is making exhibits appear 14 as if this witness has done the work himself, and when he 15 hasn't done the work himself, he is not permitted to cite the 16 data produced by others. We can't cross-examine these others, 17 we can't go into this man's brain and see if he is accurately 18 giving this data forth, and as counsel knows, this is an 19 improper way. He is an expert, but his expertise has to be 20 based upon his knowledge and not upon the knowledge of 21 others. 22 THE COURT: Objection is sustained. 23 MR. HEINEMAN: Your Honor, we have never 24 represented through this witness that he prepared any of the 49 1 data. He specifically testified to the contrary/ and Mr. 2 Carr has copies of every article he is referring to and has 3 been cross-examining him on. 4 THE COURT: In the course of your examination he has 5 adopted that data in the manner that Mr. Carr suggested is 6 improper and I agree is improper. The objection is 7 sustained. You're going to have to rephrase your questions 8 to stay in the permissible scope of examining your expert 9 witness. 10 Q. Dr. Dost, how much -- how much of the dioxin that 11 would be attached to soil if it were fed to an animal is 12 absorbed through the gut? 13 A. About 30 percent. 14 Q. Now/ what happens to the other 70 percent? 15 A. It remains in the digestive tract and continues and 16 is lost in the feces. 17 Q. All right. Now# with respect to the 30 percent 18 that enters the body through the digestive tract/ is that the 19 TCDD that is acted upon in the way you have described in, 20 Defendant's Exhibit 1271? 21 A. Ultimately/ yes. 22 Q* Now, how much of the TCDD that actually gets into 23 the body through the digestive tract, the 30 percent, is 24 disposed of in the way you have described in that exhibit? 50 1 A. Eventually all of it. It is a slow process. 2 Q. How quickly does it occur? 3 A. The half time is in various animal species on the 4 order of 30 days, that is, half of it will have been disposed 5 of in 30 days, half of what remains will be disposed of in 30 6 days and so on. 7 Q. All right. Now, you recall, sir, do you not, that 8 Dr. Silbergeld testified that a person who was exposed to 9 dioxin at low levels for years could accumulate ten thousand 10 to forty thousand times the exposure level; do you recall 11 that testimony? 12 A. I do recall that. 13 Q. I*m sorry? 14 A. I do recall that. 15 Q. Do you agree with that statement? 16 A. No, I do not. 17 Q. Why not, sir? 18 A. There is no evidence of any kind that suggests 19 that. The existing evidence on concentration of TCDD from 20 the environment provides numbers much, much lower than that. 21 The best example that I can suggest is the -- are the long 22 term feeding studies. 23 MR. CARR: Your Honor, we object to this. 24 Obviously this is not work this witness has done. I object 51 1 to it. It's hearsay. 2 THE COURT: Objection is sustained. 3 MR. CARR: Unless this witness has done this work. 4 MR.HEINEMAN: You object to it even if he's 5 referring to it as literature? 6 MR. CARR: Yesr I do indeedr counsel. Anybody can 7 read the literature. 8 THE COURT: Rephrase your question please. 9 MR. HEINEMAN: This is the same way that Dr. 10 Silbergeld testified and-- 11 THE COURT: I have ruled. You don't have to argue 12 among yourselves. I have made ray ruling. Now rephrase the 13 question or go to another point, Mr. Heineman. 14 Q. Dr. Dost, why do you believe that a person who was 15 exposed to dioxin at low levels for years would not 16 accumulate ten thousand to forty thousand times the exposure 17 level? 18 A. Because I know that the concentration in animals 19 doesn't exceed 25 times the concentration in the diet. That 20 is probably the best evidence. They also -- I know of 21 evidence in environmental studies in which other species that 22 have-- 23 MR. CARR: Your Honor, the witness is doing it 24 again. 52 1 THE COURT: Objection is sustained. 2 Q. Doctor, is there any evidence to demonstrate that 3 such an accumulation can occur in human beings? 4 A. No. 5 Q. Ten thousand to forty thousand? 6 A. No. 7 Q. Now, you recall, do you not, in Dr. Silbergeld's 8 testimony that she referred in determining that ten thousand 9 to forty thousand times to certain data relating to fish. Do 10 you remember that? 11 A. Yes. 12 Q. Now, what is that information? 13 A. EPA made an estimate based on -- not on data in the 14 environment not on experimental data, made a estimate based 15 on what is known as the partition coefficient for TCDD. By 16 the partition coefficient, I mean the relationship between 17 the amount of TCDD that is in solution in water and the 18 amount that is in solution in a fat solvent, and that 19 represents oftentimes at least a model of the distribution of 20 a chemical between the fat and the water compartment in an 21 organism. TCDD is not really very fat soluble, but is more 22 fat soluble than water, and when you have a substance that is 23 soluble in water only to the extent of perhaps ten parts per 24 trillion, it doesn't take a very large multiplier in the 53 1 other phase, in the lipid soluble phase, to create a very 2 large differential and the partition coefficient between 3 water and a solvent for TCDD is about a 40 thousand fold 4 difference. 5 There was apparently a leap of faith made because 6 the simile between water surrounding a fish -- this data was 7 originally related to the concentration of TCDD that might 8 arise in fish that are in a water body that is contaminated 9 with TCDD. The data on fish don't reflect this. There is no 10 such concentration in fish. The -- so there was a direct 11 extrapolation from a laboratory bench test of the respective 12 solubility of TCDD in water and a solvent called octanol, and 13 that was applied directly in this case, as nearly as I can 14 see, because the numbers are the same, it was applied 15 directly to some relationship in the human, and I don't Know 16 how it was arrived at, but it's not a proper deduction. 17 Q. Now, I'd like to ask you about the mechanism of 18 action of TCDD in the animal. What -- would you explain the 19 concept, first of all, of mechanism of action, what does that 20 mean? 21 A. Mechanism of action refers to the ultimate 22 biochemical events that take place when a chemical is 23 exerting its toxicological or pharmacological effect. What 24 is really happening in the cell as a result of the 54 1 introduction of that chemical, and that really -- sometimes 2 mechanism is referred to in more superficial terms on a whole 3 organ basis/ but really to provide valid information the 4 mechanism is the biochemical interaction that the chemical 5 causes in the cell* 6 Q. Does -- does dioxin by means of this mechanism of 7 action interfere with energy metabolism? 8 A. NO/ it does not. 9 Q* Is its mechanism of action dependent on a depressed i 10 immune function? 11 A. There is no evidence that the mechanism of action/ 12 the mechanism of lethal action has any relation to immune 13 function. 14 Q. Now you said the mechanism of lethal action. What 15 are you talking about there7 16 A. The mechanism by which it causes animals to die* 17 Q. All right. Nowf does that have anything to do with 18 the immune function? 19 A* Animals that are maintained for very long periods 20 of time on TCDD at levels that cause substantial pathology do 21 not show any evidence of increased infectivity. There is no 22 increased instance in respiratory infections and so forth. 23 Respiratory infections are particularly important because 24 laboratory animal colonies have a great deal of problem with 55 1 respiratory infection. 2 Animals that die of TCDD intoxication do not show 3 any evidence of increased infection. They don't die of 4 infections. Animals that are maintained in a germ free 5 status -- it's possible to breed laboratory animals in a 6 completely germ free environment, even -- it's possible to 7 exclude even the bacteria that normally inhabit the digestive 8 tract. It's very difficult to dor but it can be done. 9 Animals in that condition are no more -- have no better 10 resistance to TCDD than animals that are grown under normal 11 circumstances. To be sure* TCDD does have an impact on the 12 immune system, but it is not the means by which animals' 13 lives are shortened or by which they die. 14 Q. Now what thyroid or endocrin effects? 15 A. Again, there are a number of endocrin effects 16 caused by TCDD. None of them is responsible for the lethal 17 effect of TCDD. % 18 Q. Does -- do toxicologists know, have they been able 19 to determine exactly what it is that kills animals exposed to 20 2,3,7,8-TCDD? 21 A. No, sir, this is still not known. 22 Q. With respect to enzyme induction and its connection 23 with mechanism of action, how are you -- how do enzymes and 24 enzyme induction relate to the mechanism of action of TCDD? 56 1 A. The induction of enzymes by TCDD was found to be 2 part of a, if you will, a genetic battery. There are -- the 3 stimulus that causes induction is associated with -- with a 4 number of other effects that relate to the so-called AH 5 locus, the -- which is a section of the -- of the gene -- 6 it's a gene, a collection of genes in the -- in the animal 7 that respond in a characteristic way to TCDD. One component 8 of that characteristic response is the induction of aryl 9 hydrocarbon hydroxylase, which we discussed earlier, and one 10 of reasons that these two strains of mice behaved differently 11 is because they are less responsive to TCDD interaction with 12 that particular group of genes. 13 Because many of other effects were associated with 14 -- with the responsiveness to the induction and other -- 15 other effects that are related to this particular collection 16 of genes. For example, in mice of different sensitivities 17 the highly sensitive animals are also more sensitive 18 apparently to the teratogenic effects of TCDD, its ability to 19 cause birth defects, and for a while there was a great 20 interest in -- many people were convinced that this function 21 was really responsible for all of the major toxicity of TCDD 22 and responsible for its lethal impact. It turns out that 23 while there are a number of related -- genetisists would call 24 this coordinated functions that are affected by TCDD in this 57 1 -- this system* They are not'responsible either for the 2 lethal effect of TCDD. 3 Q* The -- is enzyme induction itself a toxic response? 4 A. Nor it is not. It is a response, it's a protective 5 response to an intoxicant. 6 Q. Is it an adverse reaction? 7 A. No, it is not. 8 Q. Now, is -- let me -- I'd like to talk to you about 9 the limitations that exist on the levels of enzymes induced. 10 MR. HEINEMAN: Your Honor, may counsel approach the 11 bench for a minute? 12 THE COURTs Sure, come on up. 13 (At this time a conference was had at the bench out 14 of the hearing of the jury.) 15 MR. HEINEMAN: Now, am I to understand that, and I 16 want to be sure I understand this. We are prepared to go 17 through with this witness a series of diagrams that are 18 prepared either by him or taken from a study, the purpose of 19 which is not to put in evidence the data in the study or the 20 data that's reflected in the diagram, but to demonstrate the 21 principle that he is talking about, limitation of effect of 22 TCDD on producing enzyme induction or the dose response 23 curve. 24 T H E COURT: A re t h e s e d i a g r a m s f r o m the s t u d y or are 58 1 these diagrams that he prepared from data in the study? 2 MR* HEINEMAN: In some respects -- well, some are 3 both -- some are one or the other. 4 THE COURT: So they're both kinds? 5 MR. HEINEMAN: Both kinds. Now do I understand the 6 Court is going to exclude his testimony or exclude the 7 admission of either of those types of diagrams based upon the 8 objection that Mr. Carr has made to this point? 9 MR. CARR: If I might respond to that, your Honor? 10 My understanding of the rule that we have been confronted 11 with here is that this witness may not testify to data from 12 hearsay sources. He can testify to data from works that he 13 has done himself. I do not object to him drawing a graph 14 based upon his theory of what's done. I do object to a graph 15 drawn from data or a graph drawn from some -- taken from 16 other hearsay source that has not been introduced and cannot 17 be introduced into evidence, and so if he has a graph from a 18 hearsay source that's not admissible in evidence or if he has 19 a graph from data based upon a hearsay source that's not 20 admissible in evidence, I will object to both of those, 21 either of those kind of graphs. 22 THE COURT: Have you seen these? 23 MR. CARR: No, I haven't seen them. 24 T HE COURT: H ow m a n y of t h e m a r e t h ere? 59 1 MR. HEINEMAN: There aren't that many here. 2 THE COURT: Do you have some small copies of them? 3 MR. HEINEMAN: Umhm. 4 THE COURT: Why don't you bring those up, and w e 111 5 talk about them. 6 MR. HEINEMAN: All right. Here are the ones that 7 we're talking about. 8 MR. CARR: All right, d e a r l y shows it's 9 apparently it's -- clearly shows it's from this work. 10 THE COURT: Now, is that from this work or 11 constructed from the work, this one? 12 MR. HEINEMAN: I'm not -- I'd have to look at the 13 paper and see. I'm not sure in this instance. I can find 14 that out and let you know in each one. 15 MR. CARR: This one is also cited as a source from 16 some work. It's a source from somewhere. 17 MR. HEINEMAN: Here's another one. 18 MR. CARR: They've all got. 19 THE COURT: They're all in that position, but that 20 doesn't tell us whether it's constructed from it or comes 21 from it. 22 MR. HEINEMAN: This one I know. This one comes 23 right out. This is -- this is a figure in the paper. 24 T HE COURT: Okay. 60 1 MR. HEINEMAN: This S-L-A-G-A on two-stage 2 carcinogenesis. 3 THE COURT: Pine. 4 MR. HEINEMAN: This comes right out of the paper. 5 MR. CARR: In either event, we object to it on the 6 basis of hearsay. 7 MR. HEINEMAN: I know that.Thigpen, T-H-I-G-P-E-N, 8 this mortality of mice* This is one that he has created from 9 data from a data table. On the -- on the Smith chart 10 relating to dose response on porphyrins and on the Poland and 11 Glover chart relating to response for induction of hepatic 12 aryl hydrocarbon hydroxylase, I don't know as I stand here 13 whether these were created by him or whether they come out 14 directly from the paper. I'd have to pull the paper to 15 look. 16 MR. CARR: Our position is the same, Your Honor, 17 that these are all hearsay documents. They're either a graph 18 from a hearsay document or its data is a graph that he 19 prepared from data in a hearsay document. It's not admitted 20 or admissible into evidence, and he cannot testify to 21 hearsay. He can testify to his opinions, but he can't 22 testify t6 hearsay unless I don't know the hearsay rule. 23 MR. HEINEMAN: Your Honor, I think that -- that he 24 is certainly entitled to use illustrations of this kind to 61 1 explain his opinions and the basis for his opinions. He is 2 not offering the data itself into evidence to prove 3 anything. He is just trying to demonstrate that there -- 4 that these things exist. 5 THE COURTS I am sustaining the objection. I think 6 they are hearsay. He is using them in a manner which as a 7 practical matter gets them before the jury in the context 8 which violates the hearsay rule. He is not foreclosed in his 9 testimony of making original drawings illustrative of the 10 same type of points in a visual nature with a visual aid in 11 order to illustrate graphically in front of jury the point 12 that he is making. This is sort of a back door way of 13 getting this data in* and I don't think it would properly be 14 used in this context. I am sustaining the objection to these/ 15 which probably should be marked so that we can refer to 16 them. Could you just mark these in sequence/ and I will read 17 into the record that these-- 18 MR CARR: Another one. 19 MR. HEINEMAN: This one is actual data taken from a 20 table. 21 THE COURT: Again to be used as illustrative 22 purposes. 23 MR. HEINEMAN: Yes. 24 T H E COURT: Marsha, c o u l d you mark all these. 62 1 (Defendants Exhibits 1273 to 1277 were marked for 2 identification,) 3 THE COURT: On the record this -- the objections are 4 sustained to Defendant's Exhibit 1273, 1274, 1275, 1276, and 5 1277, which are the smaller copies of blowups, 6 (The following proceedings were had in open Court.) 7 Q. Dr. Dost, is there a limitation on the level of 8 enzymes that can be induced in this way? 9 A. Yes, there is a very clear limitation. 10 Q, All right. And how does that come about? 11 A. It comes about because of the capacity of the cell 12 to produce the enzyme, and the location of the limitation is 13 known, has been worked out biochemically, but in essence the 14 cell has a limited ability to respond to the stimulus to 15 produce the enzyme. 16 Q. And is that true regardless of what the chemical is 17 that's stimulating the induction? 18 A. That's correct. The chemical that -- that will 19 induce -- and as a matter of fact, this has been very clearly 20 with aryl hydrocarbon hydroxylase by Allen Poland, 21 MR. CARR: Your Honor, the witness insists upon 22 doing that which he may not do. I object to it. 23 THE COURT: Objection is sustained. 24 MR. CARR: Ask the jury be instructed to disregard. 63 1 Counsel knows it, the witness knows it. I don't understand 2 this. 3 THE COURTi Objection is sustained. Jury is ordered 4 to disregard it. It's about five minutes to noon. We are 5 going to break for lunch at this time, and, Mr. Heineman, I'd 6 like you to explain exactly what the problem is to this 7 witness so that we can avoid it in testimony after lunch. 8 We'll break for lunch now. We'll resujme at 1:15. 9 The admonishments that I've given you earlier will apply 10 during this lunch break. Court's in recess for lunch. 11 (At this time a short recess was taken.) 12 13 (The following proceedings were had in open Court.) 14 MR. CARR: Your Honor, at this time the plaintiffs 15 would like to withdraw the objection to Exhibit 1271 that we 16 made this morning, and in addition, advise counsel and the 17 Court that we are withdrawing other objections that we made 18 to the testimony of Dr. Dost as long as the exhibits that 19 they use identify the source and as long as the witness 20 identifies the source of the data upon which he may or may 21 not -- that he is basing his .opinion, and we will, therefore, 22 withdraw those objections so that counsel can show what he 23 wants to show in these areas. 24 T HE COURT: No w 1271, 1271 w as a d m i t t e d w i t h o u t 64 1 objection. 2 MR. CARR: 1270 then, Your Honor, would be the 3 exhibit that we withdraw the objection to. 4 THE COURT: Okay. 5 MR. CARR; And, of course, the Court, I think, 6 instructed the jury to disregard that exhibit, and the Court 7 should then, since I have withdrawn the objection, if w e 've 8 withdrawn the objection, the jury should be instructed that 9 they may consider that. 10 THE COURT: The jury is so instructed, so you can 11 disregard my admonition to'disregard the testimony. 12 Q. Dr. Dost, with respect to the limitations that 13 exist on the level of enzymes induced, is there a chart that 14 you have had prepared that would assist you in the 15 description? 16 A. That is correct. 17 Q. -- of this subject matter to the jury? 18 A. Yes. 19 (Defendant's Exhibit 1278 marked for 20 identification.) 21 Q. Let me hand you, sir, what's been marked as 22 Defendant's Exhibit 1278. Would you examine that and 23 identify it for me, please? 24 A. That is a-- 65 X MR. CARR: May I see it first, counsel? 2 MR. HEXNEMAN: Certainly* 3 MR. CARR: Do you have a little one for us? 4 MR. HEINEMAN: Yes. 5 Q. Go ahead, sir. 6 A. That is a graph, the graphic portion of which has 7 been copied directly from an article by Allen Poland and 8 Edmund Glover in Molecular Pharmacology, the Journal of 9 Molecular Pharmacology, Volume 10. I believe that was in 10 1976, but we don't have the date on it, I'm sorry. The 11 caption was moved from below the graph up on top, but the 12 graph itself was copied directly from the article. 13 Q. All right, sir. I wonder if you would be so kind 14 as to come down here and discuss this exhibit with the jury. 15 A. I had earlier shown a graph that showed two curves, 16 and one of them was what I called a responsive mouse, and the 17 other was in a strain of mouse that -- this graph right here, 18 the responsive and the nonresponsive, and the difference is 19 that the dose required to produce this effect is greater in 20 one than in the other. 21 The reason that this work was done was because it 22 was known for some time that this strain of mouse did not 23 respond with induction to a compound called 24 3-raethylcholanthrene, which is a fairly effective inducer. 66 1 Dr. Poland in his group did an experiment in which they 2 compared the actual doses required to produce a dose response 3 curve with TCDD on this enzyme aryl hydrocarbon hydroxylase 4 and with 3-methylcholanthrene. The reason that they did that \ 5 was because they wanted to learn whether the mechanism 6 essentially was the same. Now all of the details of the 7 mechanism would not emerge, but at the very least if the dose 8 curves were parallel, it would be reasonable to assume that 9 there at least was strong similarity in the -- in the 10 mechanism. What they found was that there was a clear upper 11 limit in the ability of TCDD to induce this enzyme, and the 12 upper limit is the same as it is with 3-raethylcholanthrene, 13 Now, the reason that this curve doesn't go up any farther is 14 because, as I explained earlier, this is a very toxic 15 substance, and when we get up to high doses like this, the 16 animal simply cannot tolerate them. 17 What is interesting is that with 18 3-methylcholanthrene, which has always been thought to be a 19 very potent inducer, you see we have a logarithmic expression 20 of dosage here, and we have almost one, two, three, four, 21 five orders of magnitude difference. There's actually a 30 22 thousand fold difference in the potency of these two 23 compounds. Now, when I say potency, I mean the amount that 24 it takes the number of molecules really that it takes to 67 1 accomplish a specific amount of activity. 2 So that TCDD is, in fact, a exceedingly potent 3 substance, when we consider it in terms of the number of 4 molecules it takes to induce the effect. But the capacity 5 for the cell to respond is the same in both cases. In other 6 words, TCDD doesn't make 30 thousand times more enzymes, for 7 example. It causes the same response, and this response 8 limits -- even though we don't have the data up here, from 9 the shape of the curve here and through here compared with 10 this, we can be quite certain that 3-methylcholanthrene is 11 not going to produce any more. Certainly at this level you 12 see the comparison is the same at this level, at this level, 13 so the relative potency of the two compound is constant 14 throughout the dose response curve. 15 What this demonstrates is clearly there is a limit 16 to the capacity of the system to respond, and it also is 17 strongly suggestive that the two are acting by the same 18 mechanism. 19 Q. Now, Dr. Dost, you recall -- I'll ask you if you do 20 recall the testimony of Dr. Silbergeld in court here on April 21 12th when she told the jury that AHH can be induced by ten 22 thousand times. Do you recall that testimony, sir? 23 A. Yes, I do. 24 Q. Do you agree with that? 68 1 A. No, I do not. 2 Q. Why not, sir? 3 MR. CARR: What page would that be on, counsel? 4 MR. HGINEMAN: I think it's Page 137? 5 A. The reason that I donrt agree is because in all 6 studies of this enzyme and related enzymes for that matter 7 there is a clear upper limit to the ability of the cell to 8 respond to induction, even to TCDD, and that limit -- partly 9 when we say so many, you know, times, in this case we are 10 talking about something on the order of 25 times, you can see 11 that if the resting level was only half, which it could have 12 very well have been in another experiment, it would have been 13 a fifty-fold difference, but we don't get massive 14 differences. In many situations the maximum induction is 15 only two or three-fold. It depends in part on the organ -- 16 this is in liver, and it depends in part on the organ and to 17 some extent the species, but there is a very clear upper 18 limit. 19 Q. And that upper limit is approximately what? 20 A. On the order of 25 fold. There is difference from 21 organ to organ in its ability to synthesize this enzymes, but 22 these are not large numbers. 23 Q. Does that -- is that limit reached by other 24 chemicals besides TCDD? 69 1 A, Well, yes, surely. We have that situation here 2 with 3-methylcholanthrene. Other inducers such as 3 phnobarbital, which induces somewhat different pattern of 4 enzymes than TCDD. Also with all the enzymes that it induces 5 there is a clear upper limit to the-- there is just so much 6 capacity to make the enzyme. 7 MR. CARR: Your Honor, I'd object to the question 8 asked the witness, because it's -- Dr. Silbergeld did not say 9 that dioxin makes ten thousand times. The reference that 10 counsel gave me was she illustrated that the dioxin receptor 11 gets on the line and says I'm going to give you a 12 modification of the message, don't make ten units of AHH, 13 make ten thousand. 14 Now that doesn't say make ten thousand times, it 15 says the message -- just modifies the message, don't make ten 16 units of AHH, make ten thousand units of AHH. That's not at 17 all what counsel gave to the witness. 18 THE COURT: Gentlemen, could you approach the bench. 19 MR.HEINEMAN: That's exactly what it says. 20 MR. CARR; You said ten thousand times. 21 THE COURT: Let me see that. 22 (At this time a conference was had at the bench out 23 of the hearing of the jury.) 24 MR. HEINEMAN: Your Honor, may I respond to the 70 1 objection? I know exactly what you're talking about. Your 2 Honor, if in fact scientific evidence is that it goes up 25 3 times, what she is implying here is that the -- it can make 4 ten thousand units. 5 THE COURTi Not at all. All she is saying is that 6 message is delivered, she in no place in that language 7 implies that that in fact is what happens, and that's what i 8 you represented to the witness in the question. Your 9 objection is sustained, and I will order the jury to 10 disregard it. 11 (The following proceedings were had in open Court.) 12 THE COURT: Ladies and gentlemen, the objection is 13 sustained. The question to Dr. Dost and the contents of that 14 question as far as Dr. Silbergeld testifying as to ten 15 thousand times and the response that it brought from Dr. Dost 16 is to be disregarded by the jury. You're so ordered. You 17 may continue, Mr. Heineman. 18 MR. HEINEMAN: Very well, your Honor. 19 Q. Dr. Dost, let me read to you what Dr. Silbergeld 20 said, and I would ask you if you agree with this statement* 21 So using that analogy, what the dioxin receptor 22 complex doeB is gets on that telephone line. DNA has called 23 up RNA and said would you please make a certain amount of 24 this enzyme, AHH we been talking about, but dioxin receptor 71 1 gets on the line and says, don't listen to that message. I'm 2 going to give you a modification of the message. Don't make 3 ten units of AHH, make "ten^thousand. And what happens is you 4 get an overriding of this message, and that is indeed what 5 goes on, what we call enzyme induction. 6 Now, do you agree with that statement sir? 7 A. I think that's incorrect. 8 Q. And for what reason? 9 A. Well, the implication in that statement is that 10 TCDD acts differently from other substances that induce AHH. 11 The only difference is that TCDD is more potent. The message 12 is the same, the message is the same, and it depends on the 13 amount of the inducing chemical that binds to the receptor in 14 the cell. 15 Now, it takes a much higher concentration of 16 3-methylchlolanthrene to bind sufficient numbers of receptors 17 to produce the induction, but once that binding to receptor 18 is accomplished, the complex of the receptor and the ligon, 19 the chemical that binds to it, accomplishes the same thing 20 either way. 21 Q. And that limit is what, sir? 22 A. The limit is on the order of, depending on the 23 organ and the tissue and immediate circumstances, it's going 24 to be around 25 fold. It could be as high as 50. 72 1 Q, Nov;, Dr. Dost, we talked this morning about 2 absorption rate in the body. Does the dose have an effect on 3 the absorption rate? 4 A. In the case of TCDD? 5 Q . Yes. 6 A. Yes, it does. It's interesting. One would 7 logically expect that the -- that the same fraction would be 8 absorbed with a lot or a little. When I say a lot, I'm 9 assuming that we haven't overloaded the system so far that it 10 simply can't be absorbed. What turns out to be the case is 11 that on both the skin and in the digestive tract that as the 12 -- as the actual dose decreases, the percentage of the dose 13 that is absorbed decreases faster than the dose. 14 In other words, if there's two doses tenfold a 15 part, the amount that is absorbed from the low dose is a far 16 smaller percentage of the one percent than the percentage of 17 the ten percent that is absorbed, which is very interesting 18 and unusual even and very fortunate. 19 Q. So that the lower the dose the less the absorption 20 rate? 21 A. The lower the dose the smaller the fraction of the 22 dose that's absorbed, so the amount absorbed decreases faster 23 than the dose. 24 Q. Now, I'd like to direct your attention to another 73 1 statement of Dr* Silbergeld when she testified that the 2 induction of P-450 enzymes-- 3 MR. CARR: What page# counsel? 4 MR. HEINEMAN: 148 and 149. 5 Q. That the induction of P-450 enzymes and the 6 consequent metabolism of dioxin results in a new molecule 7 that is highly reactive and can interact with DNA. Do you 8 recall that testimony# sir? 9 A. Yes, I recall it. 10 Q. And do you agree with that? 11 A* No, I don*t. It's a similar statement to one that 12 I believe we heard earlier. It is not correct. 13 Q. And upon what do you base your opinion that it's 14 not correct? 15 A. I base my opinion on research that ha3 shown that 16 as the metabolism, as the capacity for metabolism increases 17 toxicity decreases. On research that -- that shows the 18 nature of the TCDD metabolite, on research that shows that 19 the metabolite of TCDD is in fact much less toxic than TCDD, < 20 per se, and also the fact that TCDD does not demonstrate any 21 of the characteristics of a substance that goes through the stage of a reactive intermediate, where there is a form of the chemical that is highly reactive and has the potential for interacting with DNA. 74 1 Q, Now, well, does TCDD react with DNA? 2 A. No, it does not* 3 Q* Now, whose work establishes that, sir? 4 A. Primarily the work of Allen Poland, and others have 5 studied the problem Thomas Gunther at the National Institutes 6 of Health, the work of, as I mentioned, Poiger and Schlatter 7 in Switzerland identifying the metabolites, and, of course, 8 in Poland's work he used both uninduced and induced animals 9 and was unable to find any difference, any increased DNA 10 binding in animals that did not -- that had excess, if you 11 will, capacity to metabolize, so that in both induced and 12 uninduced animals, there was no response, and, of course, the 13 indirect studies on micro-organisms seeking DNA damage in 14 various ways also do not show an interaction with TCDD. 15 Q. Now at the same time in her testimony Dr. 16 Silbergeld told the jury that dioxin enhances its own 17 toxicity and creates a vicious biological cycle. Do you 18 recall that? 19 A. Yes. 20 Q. And do you agree with that, sir? 21 A. No, I do not. 22 Q. For what reason? 23 A. Well, I really have outlined the reasons already, 24 the fact that TCDD metabolites are less toxic. In addition, 75 1 there is again from Dr. Poiger and Dr. Schlatter more recent 2 work, which shows -- in which they have treated animals with 3 TCDD, they have used TCDD as the inducing compound, if you 4 will, and then followed it later with radioactive labeled 5 TCDD and have shown that the rate of TCDD metabolite 6 production is about double. In other words, TCDD is being 7 metabolized and shipped out of the body at a much higher rate 8 in animals that have had a pretreatment with TCDD. 9 Q. What's the significance of that with respect to 10 enhancement of toxicity? 11 A. We've already learned that in the same laboratory, 12 as a matter of fact, that the TCDD metabolites, per se, are 13 much less toxic than TCDD itself. 14 Q. So that if there is more of the metabolites 15 created, then the toxicity is going down rather than up? 16 A. That's correct, that's correct. 17 Q. Now, let me hand you, sir, what's been marked as 18 Plantiff's Exhibit 1627 and ask you to examine that and 19 identify it for me, please? 20 A. This is an EPA document. It is identified as May, 21 1984 External Review Draft of the Health Assessment Document 22 for Polychlorinated Dibenzoparadioxins, and this is Part 1 23 apparently of two parts of this document. 24 Q. Does the document reflect who the reviewers of the 76 1 material are? 2 A. Most documents have a -- here we are. The 3 polychlorinated dibenzoparadioxin peer review panel members 4 -- this group is chaired by Dr. Mukerjee and Dr. Stera. 5 They're both EPA scientists. 6 Q. Is Dr. Silbergeld on that list? 7 A. Yes, she is. 8 Q* All right. Let me direct your attention to page 79 14 of that document. What is the date of that, by the way? 10 A. This is May, 1984. Did you say 7- 14? 11 Q. 7- 14. 12 A. I have that. 13 Q. First paragraph, last sentence. Would you read 14 that aloud to the jury, please? 15 A. A comparison of the mortality data at five weeks 16 after dosing indicated that the acute toxicity of 2,3,7,8 17 TCDD to guinea pigs was at least one hundred times higher 18 than was the acute toxicity of its metabolites. 19 Q. Now, what does that -- is that statement consistent 20 with or inconsistent with a statement that TCDD enhances its 21 own toxicity, creates a vicious biological cycle, and creates 22 reactive intermediates that react with DNA? 23 A. I would have to say that it is inconsistent. 24 Q. Now, sir. Dr. Silbergeld also told us that dioxin 77 1 creates a reactive metabolite which initiates cancer as shown 2 by Dr. Eisen. Are you familiar with that testimony, sir? 3 A. Yes, I -- yes. 4 Q. All right. Are you familiar with the Eisen study 5 that she's referring to? 6 A. 'I'm familiar with a review that Dr. Eisen prepared 7 that, I believe, is the document. She doesn't specify the 8 document, but I believe that it's a review that Dr. Eisen 9 developed in attempting to bring together an explanation of 10 the general question of the-- AH genetic locus. 11 Q. Do you agree with her statement about what Dr. 12 Eisen is showing in that article? 13 A. Dr. Eisen didn't specify anything. He discussed 14 TCDD in the article, but he did not identify a mechanism for 15 TCDD. 16 Q. Let me show you, sir, what's been previously marked 17 as Plantiff's Exhibit 233, and ask you if you can examine 18 that and identify it for me, please? 19 A. I recall that as a figure that was in the Eisen 20 article that I referred to, the review article. 21 Q. Now, does this exhibit show that dioxin creates a 22 reactive metabolite which initiates cancer? 23 A. This exhibit, this diagram does not show that. 24 Q. All right. Could you tell the jury what it does 78 1 show? 2 A. Well, this is intended as a -- Dr. Eisen is a -- is 3 a very capable biochemist, who has been interested for years 4 in the concept of receptors. I mentioned earlier, I believe, 5 that we depend on receptors for the action of all of our 6 hormones, insulin, for example, in the adrenal cortical 7 hormones as well as for other functions, and one of the 8 characteristics of the AH receptor that starts all of these 9 mechanisms, all of these processes that we been talking about 10 is that it resembles in many ways the receptor for the 11 adrenal cortical hormones, and Dr. Eisen had become 12 interested in this receptor, both for its own sake and as a 13 possible model to help him understand the glucocorticoid 14 receptor, and what he's done here is to try to prepare a 15 generalized scheme that -- into which all of the compounds, 16 all of the polycyclic aromatic compounds might fit in some 17 way. 18 He is not saying that every one of them follows the 19 whole road map here. What he is trying to do is put together 20 an integrated system and provide a means of relating 21 individual compounds. Now he was, as a matter of fact, my 22 recollection in the article is that his reference to reactive 23 intermediates was one based on his work with 24 712-dimethylbenzanthrozene, which is a compound that does go 79 1 through a reactive intermediate change/ and what he is 2 showing is that a route among the variety of prospective 3 routes, the compound comes into the body and interacts with , 4 the receptor, and it activates a group of genes, and that 5 causes the synthesis of these enzymes that we've been talking 6 about among them AHH and a variety of others that have 7 generally related functions. 8 One of the thing that that group of enzymes can do 9 with some compounds benzanthrozene is one, 10 3-raethylcholanthrene is another, is make a reactive 11 intermediate, because this process of making it more soluble 12 has a step where one of intermediates is very reactive, and 13 unless that hydroxyl group can be put on quickly, it could 14 conceivably escape from the point in the cell where it's 15 being made and move to DNA or some other site and cause 16 harm. 17 It happens that TCDD does not go through that 18 process. It is able -- the organism is able to interact with 19 it and convert it all the way to the hydroxyl without going 20 through this stage. So what he's done is account for the 21 kinds of substances that can be -- that can be interacted 22 that way. The compound not only induces the receptor, but 23 then it interacts with the enzymes with the potential for a 24 reactive intermediates, and we know that those kinds of 80 1 intermediates can cause DNA damage dnd cause all kinds of 2 problems in the cell, at least potentially. 3 There are question marks, here, because noone is 4 really sure where, other than at DNA and a few protein sites, 5 where that reactive intermediate might really be exerting 6 effect. There's evidence of cell membrane effect, there is 7 evidence of interference with the receptor for epidermal 8 growth factor, which is another substance that's synthesized 9 in the cell, in the body to interact at specific sites. 10 Well, if that happens there are a number of things 11 that might very well occur. Epidermal hyperplasia, that is, 12 where the skin would perhaps tends to thicken? 13 immuno-suppression, we know that there is immuno- suppression 14 with TCDD, for example, and other substances of this sort if 15 the dose is raised sufficiently. 16 The role in promotion of cancer is not clear. With 17 respect to this there is some evidence that certain kinds of IB hyperplasia are associated with promotion. A number of other 19 kinds of enzymes that are not part of P-450 pattern that do 20 induce, whenever you induce, whenever you start this process 21 with the AH receptor. 22 So this is really an excellent description of the 23 overall general process. This group of enzymes, I mentioned 24 the other day that I have done work with ornithine 81 1 decarboxylase, but not in this context* There are other 2 things that induce ornithine decarboxylase* So this is just 3 a general scheme. It does not refer specifically to TCDD or 4 for that matter to any other chemical. It's intended to 5 account for the way* to provide a scheme that will allow us 6 to generalize about the way that this group of compounds can 7 have an impact on the cell. 8 Q. And does that scheme as reflected by Dr. Eisen 9 there relate to the actions of 2,3,7,8-TCDD? 10 A. To this extent it does* We know that in 11 interaction with the receptor, the cytosolic receptor, we 12 know that it activates these genes, at least we know that 13 indirectly, and the evidence is so clear because of the 14 nature of the enzymes that it induces, and these things all 15 apparently happen. There is a real question about this, but 16 these enzymes are apparently -- well, these are -- these are 17 questionable, but this one probably is induced to some 18 extent 19 These certainly are, and, of course, all of the 20 P-450. That's the reason there are question marks here 21 because the evidence is just not sufficiently clear yet to 22 really make a statement about them* So to that extent, yes, 23 and then there are questions here, is there a direct action 24 of some of these chemicals on some of these factors? There is 82 1 some evidence, but it's really speculative at the present 2 time. So, yes; some of the things TCDD does relate to, and 3 some of them it does not. It does not -- it doesn't relate 4 to the reactive intermediate idea. 5 Q. Does it demonstrate a initiation of cancer? 6 A. No, it does not. 7 Q. Does this chart demonstrate-- 8 A. This chart does not either, except where certain 9 compounds that are reactive intermediates we have to assume 10 that they have at least the potential for initiation of 11 cancer 12 Q. Now, with respect to those parts of the body in the 13 animal that are affected by TCDD, I'd like you to discuss 14 those, if you would, and would you start with the skin 15 please. What does TCDD do to the skin? 16 A. Well, TCDD in certain species of animals has a 17 effect on the skin. It will cause chloracne, which is a 18 hyperplasia and swelling of cells on the skin. It increases 19 the amount of keratin, the flaky material on the skin, causes 20 inflammation in sebaceous, that is, sweat glands, and 21 ultimately the formation of big blackheads. It does this in 22 the rabbit. It is the same response apparently that is observed in humans with chloracne. Chloracne is caused by other substances than TCDD. There is a genetic strain of 83 1 mice that doesn't have any hair and TCDD will cause a lesion 2 that is something like chloracne, not exactly the same. 3 There is an investigation going on to see whether that would 4 be a appropriate model to use for studying chloracne. 5 Q. Is there -- well, let me ask this. What animal 6 presents the closest model for human skin with respect to 7 reaction to TCDD? 8 A. Clearly the rabbit, the rabbit ear. 9 Q, And has there been a no observed effect level found 10 with respect to chloracneiform lesions and the rabbit ear? 11 A. Yes, there has. 12 Q. Okay. Who has done that work, sir? 13 A. That work has been done by -- probably the first 14 people who really quantitated it properly where Jones and 15 Krizek back in 1962, in which they worked out a dose response 16 in the rabbit, found a clear dose response relationship and a 17 no effect dose that I believe was at the level of about 2 18 tenths of a microgram, and later on Schwetz and his coworkers 19 at Dow Chemical have also done somewhat similar work and have 20 found a similar dose response, and I believe that the no 21 effect level that they found was on the same order as about, 22 and that is not a dose per kilogram, by the way, that is a 23 dose per site. And in those experiments they treated the 24 animals for an extended period of time. They treated them 84 1 five times a week for four weeks applying new material each 2 day. 3 Q. Now, with respect to -- to humans, has chloracne 4 been observed in humans? 5 A, Oh, yes, it has. 6 Q. Has there been any work done on the dosages 7 involved with producing chloracne in humans? 8 A. 1 know of only one study in which known doses were 9 applied to human skin. 10 Q. And which is that, sir? 11 A. That was a study that was reported by Dr. Roe of 12 Dow Chemical to the EPA hearings on 2,4,5-T, the 13 administrative law hearings that were in progress over about 14 a year and a half until relatively recently. I think that 15 that work was presented, I'm not certain, in 1984, I think, 16 to the hearing, *83 or *84. I'm sorry, I would have to look 17 at it and see the date, I just don't remember right at this 18 instance, but it was a reports of work that had been 19 commissioned by Dow Chemical with the intent of trying to 20 learn where the threshhold for chloracne and other effects 21 might be for TCDD on the skin. 22 Q. Who was the one that conducted this work? 23 A. The person who actually did the work was a 24 professor of dermatology at the University of Pennsylvania 7 X named Kligraan. 2 Q. And what was found with respect to a dose response 3 level and chloracne and other effects? 4 A This was an experiment that was done -- it was 4n 5 experiment that was simple in concept# and the execution 6 required some care and timing# because the TODD was 7 administered in fractional doses* At the low doses they 8 administered half of the dose on one day and then the oth^r 9 half a week later. 10 At the higher dose# which is what we are primarily 11 concerned with# a dose of 16 micrograms applied in a 12 location. The first dose was 8 micrograms# and then they 13 applied 8 -- they applied that in two halves? in other words# 14 4 micrograms today 4# micrograms tomorrow, and then starting 15 in a week# two weeks# administered one microgram doses a day 16 for 8 days* It's a simple experiment with a complex dosage 17 schedule. 18 Q. Leading to a total dose of what? 19 A* 16 micrograms on that site over that period of 20 time. 21 Q. Now# you mentioned before the difference in the 22 rabbit experiments# I think# with site dosing as opposed to 23 overall dosing through feed and that sort of thing? 24 A Yes 86 1 Q. What is the effect of site dosing? 2 A. Well, this concentrates -- it's possible to cause 3 chloracne, at least it appears possible in the human to cause 4 chloracne by systemic administration* If the dose is high 5 enough it's possible to cause these kinds of skin lesions by 6 means other than putting it directly on the site. It appears 7 to be possible in the rabbit, although the data is not clear. 8 The difference is that the material is concentrated in one 9 site rather than being distributed through the body. It was 10 placed on the skin in a very small patch, so that it's the 11 effect on the skin at that site is going to be very 12 substantial. 13 Q. Is there any kind of a solvent used in order to try 14 and get the material through the skin into the animal? 15 A. In those experiments -- they generally use an 16 alcoholic solvent, methanol, ethanol. Ethanol is alcohol, 17 the kind that you get in drinking alcohol. Methanol is the 18 kind that's usually in rubbing alcohol. 19 Q. Which did Dr. Kligman use? 20 A. Dr. Kligman used, he used a mixture of alcohol and 21 chloroform to carry the substance. Because of the relative 22 insolubility of TCDD that was considered by the chemists to 23 be the best solvent, so that it would be certainly dissolved 24 and then distributed uniformly over the spot where it was 87 1 being applied. 2 Q. And at 16 micrograms -- now, you said there was 8 3 administered at first in 4 and 4? 4 A. That's correct. 5 Q. And then there were 8 one microgram doses? 6 A. In succession, that's correct. 7 Q. In 8 consecutive days? 8 A. That's right. 9 Q. So a total dose was what over what period of time? 10 A. 16 micrograras over a period of -- total would total 11 about three weeks. 12 Q. Was that administered at the very same spot on the 13 body? 14 A. Yes, it was placed right on the same location. 15 Q. Now, what was the result? 16 A* There was no evidence of chloracne. 17 Q. Was there any evidence of any other systemic 18 result? 19 A. The investigator. Dr. Kligman, had -- I should 20 point out that there were five other groups at lower doses 21 that were run through a somewhat similar dosage schedule. 22 All of those people were given physical examinations, and a 23 number of laboratory tests were run on those animals -- on 24 those individuals, and there was no evidence of any 88 1 abnormalities in the clinical chemistry. 2 I should mention that these were volunteers from a 3 prison population, and the-- the creatinine clearances 4 probably were a tiny bit low, but probably reflect the rather 5 low protein diet. I wouldn't be surprised if they were on a 6 low protein diet, but they were really not outside the range 7 of normal. So there was really no evidence in that of any 8 abnormality that would be detected on the basis of those 9 kinds of tests. 10 Q. Was there -- was there a subsequent test done that 11 did cause chloracne? 12 A. This is a -- this is an interesting situation, 13 because Dr. Kligman instead of awaiting instructions from Dr. 14 Roe went ahead with another protocol and used a much, much 15 higher dose of TCDD, and he did cause chloracne in roost of 16 the -- I believe he looked at ten subjects, and I believe he 17 caused chloracne in eight of those. In any case it was a 18 high fraction of the individuals. 19 Q. And was the -- what was the dose that was used? 20 A. The total dose amounted to 7500 micrograms. 21 Q. Would that be 7.5 what? 22 A. Milligrams. 23 Q. Milligrams. And he produced chloracne in eight out 24 of ten. 89 1 A. Something of that order. I'm sorry that I cannot 2 remember the exact numbers, but he produced in chloracne in 3 most of individuals. 4 Q. And was there any other findings reported other 5 than the chloracne? 6 A. There was no data presented. There was a statement 7 by him that was included in the document presented at the 8 hearings, which said that there were no significant adverse 9 findings other than the -- other than the chloracne. 10 Q. Now, based upon the animal data that you have, have 11 you -- have you determined a dose that could be obtained from 12 exposure to soil at the Sturgeon spill site? 13 A. The only way that I know to do that would be to -- 14 would be to take soil that was under the car when it came to 15 rest, where the material came spilling out, because there was 16 a -- at that point the concentration of the chlorophenols was 17 very high, as you might expect, that is, the concentration in 18 the soil, and if one were to assume that all of the TCDD that 19 was in the tank car remained at the same concentration in the 20 phenol, one could calculate a concentration of TCDD in that 21 soil, and then make assumptions about rubbing the soil on 22 one's skin or something like that. 23 Q. All right. I wonder if you could use the easel and 24 perform the calculations? 90 1 A. Well, all right. My arithmetic has been known to 2 fail on' occasions, so perhaps you might keep track of -- the 3 concentration of TCDD in the tank car was represented as 45 4 parts per billion in the phenols, there was both 5 orthochlorophenol and phenol in that, and the concentration 6 of the phenol in the soil was about 7 percent. I think it 7 was slightly under that, but let's say 7 percent, both the 8 total phenols. 9 MR, CARR: Counsel are you identifying a sample of 10 soil taken under the tank car as the witness is saying or 11 would you identify the facts that the witness is using 45 12 parts per billion TCDD, where is he getting that fact and the 13 other findings that he is testified to. Would you do that 14 please. I object to it until it's done, your Honor. 15 THE COURT: I'll give you an opportunity to do so. 16 Q. I have asked to you assume 45 parts per billion as 17 the highest level found of 2,3,7,8-TCDD, the highest level 18 reported in a reserve sample tested by Dr. Christopher 19 Rappe. I'd like to you assume, Dr. Dost, that there are a 20 number of other levels reported which are lower than that, 21 but that that's the highest one reported. 22 A* Okay. 23 Q. All right. And in addition to that, I'd like you 24 to assume the 7 percent concentration as having been reported 91 1 by Environmental Protection Agency on or about January, I 2 believe, 23rd, 1979 at a location directly at the spot where 3 the car came to rest, 4 MR, CARR; Well now, you mean under the car? 5 MR. HEINEMAN: Yes. 6 A. If we have a concentration of 45 parts per billion, 7 that means that there is a 45 -- if it is -- if it's parts 8 per million, it would be milligrams per kilogram, or it would 9 be micrbgrams per gram. If it's parts per billion, it would 10 be nanograms per gram of OCP, so we've got 45 nanograms of 11 TCDD per gram of TCDD, and then in this -- I beg your pardon 12 -- per gram of the mixed phenols. And then we have -- if it 13 is 7 percent, that means that there is 70 milligrams of OCP 14 per gram of soil. And we could say -- you want to give me a 15 number or shall I assume one, amount of soil? 16 Q. Ten grams of soil. 17 A. All right. If we -- let's say then that we've 18 taken ten grams of soil, and we've picked it up and rubbed it 19 on the skin. Mow, understand that with this much phenol in 20 it, it is not going to be very comfortable. It's probably 21 going to cause severe skin damage, but we ignore that, so 22 what we have is 70 milligrams of OCP per gram of soil. We've i 23 taken ten grams, so that means that we've got 70, 700 24 milligrams or 0.7 grams of OCP in that, and -- and in one 92 1 gram there's 45 nanograms of OCP, and so in ten grams there 2 would be -- there would be .7 times 45, which is -- which is 3 31,5 nanograms of TCDD. Does that square with -- I hope I 4 haven't lost a decimal place somewhere 5 In any case that's the amount of TCDD that would be 6 carried in that ten grams of soil. The absorption would 7 probably be on the order of one percent, about one percent of 8 it would probably get into the -- across the skin, and that 9 means then that -- that we have -- that will give us zero 10 0.315 nanograms absorbed from that ten grams. So that's the 11 total amount that we might expect to go in 12 If -- if we are talking about a 70 kilogram person, 13 70 kilograms is 154 pounds, so that's not a-- that might be 14 considered a reasonably average rate. Obviously with a 15 larger person, why, it's a bigger number, and a smaller 16 person smaller, but if we were to divide this by 70, it would 17 be -- it's not a complex -- I just done it wrong. The dose 18 would be .0045 nanograms. We are often accustomed to talking 19 about micrograms If we wanted to refer to this as 20 micrograms, we would bring the decimal back three places and 21 it would be -- it would be that many micrograms per 22 kilogram. What we have done here is to relate the dose* Now 23 what you can see is that even if the one percent is off by 24 two or three fold or if we are talking about a very small 93 1 person, we still end up with a very small number here in 2 terms of a dose per kilogram. 3 Q. All right. Now, how does that compare with the no 4 effect level in rabbits found by Jones and Krizek? 5 A. That no effect level -- okay, we can consider it in 6 two ways. Ten grams of soil would probably spread over maybe 7 six square inches if you were to really-- I don't know, you 8 probably should have a child who plays in the mud advise us 9 on how much surface that would cover, but let's say about six 10 square inches, and we've spread this amount here, this is 11 this total, we have spread that over an -- over a single 12 area. 13 Q. More appropriately perhaps we should consider it in 14 terms of dose per kilogram, and the problem with the Jones 15 and Krizek data is that they administered .2 micrograms to 16 the site and if we assume that a rabbit weighs a kilogram and 17 a half, three pounds, that's a fair sized rabbit we could 18 deduce that the dose is something less than 2 tenths of a 19 micrograra per kilogram, maybe 1.6 or something like that. 20 Here, if we were to go through a -- that's assuming 21 that it was all absorbed, and we do not know what the 22 absorption rate is in the skin of the rabbit. We got a good 23 idea what it is in the rat, but in the rabbit we don't know 24 necessarily. So there are a number of ways we can consider 94 1 this# but in any case there is a much# much lower dose either 2 if we consider it on the amount that goes on the skin -- this 3 is -- 31,5 nanograms is -- is .0315 micrograms# okay. That's 4 about roughly tenfold lower# perhaps a little less than 5 tenfold lower than this dose we're talking about on the 6 rabbit. At the same time it's spread over a larger area# 7 because in that rabbit they concentrated, they made a very 8 small target. 9 Q. All right. So which would be lower# the dose from 10 the Sturgeon soil or the dose for the no effect level? 11 A. Well# the dose from the Sturgeon soil would be 12 vastly lower. 13 Q. All right. Now# with respect to the Kligman data 14 you just talked about# the 16 micrograms# that were 15 administered# would this dose from the Sturgeon soil that you 16 calculated be lower than that? 17 A. Well# I guess the thing to do# since we don't the 18 know the absorption rate in the Kligman data is to assume 19 that the absorption here is going to be the same as the 20 absorption there. They applied 16 micrograms to that skin 21 over a period of three weeks# I guess. Here we're talking 22 about a dose over a somewhat larger area# because in the 23 Kligman study they confined it either on the forehead or on 24 the back in this area perhaps so square# and the dose that 95 1 we're talking about here compared to in the Kligman study 16 2 micrograms, Here we're talking about .0315 micrograms, and 3 if we were to divide that# it would tell us how much 4 different they are. The difference is about five hundred 5 fold, 6 Q. Now the -- in the Kligman experiment you said the 7 solvent was used to enhance the material going through the 8 skin? 9 A. Well, the solvent has two roles there* One thing 10 you've got -- TCDD is not very soluble even in materials that 11 dissolve it, and so you have to -- you have to get it in some 12 form so that's it's distributed, because if you just put TCDD 13 crystals on the skin, it's, not likely that much would 14 happen* It probably would not be absorbed very well even 15 into the superficial layers of the skin* So the solvent will 16 have an effect of helping to carry it through the skin, but 17 it also distributes it, and that's really the role of the 18 solvent as it was used in Kligman* 19 Q* What if you had TCDD in a 7 percent combination of 20 orthochlorophenol and phenol in soil which you were rubbing 21 on the skin? How would that affect the ability of the TCDD to get through the skin and into the body? A. Orthochlorophenol is not really the best of solvents, particularly for TCDD, but the soil itself will 96 1 have the greatest influence# because there is a large amount 2 of soil, and soil binds TCDD very effectively. TCDD likes to 3 attach to both inorganic and organic solid, inert materials, 4 and it would -- it would diminish its -- it would diminish 5 its potential to get across the skin or to get even into the 6 skin. W e 're really talking here about a local effect, where 7 the material gets into the skin, and to be sure some is going 8 to go beyond and become systemic, but the main effect is 9 going to be at the site of application in both cases, but 10 that a solvent would enhance the movement, because the skin 11 cells, cells and their membranes tend to be somewhat 12 lipophilic, they have more attraction for fats or fat soluble 13 substances than water, than they do for water, and they would 14 tend to -- to be -- a fat soluble substance in a fat solvent 15 would tend to move across the skin at least a little more 16 easily than otherwise. 17 Q. All right. Now, sir, the next organ that I 'd like 18 to discuss, really a group of organs, I guess, is the 19 reproductive system. r 20 THE COURT: Before you get into that, is this a good 21 point for a short break? 22 MR.HEINEMANi Yes. 23 THE COURT: We'll take a short recess at this time 24 and then resume testimony. The admonishments that I've given 97 1 you will earlier will apply during this break also. Court1s 2 in recess. 3 (At this time a short recess was taken.) 4 (The following proceedings were had in open Court.) 5 Q. Dr. Dost, before we leave the skin there's another 6 thing that I wanted to ask you about. You mentioned in your 7 previous testimony that today or Friday about a no effect 8 level in Dr. Kociba's studies. Do you recall that, sir? 9 A. Yes, I do. 10 Q. Okay. What was the no effect level in Dr. Kociba's 11 studies? 12 A. That was a extended study in which the material was 13 applied -- oh, in Dr. Kociba's study, I beg your pardon. We 14 were talking about two studies. One was a study carried out 15 over 13 weeks, which was used as a range finding study for 16 one that was conducted over a period of two years for the 17 purpose of determining the carcinogenicity of TCDD and also 18 for seeking other effects, and as I believe I pointed out, 19 they did a great deal of clinical chemistry, hematology, 20 pathology and so forth in those animals, and the no effect 21 dose for -- in that experiment of lifetime exposure -- so 22 when I say a no effect dose, I'm talking about the daily dose 23 that they were given was .001 micrograms per kilogram or one 24 nanogram per kilogram per day over a lifetime. 98 1 Q. All right. Nowr how would, how would that no 2 effect level relate to the one you've calculated for the ten 3 ounces of Sturgeon soil -- Ten grams, excuse me? 4 A. Well, X guess the way to tell the relationship 5 would be that we're talking about -- let me -- the no effect 6 level in Dr. Kociba's study was .001 micrograms per kilogram 7 body weight per day. The -- the number that we're talking 8 about here, and we put it into the same terms again, 9 micrograms, is .0000-- when you start getting all these 10 zeros, it's very easy to -- micrograms per kilogram, and it's 11 evident that this is a smaller number by quite a bit, and we 12 can find out how small by just'dividing one into the other. 13 The difference between that single dose that we 14 talked about in this hypothetical situation, where we would 15 run -- we would smear the contaminated soil on the skin is -- 16 and the daily dose that caused no effect in the rat is 222 so 17 the difference is 222 fold. 18 The no effect level in the rat, the dose, the daily 19 dose that has no effect in the rat is 222 times larger than 20 the dose that would be acquired in this situation, and again 21 if the weight is a little different, absorption rate a little 22 different, it would make some difference, but it's a very 23 small fraction. 24 Q. Now, has this Kociba level been adopted by anyone 99 1 other than the Center for Disease Control, as you mentioned 2 earlier? 3 A. The Food and Drug Administration considers the no 4 effect dose for TCDD to be .001 micrograms per kilogram, and 5 they use that as a basis for regulatory activity with respect 6 to contamination in food for human consumption. 7 Q. What would that amount to for a 70 kilogram man? 8 A. That amounts to 70 nanograms. We're talking about 9 one nanogram per kilogram. A 70 kilogram person would -- 10 that would mean a dose of 70 nanograms. 11 Q. Now, and the-- and the Jones and Krizek rabbit ear 12 had a no effect level of what, sir? 13 A. Well, I'm sorry, I made a mistake. I used the 14 lowest effect level. The no effect level rather than being 15 2 micrograms was .03. I misstated the no effect level. 16 Q. Now, is that point .03 micrograms? ./ 17 A. .03 micrograms, yes, 30 nanograms. 18 Q. Now, would that be pretty close to what you 19 calculated from the ten grams at Sturgeon, isn't it? 20 A. Yes, as a matter of fact, yes, in terms of a total 21 dose. We -- for those ten grams of Sturgeon soil 31 and a 22 and a half nanograms according to the calculation. This is 23 without accounting for absorption or anything else, the 24 amount that actually reached the skin or that it will assume 100 1 that everything was in the soil was in contact with the skin 2 and disregarding the effect of soil on absorption as 3 distinguished from the experiment in which they were putting 4 it on in solvent, but the actual amount in nanograras was this 5 -- approximately the same. 6 Q. Was just about- at the -- 7 A. No effect level ignoring all these other factors. 8 Q. Okay. Now, if we could move on to the reproductive 9 aspect. Would you tell us what the -- what are considered to 10 be the reproductive effects of TCDD in animals? 11 A. We would be concerned obviously with anything that 12 interferes with the reproductive process, which means that 13 anything that would cause any adverse effect on the 14 offspring, and that might be identified as small size, fewer 15 numbers. Rats have, for example, very large litters, 16 sometimes 12 or 15 in a litter. If we had an effect that 17 over time caused them to only have ten or nine in a litter, 18 that would be an observable effect. The weight of the pups at 19 the time they're born, any problems with fertility. 20 The other general kind of effect that we concern 21 ourselves with are birth defects, teratogenesis; in other 22 words, this -- in this context we're talking about the kinds 23 of effects that a chemical can cause on the developing fetus 24 directly that will cause deformities, and this is an impact 101 1 that takes place in a relatively narrow time window in 2 development. It has to take place at a time when the fetus 3 is at the point where the limbs and the organs are 4 developing. It doesn't happen late, for example, in the 5 gestation period. 6 Q. All right. Are there any other effects? 7 A. The fetus has the same potential for intoxication 8 that the adult has assuming that a chemical can actually 9 reach it. Some chemicals are not able to reach the fetus 10 very easily, some reach it fairly well, and the fetus is 11 subject to the same kinds o toxicity as the adult is except 12 to the extent that certain organs are not adequately 13 developed if the toxicity depends on that organ, so there are 14 some aspects of toxicity that don't appear in the fetus, but 15 in general, we can say, we use the term fetotoxicity, the 16 general kinds of intoxication that affect the fetus. 17 Q. All right. Now, these subjects have been studied 18 in animals? 19 A. Yes. 20 Q. What about the genetic impact, are there any 21 effects in that way? 22 A. Oh, yes. Obviously the genetic effects that are 23 the genetic strengths and weaknesses that are built into a given species or a given strain appear, and then there are 102 X individual genetic changes that might take place that can 2 result, and when we talk about genetic effects, we're 3 speaking about changes that can arise in one or both parents 4 and then be transmitted forward into some descendant, whether 5 it's the immediate offspring or generations later, so this 6 means genetic effects that take place in the reproductive 7 organs, A genetic change that takes place in the liver only, 8 and that can happen, is not going to have a impact on the 9 offspring. So we have -- we have, as a matter of fact, birth 10 defects that may arise from that kind of cause, and then 11 there are bound to be a variety of other effects that really 12 relate to other kinds of, if you will, weaknesses that could 13 arise genetically, 14 Q. Well, can't -- can these reproductive effects be 15 studied both in the male and in the female? 16 A, Yes, they can be studied in both sexes separately 17 or together. 18 Q. All right. Now, I'd like to review with you what 19 the animal studies demonstrate with respect to reproductive 20 effects and start with the male test results first. What do 21 they show about the effect of TCDD on male dominated effects? 22 A. The way that -- the way that these kinds of studies 23 are conducted is to treat a male either with a single dose or 24 over a extended period of time and then mate them with 103 1 females periodically for perhaps 8 weeks.is a good number. 2 Most species have a period of 8 or 9 weeks through which the 3 sperm develops and if -- and, of course, at any given time 4 there are sperm at any stage in a that development scheme, so 5 that if animals, if males are treated at a specific time, and 6 there is a effect, it is going to appear in the offspring 7 that arise from sperm that were at that particular stage of 8 development. So it may be five weeks, 8 weeks, or none at 9 all. So the animals are treated at doses up to the tolerable 10 dose. In other words, it's desirable to see some toxicity in 11 the test animals at the top dose, so that we know that the 12 maximum dose that is practical to use has been achieved and 13 then several lower doses, and the process is relatively 14 simple. The animals are treated they are mated, and then the 15 offspring are examined. In the case of a dominant lethal 16 test, which seeks certain kinds of genetic injury in the -- 17 that would be manifested in the sperm, the -- the offspring 18 simply don't survive. The conception takes place, but the 19 genetic damage is so severe that the offspring don't survive, 20 and the evidence can be seen in implantation sites in the 21 uterus of the female. So that when the uterus, when the 22 female is surgically examined, the sites where development 23 started and then stopped can be observed. So this is one 24 means, A probably more sensitive means is to use a typical 104 1 three generation reproduction test, in which usually in the 2 first test the -- both males and females are maintenance on a 3 diet at several doses, at several daily intake rates over an 4 extended period of time prior to mating- They are mated, 5 they continue on the diet so that the off spring and the 6 whole reproductive tract are subjected to the chemical 7 throughout the gestation period and throughout the nursing 8 period, and at the end -- and when the animals are weaned, 9 the original parents are usually taken off the experiment, 10 although not always, but generally they would be taken off 11 the experiment and examined carefully. The offspring are 12 going to be maintained on exactly the same diet throughout 13 their reproductive lifetime, and then their offspring will go 14 through the same cycle so that we have three-fold generation 15 of animals that have been, they and their parents or they and 16 their offspring, if you will, have been subjected to the same 17 diet throughout their entire reproductive cycles. 18 Now there are variations. Sometimes they'll do two 19 litters from each of these animals and sometimes only one. 20 If nothing appears, that is really sufficient information. A i&K 21 If there is a finding that suggests that something has * 22 occurred in either the male or the female, you may go back 23 and do this experiment again by administering the test diet 24 only to the males throughout or only to the females. So -- 105 1 but usually it's done with both species. If nothing appears, 2 then there is a.good certainty that neither the males nor the 3 females were affected. 4 Q. All right. With respect to specifically the effect 5 of TCDD on male animals, has there been any effect seen in 6 the tests? 7 A. Not from -- not as a result of -- that can be 8 identified with a male animal, no, sir. 9 Q. What about mutogenic effect on sperm and that sort 10 of thing? 11 A. The dominant lethal studies do not show any effect 12 at doses that the animals can survive. 13 Q. Are there any particular articles that you can -- 14 that you can cite to us? 15 A. Well, probably the best is a study by Lamb and 16 colleagues at the National Institute of Environmental Health 17 Sciences, in which they specifically were seeking evidence of 18 effects of TCDD, 2,4-D, and 2,4,5-T. Their objective was to 19 simulate Agent Orange at various concentrations, so all of 20 these animals had not only TCDD, but the herbicides as well. 21 Q. All right. 22 A. The other study that is -- well, that's the one, 23 the most definitive one that deals with males specifically. 24 There is another done by Kera and Ruddik that also was 106 1 negative. 2 Q. And about when was the Kera and Ruddik work done? 3 A. I believe that was done in the early *708. 4 Q. Okay. Now have there been -- what are the results 5 of the three generation reproduction studies on dioxin? 6 A. They have been -- they have been negative in 7 essence. The consensus of the people who have evaluated 8 those studies as well as the authors is that at .001 9 micrograms per kilogram per day, which incidentally is the 10 same dose rate that was a no effect level in the Kociba 11 study, that at that dose rate there was no evidence of a 12 compound related effect on the -- > on the animals. 13 Q. Is that for either males or females? 14 A. Well, that study -- in that study both males and 15 females were treated. If either had been influenced, an 16 effect would have appeared. Now, had that happened and 17 nothing done to follow it up, with just that information 18 alone one would not know whether it was males or females.. 19 Since nothing happened with both, it is accepted to -- to -- 20 that there was no impact on either females or males. 21 Q. At that dose? 22 A. At that dose, yes, sir. 23 Q. Okay. Now, who's the author that's written 24 primarily on the three generation studies? 107 1 A. Well, this was a study done by a scientist of Dow 2 Chemical* The senior author was a man named Murray. 3 Q. Now, I'm trying to differentiate between the male 4 and the female studies. Do I understand that the male 5 dominated effects have not appeared at any dose? 6 A. At any dose that's been -- yes, that's essentially 7 correct. 8 Q. Now, what about female dominated affects? 9 A. Well, TCDD is clearly a teratogen; in other words, 10 if it is given -- at least in certain species. It's 11 difficult to induce teratogenesis in the rat. In the mouse 12 it's possible to cause cleft palate and some kidney 13 deformities by treatment with TCDD. 14 Q. Now, in the Murray study were teratogenic effects 15 found there? 16 A. The study was not designed to specifically look for 17 teratogenic effects; however, they would have appeared, and i 18 it is my recollection that teratogenic effects were not seen 19 in the Murray study at any dose that was used. 20 Q. All right. Well, are teratogenic effects dose 21 related? 22 A. Oh, yes, definitely. 23 Q. What dose do you have to administer to the animal 24 in order to find the teratogenic effects? 108 1 A. For some reason the no effect dose for 2 teratogenesis, per se, has slipped my mind. It's up into the 3 micrograms per kilogram, however. 4 Q. You mean whole numbers? 5 A. , Oh, yes. We're not talking about these very, very 6 small fractions of a microgram. 7 Q. Now, in those studies that are designed to find e teratogenic effects, how many doses are normally given? 9 A. The way the experiment is done is to administer the 10 chemical daily over what we know as the developmentally 11 sensitive period Now, in the rat that's a period that starts 12 at about day six of gestation and ends at generally on the 13 fifteenth or sixteenth day. 14 The rat is born at an age of about 21 days. What 15 you must remember is that the rat is born at a stage of 16 development that would be roughly equivalent to a human fetus 17 at about two and a half months of term; in other words, less 18 than a third of gestation period. The rat is cast out upon 19 the world in a very, very primitive way and requires -- it 20 requires enormous care by the mother, you see, so this dose 21 that is administered comes relatively late prior to the birth of the animal, but the birth takes place very early in development. The period of sensitivity in the human is -- ranges 109 1 between about fifteen or eighteen days after conception to 2 perhaps, 60 days and for a few structures a little longer than 3 that, so this window, so to speak, of time in which an 4 organism is sensitive to developmental effects on the fetus 5 is relatively narrow. However, the impact in terms of 6 general toxicity, that goes throughout the entire -period of 7 gestation* 8 Q* Now, as I understand it, in the Murray study, which 9 is the three generation study, there is a no effect level 10 found in both males and females of 001 micrograms per 11 kilogram per day? 12 A. That would be a correct inference yes,. 13 Q. Now, in the teratogenicity studies in which 14 multiple doses have been given there is a no effect level 15 that's around-- that's in the microgram per day level? 16 A. Yes. 17 Q. Would that be higher than the -- what the Murray 18 study demonstrated? 19 A. Oh, yes, yes, but it's a much less sensitive-- 20 it's a much less sensitive kind of parameter, much less 21 sensitive effect* It takes doses in -- for many, chemicals it 22 takes doses up into the range where the mother is seriously 23 injured to cause teratogenesis* 24 Q. How does that dose relate to what you have 110 1 calculated for the Sturgeon dose in rubbing the soil on the 2 arm? 3 A, The .001?' 4 Q. Yes# sir. 5 A. Well, if we -- well, all right. We did that with 6 respect to the other study, the Kociba study that we were 7 considering. The no effect dose on a daily intake basis is 8 the same, as it turns out, and so we still have this same 9 relationship here with a difference of something on the order 10 of 200 fold lower than the no effect dose as seen in these 11 studies. 12 Q. So the Sturgeon dose would be lower? 13 A. Yes, you know, with the dose with these assumptions 14 that we've made about smearing it on the hand'and so forth. 15 Q. How, let's assume that the teratogenic no effect 16 level would be as low as one microgram per kilogram. 17 A. Well, one microgram is a thousand times larger than 18 a thousandth of a microgram. One could then multiply this by 19 a thousand, or if the no effect dose is down in the fractions 20 of micrograms, let's say, half a microgram per kilogram, then 21 it would be a five hundred fold difference. Q. So you multiply that figure 222 by either a thousand or five hundred? A. Yeah, depending on the -- and I have to apologize, 111 1 because the no effect doses for teratogenesis have simply 2 fallen out of my mind momentarily. 3 Q. But it would be, if it were one microgram, it would 4 be a thousand times more? 5 A. That's correct. 6 Q. And that Sturgeon would be that much lower? 7 A. There would be 222 thousand fold difference. 8 Q. Now, Dr. Dost, if Dr. Silbergeld had testified here 9 in court that a strong assumption exists that no threshhold 10 exists for male reproductive effect, would you agree or s 11 disagree with that statement? 12 A. Well, I would disagree with it. 13 Q. All right. Based upon what, sir? 14 A. Because we can't find evidence of any kind that 15 male reproduction is affected by TCDD. 16 Q. Has -- has Dr. Silbergeld written on this subject 17 herself, sir? 18 A. There was an article, a review really, that was 19 part of a symposium in late 1983, I believe, at Rockefeller 20 University, that -- in which she discussed the reproductive 21 -- in general terms the reproductive potential or the 22 reproductive toxicity potential for TCDD. 23 Q. What did she suggest for male dominated toxicity? 24 A. Her suggestion was that there really wasn't much 112 1 evidence, if any, that male mediated toxicity by TCDD was at 2 issue, and I can't remember her statement exactly, but there 3 is very, very little data to support any evidence of effect. 4 Q. On the male? 5 A. On the male, on the male. 6 MR. CARR: Counsel, where was it that you quoted 7 Dr. Silbergeld, what page. 8 MR. HEINEMAN: I think it was -- I have it right 9 here. It's 12th of April, Page 127. On 13th of April, Page 10 34. 11 Q. Doctor, I'd like you to move on now to liver 12 effects, if you would, and discuss with the jury the function 13 of the liver in the animal and what it has to do with 14 detoxification? 15 A. The liver is an-- is a extremely interesting organ, 16 because it has so many functions. It's -- it's -- it has a 17 major function in processing energy substrates for -- so that 16 we can get energy out of our -- the food we eat. The cells 19 -- in each individual cell, of course, does a good part of 20 this work, but when there is an excess of one kind of, say, 21 an excess of carbohydrate or an excess of fat, the liver 22 often has responsibility for making conversions that make it 23 possible to use these various substrates. 24 The liver is a -- because it has such an enormous t 113 i 1 capacity serves in starvation as a protein reserve so that if 2 we are starving, why, when we begin to need protein, we would 3 tend to mobilize it from the liver without losing any 4 function* 5 It is extremely important in the metabolism of 6 foreign substances as well as substances that are secreted in 7 the body* The steroid hormones, for example, have to be 8 disposed of somehow, because we can't leave them in the 9 organism to just keep v/orking all the time. They have to be 10 disposed of. Foreign compounds are metabolized primarily in 11 the liver and either converted to soluble forms, as I've 12 mentioned many times, or else moved into the bile in a 13 slightly different form and excreted into the digestive 14 tract* 15 So I guess in very general terms that would 16 summarize the functions of the liver. It manufactures bile, 17 which is necessary as a digestive aid, because it's a 18 detergent, bile is a detergent, it breaks up the fat that we 19 eat into very tiny globules so that the enzymes in the 20 digestive tract can get at it and break it up enough that it 21 can be absorbed. 22 Q. You previously described for us the metabolic 23 detoxification effect* Does that occur in the liver? 24 A. That occurs primarily in the liver* Many other 114 1 tissues have some capacity, but the liver performs most of 2 that particular kind of work, Sven the skin can do it to an 3 extent. 4 Q. Now, how is it that this organ -- what percentage 5 of body weight of the entire animal is the liver? 6 A, The liver in an -- in an adult human probably 7 weighs about a kilogram, about 1 and 3 quarters kilograms, so 8 that would be in pounds about what, four pounds. 9 Q. Now, the blood supply -- 10 A, Roughly four pounds. 11 Q. All right. As the blood supply is circulated by 12 the heart, does it -- it pumps the blood through the liver? 13 A. It pumps blood through -- the liver -- the blood 14 enters the liver actually in two ways. It enters in the 15 hepatic artery, which is the artery that supplies the blood 16 from the general circulation; in other words; that you might 17 say is the main line from the heart. There is also a vessel 18 called the portal vein that comes from the digestive tract 19 and brings material that's absorbed from the digestive tract 20 to the liver, so blood is coming to the liver from two 21 different directions. 22 Q. All right. Now as rapidly as blood circulates 23 through the body how does the liver do all of this function 24 in what is apparently a pretty short period of time as the 115 1 blood is going through? 2 A. Well, actually the circulation time of the blood is 3 not necessarily related. It only takes -- if we were to 4 inject a dye into a vein right here, it would take about a 5 minute before we could find it coming back. It will have gone 6 all the way through the circulation and at least some of it 7 would get back to this point. So blood moves very quickly 8 through the body. What we are concerned with as it goes 9 through the liver, you can think of the liver perhaps as a 10 big filter. There is a lot of circulation going into the 11 liver and each pass -- and as a matter of fact, physiologists 12 use the term first pass, second pass. The idea of the first 13 pass is that how much of material -- and if you inject it 14 into a vessel coming into the liver, how much of it is still 15 there on the other side. 16 Obviously the liver does not simply clean 17 everything out of blood every time it goes through. It 18 continually takes out more and more and more and more and 19 processes it. Now, if it can't process it very fast, it's 20 not going to take it out of the circulation very fast. If it 21 is working on a foreign chemical, and you recall the diagram 22 I showed with equilibrium related to TCDD, the liver is 23 working relatively slowly. The material that the liver is 24 going to work on is not just simply stacked up outside the 116 1 liver waiting its turn It's only going to come in to the 2 extent that it can be handled It's possible in the kidney* 3 on the other hand* if there is a lot of soluble material 4 that's available to go through the kidney, the concentration 5 in the kidney may become very high. That's different. 6 So what we're talking about here is functions that 7 relate to the ability of the liver to do its work and to take 8 material out of the blood. 9 Q. Now* the liver works on or filters the blood that 10 the body is sending through it* correct? 11 A. In a sense* yes. 12 Q. Now* does it get its own blood supply with the 13 cells that are in the liver? 14 A. Exactly* in the same way. In other words, the 15 blood that's coming into the liver and ultimately that's 16 bathing a liver cell both brings the necessary* the staples* 17 if you will, that the liver requires to live on as well as 18 the work that the liver is supposed to be doing It's 19 bringing glucose to either be burned or stored, because the 20 liver is able to store a very large amounts of glucose in a 21 form called glycogen, which is a little like starch, so that 22 we have eaten* we've got a ready reserve of energy that we 23 can draw on while we aren't eating anything. But the blood 24 supply as it brings glucose in or brings amino acids for 117 1 manufacture of products is also bringing whatever materials 2 need to be detoxified or even raw materials for synthesis of 3 other substances* 4 Q. Now, when -- if there is dioxin in the blood supply 5 and it is going through the liver, can it have adverse 6 effects on the liver itself? 7 A. Well, the liver clearly is affected by intoxication 8 with TCDD* If the dose is high enough, we see a variety of 9 liver pathology, because the concentration in the liver has 10 has risen sufficiently -- I mentioned a moment ago that we 11 don't have a lot of piling up of molecules, but I'm speaking 12 of a situation in which there isn't a great mass of material 13 in the -- in the organism* When we have a large amount, when 14 the dose is high, there's going to be a lot of TCDD reach the 15 liver, and it's going to have a variety of deliterious 16 effects if there is sufficient amounts. 17 Q. If there is sufficient amounts? 18 A* If the dose is high enough. 19 Q* All right* Now, what are the effects that arise 20 when the dose is high enough? 21 A. We can see effects perhaps on the cell wall. One 22 of the things that would happen in a damaged or injured or 23 intoxicated liver cell would be that the cell wall would 24 become more permeable. Now, cells have membranes surrounding 118 1 them* and it's possible when the cell has been injured that 2 that cell may, that wall may become leaky and some of the -- 3 Q. Is that what permeable means? 4 A. That's right. That's right. Permeable, if you 5 put, if you poured this water through a handkerchief, it 6 would undoubtedly go through, it's permeable. If we poured it 7 onto a piece of paper, probably it wouldn't go through until 8 the paper got thoroughly soaked and finally became weak and 9 it started to come through, but it would come through 10 slowly. That would be high permeability, limited 11 permeability. 12 Well, cell calls are the same way, because they 13 have to bring materials in. The only way that materials can 14 come into a cell is by transport across the membrane, you 15 see. Well, there are a lot of components in the cell, 16 enzymes, which are rather large molecules, for example. Such 17 enzymes as the glutamic oxalacetic transaminase, which has a 18 function in the cell -- I'm sorry about that name, but the 19 importance thing is that it is an enzyme that is in solution 20 inside the cell, and if the cell wall becomes sufficiently permeable, it will start to leak, and we can find it in the bloodstream in a clinical test, and that's one of the clinical tests for liver function. Pathologically, we can see if that membrane effect 119 1 is sufficient. Sometimes cells, two cells will join 2 together, and we'll see a large liver cell with two nuclei or 3 the whole cells will swell, because they will accumulate a 4 lot of fats. 5 One of the characteristics of TCDD intoxication in 6 the liver is the accumulation of fat in liver cells, and the 7 architecture, the various structures within a cell will tend 8 to become distorted and perhaps even swollen in their own 9 right. So there's a whole gamut, the end of which is actual 10 death of the cell, so that we see in a severly intoxicated 11 animal pockets of dead cells, maybe only a couple at a time 12 or perhaps several hundred, where there's just simply been 13 cell death as a result of the intoxication- This is a fairly 14 typical pattern of many liver intoxicants, as a matter of 15 fact, 16 Q, That's what I wanted to ask you about. Is TCDD the 17 only thing that has these effects on the liver? 18 A. No, there are other substances. It's -- many of 19 the effects on the liver are relatively nonspecific. Now, 20 the mechanism by which liver damage, occurs as a result of 21 high doses of TCDD is not entirely clear* We do know more 22 about some other chemicals, but this general pattern occurred 23 with a lot of different substances. 24 Q, Such as what? 120 1 A. Well, some of the solvents, solvents, common 2 solvents, even things like kerosene. Very rarely does 3 someone get a dose of kerosene, but you can induce this kind 4 of effect with those kinds of solvents. 5 An inorganic chemical that I've worked with a good 6 bit called hydrozene will cause very interesting pattern of 7 liver damage, many characteristics of which are rather like 8 what is described for TCDD. 9 Q. How about alcohol? 10 A. Well, alcohol certainly will do it. Again it's a 11 dose related phenomenon, and usually that kind of damage does 12 not occur after one drink or even after just one drunken 13 binge, but it will occur in time if excess consumption of 14 alcohol is maintained. IS Q. Now, are there -- are there gross effects that the 16 animal studies have found for liver? 17 A. Well, the liver tends to swell, the liver tends to 18 swell, and that is -- that is not 1-- there are degrees 19 involved here, because the liver in its -- in its daily work 20 tends to swell. In a well-fed individual ten percent of 21 liver weight, for example, is glycogen, this complex sugar 22 that I mentioned that is stored in liver cells. Ten percent 23 is pretty good sized increase and the liver actually it can 24 be seen to become larger. The liver when it's responding to 121 1 foreign chemicals -- a good -- a good solid induction causes 2 enough protein synthesis, increase in endoplasmic reticulumf 3 that the liver will, swell, and so this is seen. Now, there 4 is a point beyond which it is no longer a normal phenomenon. 5 Q. What do the studies show with respect to -- with 6 respect to such liver effects and TCDD? 7 A. In what sense, the nature of the damage? 8 Q. Yes. 9 A. Well, I think I have described a good part of it. 10 That is -- we see the cellular disorganizataion at higher 11 doses. We see -- probably the first thing that would be seen 12 would be the leakage of enzymes through the cell wall. These 13 are -- unless the cell -- unless the cell dies, these kinds 14 of changes eventually -- eventually recover. 15 Q. Now, in terms of the effects that can be found, you 16 talked about certain enzymes. Well, how about lipids? 17 A. Well, I mentioned the accumulation of fat in the -- 18 in the cells of the liver, and these are -- these are -- 19 apparently they are triglycerides, which is fat in the same 20 form that -- that we store it in fatty tissue, in adipose 21 tissue, and it -- the liver normally would break that fat up 22 and ship it out into the circulation for either use as a fuel 23 by muscle and other tissues or it would be -- it would be, if 24 it was in excess sufficiently, it would go to adipose tissue 122 1 and be stored* So we have an abnormal situation in which 2 lipid metabolism, fat metabolism in the liver cell is 3 deranged in some fashion* 4 Q* Is there a -- does the dose response -- 5 A. Oh, yes. 6 Q. -- Principle apply to this as well? 7 A. It certainly does. 8 Q. And is there-- What dosage is required for -- you 9 mentioned necrosis or dying of the liver cells. What -- 10 versus the finding of lipids or versus enzyme changes and 11 that sort of thing? 12 A. One of the difficulties is that the -- much of the 13 pathology has been done on -- with single doses that are 14 known to cause an effect* However, we have a lot of evidence 15 in chronic studies which really probably are more -- are more 16 pertinent. 17 The Kociba study that we've talked about already 18 shows liver pathology in the higher dose animal, relatively 19 little at a dose of a hundredth of a microgram per kilogram, 20 and nothing that can be described as pathological at a 21 thousandth, at one nanogram per kilogram. So there is a -- 22 Q* Has a no effect level, N-O-E-L, been described? 23 A. In the chronic sense, yes. I can't bring to mind 24 specific dose response data. It seems to me that Jones and 123 1 Grigg have looked at this in acute treatment. I do not 2 remember the numbers offhand. 3 Q. The -r what about the enzyme induction that we 4 talked about before. Does that have any relationship to 5 liver damage? 6 A. Not really. That occurs at doses below those that 7 are going to cause liver pathology. That's a -- that's not a 8 pathological response. That's a normal response to low 9 concentrations of a substance with the objective of getting 10 rid of it. 11 Q. So again with respect to -- X guess if we're back 12 to Kociba study as being one of the primary studies on the 13 liver effects, is that right? 14 A. Certainly in terms of the pathology, and also they 15 did a great deal of clinical chemistry in those studies, and 16 the effects were -- existed at the highest dose of a tenth of 17 a microgram per kilogram per day, relatively little, if 18 anything, at the -- at the intermediate dose, and nothing at 19 the lowest dose. 20 Q. Well, then if we were to compare this with the 21 Sturgeon calculation that you made a little while ago that's 22 that same figure? 23 A. We're right back where we began here keeping in 24 mind that we are talking about a chronic exposure to this 124 1 dose as differentiated from this single adventure with the 2 contaminated soil. / 3 Q. What has been found with respect to whether or not 4 liver effects are reversible? 5 A. There have been -- there have been studies of the 6 pathology of the liver. Bruce Fowler has done such a study 7 in which he's used electron microscopy, which is, of course, 8 much more sensitive then using a light microscope, and he has 9 defined a time course through which in rats the liver after 10 -- after showing such signs as crowding of the -- of the 11 mitochondria, accumulations of fat. The other things that we 12 recognize as being part of the -- part of the pattern of 13 toxicity of TCDD in the liver have reversed over a period of 14 time. 15 Q. Well, how much time does it take? 16 A. It would take -- they are well under way, after a 17 single dose they are well on the way toward normalcy by 16 or 18 20 weeks, and after about 32 weeks they seem to be reasonably 19 well recovered. 20 Q. Now, are there other effects that are -- on the 21 liver that are also reversible? 22 A. Well, the enzyme induction, of course, reverses. 23 It is a long -- it persists for an extended period of time, 24 but it does, in fact, it does in fact return to normal. I 125 X suspect that the reason that the induced enzymes stay up is 2 because the TCDD remains in the animal for a longer period of 3 tirne^ and that induction is more or less maintained. If it 4 went in and left# those enzymes would drop more rapidly as 5 they do following other kinds of chemicals. 6 Q. Now, I 'd like to move next. Dr. Dost, to the 7 discussion of heme synthesis, and I wonder if you would 8 explain and describe the heme and the heme synthesis process 9 to the jury. 10 A. Well, heme is a -- I guess by itself we can 11 consider it a molecule, but its real function is to serve as 12 part of a large number of other kind of molecules. Heme is 13 the component of hemoglobin, which is in our blood that 14 carries oxygen. It's an iron bearing compound that is 15 incorporated in hemoglobin. It's, part of a substance called 16 myoglobin that is very, very similar to hemoglobin that's 17 found in muscle. 18 There are a number of other heme proteins. There 19 are a group of cytochromes so called that are used in the 20 cell as part of the process of transforming energy sources 21 into energy. We call them electron transport proteins. 22 There are a number of enzymes, an enzyme called catalase, 23 which has the function of getting rid of hydrogen peroxide, 24 which is made in large quantities in all cells. A enzyme 126 1 called peroxidase, which has a similar function. The P-450 2 enzymes that have been mentioned any number of times, the so 3 called microsomal drug metabolizing enzymes are cytochrome 4 enzymes. So there is a wide spectrum of proteins in the body 5 that need heme as -- in order to function, and the heme is 6 incorporated in the -- in the molecule. It's a small part of 7 the molecule. The P-450 proteins are -- have a molecular 8 weight of about 50 thousand, and heme has a molecular weight 9 of eight hundred something, so you can see that comprises a 10 relatively small part of the molecule. 11 The hemoglobin subunit, each molecule of hemoglobin 12 is actually four units, and each of those subunits is 16 13 thousand and each subunit has a molecule of heme in it that 14 has a molecular weight of about 830, something like that. 15 Q. Now, how is heme maintained in the body? 16 A. Well -- 17 Q. Where is it? 18 A. Well, it's in every cell. Every cell makes its own 19 heme. Heme is not something that's made in some central 20 location and shipped around the body. It's -- the bone 21 marrow would obviously make a great deal of heme, because 90 22 percent of the heme proteins or 90 percent of the heme is in 23 hemoglobin, and that's made in the adult in the bone marrow. 24 Q. When you say 90 percent of the heme, you're saying 127 1 90 percent that may be found in the body at any one time? 2 A, That's correct. 3 Q. Would be in hemoglobin? 4 A. That's right. 5 Q. But those levels will rise and fall? 6 A. Well,they would rise and fall if you're in athletic 7 training, and you're doing things that require high oxygen 8 turnover. As you become better conditioned, you would have 9 -- you'd have more red blood cells. The concentration of 10 hemoglobin within the blood cells might rise a little bit, 11 but that's pretty difficult to accomplish. You would have 12 more red blood cells. If you moved up into the Andes where 13 you are in very rare atmosphere, you -- over a period of time 14 your red blood count would rise in order to improve your 15 ability to bring oxygen from the lungs to the tissues where 16 it's needed. 17 The myoglobin in the muscle comprises about ten 18 percent of the total. Mow, what I've done, I've given you 90 19 percent and ten percent and that makes a hundred percent, and 20 we haven't accounted for all of the cytochrome enzymes. 21 Those are rough figures. The cytochrome enzymes and other 22 heme containing enzymes only account for less than 1 percent 23 of the total heme in the body, and in the places where those 24 enzymes are made heme is manufactured in that cell for those 128 1 proteins, whether we're talking about the cytochromes or 2 catalase or peroxidase or any of the heme proteins, the heme 3 containing substances* 4 Q. Now, do you recall the testimony of Dr- Silbergeld 5 when she said that the -- 6 MR* CARR: Counsel, what page please. 7 MR. HEINEMAN; Well, we've got April 12th, we've got 8 Pages 76, 112, and 109, 113. 9 Q* Where she said that dioxin affects the red blood 10 cells and strains the body's requirements for heme and by 11 induction of enzymes drains heme from the heme pool. Do you 12 recall testimony along that line, sir? 13 A * Yes * 14 Q. All right. Would you agree with what she has said? 15 A. Not at all. 16 Q. And why not, sir? 17 A. First, every cell makes its own heme. 18 MR. CARR: Your Honor, first of all, it was doctor 19 -- she's quoting Doctor Poland, and at that time on page 76 20 at least so apparently either the witness -- well, I'd like 21 to make that clear that it was Doctor Poland that was being 22 quoted there, counsel. 23 Q. All right. Would you agree with that statement, 24 sir? 129 1 A. That statement is incorrect. 2 Q. All right. And what is -- why is it incorrect? 3 A. Well, each cell makes its own heme and as a given 4 group of cells makes heme for its own needs, there is no 5 impact on heme synthesis in other cells. In other words, a 6 requirement for heme at one point does not generate a 7 deficiency in another point. 8 Q. Has Dr. Poland ever done any work on heme dynamics 9 in the cell, has he ever published a single paper on that 10 subject? 11 A. The only thing that Dr. Poland has done is to 12 investigate the induction in that -- that might be considered 13 heme dynamics. He's investigated the induction of P-450 14 related enzymes. He does not do work on heme dynamics. 15 Q. Now, what is the evidence with respect to whether 16 dioxin affects red blood cells? 17 A. Well, the evidence shows that -- that dioxin as a 18 matter of fact does not until animals are so intoxicated that 19 they are in a state of collapse and cells are failing all 20 over the body. At that point bone marrow stops making red 21 blood cells, and the animals become exceedingly anemic. These are animals that are essentially in a state of collapse, they are very far along in the process of intoxication 130 1 Q. What kind of levels of exposure cause these kinds 2 of problems? 3 A. Well, this has been seen in the monkey, and the 4 monkey is fairly sensitive to TCDD. Doses on the order of 2 5 or 3 micrograms per kilogram would -- would probably carry an 6 animal to that point. 7 Q. Now, does the induction process require a lot of 8 heme? 9 A. Not relatively, no. If we look at just the P-450 10 enzymes, and we have talked about a 25 or maybe 50 fold 11 increase, then obviously there has to be that much of an 12 increase. However, this is an exceedingly small fraction of 13 the total heme required in the body, and it's a relatively 14 small fraction of the -- of the heme demands of the cell in 15 general even with that increase. 16 What is of most interest is that the animal that's 17 intoxicated with TCDD is still able'to induce the enzyme, 18 synthesis goes forward very nicely. Animals that are iron \< 19 deficient, and iron is a component of heme, can still 20 manufacture the heme protein enzymes. 21 Q. Well, what about the existence of a heme pool. Is 22 there such a thing? 23 A. No, there is not. Each cell makes it own. I 24 mentioned that earlier. Each cell makes its heme as it needs 131 1 it, and in the cell there is not a heme pool. Certainly this 2 complex process by which heme is synthesized will at one 3 point have some heme that has not yet been incorporated into 4 a protein, but it is driven by demand except in an 5 intoxicated animal. 6 THE COURT: Hr. Heineman, is this a good point for a 7 short break? 8 MR. HEINEMAN: Sure, Judge, that would be fine. 9 THE COURT: Take about a ten minute recess and then 10 resume testimony. Court's in recess. 11 (At this time a short recess was taken.) 12 13 (The following proceedings were had in open Court.) 14 MR. HEINEMAN: Just a moment. Your Honor. I'm 15 trying to get three exhibits. Your Honor, at this point, if 16 I may, I neglected to offer Defendant's Exhibit 1278 into 17 evidence, which is the Poland and Glover member -- or graph 18 described by Dr. Dost, and I have also had these handwritten 19 pages, Defendant's Exhibit 1279, which was his computation of 20 the Sturgeon exposure, the dose, and Defendant's Exhibit 21 1280, which is his comparison of the Kociba no effect level 22 and the Sturgeon dose, and I'd like to offer those at this 23 time. 24 MR. CARR: We have no objections to them, Your 132 1 Honor. 2 THE COURT! All admitted without objection. Thank 3 you. 4 MR. HEINEMAN: If I may pass 1270 and 1278 to the 5 jury. 6 Q. Now, Dr. Dost, we were talking about the heme. 7 What is the relationship between heme and chemical porphyria? 8 A. If -- in the process of making heme there is -- to 9 start with this is a multi-step process. If for some reason 10 there is either a pathological or a genetic influence that 11 accelerates the early steps in this process, it is in a way 12 like any assembly line, if the material doesn't come off the 13 end of assembly line, faster than the parts go into the 14 beginning of the assembly line, there will in all probability 15 be a problem. 16 In the process of manufacturing heme if the enzymes 17 at the beginning of the cycle are accelerated, there is a 18 potential for a buildup of some of the intermediate products 19 before the final product. If, in addition to that, there is 20 an interference with the terminal process, then obviously the 21 accumulation of material in between is going to be all the 22 worse, and porphyria is an accumulation of the precursors of 23 heme before iron is added to the molecule. 24 Q. Now, you've talked about the fact that heme is 133 1 produced in the cells where it's needed? 2 A. That's correct, 3 Q. Where do porphyrins accumulate? 4 A, They accumulate in those same cells. However, they 5 can escape, because in an individual with porphyria 6 porphyrins are the way we identify those molecules before 7 they become heme, and if they accumulate in the cell, some of 8 them will leak out. They're after all not a large -- not a 9 large compound. They will leak out, and they will be/seen in 10 the urine. But they also accumulate in the cells wh^re they 11 are made. 12 Q. All right. Now, I'd like to ask you if you recall 13 this testimony from Dr. Silbergeld. April 12th, beginning at 14 Page 112. She said, first off since the porphyrin system 15 produces heme, that molecule which carries oxygen in the 16 body, a reduction in theamount of heme through the draining 17 off into that enzyme system that's induced, a reduction in 18 that amount of heme deprives cells of their oxygen carrier, 19 if you will. Nerve cells have the highest need for energy, 20 for oxygen of all cells in the body because they are always 21 active. Therefore, they are among the first to die or be 22 affected when there's a lack of oxygen, she goes on to say 23 another way in which altering porphyrin chemistry affects the 24 nervous system is this insulation, this fatty wrapping around 134 1 the nerves, which is made out of lipids. It's very dependent 2 on active porphyrin system in those same cells, and its 3 porphyrin system is deranged as has been shown by workers at 4 Rockefeller University. Now, do you agree with those 5 statements, sir? 6 A. Well, there is a problem -- there is a number of 7 statements, and it's a very confused, a very confused 8 description. To begin with, we don't have -- I already 9 discussed the problem of a drain on heme. Now, when 10 porphyrins accumulate, they -- they do represent some 11 quantity of the intermediate product, but they are not 12 sufficient to constitute a drain on heme, and as a matter of 13 fact, it can be demonstrated that animals that are porphyric, 14 that is, have been intoxicated sufficiently with TCDD, that 15 they do show porphyrins in the liver as deposited in ,the 16 liver and in the urine continue to synthesize, they respond 17 to induction of the P-450 series. In other words, there 18 doesn't appear to be any evidence in those cells that there 19 is a -- that there is a drain, any interference with the 20 ability of the cell to make heme proteins. 21 What is happening is that there is a diversion, and 22 some of the precursors are not being processed into the final 23 product, but there is still enough capacity to make the heme 24 proteins that are necessary. If one looks at red blood cells. 135 1 there is no impact of TCDD on red blood cells until, as I 2 pointed out earlier, the animal is in a very, very serious 3 state of intoxication, 4 Q. Whose work are you relying on there, sir? 5 A. The work particularly of Joyce Goldstein and of the 6 group headed by Cantoni in Italy, They have devoted the 7 major part of their career to this question of chemical 6 porphyria. 9 Q. Where is Goldstein located? 10 A. Goldstein is located at the National Institute of 11 Environmental Health Sciences in Research Triangle Park, 12 North Carolina. 13 Q. And Dr. Cantoni, Cantoni is from Italy? 14 A. Is, I believe, from Milan in Italy. 15 Q. I'm sorry, I didn't mean it interrupt. Go ahead. 16 A. I may ask to you read some of that testimony again, 17 because it's ver complex, and I have difficulty keeping it 16 all in my mind. 19 The question of oxygen supply to the central 20 nervous system. It is true that the brain has a higher 21 consumption of oxygen than almost any other tissue. The 22 brain consumes an enormous amount of oxygen, but there is ho 23 impact on the brain's oxygen supply in TCDD intoxication. \ \ 24 There is -- for two reasons. One is we can see that there is 136 1 no impact on red blood cell counts or hemoglobin levels, no 2 evidence of impaired oxygen transport under TCDD intoxication 3 until it has reached the case where the cells are no longer 4 being made. The -- the central nervous system and the blood 5 cells both have a limited capacity to form porphyrins, 6 because no evidence is found for porphyrin derangement in 7 bone marrow as a function of TCDD, and that's not surprising, 8 because chemicals that cause porphyria almost always are 9 limited in their effect to the liver.1. 10 Now, there are a variety of genetic porphyrias that 11 do that do have an impact in other organs. Chemical porphyria 12 only attacks the liver, and as a matter of fact, one of the 13 enzymes that is inhibited in the porphyria caused by TCDD is 14 an enzyme called uroporphyrin decarboxylase, and it comes at 15 the end of this cycle of-- it's the finishing enzyme, so to 16 speak, because it trims a lot of the useless side chains off 17 the porphyrin molecule before it becomes heme. If it's 18 inhibited, obviously production is going to pile up. There 19 will be porphyrin intermediates formed, and there will be 20 interference, some interference with heme synthesis. That 21 enzyme exists in the precursor cells for red blood cells, in 22 the bone marrow, and when red blood cells are formed in the 23 bone marrow, they're derived from cells that are able to form 24 a variety of other cells. As they differentiate, they 137 1 eventually lose much of the machinery in the cell, because 2 all the red cell really is a just a bag of hemoglobin, and it 3. lasts in the circulation for a hundred or 120 days, and then 4 it breaks up and then the material is salvaged or discarded. 5 But the enzyme, uroporphyrin decarboxylase, happens 6 to be carried along. It's left in the cell. When the 7 nucleus comes out, the mitochondria, which is an energy 8 producing mechanism in the cell, when that comes out, some of 9 the enzymes are left, and uroporphyrin decarboxylase is 10 left,and it is not impaired in red blood cells by TCDD. It's 11 only impaired in the liver. 12 I would appreciate it if you would read the part 13 again about the relationship between effects of porphyria on 14 the central nervous system. I became a little confused 15 there. 16 Q. All right. What I read was that the porphyrin 17 system produces heme. A reduction in the amount of heme 18 through the draining off into that enzyme system that's 19 induced, a reduction in that amount of heme deprives cells of 20 their oxygen carrier, if you will. Nerve cells have the 21 highest need for energy, for oxygen of all cells in the body, 22 because they're always active. Therefore, they're among the 23 first to die or be affected when there's a lack of oxygen. 24 A. With respect to that, there have been specific 138 1 experiments done, particularly by George Lucier at again the 2 National Institute of Environmental Health Sciences* That 3 group, by the way, does a great deal of research of TCDD, 4 because it is a major national concern, and they can't find 5 any evidence of an effect on energy metabolism* 6 In other words, there isn't any -- there isn't -- 7 there's no impact on the electron transport system, which 8 depends so intimately on the cytochrome system, the 9 cytochrome enzymes that are responsible for helping to 10 transport electrons to oxygen so that we can make -- complete 11 the energy retrieving cycle in the cell. 12 And there's really no evidence that demonstrates 13 any impact on the ability of the organism to transport or 14 utilize oxygen either throughout the whole body or within a 15 given cell* 16 Q* With respect to TCDD intoxication of animals, has 17 there been any evidence of brain pathology? 18 A a NO 19 Q. Has there been any effect shown on porphyrin 20 synthesis in the brain? 21 A. Well, the brain -- no. 22 Q* Has there been any effect on hemoglobin synthesis 23 in animals? 24 A. No, only, as I said, until at the time the cells in 139 1 the bone marrow are ruined, and they simply are unable to -- 2 and that's probably a nonspecific effect of some kind. It 3 certainly isn't anything that's attributable to a direct 4 effect on heme synthesis in the -- or hemoglobin synthesis, 5 either heme itself or the ultimate heme protein in the bone 6 marrow, where blood is made. 7 Q. Now, what particular studies do you rely upon to 8 reach those conclusion? 9 A. A variety of pathology studies particularly, 10 because -- and evidence also by Cantoni again, where .11 porphyrin metabolism or porphyrin accumulation in the central 12 nervous system as well as the activity of the uroporphyrin 13 decarboxylase, this terminal enzyme in this chain, were 14 measured, and there is not an accumulation of porphyrins in 15 the cells of central nervous system. 16 It may be useful to point out that there is in 17 individuals who have genetically derived porphyria, 18 neurotoxic symptoms, neurological symptoms that are 19 apparently a function of the development of porphyria. This 11,11 a mil a^ ^ m+ ,,pTr-MJifn ^ 20 is not seen in the chemical porphyrias. It also only arises 21 after a long history of porphyric episodes that are 22 genetically based. 23 In the case of TCDD there is no evidence at all for X------- 1-- ------ " ' 24 a -- a porphyric impact in the central nervous system as a 140 1 result of TCDD intoxication* Now* as far as the pathology of 2 the central nervous system is concerned, the brain is always 3 a primary organ that is examined in any pathology study, in 4 any toxicology evaluation. Again Kociba in animals treated 5 for afi\^ntire lifetime, both in the studies that they did for \ 6 two years and the studies that they did preliminary to that 7 for 13 weeks at which they actually studied pyradosis. James 8 Allen's work in monkeys, for example, showed no brain 9 pathology. The only thing that appears in the nervous system 10 of heavy intoxicated animals is capillary failure, because at 11 high doses the capillaries become more permeable, and they 12 start losing blood, and in the central nervous system this 13 will occur around nerve cells. It's a circulatory lesion, 14 not a nervous lesion. 15 Q* Now, if I may I'd like to read you another 16 statement that Dr. Silbergeld made. Well, I guess I already 17 did. It had to do with the myolin? 18 A. , Oh, I missed that. 19 Q. All right, let me read it. Another way in which 20 altering porphyrin chemistry affects the nervous system is 21 this insulation, this fatty wrapping around the nerves, which 22 is made out of lipids. It's very dependent on an active 23 porphyrin system in those same cells, and its porphyrin 24 system is deranged, as has been shown by workers at 141 1 Rockefeller University and elsewhere. Some of the first 2 cells to die are these wrapping cells, what's known as myolin 3 around the nerves. Do you recall that testimony? 4 A. I do recall it now, yes. 5 Q. Do you agree with it? 6 A. No, I'm afraid not. 7 Q. Why not, sir? 8 A. Well, to begin with the pathology studies do not 9 show any evidence of demyelination. Derayelination is very 10 easy to see under a microscope. I've observed it in my own 11 work on hexachlorophene. Hexachlorophene happens to be a very 12 effective demyelinator. That is in part the reason why it 13 exerts pathology, why it has caused neurological damage in-- 14 in humans, and why it is no longer used as a skin 15 disinfectant. So there is no evidence to begin with for any 16 myolin lesions, any lesions of the myolin sheath. Now, the 17 myolin sheath is a structure that surrounds the nerve fiber, 18 and it is relatively inert metabolically, it doesn't do much, 19 but it's there as insulation, and it's sustained by a cell 20 that is located -- cells are located periodically along the nerve fiber that -- and this is not a nerve cell, but it's a separate cell called a schwann cell, and it is responsible for maintaining the myolin sheath, which is really a wrapper. It would be very much like taking a pencil and 142 1 making several wrappings of a membrane around it, and in 2 between each layer of the membrane is this material, myolin, 3 which is more or less homogenous, that is, it doesn't have 4 much structure. 5 I have been trying to find evidence of any role for 6 porphyrin synthesis or heme synthesis in maintaining rayolin, 7 and other than the necessary heme proteins that are needed 8 for the maintenance of energy metabolism in any cell, 9 including the schwann cell, even a fat cell has these kinds 10 of functions. I cannot find anything that is specific to 11 effects on maintaining myolin, but the bottom line, if you 12 there 1 *' *' TCDD causes 13 ^ ______ around nerve fibers in any experimental animal. 14 Q. Whose work do you rely on? 15 A. Again, I rely on the pathological studies done by 16 Allen on monkeys, on Kociba, and particularly -- there are a 17 number of others, the brain is routinely examined in any 18 pathological screening, but those are perhaps the most 19 important. 20 Q. Has there been any work done in the field by a man " 21 named Calder? 22 A. Yes, as a matter of fact. Calder is a researcher 23 in New York who in contemplating the problem of TCDD and what 24 appear to be some evidence of peripheral neuropathy or at 143 1 least suggestions that TCDD can cause a peripheral nerve 2 derangement that could take many manifestations, but 3 particularly tingling and pain in the peripheral nerves, 4 treated rats specifically for the purpose of doing pathology 5 on the peripheral nerves, and by peripheral nerves, I mean 6 the nervous system outside the brain and spinal cord. The 7 nerves that serve ray fingers are part of the peripheral nerve 8 network. 9 Calder and his associates were not able to find 10 evidence of the -- that they could attribute to-- that would 11 be associated with peripheral neuropathy, particularly no 12 demyelination, no other lesions that they could associate 13 with such an effect in the rat. 14 Q. Now, with respect to porphyria and the causes of 15 it, you mentioned chemicals such as hexachlorophene? 16 A. Not hexachlorophene. We know that TCDD will cause 17 porphyria. Hexachlorobenzene will cause porphyria. 18 Q. All right. How about any metals or any other 19 materials? 20 A. Lead will cause porphyria. Q. You mentioned the genetic cause? A. And the genetic problem, which is really defect in the -- somewhere along the enzymatic trail of porphyrin synthesis. 144 1 Q. Has there been any investigation to see whether -- 2 well, let me start over again. What is a subclinical 3 porphyrin result? 4 A. A subclinical resultr I would say, is one that can 5 be measured if one examines the urine, and usually urine is 6 the body fluid in which we would attempt to detect 7 porphyrins. There are going to be porphyrins appearing in 8 the urine all of the time anyway, and one would, I presume, 9 look for a change in the pattern or a change in the amount. 10 I would say that a subclinical change in porphyrins would 11 fall within the realm of those that are occurring just as a 12 result of our daily life without any particular evidence of 13 toxicity, illness, or other effect. If it were clinical, 14 clearly it would be associated with some kind of 15 symptomotology that made us go and look for it. 16 Q. What is porphyria cutanea tarda? 17 A. Porphyria cutanea tarda is a form of porphyria in 18 which porphyrins accumulate in the skin, and this creates a 19 problem, because it causes a stiffening of the skin, it 20 causes, but the main reason that it is -- that it causes an 21 effect is because porphyrins are -- have the ability to trap 22 light. In other words, they are -- and in the skin they tend 23 to cause an excess of ultraviolet damage in the skin, and 24 that combined with the excessive amount of those materials 145 1 creates some pathology in the skin, results in discoloration, 2 extreme sensitivity, skin injury in the sunlight and so on. 3 Q. Is porphyria cutanea tarda found in humans? 4 A. It is found in humans, yes. 5 Q. Is it found in animals? 6 A. I don't believe that there is an adequate model in 7 animals for porphyria cutanea tarda. 8 Q. What -- what are the doses which have been used to 9 give rise to abnormalities in the porphyrins in animals? 10 A. Well, Goldstein was able to produce porphyria at 11 doses of a tenth of a microgram per week per kilogram. 12 Cantoni, I believe, was -- I'm afraid that I would want to 13 look at the papers to confirm that, but it seems to me that 14 Cantoni conducted an experiment over a 45 week period with 15 somewhat similar doses that were at the -- were porphyria -16 forgive me -- porphyrin accumulation in the liver was 17 beginning to show up at marginal levels. 18 Q. Has there been a no effect level discovered in 19 animals for abnormalities in porphyrins? 20 A. In all of the studies that show a sufficient dose 21 range, yes, a no effect level has become apparent. Clearly i 22 there is a dose response, and if there is a wide enough range 23 of doses, a no effect level becomes evident. 24 Q. The -- what is the difference between urinary 146 1 porphyria and hepatic porphyria? 2 A, Urinary porphyria is simply the discharge of 3 porphyrins that have escaped the liver cell. We're talking 4 here about a chemically induced porphyria, which is 5 restricted to disorder in the liver. Porphyrins that escape 6 the liver cell and then find their way into the circulation, 7 they are relatively small, are excreted in the urine are th 8 urinary porphyrins, and the accumulation that remains in the 9 cell is, I guess what we would describe as hepatic porphyria, 10 but they both arise from a liver disorder, a hepatic 11 disorder. 12 Q. I'd like to deal with the cardiovascular system. 13 Dr. Dost. Would you describe what constitutes the 14 cardiovascular system? 15 A. The cardiovascular system is the entire -- well, 16 it's the system that transports, pumps arid transports blood 17 throughout the body, it's the plumbing. It includes the 18 heart, and all of the blood vessels. Interestingly enough, 19 in an ideal sense we don't consider blood to be part of the 20 cardiovascular system, we think of it separately. It's 21 carried by the cardiovascular system. Without blood there 22 would be no point in having a cardiovascular system, but the 23 system itself is really the heart and the arteries and the 24 veins coming back and all the capillaries in between. 147 1 Q. Now, in terms of dioxin exposure to animals, has 2 there been seen any effect in the cardiovascular system? 3 A. At higher doses the capillary bed tends to become 4 leaky. I think I mentioned earlier how at high doses there 5 is -- are capillary hemorrhages found around nerve fibers, 6 around nerves. This is -- this is fairly characteristic. 7 The cardiovascular system structurally does not suffer a 8 great deal from -- from TCDD intoxication other than this 9 increased permeability of the capillaries at high doses. 10 Q. When you say high doses, what -- how high are you 11 talking about? 12 A. Well, those kinds of doses are up into the -- in 13 the really long chronic experiments on the order of a tenth 14 of a microgram per kilogram per day. Shorter term 15 experiments, single doses up into the micrograms per kilogram 16 per day. 17 Q. So it would be from a hundred to a thousand times 18 higher than the no effect level? 19 A. Yes, yes. 20 Q. Now, has there been observed any degeneration of 21 heart muscle as a result of dioxin exposure? 22 A. Well, okay. That has been seen in -- at again to 23 some extent in sustained -- in sustained intoxication at 24 higher levels. 148 1 Q. All right. In what kind of animals? 2 A. Well, in -- I believe in the rat. 3 Q. Now, we talked before about elevated serum lipids 4 in connection with dioxin exposure? 5 A. Yes. 6 Q. Is that really a cardiovascular effect? 7 A. Well, it has its impact ultimately or at least -- 8 let's put it this way. Elevated serum lipids and serum 9 cholesterol represent a risk factor for cardiovascular 10 disease. All right. In terms of the effect of TCDD, it's 11 probably an impact on the liver, although it could very well 12 be -- it could be an effect to some extent on metabolism out 13 in the periphery where lipids are being used for energy. 14 There is no evidence of that, but the derangement in the 15 liver that results in increased serum lipids if it were 16 sustained for a period of time, could imaginably be 17 associated with an increased risk factor for some kind of 18 cardiovascular disease, because we know that particularly 19 elevated serum cholesterol is associated with increased 20 arteriolosclerosis, which is a factor in heart and artery 21 disease. 22 Now it has to be kept in mind that that 23 relationship applies primarily to sustained situations; in 24 other words, in which the individual has a naturally 149 1 occurring -- well/ naturally it's going to be a function of 2 diet, lifestyle, or even genetics, so that the elevated blood 3 lipids are maintained over an extended period of time, and 4 that would increase the risk of cardiovascular disease. It's 5 questionable whether a short period of elevation would have 6 that much significance* Vie have to consider it at the same 7 time in that light* 8 Q* When you're talking about a short period, what 9 period of time are you talking about? 10 A. Well, if it were to occur as a result of an 11 intoxication, a few weeks, a few months* 12 THE COURT: Mr* Heineraan, is this a good point to 13 break for the day? 14 MR* HEINEMAN: Yes, sir, that would be fine. 15 THE COURT: We'll break for the day at this time*. 16 We'll resume again tomorrow morning at 9:30* I would remind 17 you that you're not to read, listen to, or watch anything 18 about this case in particular or the subject matter in 19 general in any of the media* Thank you for your attention 20 and cooperation* Court's adjourned* 21 22 23 24 150 1 STATE OF ILLINOIS } ) SS. 2 COUNTY OF ST. CLAIR ) 3 4 If MARSHA SCHNIPPER, certify the foregoing to be a 5 true and accurate transcript of the testimony and proceedings 6 in the above-entitled cause. 7 Dated this ____ day of November, 1985. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 151 1 STATE OF ILLINOIS ) ) SS. 2 COUNTY OF ST. CLAIR ) 3 4 5 If Richard P, Goldenhershf one of the Judges in and 6 for the Tv/entieth Judicial Circuit/ do hereby certify that 7 the foregoing transcript is a true ahd correct transcript of 8 the proceedings had in said cause. 9 Dated this ____ day of November/ 1985. 10 11 12 13 RICHARD P. GOLDENHERSH, JUDGE 14 15 16 17 18 19 20 21 22 23 24