Workshop on Plasticizers
Scientific Issues in Blood Collection, Storage and Transfusion (Plasticizers in Blood Bags)
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UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
CENTER FOR DEVICES AND RADIOLOGICAL HEALTH
WORKSHOP ON PLASTICIZERS
SCIENTIFIC ISSUES IN BLOOD COLLECTION,
STORAGE AND TRANSFUSION
(Plasticizers in Blood Bags)
MONDAY,
OCTOBER 18, 1999
The Workshop took place in the Masur Auditorium, National Institutes of Health, Bethesda, MD at 8:00 a.m., Jaroslav Vostal, Chair, presiding.
PRESENT:
JAROSLAV VOSTAL, M.D., Ph.D., Chair
TRACI HEATH MONDORO, Ph.D., Session Chair
RONALD BROWN, M.S., DABT, Session Chair
SUKZA HWANGBO, R.Ph. DABT, Session Chair
MELVIN STRATMEYER, Ph.D., Panel Chair
PAUL NESS, M.D., Speaker
JAMES AUBUCHON, M.D., Speaker
EDWARD SNYDER, M.D., Speaker
MICHAEL CUNNINGHAM, Ph.D., Speaker
ROBERT CHAPIN, Ph.D., Speaker
JOHN BUCHER, Ph.D., Speaker
VIRGINIA KARLE, M.D., Speaker
RAYMOND DAVID, Ph.D., Speaker
JOY ANDERSON, Ph.D., Speaker
RALEIGH CARMEN, Speaker
JEFF MIRIPOL, Ph.D., Speaker
MICHAEL SHELBY, M.D., Ph.D., Speaker
DALAND JUBERG, Ph.D., Speaker
JOEL TICKNER, M.Sc., Speaker
NAOMI LUBAN, M.D., Panelist
KATHERINE SHEA, M.D., MPH, FAAP, Panelist
SCOTT PHILLIPS, M.D., Panelist
PETER ORRIS, M.D., MPH, Panelist
MAY JACOBSON, Ph.D., Panelist
Introduction
Jaroslav Vostal, M.D., Ph.D.
SESSION I: Plastic Blood Bags
Chairman, Traci H. Mondoro, Ph.D.
Historical Perspective and Overview
Paul Ness, M.D.
Effects of DEHP on Red Blood Cells during Storage for Transfusion
James AuBuchon, M.D.
Effects of Blood Bag Plasticizers on Platelet Storage and In Vivo Performance
Edward Snyder, M.D.
SESSION II: Toxicology Issues of Phthalate Plasticizers
Chairman Ronald Brown, MS, DABT
Rodent Carcinogenicity of Di(2-ethylhexy)Phthalate
John Bucher, Ph.D.
Mechanisms of Toxicity
Michael Cunningham, Ph.D.
Reproductive Toxicity
Robert Chapin, Ph.D.
Pediatric Toxicology
Virginia Karle, M.D.
IV Toxicity
Ronald Brown, M.S., DABT
DEHP Physical Characteristics/Current Research
Raymond David, Ph.D.
SESSION III: Alternatives to Current Blood Bag Plastic and Plasticizer Materials
Chairman, Sukza Hwangbo, RPh
Alternatives to Conventional Blood Container Materials: What are the Challenges?
Joy Anderson, Ph.D.
Pall/Medsep: Alternative Plasticizer Efficacy/Toxicity
Raleigh Carmen
Terumo: Benefits of Using DEHP
Jeff Miripol, Ph.D.
SESSION IV: Current Risk Assessments of Plasticizers
Chairman, Jaroslav Vostal, M.D.
Center for Evaluation of Reproductive Risk
Michael Shelby, Ph.D.
The American Council on Science and Health: A Scientific and Multidisciplinary Assessment of DEHP in Medical Devices
Daland Juberg, Ph.D.
U Mass Lowell Center for Sustainable Production
Joel Tickner, M.Sc.
SESSION V: Panel Discussion
Melvin Stratmeyer, Ph.D. , Chairman
James AuBuchon, M.D. Dartmouth/Hitchcock Transfusion Medicine
Edward Snyder, M.D. Yale University Transfusion Medicine
Naomi Luban, M.D. Washington, DC Children's Hospital
Katherine Shea, M.D., MPH, FAAP McMillan and Moss Research, Inc.
Scott Phillips, M.D. University of Colorado/Phthalate Esters Panel
Peter Orris, M.D., MPH Cook Country Hospital/Health Care Without Harm
May Jacobson, Ph.D. Harvard University/Health Care Without Harm
FDA Closing Statement
Jaroslav Vostal, M.D., Ph.D.
PROCEEDINGS
(8:19 a.m.)
CHAIRMAN VOSTAL: Good morning. I wonder if we could get started this morning. Hello, my name is Jaro Vostal, and I welcome you to the Workshop on Plasticizers, Scientific Issues and Blood Storage and Collection. We are running a little behind time this morning. We are waiting for Dr. Zoon. Hopefully, she will show up, and when she does show up, we will have her give her introductory speech at the first break.
I am glad you are all here to help us discuss these issues. They are two very important issues to FDA and CBER. There are a number of issues that concern DEHP; however, today, we are only going to concern ourselves with the issues that arise from blood collection and storage. And because we are short on time, I think we better get started. I would like to introduce Dr. Mondoro. She will be the moderator for the first session.
DR. MONDORO: Good morning. My name is Traci Heath Mondoro, and I will be chairing the first session, which is entitled Plastic Blood Bags. I have one announcement to make before we get the session started. If you would make sure that you pick up two supplement packets that are out on the table. These are some more abstracts and biographies that can be put in your folders. There are, like I said, two packets that are paper-clipped, and they are out on the tables in the lobby.
The first session, the name pretty much says it all, Plastic Blood Bags. Our first speaker is Dr. Paul Ness. He is the Director of Transfusion Medicine Division at Johns Hopkins, and he is going to give a historical perspective and overview. As he is coming up, I would also like to remind you that today's meeting is being transcribed. So that if you do come to the microphone to ask questions, we ask that you state your name and your affiliation, so that it will be part of our public record. Thank you.
DR. NESS: Good morning. It is nice to be here although I had a lot of second thoughts after I agreed to give this talk. I guess I have reached the point in my career where I am asked to do a historical introduction rather than trying to present anything I really did myself. But as you will see as I give my remarks, this has been something that I have been interested and involved in for quite some time. So I am actually very happy to be here.
When I started trying to do the idea of doing a historical introduction about DEHP and blood bags, I looked back into some of the medical literature for sort of reviews of this topic. Because if you look in the current blood bag text, you won't find very much in terms of the issue of phthalates in blood bags. People seem to think that the problem has gone away and it no longer really needs to be discussed.
So I picked up this book. It is a book called The Red Blood Cell, which was edited by Dr. Douglas Surgenor, and I will read to you a section from it briefly in what was called "The Historical Introduction." It says, "It is necessary to incorporate a plasticizer with polyvinylchloride polymer to provide the flexibility, toughness, ease of sealing and manipulative qualities needed in a blood bag. The added plasticizers have been in the phthalate group with DEHP a common choice. Adverse findings which demonstrate that significant quantities of phthalates leach out from the material of the bag have directed a search for other materials for bag fabrication. There have been many alarming reports that phthalates can migrate from polyvinylchloride blood bags into stored blood and localize in human tissues. The ability of man to metabolize phthalates remains unclear, and the overall biologic impact of the phthalate plasticizer is still unresolved. Acute effects of phthalates have not been clearly demonstrated, but potential teratogenic and other long-range toxic effects are of great concern."
This was published in the early 1970's, and I thought it was a very well written statement now and actually at the time, because I actually wrote it. This is the first thing I ever wrote as a person who came to this campus and worked in what is now the National Heart, Lung and Blood Program. And I think you will see that we haven't actually moved that far beyond that unfortunately.
So in reviewing sort of the real early history, I think most of us in this audience are aware -- there may be a few people who don't know that much about blood bags, but just to cover them. The early history is that vinyl plastic bags were introduced sometime around 1950. Walter is given credit for that. It was shown that the survival of red cells stored in these bags was actually improved compared to glass bottles. And we all have seen that there are major advantages in collection, processing, storage and dispensing of blood components, particularly in platelet concentrates as a result.
An old friend here for some of us -- I guess I was in the field long before we were using this, but some of you out there may remember these more fondly, and obviously we have now moved to this type of arrangement with plastic bags, different plastics to facilitate, for instance, platelet storage as opposed to red cell storage, and it really has allowed us to make a number of different blood components from whole blood. It has allowed us to facilitate aphoresis collection for various blood components, stem cell collections and a whole host of other kinds of medical things that have made transfusion medicine a very growing discipline.
Again, to review, unfortunately though a plasticizer needs to be incorporated. So that for the blood to be pliable, vinyl plastic containers require the addition of a plasticizer at levels of up to 20 to 30 percent of the final weight. And DEHP, di(2-ethylhexyl)phthalate, is a common choice for most of the medical plastics. DEHP is not chemically bound, but is dissolved physically in the plastic film. Initial studies when these bags came out implied that there were trace amounts of these materials which went into the bags when they were filled with anticoagulants. These initial results seem to be reassuring, but later other results came out which were a little bit more alarming.
This is a slide that actually I was able to borrow from Bob Rubin, which shows some of the original work that he and a graduate student, Rudy Jaeger, at Johns Hopkins did a number of years ago. He was looking, actually, at an isolated chamber to isolate livers and do profusions of the livers and using a chromatographic technique when he found what he called in one of the profusion studies an unidentified compound compared to these other peaks that had easily been identified. To hear Bob tell the story, which is always a very entertaining event, this unidentified compound had been obtained from a profused rat or mouse liver, so the amount of blood in which to do biochemistry on this was very, very small. And biochemistry then is not what biochemistry is now. In any event, he decided that it would probably be a good idea to try to scale up this apparatus so that they could get enough of this material to actually analyze and find out what it was. So they went into a more macro system and actually came over to the Hopkins blood bank, because he said, well, we have a lot of outdated blood there and we could use the outdated blood to profuse the system. According to Bob, these are actually some of the first bags that they borrowed or took from the Hopkins blood bank as outdated blood to study. And when they did these experiments in a larger system, Rudy Jaeger apparently came to Bob and said, well, I have good news and bad news. In the larger system, I certainly can find the compound which you were interested in, that compound X. Unfortunately, it is also there in heavy quantities in the starting material.
This then obviously became, after about a year of biochemistry, identified as DEHP, and Jaeger and Rubin reported initially in Lancet and later on in The New England Journal in the 1970's about contamination of stored blood with DEHP at levels of 50 to 70 milligrams per deciliter. It was also shown in this later article that it migrated substantially during storage, so that the migration rate they calculated was 2.5 mg/liter of blood for 24 hours of storage, such that one could get a possible dose in a bag of blood of almost 300 mg or about 5 mg per kilogram for an adult and even higher dose for a child, and these doses, as you will hear later, had been attributed or suggested that they may have some toxicity in some of the animal models.Bob went on to work with Charlie Schiffer doing some actual measurements in platelet transfusion recipients, and they reported in Transfusion in 1976 that when platelet transfusion recipients were getting platelets, they actually had an intravenous injection of 26 to 62 mg of DEHP in the platelet recipients.
This, for those of you who haven't met him, is Bob Rubin at a younger day. He is actually in the audience today, and I am sure that we will be blessed by some of his comments as the day goes on. His observations that I have talked to you have since obviously been confirmed by many laboratories around the country. What added, unfortunately, to some confusion about what was the role of DEHP in blood bags, however, were reports of widespread environmental contamination with DEHP. So how to place this transfusion problem into perspective became a difficult endeavor.
One of the things that happened in the 1970's and how I sort of got involved a little bit was that a number of studies were actually funded by what was called the National Blood Resource Program on this campus, and now it is part of -- it was part of what was then the Heart Institute, then the Heart and Lung Institute, and now the Heart, Lung and Blood Institute. But these studies were actually funded by NHLBI, which had some industrial studies, some studies by the military and studies by the private sector. Many of these have been reviewed in an international forum which was published in Vox Sanguinis in 1978.
Obviously I don't have time to go through that whole review, but you can see that there were various flavors of sort of reports that came out at that time. The industry studies showed that when they looked at tissue residues in transfused recipients, those studies were essentially negative. They showed that platelet storage did not appear to be effected. They didn't show any increased particulates in bags stored with DEHP, and they emphasized the importance of making the DEHP a solution rather than an emulsion, which sort of clouded some of the studies about the vehicle in which DEHP was administered to laboratory animals.
The military published some excretion and metabolism studies and gave the implication that since these seem to be relatively rapidly metabolized, they would not be likely to cause a problem for most human recipients. On the other hand, there was a very intriguing report by Dr. Sherwin Kevy from Boston Children's Hospital, where he used a monkey experiment and these monkeys were given chronic platelet transfusions on a schedule which was not very different from what human recipients could be given, and showed direct evidence of hepatotoxicity in the monkeys who were being transfused with platelets which had been stored in the DEHP-containing container.
So there were studies that implied not much problem, maybe a problem, and it wasn't exactly clear where to go from here.
Well, at this point in my career, I came on to Johns Hopkins and actually had the opportunity to meet and actually work with Bob Rubin directly. This was something that turned out to be a lot of fun and very intriguing in terms of this issue. When I first got there, Bob and I worked with a graduate student, who was going for a Ph.D. thesis, and he had some preliminary -- they had some evidence that when rats were given DEHP-containing infusions, they developed a DIC-type picture with fibrinogen activation and the generation of fibrin-split products.
So we decided to do some studies that compared blood which was stored in blood bags versus blood which was stored in glass bottles at the time. We had some very interesting results. When we looked at whole blood, in the bottles there were no evidence of any fibrin-split product generation or no fibrinogen activation. Whereas in the plastic bags, we did have fibrin-split products by clinical assay and evidence of fibrinogen activation. We also tried to make platelets and plasma and store them in glass bottles or plastic bags. Platelets obviously stored in a glass bottle were difficult. But we again found that fibrin-split products were found in blood stored in the plastic bags, and there were higher titers that were actually found in the platelets than in the native plasma, implying that cells in the medium had some additive effect in terms of the leaching or fibrinogen degradation.
We concluded in an abstract that we published at that time that blood stored in plastic bags in current use is not maintained in its native state. We actually presented these results at the AABB. We presented similar results at the American Heart Association in a toxicology meeting, but were never able to get them published in a peer review journal. These results sort of intrigued us and made us concerned that perhaps -- or at least me concerned that perhaps recipients of massive transfusions, where they have already been known to have a DIC-like picture sometimes, or people who had massive transfusions and had pulmonary failure, the so-called ARDS syndrome after a transfusion, that perhaps the DEHP storage media was having some sort of effect.
And I worried about this a little bit, but not too many other people worried about it too much. Everybody, at this point, started worrying about something else, which was the HIV epidemic, and I think that the sort of plasticizer issue sort of went away for a number of years. It actually went away, at least for me, for a number of years until the late 1980's, when Bob called again and said that Jeff McCullough, the editor of Transfusion had asked him to write a review on the status of blood bags. And that since he hadn't been that much involved in the use of blood bags for a number of years, would I be willing to write or help him write this article.
So we wrote an article which was published in Transfusion called "What Price Progress", and what we showed is results that I am sure many of you are already aware of. We showed or reported that there was a low, acute toxicity for DEHP. But we did say that there were pulmonary reactions in animal models that were somewhat troubling. We quoted a number of papers from around the world showing suggestive evidence of chronic effects, including infertility, teratogenicy, carcinogenicity, hepatotoxicity, and cardiotoxicity. We, on the other hand, acknowledged that even though these effects might be deleterious, it was clear that DEHP had since been shown to have some benefits, actually, for red cell storage. It seemed to enhance red cell storage, which I am sure Dr. AuBuchon will talk about in the next talk. And in the conversation or in the article, we talked about further discussion and perhaps new solutions that might be available.
Soon thereafter, a plasticizer or a plastic bag system was released by the Baxter company called PL2209, which was a plastic storage system without DEHP introduced in the early 1990's. Now I am sure the immediate assumption of anybody who read our paper was that we were being paid in some way by Baxter and were aware of this development and we were just writing this at that time to promote this release of this new blood bag system. And I can tell you that nothing was further from the truth and that when we wrote this paper, we didn't know anything at all that industry was actually working on a blood bag substitute that did not have DEHP.
I think it is fair to say, though, that even with our article, which we thought expressed appropriate concerns, there seemed to be little enthusiasm. I think I have used the term sort of collective inertia generated by transfusion services, perhaps because clear-cut human toxicity had not been identified and widespread acceptance was, at that time, inhibited by higher costs. These systems were introduced into a number of blood bags but have since been actually withdrawn from some of them or many of them because of the higher cost of implementation.
Well, I think it would serve as a good summary for this sort of historical introduction to sort of read the final paragraph of what we said in our "What Price Progress", because I think it is actually kind of an interesting summary, particularly for this meeting today. What we said was on the basis of the available data, we believe that DEHP problems should be addressed in the following ways. Because much of the data suggesting toxic effects of plasticizers remain unknown to physicians and their patients, we would suggest that these data and the resulting issues be presented and discussed at a forum such as an NIH consensus development conference. We would anticipate that this type of public exposure would result in a call for more research in this area with emphasis upon the clinical study of multiply transfused recipients to determine if any evidence of toxicity can be found in humans. Another focus of this type of meeting would be the consideration of the status of blood collection systems without DEHP. The practical and regulatory issues that would confront any new blood bag system could be addressed, and the likelihood of substitute systems becoming available in the near future could be presented.
While we proposed this meeting actually in 1989, it is now 1999, and I guess we are just 10 years too late. But hopefully it is never too late, and I personally am very pleased that we are now having a meeting to sort of discuss these issues and come to grips with what the appropriate cause or causes and courses ought to be. Thank you very much.
DR. MONDORO: Thank you, Dr. Ness, for that overview and introduction. Now we are going to get a little bit more specific. Our next speaker is Dr. James AuBuchon. He is the Medical Director of the Blood Bank and Transfusion Service and professor of pathology and medicine at Dartmouth-Hitchcock Medical Center.
DR. AUBUCHON: Good morning. If I could have the first slide, please? I too appreciate the opportunity to speak before you today. It was fun to go through some old data and some old reports, which frankly many of which I had forgotten about, to return again to the issue of what does this plasticizer do with red cells.
Depending on your point of view, this is either the villain of the story or the hero. Its characteristics certainly have not changed in the last two or three decades. We know that this plasticizer is not covalently bound within the polyvinylchloride plastic, and it can indeed leach out. And this information, as Paul reviewed, has been well known in the literature for a number of years. This is not -- this compound, obviously, is primarily lipophilic and does not dissolve very well in crystalloid solutions. But if you put protein or lipoproteins or perfectly plasma in contact with polyvinylchloride containing DEHP, this compound will very rapidly appear in the blood or the profusate.
The amount that accumulates over storage varies depending on how you assay it and exactly how the blood is stored and what blood component is being stored, but certainly a measurable amount does occur in blood during normal blood bank storage. The majority of DEHP appears in plasma, probably in association with albumin or lipoprotein, but somewhere between 5 and 10 percent does end up being associated with the red cells. And this red cell take-up of DEHP occurs quite quickly. Gail Rock was able to show that within minutes, a large proportion of the available DEHP could be found attached to red cells and approximately equal proportions of that DEHP were found in the red cell membrane and the red cell cytosol.
Of course, when the DEHP is transfused, it can be measured, as was just mentioned, and we will probably be hearing more about that today -- exactly what happens to DEHP and what it causes on its way to metabolism and disappearance.
The studies that were mentioned from Boston Children's indeed attracted a lot of attention in the blood banking world because of the potential for chronic exposure to DEHP having some detrimental affect to our patients. This was in an era where we were not used to having a lot of public scrutiny as to what we were doing in blood banking, and frankly this escaped public scrutiny as well. It wasn't until after the era of AIDS that blood bankers became very accustomed to having the public pay attention to everything that we did. But blood bankers at this point still were concerned that the chronic exposure to DEHP may have a negative effect.
But on the other side of the coin, there was clear recognition that DEHP may be doing something good, and I will be spending the next few slides going through some of the data that were available back at that time, in the 1970's and early 1980's, detailing exactly what DEHP was doing for red cells. In fact, the more recent report of the Blue Ribbon Panel concluded that DEHP imparted a variety of important physical characteristics that are critical to blood storage, and that is indeed true.
As we mentioned from the early times of plastic blood storage, it was understood that these plastic bags were at least as good as glass bottles, if not in some ways better than glass bottles for long-term storage of red blood cells. The initial studies with plastic bags when you look at them today did not necessarily meet the same scientific criteria. There were not good control groups, and to my eye anyway, it appears that the plastic bags of the mid to early 1950's were probably a little bit better than glass bottles in storing red cells, but it is difficult to say that with a P value in any true scientific confidence.
However, there are some data that we can indeed hang our hat on and that suggest that PBC with DEHP was better than glass containers. For example, after storing whole blood for 21 days in ACD and then determining at what saline concentration the red cells would completely hemolyze, it appeared that the PDC container stored red cells were more resistant to osmotic lysis than those stored in a glass container. Similarly, the plasma hemoglobin levels were found to be lower in those units of blood that were stored in the presence of DEHP than those stored in the glass containers. These are not proof absolute that the red cells are going to do better after transfusion, but they certainly are suggestive.
These initial concerns about the toxicity of DEHP and initial indications that DEHP may be doing something good for red cells prompted a number of in vitro studies. I will review a few slides here from Tim Eslep's work from Baxter. Baxter was obviously very interested in detailing exactly what DEHP was doing. And in the studies that his group performed, they took CPDA-1 red cells and stored them -- either stored them not in contact with polyvinylchloride, either with the buffer with an emulsifier or with an emulsifier that had emulsified within it DEHP. And they looked at a number of in vitro parameters in an attempt to determine what the plasticizer may actually be doing.
They noted that when the red cells were stored in the presence of DEHP but not in the presence of the buffer or just the emulsifier, that the morphology was better maintained throughout 35 days of storage, and that the plasma hemoglobin level did not rise nearly as rapidly as when DEHP was not present. Again, this was not due to emulsifier. It was due to the DEHP, it appeared. And indeed when they looked at a number of other compounds, including metabolites of DEHP and including MEHP and ethylhexanol, they were able to show that these metabolites singularly or in combination did not produce the same effect on morphology or hemolysis that the DEHP did. So it appears that the DEHP was, indeed, in some ways assisting the red cells surviving the storage period.
Interestingly, if red cells were first stored without the presence of DEHP and then DEHP was added in a solubilized form part way through the storage, the changes that were otherwise occurring were reversed. So here we see, for example, the effect of DEHP on morphology. This is the red cell morphology when DEHP is present, better maintained than when DEHP is absent. But when DEHP was added after two weeks of storage without DEHP, the morphology very quickly becomes that of the red cells that had been stored always in the presence of DEHP. That suggested that there was something physical that the DEHP was doing inside the red cell, which was not necessarily a metabolic-driven event. And indeed all of the standard metabolic indices that one looks at during red cell storage were just as well preserved with emulsifier as with DEHP. However, there was a difference in the amount of microvesicle formation during red cell storage when DEHP was added. So it appeared that in the presence of the plasticizer, there was less budding off of the membrane and less loss of membrane during storage, and that that may indeed be responsible or in some way related to the preservation of morphology and the lower hemolysis in the presence of this plasticizer.
Now the only recent study that I was able to find on this issue was published earlier this year from India looking at manufacturers' plastic bags that included DEHP compared to glass bottle storage. This study appeared to indicate that the ratio or the amount of cholesterol and phospholipids during the storage was better maintained and was more normal, I guess you would say, in the presence of DEHP than in its absence. So although the cholesterol concentration appeared to increase and the phospholipid concentration appeared to increase during storage, that increase was not as great in the presence of DEHP as in the storage without DEHP.
A number of other groups were involved as well from the New York Blood Center. Some essentially dose response studies. Whether you looked at plasma hemoglobin or osmotic fragility, that the change that was seen over storage was less in the presence of DEHP. And as you increase the amount of DEHP, there appeared to be more beneficial effect there. So the more you put into these red cells, the more plasticized they became, if you would, the happier they appeared to be during storage. Indeed, here is a dose response curve done in parts per million showing that the greater the concentration of DEHP to which the red cells were exposed, the lower the hemolysis during storage.
This prompted us to conduct an in vivo study. These were all interesting in vitro phenomenon, but did they have any bearing to what was going on in the patient. This study, actually conducted back when I was a fellow here, was interesting to review again. We took whole blood from normal subjects and stored it in PVC plasticized with TEHTM, a so-called non-leachable plasticizer paired with the same individuals storing their whole blood in DEHP plasticized plastic. In another arm of the study, these same subjects just stored their whole blood in glass or glass to which DEHP was added. These glass containers to which DEHP were added were glass bottles. We had to manufacture the CPDA-1 outside of any plastic containers to make sure that we did not have any DEHP contaminating the system through the anticoagulant. And then weekly, DEHP was mixed with an aliquot of autologous plasma that had previously been stored frozen. The DEHP was solubilized in the plasma. A measured amount of that plasma, in order to deliver the appropriate amount of plasticizer, was added on a weekly basis to those glass bottles to mimic the accumulation during storage of DEHP.
Another study performed later looked at red cells, where again in a paired fashion subjects stored their red cells in either non-DEHP plasticized plastic or with DEHP. In all of these three sets of studies, the 24-hour recovery of radio-labeled red cells at the end of a 35-day storage period was better in the presence of DEHP than when it was not included in the formulation. Some of these differences are indeed clinically significantly potentially as well as all being statistically significant.
The difference in the curves appeared in the first few minutes. If you look at the T50 of the disappearance of the radio-labeled red cell, there is a marked difference in the cures just in the first 10 minutes. The difference appeared to decline after that. So the primary difference was immediate clearance of the red cells, which was greater without the presence of plasticizers in the bag.
This is shown here that from about the -- after the first few minutes clearly there was a flattening out of these curves. And between the 60-minute and 24-hour points, the curves were almost parallel. So the difference might be attributed -- I say might, we didn't actually look at this -- to increase rigidity or some other physical factor which led to earlier removal of the non-plasticized stored, non-DEHP-stored red cells.
If you then calculate this out to 24 hours, assuming approximately the same long-term survival -- which is standard in blood banking to assume that if red cells survive the initial time period, they will probably have a normal life span, you would predict that there is a 17 percent difference in red cell availability, which is really attributable to this difference in what is occurring very rapidly after transfusion.
What exactly is going on here? This answer has never been defined Maybe that there is better preservation of phospholipid asymmetry, which is important in preventing microvesicle formation or which is associated with reduced microvesicle formation . It may be the plasticizers in some way interacting with the red cell cytoskeleton to counteract any effects of oxidation or detachment of the cytoskeleton, which would also lead to increased microvesicle formation. It may be that there is less availability of divalent cations, particularly calcium, to interact with the red cell member -- again, to cause effusion of these little microvesicle buds which can form.
So exactly what is going on here, we are not certain, but it does appear that there is some relationship between the presence of DEHP and the membrane directly.
Well, if DEHP confers some benefits to red cell storage but there are some risks associated with its use, is there something else that we can do? Could we use less of it? Is there some other plasticizer that could be used? A reduction in dose would appear ultimately to be problematic. Not only would the bags become stiffer and potential for breakage increase during component production, but the dose response curves from the New York Blood Center studies would suggest that we could get to a point where we would not see the benefits that we had become accustomed to.
What about switching to a system in which there is less plasma? That is indeed what happened about this same time, where we switched from using whole blood primarily or packed cells to additive systems. And indeed, when you go from a whole blood system to an additive system, there is less accumulation of DEHP during storage. That may well be because there is just less plasma there and much of the DEHP is solubilized in the plasma. However, interestingly, although the total amount of DEHP is lower in an additive system unit, there is more actually in the red cells. It may be that there is less competition from proteins in the supernatant and the fluid surrounding the red cells and more of the DEHP is able to get to the red cell, where it is actually providing some benefit. We are not aware of any benefit of the DEHP being dissolved in the plasma. This is entirely conjecture. We don't know this for a fact. But it is interesting that we were able to accomplish a switch to an additive system which does provide better storage of red cells and longer storage of red cells than a whole blood or a packed cell system, and possibly this is part of the reason that it does so.
Now as Paul mentioned in the previous talk, we do have other plasticizers available, and the butyryl-n-trihexyl citrate plasticizer, BTHC, which is part of 2209, has been available in the United States. Certainly, you can get 35 or 42-day storage of red cells with the appropriate anticoagulant. There appears to be no demonstrable difference between the 2209 and 146 plastic bag storage of red cells. How can that be if this is not a plasticizer that is doing the same things as DEHP. It clearly is not seemingly doing anything inside the red cell. These metabolic parameters are the same as one would expect with DEHP. The hemolysis is the same as one would expect with DEHP, and the recovery is about the same. Is there something else going on? That has never been finely determined. But it does appear that there is at least one plasticizer which does the same thing or provides the same environment for red cell storage that DEHP does.
We have some problems with this plasticizer, as was mentioned. It does have -- the bags do have what some regard to be an objectional odor. There is an increased cost and it does have increased oxygen permeability, which is not necessarily a down side for red cells, per se, but it is just a different characteristic, and blood bankers did have to get used to having blood bags that were bright red as opposed to darker red with the use of 2209.
The question of which plastic to use is one which I think others will be talking about later. But PDC, plasticized with DEHP, is one that we have come to know and learn how to use very well in blood banking because of a number of very positive characteristics. These same characteristics are not present in other plastics that are available to us. So it does appear that the polyvinyl chloride family is one that we have been able to use successfully over the last three decades. The question of which plasticizer should be in that polyvinylchloride is another issue. Clearly, the DEHP which has been there for the last several decades is providing a benefit for red cells, and we cannot immediately remove DEHP and replace just any other plasticizer or use a non-leachable plasticizer. Because the red cell storage characteristic will indeed change, and we will not be able to store red cells for as long as we have in the past or with as good an outcome after transfusion.
So we clearly have benefits associated with DEHP. We have risks that are toxicologic in nature. The Alternatives are not perfect and I look forward to today's discussions to determine where we should go next. Thank you very much.
DR. MONDORO: Thank you, Dr. Aubuchon. Our next talk is the final talk in this session on plastic blood bags, and it will be given by Dr. Edward Snyder. Dr. Snyder is a professor of laboratory medicine at Yale University Medical School and Director of the Blood Bank aphoresis service at Yale New Haven Hospital. I would also ask at the end of Dr. Snyder's talk if all three speakers could be seated at the panel table so that we can have a short question and answer period after that.
DR. SNYDER: I'm talking about platelets. This -- my talk is about plasticizers and platelets. For several years, there have been a variety of alternative plastics available for platelet storage. What I am going to do is to go through in my usual rapid flicker-fusion type of approach to try to cover as much data as I can, the purpose of which is to show the industry and the public that the blood bank community has available several different types of plastics and plasticizers which are shown to appropriately store platelets, and I wanted to provide some of that data and put some of this in perspective.
This is a picture of a platelet. What we are concerned about is not only all the biochemistry inside the platelet, but what effects there are on the membrane. There does not appear to be same effect on platelet membranes as there is on red cell membranes. That is, it has not been shown that DEHP has a beneficial effect on platelets and platelet survival.
This is just electron micrograph showing similar kinds of things. We are concerned about not only attachment to receptors in the membrane, but also the release reaction whereby the various hemostatically active materials in the alpha granules and also the dense bodies, ADP and serotonin, can get to the outside by merging with the surface collecting system, which although it looks like a vacuole actually is an evagination of the membrane. The whole purpose of platelet storage is to collect the platelet, store it in a plastic bag, and then have it function during transfusion as well as it would if it were a fresh platelet.
Platelet storage bag suitability has a variety of characteristics that have to be evaluated. It has to have acceptable O2 and CO2 gas exchange, which is critical. The pH should be above 6.0 at the end of the storage period. Right now in the CFR, it is 6.0. But in new guidance that has been let out to the industry for comment, the suggestion was made by the FDA that this should be raised to 6.2, which people working in the field applaud, because 6.0 is too low.
In vitro characteristics need to be measured. Radio-labeled in-vivo characteristics are also evaluable. In-vivo post-transfusion corrected count increments, although corrected count increments are falling somewhat into disfavor but I think they are still useful. And possibly hemostatic efficacy. So any changes that might occur as a result of this or other meetings where different plastics or plasticizers would need to be used, we have the tools to evaluate how platelets would store and whether the changes are acceptable.
The platelet assays are myriad. This is just a Whitman's Sampler of some of the major ones. There are other slides from the BEST Committee which have about 45 or 50 different tests. The fact that there are so many implies that there is no one test which gives you an in vitro evaluation of how platelets function in vivo. To do that, you still need to do radio-labeled survivals and patient transfusion studies. So any data that shows an in vitro change would have to be modified by saying, well, that is great, but what is the radio-labeled survival study show in normal volunteers and what does it do in patients once the cells are infused or the platelets are infused.
More than the plasticizer have an effect on the platelet, the plasticizer's main effect in my opinion is on the ability of gas exchange to occur in the bag. PVC is a vapor barrier. It is a solid plastic. In order to make it flexible and malleable, plasticizers are added, and that changes gas exchange properties. And any other changes in any other kind of plastic, be it polyolefin or any other kind of plasticizer, alters gas exchange. And for platelets, that is the key. It is not the plasticizer having a good or bad effect necessarily as much as it is gas exchange, which has to occur across this container. If enough oxygen comes in for the number of platelets in the bag, aerobic metabolism through the Krebs cycle will occur resulting in CO2 being produced, which can diffuse out of the bag maintaining proper pH. If there is insufficient oxygen because you have a bag that cannot have good gas exchange or there is too many platelets for the gas, glycolysis will occur through the Embden Meyerhoff pathway with lactic acid. Eventually the bicarbonate will be used up, the pH will fall, and the platelets will die. So the plasticizer's effect mainly, in my opinion, is for gas exchange across the wall.
The key thing is for the mitochondria to function. That is where the Krebs cycle occurs, and if you have healthy mitochondria spewing out little green balls, everything is fine. If they switch to bad red balls or you have bad mitochondria because of lack of oxygen, the platelets will not store well. That is what needs to be evaluated. The trouble is there are not a lot of mitochondria in platelets. This is a slide from our lab where platelets were stained with JC-1, which lights up mitochondria. And then they were false stained with red to show the outside of the platelet. There is about four to five mitochondria in a platelet, as opposed to brain cells, which have hundreds of mitochondria. So what you are looking at is basically you can actually count the mitochondria in some of these. There is not very many. So any damage to the platelet that occurs from hypoxic storage would result in the potential death of the platelet.
Now plastic bag storage variables -- and I refer you to a excellent paper written by Raleigh Carmen in Transfusion Medicine Reviews in 1993, where he discusses the types of variables. Plastic sheet, and therefore bag wall thickness, surface area, type of plastic, type of plasticizer, amount of plasticizer, and permeability of the label all relate to gas exchange for platelet storage. And Raleigh and his group certainly have done a tremendous amount of work. These are slides taken from that paper, and it lists a variety of manufacturers and plastics, some of which are not around -- some of the companies are not around. Basically, there is polyvinylchloride, which was mentioned, as a solid plastic, DEHP, which allows it to be malleable and flexible. There is the trimeletate plasticizers. Baxter had a PL732 polyolefin bag without a plasticizer and without PVC. And since this slide has been made, there have been the citrate-based plasticizers and several other types of bag, ethyl vinyl acetate and so forth. And as we get into the age of pathogen and activation as yet another net for safety of the blood supply, one would have to evaluate bags that are permeable to various types of light to see whether they would be acceptable for use in various types of photoinactivation technologies.
Now, again, from Dr. Carmen's paper, various bags which are Baxter bags and Cutter bags and Terumo bags showing oxygen transfer rate. As you can see, the PL146, which was the early plastic PVC with DEHP, only had 4 micromoles per hour. This is a Terumo bag, which is also PVC with DEHP, but it is a thinner bag and it has some other changes to allow better gas exchange. So there are ways of working around that. And then there were - these are the trimeletate plasticizers. PL1240 is also trimeletate. This is the polyolefin bag, which was the winner at that time that this was done.
This is a slide which I did obtain from Baxter showing oxygen permeability. This is PL146, which has the DEHP and the PVC. This is a trimeletate bag. This is a citrate-based plasticizer. Here is the polyolefin. And this is another bag which is also -- it is a different type of bag that doesn't seem to have a plasticizer, PL2410. Here is yet another bag, 3014, which is a bag that has a very high amount of citrate. You really need a score card to be able to keep these in mind. But the comment that Jim Aubuchon said, the more oxygen that comes in a platelet bag, at least for now, the better. It allows you to store platelets for longer periods of time. There may be a point where oxygen toxicity may occur, but I don't know if we know anything about where that would be. And if oxygen can diffuse in, CO2 needs to be able to diffuse out, and this is a similar type of bag. Again, this slide was obtained courtesy of Baxter.
Now this is a slide again from Dr. Carmen's paper showing oxygen transfer based on the amount of trimeletate plasticizer, which does leach out into plasma, but not to the same degree that DEHP does. And as the plasticizer content increases, it is as if you are making more pores in the bag and more oxygen can diffuse in and CO2 out. So, again, this was one of the comments, that the ability of a bag is necessarily based on the plasticizer, only it is the thickness of the bag and the amount of plasticizer content, and this shows this very nicely.
Now 2-DEHP, and a lot of this was shown in this classic paper by Rubin and Ness, that it is 30 to 40 percent by weight and it does migrate into plasma. DEHP, however, has been associated with some decreased platelet function in vitro. Acute toxicity is low and many other types of bags exist.
A paper by Labow in Transfusions showed that there was no specific binding site on platelet membranes for DEHP, but clearly it does bind to the membrane. About 95 percent binds to the membrane and 5 percent is in the cytosol, and it migrates into the plasma and sets up an equilibrium. If you do an SDS gel, you will see the DEHP migrating in the front of the dye as a lipid would. And the membrane bound to platelets is proportional to the amount in plasma, which you would expect. And the actual data shows that looking at the platelets over here, you can get
-- in two days, you can get 19 mg/100 ml and certainly lots more, as has been reported.
Interestingly, there is a higher concentration in the platelet pellet, 37 mg/dl, as opposed to the platelet-poor plasma, only 16. But the amount recovered is much lower in the PC because there is so much more plasma than there are platelets. So the percentage of binding is greater in the plasma, although it is concentrated in the platelet. And a 5 to 10 unit pool, as Jaeger and Rubin commented on, could give you well over 114 mg of DEHP.
This is a paper by Dr. Ishikawa, where he used what is called the glow discharge technique. He took a PVC DEHP bag and treated it with radio frequency to form cross links and prevent the migration of DEHP, which was the glow discharge technology. I am not more familiar with it than that. DEHP in micrograms per ml, this is storage period. And though the control bag was leaking DEHP in its usual fashion, the glow discharge treated bag did not leach DEHP very much. So here is another possible technology. I don't know how proprietary it is, but there are ways of using the bag without necessarily having it leach in. What effect it would have on a variety of characteristics other than platelets, I am not sure.
This was a paper, again by Labow, where they showed -- they validated that most of the -- this is a percent of C-14 DEHP. The majority of it was in the supernatant and less in the pellet, although the pellet had a higher concentration. This is percent and since there is more plasma, the number was higher in the supernatant plasma.
This is a paper by Ishikawa which shows that if you took DEHP and you incubated it with platelets over time, over 18 hours, this is the change in the ADP-induced aggregation of the DEHP-treated versus a control without DEHP. And at two hours, there was no change. The various bars show increasing concentration. This is 100, 300, and then 500 micrograms per ml. And over time of storage and with increasing concentration, the amount of ADP aggregation decreased. Now what does that mean? Well, it would mean a lot if it also meant that the platelets didn't survive very well. What it meant is that the aggregation dropped, so it dropped from 100 percent down to 60 percent. Does that give you a platelet that will still correct a bleeding time and stop somebody from bleeding despite the fact that it is somewhat less? We see aggregation studies all the time during regular storage in all kinds of bags that do drop. So I was not as impressed with this. But it still, nevertheless, points to the fact that in some in vitro systems, you can show an adverse effect of DEHP, although not a fatal flaw, if you will.
This is another paper by Ishikawa in 1984, which shows no effect of glow discharge where the DEHP would not leach versus a control bag where the DEHP would leach on pH. But here it shows that in control bag or in a bag exposed with the methanol vehicle, there was no change in hypotonic shock response. Whereas as you use increasing amounts of DEHP, either 150 or 300, you get a drop off in the hypotonic shock response in platelets over or up to about 20 hours. We see a drop off in hypotonic shock response with platelets that are stored in polyolefin bags with no plasticizer as well. These platelets correct bleeding times. They give good corrected count increments. And not damning, but again some evidence that DEHP seems to have an adverse effect.
However, Bob Valeri, as he has always want to do, published 10 years earlier that he didn't find any changes. He stored platelets with DEHP, millimoles as opposed to micrograms, and showed that for aggregation, there was no change with collagen, ADP or epinephrine, whether the platelets were stored fresh or with varying amounts of DEHP. You could say, well, it needed to be incubated for longer period of time and perhaps so. But he, at least, found data that there wasn't a change. And also effects of addition of DEHP on platelet aggregation to epinephrine one micromolar. Again, no change with increasing doses of DEHP versus a fresh control. So you can pick whichever study you wish.
Other studies have shown that when platelets store, they undergo the release reaction and you get a variety of microvesicles and platelet debris and pseudopods, and there is a whole scoring system that was developed. Dr. Fratantoni pointed out that platelets that are stored in polyolefin bags, however, have in addition to the kinds of pseudopod formation and so forth, as you can see in B, C, D, E, F and G, which is this paper by Labow in 1986, you see holly forms and ring forms and a variety of bizarre unclassifiable shapes. Dr. Fratantoni raised this as a question. This was data that was repeated by Labow and refers to Dr. Fratantoni's work. We don't know what this means. These were in the polyolefin bag. The survivals were acceptable. Corrected count increments were good. So what does this mean? It is not sure. Was it a lack of plasticizer? Is it oxygen? Is it something else in the polyolefin bag? Like Dr. AuBuchon mentioned, there were other things that occupied our attention and we never really pursued this. If it turns out that polyolefin becomes a much more important issue, we would need to go back and look at this again. But we do have some information. We are not at square on. We are at square two or three.
This is a paper by Valeri again. I apologize for not putting the name in. But this was a xerox of a xerox done at the last minute before I left when I just found this paper. But this is Dr. Valeri's paper. This shows the percent of infused radioactivity, which is the radio-labeled recovery, and this is survival in days. For platelets that either are fresh or stored in DEHP plastic for 24, 48 or 72 hours. And this is believed to show that the recovery of fresh platelets is about 65 percent here. It goes down to -- this is the mean and the standard error of the mean bracketing it. About 50 percent, about 40 percent, and about 30 percent as the platelets store for up to three days. This is about what we see. We see about 40 percent plus or minus for platelets stored in any kind of a bag at about day five. That is pretty much what we see. Whether this is a plasticizer effect, unlikely. Because 732 bags give you the same results and it doesn't have a plasticizer. So when you do these studies, you have to compare storage and the storage lesion changes with what plasticizer effects might be. Regardless, all the platelets seem to have a survival of about 7 to 8 days, which would imply that of the surviving platelets -- and this is at time zero -- whatever platelets are left right after infusion in an autologous survival model, they do survive the same length of time as fresh platelets would. So you get less recovery, but the platelets that do survive and are not damaged do circulate.
Now this was a paper by Hogge, et al. in Transfusion, which looked at corrected count increments in fresh platelets versus platelets stored after three days or seven days. And what they found was that the corrected count increment in fresh platelets after one hour was 20,000, but after three days of storage in either polyvinylchloride or 7 days in a trimeletate plasticizer, you had the same result of 10,000 to 12,000. There was no difference between these two, but there was a difference between fresh. We know fresh platelets is an anachronism. We don't have that anymore. It is merely for information. The point is that whatever changes occur, it occurs relatively frequently in the PVC bag and also in the trimeletate bag by day 7, but it doesn't seem to get any worse. So this bag is PVC with a trimeletate plasticizer, showing that we can take DEHP out and still have the same type of responses that we get. In fact, we don't use PVC with DEHP in this kind of a bag any more. Terumo does, but again they have modified it so it has better oxygen characteristics. And the 24-hour gives you the same thing at a slightly different level. So we do have ways of evaluating changes in plastics.
This is the paper by Valeri, and all of Valeri's data came from this Environmental Health Perspectives, 1973, Volume 3, page 103. What he did
-- and again, I apologize. I was trying to show you the slopes, which was all I was really interested in. This was platelets that were stored with about 20 mg/dl of DEHP, and this had about 35. This is a polyolefin plastic with very minimal DEHP, less than 1 percent. What he did was he looked at bleeding times. He gave a normal volunteer -- and it is the same volunteer in all the panels -- aspirin over here, and then he let the control go. The control is over here showing that the bleeding time went from normal up to about 12 to 14 or 16 -- it is hard for me to see -- and came down over four days to this level. The same thing here -- aspirin, control and the bleeding time goes down. When he gave platelets that were plasticized with DEHP with about 20 mg, he found that after 24 hours the bleeding time corrected after transfusion. With the polyolefin, it also corrected somewhat better. And with DEHP that had 35 mg, again the bleeding time corrected.
So what was the difference with all of the Ishikawa information showing that the aggregation studies were impaired? Well, it may be impaired but an in vivo assay, which is the bottom line as it were, didn't seem to show in Valeri's work a problem. It corrected bleeding times whether there was DEHP, either in relatively low or higher amounts, or no DEHP. They still seemed to work. In fact, he pointed out that this was the only one at two hours that actually improved the bleeding time down to about -- I think it says 8 minutes from about 14 or so after two hours, whereas without the plasticizer, it actually took longer to get the correction.
So, again, is it helping or not? It appears that it doesn't seem to have a problem in vivo, even though in vitro it might.
Other things to be considered was a paper we published many years ago looking at 1240, which is a trimeletate plasticizer from Baxter comparing it with the trimeletate Cutter product, and this is the polyolefin. We did radio-labeled survivals in normal volunteers for platelets stored on an elliptical 1 rpm rotator, a circular 2 rpm, a circular 5 rpm, or an elliptical 6 rpm. And this had to do with the sheer stress. What we found -- this is the mean, and again it is about 40 percent recovery is what you get after five days of storage and one standard deviation. The one that lost was the PL-732 bag with the 6 rpm elliptical rotator. This is the end. These are not days of storage down here. So what that meant was that some plasticizers or lack of plasticizer with certain types of sheer stress associated with an elliptical rotator may give you unacceptable characteristics. There is no gold standard for platelets like there is a red standard, if you will, for red cells, where you need 75 percent survival 24 hours after infusion on the last day of storage to get an acceptable red cell. For platelets, however, most people consider 40 percent recovery plus or minus one standard deviation to be a reasonable number. But the 732 and the 6 rpm elliptical rotator failed to meet that standard. All the other ones did. This was similar to -- the multiple hit survivals showed that the survivals were roughly the same regardless of the type of rotator, which was shown by the other study that was done by Valeri years ago, again, as is always the case, that those that survived circulated normally, even though fewer may have.
This classic paper by Dr. Scott Murphy and others, which basically showed that -- and this was published shortly before ours was -- this PL-732 on an elliptical rotator had an in vivo recovery of less than 40 percent, again this semi-magic number, whereas those on a tumbler did very well. Which is why we no longer -- we do not store PL-732 on elliptical rotators. In fact, not many people use 732 very much because other bags are being used. But this kind of work shows that maybe the plasticizer in conjunction with sheer stress or the lack of plasticizer had some effect. And this would need to be looked at again further.
So the last couple of slides. Patient transfusion studies. Trimeletate plasticizer with PVC or polyolefin, looking at corrected count increments. The increments, 46,000 with the trimeletate and 58,000 with trimeletate, and 63,000 in comparable patients getting the polyolefin PL-732 without plasticizer. Corrected count increments were all in the same range. So what this shows is, again, despite in vitro studies, which may show some problems with PVC or with other types of things -- these are the trimeletates -- without a plasticizer in the polyolefin bag, you get good corrected count increments, and in vivo it appears to be acceptable.
So what are the final things we need to look at? Again, we refer to Dr. Carmen's paper. If we are going to, as a result of this conference, store platelets in some other type of bag, what the manufacturers will need to work with the public and to some degree the industry, that is, the laboratories that evaluate this, is flexibility, so that they can fill and transfer. Temperature resistance is required so you can store them in frozen red cells or frozen plasma. The strength is required for centrifugation. Whatever new combination would have to have safety and compatibility. Various manufacturing issues, which we may here from from the manufacturers. Dr. Carmen is in the audience. And we have the ability to evaluate this and we will do it by in vitro analysis, radio-labeled survival studies, and eventually in vivo patient transfusion studies. So we have the capabilities to evaluate this. And from my perspective, we could lose PVC and we could lose the DEHP and platelets would survive very nicely in other types of bags available. The question is, are we trading the devil we know for the devil we don't know? Thank you.
DR. MONDORO: I'd like to thank the speakers very much for getting us focused on blood bags before we get into any other issues. We do have time for a short question and answer period if anyone would like to come to the microphone. I would like to remind you to state your name and affiliation for the record. Thank you.
PARTICIPANT: Herb Cullis, American Fluoroseal Corporation of Gaithersburg. I want to add to Dr. Snyder's comments that in 1998 and 1999, an additional plastic fluoroethylenepropyline was evaluated by the Phorcenias Corporation and eventually obtained approval for the storage of platelets in the United States. It has ten times the oxygen transport of PVC plastics and six times the CO2 transport and was found to be able to support platelets at twice the concentration of the 732 plastics.
CHAIRMAN VOSTAL: Vostal, FDA. Dr. Aubuchon, those survival studies were, I think, 35-day red cell storage. Does the DEHP beneficial effect hold up in 42-day stored red cells?
DR. AUBUCHON: I have not seen a study comparing storage of red cells in an additive system of 42 days with and without DEHP. I would think they would. I would predict that you would see the difference and I would think that red cells would not be able to be stored without DEHP for that time period, but I have not actually seen the exact comparison. Certainly at 35 days, one is not able to store red cells to meet the 75 percent criterion of 24-hour recovery without DEHP, and I don't think we would have much hope unless there is another approach, such as with the citrate plasticizer.
DR. MONDORO: I have one question for all of you if you would like to comment. One of Dr. Snyder's last point was that of temperature, and I was wondering how DEHP stacks up against alternative plasticizers with regard to the colder frozen storage of blood components as far as thawing. Is there any one that is better or has that been -- have temperature effects been studied?
DR. SNYDER: I don't know that much about it, which of course has never stopped me from commenting in the past. But I think there is the concept of a glass transition phase in a plastic, and I do believe that some of the non-DEHP plasticized bags have better glass transition characteristics. Because that has been a problem with breakage of fresh frozen plasma, as you might imagine, during storage. So I think there are some that have improved characteristics, and that is not a major problem. If I am incorrect on this, somebody please correct me.
DR. MONDORO: Please come to the microphone, yes.
PARTICIPANT: Bob Rubin, Johns Hopkins University. I particularly liked the way the topic was introduced, I think it was by Dr. Snyder, about depending on your perspective, we've either got the hero or the villain here with DEHP. Now a large part of the evidence on the toxicity of DEHP is going to depend on in vitro studies. And I would like to emphasize this point about such studies. Some of it was reflected in these early talks. Maybe we will see more on the toxicity or toxicology presentations. And that is the nature of the solubilization of the DEHP. Now I think Dr. AuBuchon had some data where he used sort of natural solubilization. You use a system of having a subset of plasma that you added DEHP to. Dr. Snyder, you had some data that as near as I could see used methanol as a solubilizing agent. In the Ishikawa studies, I don't think I picked up exactly how it was solubilized.
My comment, bottom line, and I would like to hear comments from the group, is the nature of the solubilization of DEHP. There are a number of critical examples where we can demonstrate either a positive effect or a negative effect of DEHP, depending on how it is solubilized. And we should keep that in mind in designing any further experiments.
DR. SNYDER: The Ishikawa also used methanol, I believe, as well.
PARTICIPANT: (Bob Rubin) If I can just follow that up and point out the major difference. Again, it may be most important in toxicology. It is using naturally solubilized DEHP, we were able to show this shocked lung or acute respiratory distress syndrome in experimental animals. In Baxter's solubilized DEHP in ethanol, not methanol, they were not able to reproduce that effect. That is the key one that I would be concerned about.
DR. SNYDER: One of the things I think we have to be cognizant of is not only the experimental conditions for bags that are being stored, but also the effect of other external attributes, if you will, such as gamma radiation, ultraviolet radiation, effects of freezing and thawing, and even physical shaking and so forth. So when these studies are designed for future plastics, all of these various iterations and permutations would need to be taken into account, which leads you to a branch chain that can be quite labor intensive and expensive. But I think that is the challenge for the industry and for the community.
DR. AUBUCHON: Even such seemingly mundane issues as the ability to adhere a label to a plastic as it is being frozen and thawed in a waterbed.
DR. MONDORO: Any more questions or comments? Dr. Ness?
DR. NESS: Yes, I had a question actually for Dr. AuBuchon. The data you showed implied that some of the effect of DEHP in terms of red cell storage is really immediate, which led me to wonder whether anybody has looked at storing or collecting red cells in the DEHP media and then transferring them to a non-plasticized bag to see if the effect is maintained without the leaching from the bag during the storage.
DR. AUBUCHON: All of the studies that have been reported, sort of mixed media studies, have been the other way around, where the red cells have been stored without DEHP, as you saw from the work of Tim Estep. I am not aware of anyone who has attempted that. Clearly, Gail Rock has shown that DEHP is picked up very quickly from a plastic bag. But whether over time the DEHP might diffuse to other components and the effective concentration within the red cell membrane might be inadequate to achieve these effects over time is unknown.
DR. MONDORO: We will take one last comment from Dr. Snyder.
DR. SNYDER: Yes. I would be interested as the day goes on to hear from the representatives of the pediatric community. Some of our pediatricians, for example, are still reluctant to use additive solution red cells because they are concerned about the adsol lo all these many years. So the idea of changing different plastics and plasticizers as far as the pediatric and the neonatal group, I think their comments would be extremely important in this regard.
DR. MONDORO: Thank you very much. I would like to thank the speakers. You will be seeing them on our panel at the end of the day. As I said, we have now focused your attention onto blood bags, the focus of the workshop, and our next session is going to be a more general -- of more general interest and that will be chaired by Ron Brown.
MR. BROWN: Good morning. My name is Ron Brown. I am a toxicologist at the FDA Center for Devices and Radiological Health. As we heard in the first session, the use of DEHP as a plasticizer for blood bags clearly confers some benefits, particularly when we are talking about red blood cell storage. However, as each of these speakers this morning has eluded to, exposure of experimental animals to DEHP has been shown to have adverse or toxic effects. Those are the effects that we would like to focus on here.
I was struck by a comment that Dr. Ness had in his opening comments, particularly that some colleagues had expressed to him surprise that we thought the DEHP issue had been addressed already. I think partially that is a function of sort of the biphasic nature in which the literature has been developed. Certainly, there was considerable interest in the 1970's, largely to the work of Dr. Rubin and his colleagues, with the pioneering work on DEHP toxicity. And then it appeared in the 1980's that there was a bit of a lull in terms of the research effort that had gone on. Clearly in the past several years, there has been an explosion of research on DEHP toxicity, and we are fortunate that we will have a number of speakers that will describe some of that research for us.
What I would like to do is to let you know that we have reordered the order of speakers in this session to allow the talks to flow more logically from one to the other. First, we are going to hear from Dr. Bucher, who is going to describe the rodent carcinogenicity studies. Then we will hear from Dr. Cunningham, who will describe the mechanisms of toxicity and carcinogenicity, particularly as they relate to the rodent studies. Then we will have a short break followed by Dr. Chapin, who will discuss the reproductive toxicity of DEHP. Then we will hear from Dr. Karle, who will discuss her recent study particularly, but in general pediatric effects of exposure to DEHP, and whether or not children and neonates represent a sensitive subpopulation. I will sort of have a catch-all talk trying to pick up on endpoints that the previous speakers had not addressed, looking at other effects produced following IV exposure to DEHP. And finally, we will hear from Dr. Ray David from Eastman Kodak on some work that the chemical industry has sponsored.
So let me introduce Dr. Bucher as our first speaker. Dr. Bucher is the Deputy Director of the Environmental Toxicology Program at the National Institute of Environmental Health Sciences, with particular expertise in the conduct of rodent carcinogenicity studies.
DR. BUCHER: Thank you. I just walked in and discovered that we had reordered the talks. That is okay. I would like to thank Bob Chapin for running my overheads here.
I was asked to address some of the issues related to the rodent carcinogenicity studies of DEHP. There is a fairly long history of rodent studies with DEHP. There were three studies that were performed before 1982 that were considered to be inadequate evaluations by IARC when they last looked at DEHP.
The first positive studies of DEHP were the National Toxicology Program studies reported in 1982. These were of standard designs using Fisher rats and B6C3F1 mice receiving diets of up to 12,000 ppm's for rats or 6,000 ppm's for mice for 103 weeks. The doses for these studies were selected based on 13-week studies using dietary concentrations much higher or higher than that, up to 25,000 ppm's for rats and 12,500 for mice. In rats, the only real effect that limited the dose used in the chronic study was an unacceptable body weight gain at 25,000 ppm. There was also testicular atrophy seen in the 13-week studies in males at 12,500, but was not considered to be -- would not be considered to have an impact on the chronic study. For mice, body weight gains were variable at 1,600 parts per million and higher concentrations, but they lacked a dose response.
In the rat study, as I said, the doses went up to 12,000 ppm's. Body weights at 6,000 and 12,000 ppm groups were less than controls in males and were also somewhat less than controls in females at the top dose only. Survival was pretty good in both studies, and there was, in terms of neoplastic effects, not a lot of liver effects. But there was an increase in clear cell cytoplasmic change, a slight increase in males. There was the expected testes degeneration and atrophy, especially at the top dose in males, and there was probably a related effect to this. The anterior pituitary hypertrophy probably reflecting an increased need for LH release from the anterior pituitary given the loss of testosterone feedback on the anterior pituitary.
In terms of chronic neoplastic effects in the NTP rat study, there was a modest increase in neoplastic nodules in males and females. This was statistically significant in females with a trend. There was an increase in hepatocellular carcinoma in both sexes and the combined incidence of neoplastic nodules and hepatocellular carcinomas was increased and showed a dose response in both males and females.
At this time, the NTP declared the studies either positive or negative, and there were not the levels of evidence that we use today. These two studies were considered positive for liver tumor effects.
There were also decreases in neoplasms. There was a decrease in anterior pituitary neoplasms in males. There was a decrease in testicular interstitial cell tumors in males. And there was a decrease in mammary gland fibroadenomas in females.
In the mouse study, as I indicated the doses went up to 6,000 ppm in the feed. This was half the doses that were given to the rats in terms of dietary concentration. The 3,000 and 6,000 ppm groups had a slightly lower body weight gain than the controls in males and a little more of a body weight decrease when compared to controls in female groups in mice. Survival, again, was not too bad and not affected by treatment. In terms of non-neoplastic effects, there was an increase in testes degeneration and atrophy, although this was very slight.
The two-year study findings -- neoplastic findings in mice included a slight increase in hepatocellular adenomas in males, a mid-dose effect in females. There was more of a marked effect on hepatocellular carcinoma in both males and females, and the combined tumor rates were increased in a dose-related fashion in males and females. Both of these studies, the male and female studies, were considered positive for carcinogenicity. And there were no decreases in neoplasms in this particular study, the mouse study.
After the 1982 studies, there were a couple of confirmatory smaller studies that were performed. Rao, et al., found an increase in hepatocellular neoplasms -- he found hepatocellular neoplasms in 11 of 14 male Fisher rats fed diets at 20,000 ppm DEHP. This is higher than the NTP doses. And that was compared to a rate of 10 percent in controls. Also at CIIT, Cattley and Popp, et al., found tumors in 6 of 20 Fisher rats, these were liver tumors, given diets containing 12,000 ppm DEHP for two years compared in zero of 18 controls.
There have been a number of more recent studies that have been reported partially. These are studies by Dr. David, who will have a chance to comment on them later. They were reported as abstracts at the SOT meetings in 1996 and 1997. These studies expanded upon the NTP studies by providing lower doses of 100, 500, 2,500 or 12,500 ppm and given to male and female rats for two years. One of their groups received 12,500 ppm for 78 weeks, and some animals were evaluated at this time, and some of that group were held until 104 weeks to look for potential reversibility of liver tumors.
The findings of this study as reported in the abstract were that liver and kidney weights were increased and testes weights decreased at the higher doses. There were hepatocellular carcinomas increased in the 12,500 ppm groups at 78 and 104 weeks and the adenoma incidences were not reported. The NOAEL was reported for carcinogenic potential, and I presume that this includes adenomas and carcinomas, but it was determined to be a NOAEL at 500 ppm for this endpoint. And there was a statement that the tumor incidence dramatically reduced in the recovery group and that is the comparison of the adenoma and carcinoma incidents at 78 weeks as determined in similar groups of animals evaluated at 104 weeks after stopping dosing at 78 weeks.
There was also an increase in mononuclear cell leukemia in dosed males, but this was also accompanied by a low incidence in the control rate.
Eastman Kodak in 1997, I believe, also reported their two-year findings from the B6C3F1 mouse study of DEHP. Again, they used the 6,000 ppm group, which was the high dose used in the NTP study, and they went down from there down to 100 ppm. Also, the same design was used here where the high dose of 6,000 ppm was given for 78 weeks. The dosing was stopped and an attempt of looking at the disappearance or regression of tumors was done at 104 weeks.
In this particular study, liver weights were increased and testes weights decreased at the higher doses. There is a report that hepatocellular carcinoma increased in the 1,500 and 6,000 ppm groups at 78 and 104 weeks. And, again, the adenoma incidences were not reported. The NOAEL for carcinogenic potential was, again, 500 ppm, the same as in the rat study. And the tumor incidence was reduced in the male recovery group at 104 weeks compared to that incidence at 78 weeks, but it was not reduced in the females given that same design. A reduction in liver tumor incidence in sort of a stop-study paradigm has also been seen with some other peroxisome proliferators by other folks.
There have also been some studies where DEHP has been evaluated in hamsters, and these were a quite different design. There were smaller groups of 25 male and female Syrian hamsters receiving 3 grams per kilogram by IV injection on varying weekly schedules for up to 32 weeks. Syrian hamsters were also, by the same group, exposed to air or saturated atmospheres of DEHP for a lifespan and no carcinogenic effects were reported from either study. Both of these routes of administration bypass the gut. Therefore, the presumed MEHP metabolite and 2-ethylhexanol metabolites which are presumed to be more powerful peroxisome proliferators in DEHP would not be formed by either of these routes of administration. So it is not clear from this particular study whether the Syrian hamster is simply less sensitive to the formation of liver tumors than are rats and mice, or if in fact the proximate carcinogens, which would in this case be presumably MEHP or 2-ethylhexanol, were not formed.
There was also a study, a BASF study, reported of the metabolite 2-ethylhexanol. This was a standard design of 50 male and female Fisher rats and B6 mice. The study was done by gavage at 50 up to 500 ppm per kilogram for rats or up to 750 mg/kg for mice for 18 months. These doses were clearly high enough. Body weight deficits and increased mortality were seen at the higher doses. There was no neoplastic response reported for rats and there was no increase in hepatocellular adenoma reported in mice, but the data were not shown in the paper. There was a small increase in hepatocellular carcinomas in females, especially when compared to the historical rate in a 78-week study. Their conclusion was that 2-ethylhexanol is a weak carcinogen in female mice and may account in part for the carcinogenicity of DEHP.
In terms of genetic toxicology, DEHP is considered negative in almost all kinds of studies evaluated. It is negative in salmonella with and without metabolic activation as are the MEHP and 2-ethylhexanol metabolites. It is negative in the mouse lymphoma assay as are the metabolites. It is negative or marginally positive in the Drosophila sex-linked recessive lethal assay. MEHP was negative in this assay. It is negative for hepatocyte or CHO cell DNA single strand breaks and UDS in in vitro studies. It is negative for unscheduled DNA synthesis in the liver in vivo in studies in rats and it is negative for DNA alkylation in rats in vivo.
There are some positive studies looking at chromosomal aberrations or induction of aneuploidy with DEHP or MEHP in fungi and mammalian cells in vitro. It appears to be negative for micronuclei formation in peripheral mouse blood in in vivo studies.
In cell transformation assays with DEHP, it seems to be positive in transformation systems using SHE cells, embryonic mouse fibroblasts, and Fisher rat embryo cells. In a paper that is important for me to mention because it is authored by my scientific director, they compared the various peroxisome proliferators with DEHP and MEHP for their ability to induce morphological transformation, chromosomal aberrations, and peroxisome proliferation in SHE cells, and there was not a clear relationship established between these endpoints. So cell transformation may not follow directly with peroxisome proliferation.
Another group looked at the decrease that DEHP tends to give in GAP junction communication as a means of explaining the DEHP-induced transformation of SHE cells. And while it was decreased slightly, it wasn't considered sufficient to transform those cells.
There have been a number of proposed mechanisms of DEHP carcinogenesis. In most initiation promotion studies, DEHP is not an initiator, but it consistently promotes DEN-initiated altered liver foci and tumors in mice. Peroxisome proliferation is, of course, induced by DEHP metabolites, the MEHP and 2-ethylhexanol, more so in rats and mice than other species, likely through a peroxisome proliferation activated receptor alpha retinoid X receptor activation complex. This is a receptor-mediated activity. It is accompanied by liver enlargement, induction of peroxisome and microsomal fatty acid metabolism and cell turnover in the liver.
DEHP is a moderately potent inducer of peroxisomes when compared with the whole range of chemicals that induce peroxisomes. It has been shown by a number of investigators that peroxisome induction potency does not equal cancer potency. On the other hand, studies that have been done with the PPAR, the peroxisome proliferator activated receptor, in knockout mouse treated with a Wyeth compound 14643, which is a very strong peroxisome proliferator, did not show liver tumors. So that would indicate that there is a strong involvement of the PPAR receptor in the liver tumor response.
More on proposed mechanisms of peroxisome proliferator carcinogenesis. Of course the classic idea is that peroxisome-induced oxidative damage is the cause of proliferation, although DEHP is not a positive initiating agent. It does seem to be a promoting agent. The oxidative damage there is that the peroxisomes induce enzymes that generate hydrogen peroxide more so than they induce enzymes that take care of hydrogen peroxide - catalase and other things like that -- such that there would be oxidative damage to the cell.
Kaufman at UNC and their colleagues have found that if they poison the Kupffer cells in the liver, you do not get hepatocyte proliferation when treated with DEHP. So there is apparently a role for Kupffer cell mediated mitogenic factors in this hepatocellular proliferation.
Cattley and Popp have proposed that the promotion activity of DEHP on basophilic growth foci is stronger than on other liver foci. And it has been proposed by Roberts, et al., that they found that DEHP-treated rodent hepatocytes show an inhibition of apoptosis, and in their hands DEHP stimulates apoptosis in human hepatocytes.
Hayashi, et al., may have found at least partial explanation for the effect on apoptosis. They have found that Poly(ADP-ribose) polymerase is induced by DEHP in rodent hepatocytes. This enzyme apparently has a lot of functions, but one of them there is a requirement that this enzyme decrease for apoptosis to occur. So an induction would be an anti-apoptotic signal. There have also been proposals that the peroxisome proliferator carcinogenesis might be due to altered sex hormone metabolism. You will be hearing much more about the sex hormone effects and reproductive effects later. And there has been a proposal that it reduces serum ceruloplasm and that there might be some involvement of copper toxicity. These are much less well understood.
And I would like to finish up by pointing out that there has also been a nice paper put out recently in Critical Reviews in Toxicology that goes over the extraperoxisomal targets of peroxisome proliferators. There are many, many extra peroxisomal targets and peroxisome proliferators. This isn't necessarily all in relation to DEHP, but there are effects on mitochondria-inducing proliferation and changes in mitochondrial enzyme activities. Succinate dehydrogenase is affected by DEHP. There are changes in microsomal enzyme activity changes in addition to those that are known with cytochrome P4504A system that is induced obviously by the peroxisome proliferators. There are changes in cytosolic enzyme activities. There are changes in hormonal pathways, and there are changes in intracellular ion homeostasis. Calcium ion, for example, is accumulated in hepatocytes treated with peroxisome proliferating agents. And there is an emerging body of evidence that would indicate there is at least the possibility that peroxisome proliferator-induced changes in a cell can lead to changes in signal transduction pathways.
So I would encourage you all to look at this reference if you are interested in alternative explanations for the peroxisome proliferation-driven hepatocyte proliferation mode of action of carcinogenesis of the peroxisome proliferators. Thank you. Any questions?
MR. BROWN: Thank you, Dr. Bucher. As you can imagine, whenever you have a compound that produces a carcinogenic effect in rodents, there may be some significant public health or regulatory implications of those findings. I think these results have prompted a lot of research into the mechanisms by which DEHP exerts this carcinogenic effect. Dr. Bucher described some of them and we are going to hear in a little bit more detail from Dr. Michael Cunningham. Dr. Cunningham is a toxicologist at the National Institute for Environmental Health Sciences. And I think importantly, he is the team leader for the peroxisome proliferation initiative. So we are going to hear more about the mechanisms of DEHP effects.
DR. CUNNINGHAM: Thank you and good morning. I am going to restrict my comments to the mechanisms of the toxicology of phthalate acid esters in rodents and humans comparing and contrasting common features between the two species and especially in relationship to the hepatic peroxisome proliferation and hepatocarcinogenicity.
DEHP belongs to the class of chemicals referred to by Dr. Bucher as peroxisome proliferators. Peroxisome proliferators have generated extensive interest during the last 20 years. This increased interest has come about largely by the reproducible association of the induction of peroxisomes and liver tumor formation in the rodent. Since rodent carcinogenicity is widely used as a factor in assessing human risk, there is intense interest in understanding the biochemical, cellular and molecular basis for this carcinogenic effect.
The fact that peroxisomes are induced by a large number of chemicals of various chemical classes has been used as a common mechanism to understand the basis of carcinogenicity for this class of compounds. Although as Dr. Bucher pointed out, a strict linear relationship between peroxisome proliferation and hepatocarcinogenicity has been difficult to support.
Recent data has provided focus for the hallmark effect in the rodent liver of the peroxisome proliferation phenomenon, which has been shown either not to occur or occur in a very limited extent in the livers of humans. It has also become that chemicals in this class of peroxisome proliferators vary widely in potency for this effect, from parts per million to parts per hundred.
I put this slide up to show the various examples of compounds that have been shown to produce peroxisome proliferation in rodents. Certainly many therapeutic agents that have been in the clinic for a great deal of time and have been proven safe and effective induce peroxisomes in rodents. Steroids, herbicides, and the plasticizers that we are discussing today generally all induce peroxisome proliferation in rodents although fairly weakly compared to some of the therapeutic agents. And certainly there is a whole variety of solvents and industrial chemicals as well as food products and natural products that produce this response.
I hope you can see some of the structures. This is put up for a couple of reasons, one of which is to demonstrate the wide variety of structures that produce peroxisome proliferation from larger therapeutic type agents. Straight chain or halogenated compounds can produce this as well as some endogenous compounds such as arachidonic acid and prostaglandins have also been demonstrated to induce peroxisomes in the rodent liver.
The hallmark structural feature is that the compound has to either posses a carboxylic acid functional group or a metabolite of the compound produce a carboxylic acid functional group such as -- although DEHP does not produce a carboxylic acid group, the MEHP metabolite, which is thought to be the proximal peroxisome proliferating compound, does produce that.
In general sense, the term peroxisome proliferator denotes a drug or a xenobiotic that induces proliferation of the cytoplasmic organelle, the peroxisome. This is an electron photomicrograph of the normal liver. Peroxisomes are constitutive in the normal liver. They are usually identified by their very dark opaque structures on an electron micrograph. Peroxisomes historically had been referred to as microbodies. Those two terms are interchangeable. These microbodies or peroxisomes are single membrane limited cytoplasmic constituents. They appear as a finely granular matrix and are ubiquitous in both plant and animal cells because they function in the intermediate metabolic pathways for the beta oxidation of fatty acids for the homeostasis of lipid metabolism.
Under conditions of peroxisome proliferation, by for instance DEHP, one can see an enormous increase in the number of peroxisomes. You can see the increase in the size as well. It may not be obvious, but the cell is also very much larger. And as Dr. Bucher pointed out, there are actually more cells in the liver. There is a combination both of hypertrophy as well as hyperplasia observed following exposure to a peroxisome proliferating agent.
The biochemical composition of peroxisomes are mainly hydrogen peroxide-generating oxidases as well as catalase, which degrades hydrogen peroxide. Often there is an imbalance in the amount of hydrogen peroxide produced versus the amount of catalase which is present. There is also other oxidases, including alpha hydroxy acid oxidase, D-amino acid oxidase, urate oxidase, isocitrate dehydrogenase, carnintene acetyl transferase, as well as all the enzymes responsible for the beta oxidation of long chain fatty acids.
As a brief caveat, peroxisomes should not be confused with lysosomes, which contain proteolytic enzymes, acid hydrolases. They are very distinct, both in their form as well as their function.
Peroxisome proliferation has been postulated to produce an oxidative stress implicated as a possible mechanism of hepatocarcinogenicity. Peroxisome proliferators are thought to produce secondary genetic toxicity by stimulating the biosynthesis of peroxisomes, which in turn increase all these oxidase enzymes resulting in an increase or over-production of hydrogen peroxide, which is thought to react via the femptin chemistry mechanism to produce hydroxyl radical and may result then in the genetic lesions that are observed and may possibly contribute to the hepatocarcinogenicity, which is very common in long-term exposure to these class of compounds.
I think there is a great deal to learn from the therapeutic peroxisome proliferators, and there has certainly been an enormous amount of work done with those that are used clinically, such as the fibrate hypolipidemic agents as well as the thiazolidinedione anti-diabetic agents. Much of the research that has elucidated common mechanisms has come from studies using those compounds, and I would like to use that data as a parallel for what a generic peroxisome proliferator such as the phthalates might do in rodents and contrast that to what they might do in humans.
I have already discussed all the types. This is the history of peroxisome fatty acid oxidation. You can read it as well as I can. But the point of this slide is that much of this is fairly recent. The toxicity of peroxisome proliferators is an ongoing research effort, and there is still a great deal to be learned, both on the biochemistry as well as on the toxicity of these types of compounds and certainly the relevance of peroxisome proliferation to potential adverse human health effects.
But in general, as Dr. Bucher had pointed out, the mechanism whereby a xenobiotic induces peroxisome proliferation is similar. The peroxisome proliferator in a rodent or a human has to interact with a peroxisome proliferator activated receptor in conjunction with the RXR retinoic acid binding receptor. These two have to simultaneously bind on a response element in the gene in order to effect any transcription. In the rodent, this binding results in peroxisome proliferation. The hypertrophy and hyperplasia that I indicated before, a decrease in apoptosis, and in the rodent ultimately tumorigenesis.
Humans possess the PPAR activated receptor. Again, this is just to reiterate that the peroxisome induces hydrogen peroxide, which may interact with femptin chemistry to produce hydroxyl radical and produce DNA damage via this indirect mechanism. As stated before, there is a variety of other hypotheses, such as increase in lipid peroxidation, which may induce DNA damage by itself or membrane damage that results in lipofuscin deposition that has commonly occurred. Although this is studies for ongoing research, we have very recently generated data in our laboratory that this seems to be the predominant pathway with peroxisome proliferators inducing DNA damage, very much similar to what one would expect a hydroxyl radical type chemistry to produce and probably less likely to be through the lipid peroxidation pathway.
This slide shows the occurrence in humans of the PPAR receptor. The PPAR receptor has several subtypes -- alpha, which is very common in the liver. Let's see, where is the liver? I can't see my own slide unfortunately. It is here. You can see the PPAR alpha content in human liver is quite significant. The PPAR gamma isoform is common in human adipose tissue. There is some reports that the levels of PPAR are significantly lower in humans and that may result in a lower sensitivity to peroxisome proliferators compared to rodents. But they do exist and are significant and are able to activate certain genes. So although they may be in lower amounts, they are certainly still active in human tissue.
There is a differential activation by fibrates which interact mainly with the PPAR alpha subtype, and so they are mainly liver active, whereas the thiazolidinedione anti-diabetic agents are thought to mainly interact with the PPAR gamma isoform and activate transcriptional events in adipose tissue more than in liver. And conversely, the clofibrate type compounds activate transcription in the liver and not in adipose tissue.
This is a schematic then of what is thought to occur upon activation of PPAR with the retinoic acid binding receptor. These bind both in human as well as in rodent at the peroxisome proliferator response element. This is the common feature between rodents and humans. The place where they diverge then is the location of this PPARE response element to induce downstream transcription at different gene products. So even though this is common between rodents and humans, the location of this response element is key to understanding the differences in the types of gene products that are induced between the two species.
The response element has been reported in a number of laboratories either to be similar -- this is the rodent or the rat PPRE -- very similar to the human PPRE in this paper. A more recent paper demonstrated there were possible genetic polymorphisms in humans where there are actual sequence differences in the human PPRE compared to the rodent PPRE. The major common feature is that the human, both from Jan Reddy's lab as well as I think this is Ruth Robert's lab, both localize the human PPRE very much different in the relationship to the ACO co-a-oxidase and the beta oxidation gene. So that these are so far away that this is thought to explain why activation of the PPRE in humans does not result in a transcription of the ACO co-a-oxidase. Whereas in the rodents, it is very much closer and may result in the differences in the induction in the entire peroxisome proliferation response between rodents and humans.
They do have an entirely different set, then, of gene products that humans produce upon activation of the PPAR receptor and stimulation of transcription at the PPRE response element. As you can see in fibrates in the liver or thiazolidinediones in adipose tissue, instead of inducing the peroxisome proliferation response observed in rodents, they induce APO C-III gene products. They increase lipoprotein lipase activity. They increase APO-A-I and II. They both end up having lipolytic activity basically because of the lipoprotein lipase activity, and then they have their effect to decrease the triglyceride component in the plasma. Similarly to what you would see -- the end response is similar to what you would see in a rodent. But in the humans upon activation of the PPAR alpha, the transcription response is entirely different without inducing any of the peroxisome proliferation activity like you see in the rodent.
And finally, just to reiterate that and compare rodents versus humans, this is just in one gene product. Humans and rats basically do the opposite and do it through a similar mechanism. So even though we see a similar PPAR alpha expression and similar binding, the location or the response element seems to be different in rodents and humans and result in differential gene synthesis and presumably differential toxicity. Thank you very much.
MR. BROWN: Well, thank you Dr. Cunningham. We have a 15-minute break scheduled. Because we are running a little bit late, I would like to resume this session promptly at 10:30.
(Whereupon, at 10:14 a.m., off the record until 10:33 a.m.)
MR. BROWN: Clearly, the carcinogenic effects of DEHP have taken center stage in terms of, again, both regulatory and public health considerations. But it is important to keep in mind many of the non-cancer effects that have been manifested in experimental animals following exposure to DEHP. Our next speaker, Dr. Robert Chapin, is going to address one of those endpoints, reproductive effects. Dr. Chapin is head of the Mammalian Reproductive Toxicology Center at the NIHS. And also notable for this meeting, he is part of the Center for Evaluation of Risks to Human Reproduction, which is evaluating reproductive effects of phthalate esters. So, Dr. Chapin?
DR. CHAPIN: I have been asked to give a 20-minute overview of eight-and-a-half hours worth of material, so bear with me while we start cranking here. So because of the amount of data that we have got to go over, basically we are just going to be covering -- kind of hitting the high points, if you will.
One thing that was touched on lightly earlier is a concept that is important in this discussion of the IV exposures to DEHP and other phthalates. The diester phthalate with the two long side chains for reproductive toxicity appears to be -- metabolism appears to be required. So what happens is that esterases cleave one of those chains off
and turn the diethylhexylphthalate into a monoethylhexylphthalate. Those esterases are mostly in the gut and the liver. So it is the monoesters that appear to be the active moiety. As we heard John Bucher say, when you deliver it by inhalation, it basically goes straight into the blood stream and you miss that activation step. So the internal ratio of the metabolites is different, and that would be true for IV exposure, and that is going to relate to what kind of toxicities you see for reproduction.
I wanted to just get across the point that structure relates to function. Different phthalates with different side chains will have different biological activities. Nonetheless -- and different biological activities mostly in terms of potency, which is to say that those that have shorter or longer chains than DEHP tend to have -- tend to require more compounds to do the same kind of effect. We will see an example or we will see a manifestation of that in the next slide.
Basically, you can break reproduction down into male effects, female, male reproduction and female reproduction and the resulting fetus. So we are going to go racing through those in the body of the talk here. The male effects -- so if you are treating a pubertal or an adult male basically manifest as effects on the Sertoli cells, and I will show you an example of what that looks like. So these are sort of the mom and dad and the house, if you will, in the seminiferous epithelial, whereas the germ cells are the ones that grow up and leave. So if you affect the functioning of the hardware of the support system, then the germ cells will be adverse affected as in they die, and then that leads to testicular atrophy and reduced sperm count and reduced fertility. And we will see examples of that in just a minute.
The dose levels for that tend to be in the half to 2 gram per kilogram per day range. These are all oral studies. So what I am going to do is talk to you about oral studies, because those are the ones that, number one, where most of the data are, and number two, that is the effective route. The last three slides or so are going to cover the couple IV -- relatively inadequate IV studies that were done much earlier, and I will just sort of address those just so that those have been covered here. But mostly what we are going to talk about are oral dosing kinds of studies.
The female effects, we tend to see reduced fertility, which manifests as a reduced proportion of females in a group of animals getting pregnant, and they have a lower litter size, and that is due to a reduced concentration of estradiol. The developmental effects -- MEHP appears to behave like an anti-androgen, but there are also changes in cell cycle, which we won't have time to go into very much.
So this is a slide from Jerry Heindel, where he was summarizing the effects of many different phthalates in a continuous breeding study, and we are going to be looking at some of the data form the DEHP continuous breeding study, and we can see that at a given dose -- at the same dose, there is a sort of increasing effect on fertility as you approach DEHP. It tends to -- and it reduces sperm concentration and it reduces testes weight. This was not evaluated, but there are changes in estrous cycle, as we will see.
So what does the testicular effect look like? Well, this is the slide that is apparently stuck in the projector, which is a pathology slide showing the effect on the testes of a rat treated with a similar compound, dipentylphthalate, so reasonably closely related, but it produces the same kind of effect. What it finds is -- what it produces is big vacuoles in the basal part of the Sertoli cells. So we have got the seminiferous tubules in the testes, which is where spermatogenesis happens. We have got the Sertoli cells, which support those germ cells. The first structural change is -- this is sort of a testes by candlelight kind of figure. What we see here are two -- so these are the seminiferous tubules, there is one here and there is one here. This animal was treated 24 hours previously with dipenylphthalate. These two tubules look normal. So we have got basically a nice plump epithelium if you will. You can't really see it, but there are hundreds of germ cells in here with the Sertoli cells being the nearly invisible structural support in those cells. For the tubules that actually manifest the damage, you can see this basal vacuolation here. That represents an adverse response of the Sertoli cells. If you continue to dose this animal with this or any other active testicular toxicant, effective testicular toxicant, you will get testicular atrophy. The next slide shows that. Before we move on to that, I want to just for reference show you a little arteriole in-between the two seminiferous tubules, and then here is the same arteriole. So we have gone up in power now. So now these are seminiferous tubules from an animal that has received continued treatment with a testicular toxicant, and basically all that is left are the Sertoli cells and an occasional stem cell spermatogonium. So all the germ cells are gone. This animal's testes weighs a lot less than the controls. There is no sperm here, so there is no sperm output and so there is no fertility.
So that shows you both the beginning and the end, if you will, of the testicular lesion, and that has a variety of in vivo kind of correlates. So this is the -- this is one of two slides of data that I will present from this continuous breeding study, which is basically the National Toxicology Program's version of a multigeneration reproduction study. This was done and published by Jim Lamb in the mid-1980's, and they necropsied the control group and the high dose group, so the high dose animals received .3 percent DEHP in their diet. And basically what you can see is that there was an increase in liver weight, a significant reduction in right testes weight from 135 mg to 55, and then concomitant reductions in right epididymal weight and prostate weight and sperm concentration. So sperm concentration in the epididymis went from 473 down to 101, and in fact it would have gone down lower if we had continued dosing the animals. So significant reproductive effects there.
One of the capabilities of this design is that at the end of a certain amount of treatment in vivo, there is a possibility to cross-mate the group. So you can take the treated animals and mate them with control partners and vice versa, and you can see which sex is affected. That is what Jim did in this study. So the control/control mating, there were 18 out of 20 pairs that mated and got pregnant and they delivered an average litter size of about 8. When the treated males were mated with control females, only 4 of 20 females got pregnant and the litter size was six-and-a-half, so a little smaller but not significantly smaller than the controls. So there is a significant reduction in the proportion of pairs getting pregnant with treated males. With treated females, none of the treated females got pregnant, zero out of 16. So a clear female effect as well. So we have both male effects and female effects.
Before we move into the female, let me just summarize the results from this Lamb study. What he found was that there was reduced fertility, both at the high dose, which in this case gave an average consumption of about 425 mgs per kg per day, and the middle dose, which gave an average consumption of about 141. And there was a clear NOAEL, no observed adverse effect level, at 14 mgs per kg per day. This is in adult mice. So the LOAEL of 141 and the NOAEL of about 14. So remember those numbers or find them in your handout, because we are going to be coming back to this later.
Okay, so you remember that we said that there was a significant female effect and that none of the treated females got pregnant. Barb Davis at the National Toxicology Program pursued that a little bit, mostly to show proof of principle and to explore likely target sites. She gave a series of regularly cycling rats a very high dose -- a high effective dose of diethylhexylphthalate. And what she found was that on the morning of proesterase, there was this estradiol surge, which then stimulates the LH surge in the late afternoon of proesterase and that stimulates ovulation and thus her receptivity and then mating happens that night. Well, in the presence of a high dose of DEHP, the estradiol surge or the estradiol rise did not happen. So without the estradiol priming the ovary, the LH surge didn't happen. And without LH surge, there is no ovulation and so there would be no -- she wouldn't come into heat.
So Barb's interpretation was that the primary effect was on the effect on estradiol here. Well, so how might that be mediated? What might be the target process that might be affected by DEHP? So what Barb did was gave -- sort of worked her way back from estradiol through the synthesis pathway. The first thing that she found was if she gave -- and as you will recall, testosterone is converted into estradiol by the enzyme aromatase. And she found that in control animals, as you give increasing amounts of testosterone, you can produce increasing amounts of estradiol. That amount is reduced in the presence of 2 grams per kilogram of DEHP. And as you went further back up the pathway, this reduction was not aggravated. So Barb's interpretation is that the primary effect is on the enzyme aromatase, which makes the final conversion from testosterone to estradiol.
So she found those effects at this relatively high dose. Then when she did the in vitro sort of dose response, she found effects occurring at this kind of concentration, which is difficult to relate to in vivo levels. But she was finding effects in the female.
Okay. So male repro/female repro development. The phthalates have been the subject of a lot of concern for the possibility that they might effect the development of the reproductive system in developing animals, in fetuses and neonates. That puts them in the category of "endocrine disrupters" or endocrine modulators. So I need to take a two-slide sort of parenthetical, contextual setting up for you to introduce you to the concepts of endocrine disrupters so that you can put this in some kind of context.
Endocrine disrupters in general -- the concern about endocrine disrupters is that they will
-- that because of in utero exposure, there will be changes in the steroid milieu of the organism or of the fetus and that will produce changes that won't happen until much later in life. And that happens because developing organ systems depend on and are very sensitive to endogenous levels of steroid. You have got to see the right amount of hormone at the right time for that tissue to say, okay, I am a rodent prostate and this is the way I am going to respond when this animal is an adult to X amount of testosterone. Or I am the rodent brain or the hypothalamus or some part of the animal. And so if you change that setting up process, then you will forever change the function and behavior, if you will, of that organ when the animal is mature. So the concept is that by interfering with this signaling process, they can change this. And the interesting thing about the reproduction system, of course, is that that doesn't start to manifest shortly after the animal is born and you don't see it when you do a regular teratology study, which is just looking for basically the presence or absence of limbs or organs. What you are doing is you are changing the function of an organ.
For the reproduction system, of course, the function is -- that is one of the last functions to really kick in, and that only happens at puberty. So you are talking a month in mice, two months in rats, 18 years in humans. So there can be a big lag between the exposure time and the time when you can actually measure a change.
What sort of changes might you see? There are both structural and then structural changes will also lead to functional changes. But there are functional changes that lack an immediately obvious or clear, easy to find structural correlate. TCDD prevents the death of some of the cells in the middle of the vaginal folds, so you get a vaginal thread which reduces mating. So if you don't have the same amount of mating, then you get reduced fertility. You can see hypospadias compounds that behave by blocking androgen signaling to the organism will produce a series or a suite of effects, one of which is hypospadias, where the opening of the urethra is not at the end of the penis but is someplace more closer to the body along the under side of the penis. There are smaller absent accessory organs like the prostate or the seminal vesicle. There is ectopic testes, so they don't distend into the scrotum but come out someplace in the abdomen and live between the abdominal musculature and the skin, or there are un-distended testes. There is altered anogenital distance, which in the rodent is a measure of androgen status.
Additional functional changes include altered CNS sensitivity to hormones, which would lead to disrupted ester cycles, altered libido or alterations in the ability or willingness of either the male or the female to mate and concomitant with other changes you get reduced sperm output, altered numbers of Sertoli cells, an inability to mate due to either hyperspadius or this vaginal thread, et cetera.
So this kind of sets up the kind of the context for you. Like I said, compounds that interfere with androgen signaling tend to produce a suite of effects including hypospadias and altered accessory organs and ectopic testes or distended testes.
These kind of endpoints have been evaluated for DEHP only by one investigator so far and that is Earl Gray -- or have been published by only one investigator, and that is Earl Gray at the EPA, and he used a relatively high dose of DEHP and gave it to female rats as a part of a much larger study looking at both DEHP and like 7 or 8 other compounds.
What I will do is show you just one piece of similar kinds of data. These were data actually generated by Eve Micrease and Paul Foster at CIIT using dibutylphthalate, and what they were measuring was hypospadias. They found that there was basically no litters out of nine control litters that showed any hypospadias, but one litter out of eight, four out of seven, and two out of four showed them hypospadias at between 250 and 750 mgs per kg per day, and then this is the number of pups that evidenced that effect. So you can see a nice clear dose response relationship in the presence of hypospadias when dibutylphthalate was dosed to pregnant moms and then the kids were evaluated after birth. This is representative of the kind of data that Earl has produced, but not in any kind of dose response kind of fashion.
All right. So we don't really have the data that we really want in terms of good dose response and any kind of functional assessment for DEHP yet. That is going to change. Both Dr. David and myself are part of or running or overseeing very large multi-gen studies that are going to be collecting these kind of endpoints. But we don't have them yet. So what have we got as a fall-back?
The next best study, I think, is one done by Arcadi, et al., where he exposed pregnant rat dames to two different dose levels of DEHP in the drinking water only during gestation and lactation. So the three-week gestation period in a rat and then the three-week lactation period and then he stopped the exposure and started evaluating the male pups at different times up to the point where they were 56 days of age, which is a little after puberty.
All of the studies that I have talked about so far have significant drawbacks from the standpoint of being able to address sort of the global issues of reproductive and developmental toxicity in rodents. The drawbacks for the Arcadi study is that the elemental/elementary kind of data collecting that they should have done was to at least measure water consumption, and they didn't do that. So we don't know how much those animals really received. Not only did they not measure water consumption, there was no assurance of how much DEHP was actually in the water that the animals received. And this is significant because DEHP is not very soluble in water, as we saw in some of the early talks. It will go into water at very low levels, but it really helps to have lipoproteins or some sort of lipid fraction there to help haul it in. Nonetheless, if we take at face value the intended concentrations in the water and a guesstimate of how much those animals dra