Well, good morning. Practically there. Good morning, everyone. Welcome to the day two of our Chronic Hazard Advisory Panel meeting. Today, we have distinguished scientists who were invited by the CHAP to present on their latest work. And I appreciate very much the speakers joining us here today, making the trip to Washington. Unfortunately, one of our speakers, Dr. Burke, couldn't come because of death in the family. So he is not here today, but welcome. And as all our meetings, this meeting is open to the public. It is also being webcast. And so the remarks will be part of the public record on phallates. So thank you, and good morning. Phil? Thanks, Mike, and my welcome also to the speakers today who are here to help us obtain information that we need to do our task. And I'd like to start off by going over what I've listed as critical issues that we need to address as part of our charge. And this is not meant to indicate that this is an inclusive list, but these are some of the major issues that particularly Byrne and Mike and I have put together. And it may help us to focus some of the discussion today. And we've also given to the speakers a list of questions as well. I won't go over those, but those will become apparent, I think, when we go through their talks and the discussions ensuing from those talks. So first, what if any are high priority endpoints of toxicity beyond male developmental reproductive alterations? For example, are there cancers that arise from in utero or neonatal exposure? Other developmental or reproductive effects, including behavioral or cognitive effects, other cancers, chronic organ toxicity, immunological effects, or asthma, or others? Our charge is very broad. So we need to help answering that question. Two, what are the relevant populations? The fetus, the infant, the expected mothers, others that the CHAP should consider. Which phthalates are essential for a cumulative risk assessment? And how is this to be determined? And is this based on biomonitoring data, production levels, health effects, and forth? Well, fifth, are there critical biological studies that are needed before the CHAP can meet its charge, or at least substantially strengthen the CHAP's risk assessment? And these studies could be in any area. Another question we need to answer is, what are the sources of human exposure that are most relevant to prediction of human risk? In utero, neonatal, children, adolescents, adults, males, females, et cetera. And then for this afternoon's discussion, or later on, yes, this afternoon's discussion, we need to know what regulatory gaps between federal agencies make it difficult to minimize risks of phthalates to humans. And are these gaps a function of the existing regulations or priorities of the agencies? So these are some of the critical issues that we need to address. And hopefully, there will be help coming from the speakers today. So without going into any more detail, let's get started. So we have as much time as possible. And Paul, you want to start off? Well, good morning, everybody. So where to start? For someone who's worked on for more than 25 years it's always quite difficult to know what to leave out. And so Dr. Gray and I, I think we're the ones who've got the largest list of questions that were posed to us. So we did actually decide to talk to each other a little bit beforehand to see if we could divide our talks up to cover enough areas that weren't too overlapping. So I'm going to try and start off by giving you a little bit of the history, some of the effects that one might see, the classical kinds of mal reproductive tract malformations and so on and so forth. And describe to you what Dr. Gray and I have called the phthalate syndrome. And I do apologize to people that have heard this many times before, but we thought it was probably worthwhile going over that. And then in Earl's talk, I think he's going to concentrate and give you a little bit more of the newer information on structure activity, potency factors, and mixture type studies, if I remember correctly. OK. So what I'm going to try and cover is to provide you some background to disruption of mal reproductive development in mammals. A very brief background to the reproductive toxicology of some of the ortho phthalates. Describe the thenotypic pattern of lesions that we see in mal offspring following a neutral exposure to one of the phthalates. But I think that what I'm going to show you is really very typical of all of the ones that are active. And we'll talk about that as we go through this morning, I'm sure. And note some of the early changes that you could see, particularly in fetal testis, testosterone production, and specific gene expression. And these are likely to be precursors of the adverse effects that can then be noted in the adult following a neutral exposure. And then lastly, talk to you a little bit about the critical windows of development for production of these lesions in rats. OK, so it's about 15 years now since, I think, Nils Gagabec and Richard Sharp proposed the human testicular dysgenesis syndrome. And this is really a hypothesis. I think it's still a hypothesis that's about to be that still has garnered some information in humans, but I don't know that it's necessarily completely proven yet. But essentially what they attempted to do was to come up with a hypothesis that tried to describe some of the secular changes that have been noted in mal reproductive parameters and link them together to a particular sequence of events that occurs during fetal life. And what they really were talking about was changes in the activity of two of the major somatic cells of the testis. The latex cell, which is a cell of the interstitium that's really responsible for androgen production predominantly, and the satoli cell, which is actually present in the seminiferous tubules or the cords, as they are in fetal life, which really acts as a nurse function to control the normal development and differentiation of the germ cells. And what this really says is that a couple of the different factors that are made by the latex cell, and particularly in stem like factor three, I'll talk a little bit more about that later, production, androgen insufficiency could lead to other adverse consequences and changes in satoli cell function could lead to impaired germ cell differentiation. And so the adverse consequences of these kinds of effects would be cryptocutism, a failure of the testis to descend normally, which is the most prevalent human birth defect. It affects some of like 400 life births and boys. But also you're going to see lower testosterone levels in these animals. You could also see hypospadius, which is a malformation of the penis, which is the second most prevalent human birth defect, and obviously effects on spermatogenesis that would be decreased. Impaired germ cell differentiation could also lead to problems with spermatogenesis. And it has been proposed that through carcinoma in situ cells that are generated in early fetal life, that this could lead to germ cell cancer, which is the most common cancer in young men. And there are huge numbers of different things that could actually affect how these particular germ cells, or sorry, these cells in the testis could actually be perturbed during fetal life. It could be in various environmental factors. It could be into uterine growth retardation. It could be genetic defects in, for example, the androgen receptor. And there are also a number of different polymorphisms that could contribute. So it wouldn't be a single factor that could lead to these kind of effects. So what do we know about sexual differentiation in the human? So around week six of pregnancy, the fetus is termed sexually indifferent. That doesn't mean it doesn't care about sex. What it means is it has the ability to either become a male or a female at that particular time. And one of the things to bear in mind about becoming a female is that the normal development in the womb is largely hormone-independent in females. However, in the male, it's almost the opposite. The testis need to form, need to have specific hormones produced at various and specific stages of development. And you can see this is around week 7 to 8 of pregnancy. And the critical factor is that in the male, normal reproductive development is totally hormone-independent as opposed to the female. And what that means, of course, is there's a potential window of hormonal susceptibility. And in the human, you can see this would actually be over and complete by about week 15 of pregnancy. So in many instances, the critical window could be before a woman even knows that she's pregnant. So what is the same kind of things that one sees during the development of the male reproductive tract in the rat? So SRY, which is the sex-regulating gene on the Y chromosome, gets switched on about gestation day 12 in the rat. Soon after that, you see a number of the other important. So SF1 is steroidogenic factor 1. So this is the major trigger that's going to turn that undifferentiated gonad into becoming a testis. So the seminiferous chords start to form. We then start to see satoly cell differentiation and proliferation. The satoly cells then make another hormone called malaria inhibiting substance that causes the malaria and ducts to undergo regression, which is the female plumbing, if you like, what would have become the uterus and overdux and so on and so forth. We then start to see the appearance of critical steroidogenic hormones appearing. And then we see this wave of testosterone production. In fact, in the rat, it peaks around 17 or 18 days of gestation. It can reach adult levels in the testis of the fetal rat. And then you see this huge drop-off just before birth. And in the rat, the testosterone really does not begin to rise again until the animals go through puberty around 40 days of age. During this time where we see this increase in testosterone, the males under nipple regression, and that's a sexually dimorphic measure in rodents, you see differentiation of the dwarfian ducts. That's where this duct system will develop into the vas, the epididymis, the seminal vesicles. You then see trans-abdominal descent of the testis. But in rodents, true testicular descent does not occur until after birth, whereas in humans, this is a prenatal effect. And now, obviously, you're seeing growth and differentiation of the external genitalia and prostate. So you can see that most of these critical effects are actually in the latter part of gestation in a rat compared to early gestation in a human. And if you remember some of your basic teratology guideline studies, how they used to be done, the exposure was only from gestation day 6 to 15 in a rat. So most of the critical windows for looking at effects actually weren't covered in those early teratology studies. So what do we know about the esters and background? And as soon as we're dealing with toys here, I thought we'd have a rubber ducky. Clearly, these things are used extensively as plasticizers and solvents. I think it's worth pointing out that there's some evidence that some of these esters are weak ear agonists in vitro in some systems. And so the data actually is very, very sketchy to say the least. So you might see an effect in an MCF7 cell, but you won't see it in a yeast cell or vice versa. But I think the clear message that comes out of this is when you actually look for estrogenic activity in vivo, you never find any. So they really are not in vivo estrogens. As I mentioned, these are generally not teratogenic in the classical 6 to 15 studies at levels less than a gram per kilo. And I think most would consider that fairly heroic. And when you see developmental toxicity, it's usually always accompanied by a maternal tox. And so some of you don't need me to tell you that you see extensive metabolism occurs in the gut rodents to form monowesters. And these are absorbed and believed responsible for the ensuing toxicity. So one of the things that you have to remember is that you could be dealing with different mechanisms at different life stages with some of these compounds. And so when you look at effects in the testes of pubertal and adult rats, they don't seem to be the same kinds of effects that you see in the testes of fetal animals, for example. And certainly in the pubert or an adult rates, there's a certain amount of evidence that would suggest some of the effects that one sees are through a FSH mechanism directed to the Satoli sip. There's an aid sensitivity in response. And so in general, for those stelae esters that have activity, the fetal animal is more sensitive than is the neonate. The neonate is more sensitive than is the pubertal animal. And the pubertal animal is more sensitive than the adult. And by sensitivity, what you really means you need a higher dose to produce adverse effects in the testes. And even though the testicular toxicity of some of these agents has been known for more than 25 years, there are actually only a limited number of multi-generation reproduction studies that have been undertaken that have focused really on reproductive development. So many of the studies were quite old. They don't have some of the endocrine endpoints that came into the guidelines. And so we don't really have a full suite of information on all of these particular agents in appropriate studies. And I think it is worth noting that you do see effects in females, consequently, either as adults or as the fetus. But these effects tend to be at higher doses than the corresponding males. And if that's all that, the prevalence is actually much, much less than what you see for males. So you can see effects in females. And I know Earl will talk a little bit more about that in his presentation. So what are the thalic esters that we are known to affect male reproductive development in rodents? And I've tried to give you a list of the ones that I would be fairly confident of showing effects. And these are the ones that are really shown here. And I don't have a pointer, so. And that's butyl, isobutyl, pentyl, ethylhexyl, isononol. And I think it's fair to say isononol is weaker than many of the other thalates. And I think it's also worth bearing in mind that compared to these other compounds, this is not a pure chemical. It's actually a mixture of isos. It averages around C9, and it's bronched. So whereas all the others really are pure agents. And butylbenzole. And I think what's interesting is that by and large, this follows the structure activity requirements for producing effects in the pubal test that was published by someone back in 1980, where they must have been about six years of age. It wasn't me, it must have been my dad or something like that. And really in terms of metabolism, the first part really is you see deistherification to produce the alcohol on the corresponding monowester. And then what you can see, either the monowester itself gets glucuronidate for an excreted, or you can see omega or omega minus 1 hydroxylation at that side chain before glucuronidation or other forms of conjugation. So I dragged this one out from the archives. And I think it's worth, so this is a comparison of effects in the pubal test versus what you see in vitro in a Cetoli germ cell co-culture system. And remember I said that these animals seem to target the Cetoli cell. And I think the interesting thing here is, was two, several interesting things. One is you see a very clear differentiation in toxicity and that the ones that are left here, the ones that really have activity, the ones that are right are really without activity. And I also hasten to add, if you put phthalate diesters into these cultures, you get nothing. If you put the corresponding alcohols into these cultures, you get nothing. It's the phthalate monowesters that produce the effects. And the other interesting thing, because I know Earl's going to talk a little bit about this, these phthalate monowesters, you see toxicity. And the one thing where we didn't see effects with the corresponding diester was actually dien-heptile in the pubal. You can get effects with a monowester, but we didn't see any effects with a diester. And the other thing to note is that even though, for example, M-butyl produces positive effects, as does isa-butyl, if you give tertiary butyl, you don't see effects. It's negative. And the lower-chain ones, propyl ethyl and methyl, are also without effect. And I'm sure Earl will talk a little bit more about that in the figal animals a bit later. So we have actually undertaken a number of different kinds of studies where we've embraced this idea about where's the critical period for induction of mal-reproductive effects during sexual differentiation. And that really, for us, has involved dosing from about gestation day 12 to just before birth on 21. And what we've done is looked at a number of different fetal molecular studies here. We've looked at AGD, and I'll explain and tell you a little bit more about that later. We've looked at nipple retention, which I've already mentioned is 60 dimorphic M rodents. We looked at cubitii.  And now we would look at anecropsy and look at all the various developmental abnormalities that you could see. But really, many of these are not particularly obvious at earlier times. It's quite difficult to see a prostate malformation in a wingling animal or a neonate. I mean, it just hasn't developed at that time. And you really do need to look into the adults. And the other thing to bear in mind in these studies is the need to retain a lot more animals per litter in order to have confidence in detecting effects. So most of our standard reproduction studies really only retain one male and one female per litter. And in these studies, we retained all of the pups in a litter, as you would say, doing a teratology study to examine them for adverse effects. So ain't a genital distance. It's the way one normally sexes a rodent and a rat in this case. And in the male, it's the distance between the sex papilla and the anus, and it's about three millimeters in a newborn rat. If you look at a treated animal, you can see that this is a little bit shorter than the standard male, which I've color-coded blue, but obviously a bit longer than a female, which is color-coded pink, just for those of you out there. And here's an early study that we did looking at the effects of DVP on ain't a genital distance. And you can see what happens is that the distance gets shorter and it moves becoming demasculized, feminized. And the dosa flutamide that was used, which is down here on the right, actually was sufficient to produce all females. So in other words, based on ain't a genital distance, you could not tell the males and the females. They all had a female distance. And it's permanent. So this is just comparing ain't a genital distance animals that were dosally either 100 or 500 milligrams per kilogram per day on postnatal day one and when they were 180 days of age. And you can see there's actually a linear fit for this. Animals that were affected straight after birth were also affected when they were six months of age. So nipple development in the rat. So in the female, nipples started to develop about gestation day 14. It's really complete by about gestation day 17. And then female pups are born with a full complement of 12 nipples. Now in the male, the nipple and lager can actually undergo regression. And that's under the control of fetal dihydrotestosterone. And so what normally happens in a male is that these tissues undergo apoptosis.  However, if you had something that could block production of DHT like an antiandrogen, for example, you can end up with feminized male pups with nipples. And just to show you, this is what it looks like. On the left is a control male around PND 13. And on the right is a phthalate-treated male. And you can see, here's the nipples along the flanks. By the way, that's the umbilicus. Some people think there are 13 nipples there. You can see, you still see these thoracic nipples along the, and abdominal along the flanks of the animal. So what we have here is a male, but it has the nipple characteristics of a female. So it's a good non-invasive method to look at the androgen status of these animals. And again, you can see how you can get a dose-dependent induction of these particular effects, either on a pup or litter basis with dibutyl phthalate, such that you can reach pretty well a female status with a high enough dose. But really, the classic effects that we saw with dibutyl phthalate, and I think it's probably true of the other active phthalates as well, is all the major effects that you see on the testis and the epididymis. So here's a control adult testis and epididymis. Oh, thank you very much. So here's a control adult testis and epididymis.  If you expose for our 10 days in utero, this is what you see. So what we have here actually is just the fat pad. Actually, this testis was cryptalkid. It was up inside the abdominal cavity. This one, as you can see, is a little smaller and it actually doesn't have an epididymis. We got this in something like between 50 and 60% of the animals in our strain of animal, our strain of rat-produced epididymal malformations at around 500 milligrams per kilogram of DBP. When you look at these histologically, so here's what a control testis looks like. This is what you see in the high-dose DBP. I think you don't need to be the world's greatest pathologist to spot here that there actually are no sperm here. And consequently, these animals are infertile. The other thing you can probably also make out is that the interstitial cells here, this is where the latex cells reside in the interstitium of the testis, you actually see a hyperplasia. And actually, we did that morphometrically to prove that there was, in fact, latex cell hyperplasia that goes on after this exposure in utero. And a small number of the animals also develop latex cell tumors. So this is not the germ cell tumor that you see in humans, but you actually can see latex cell adenomas produced just after in utero exposure to W. Telfalli. One of the other things that we see is cryptocortism. And actually, it's quite normal to see hemicryptocid animals that appear from time to time. So here's the testis that should normally be found down in the scrotum. The other one is somewhere up here in the abdominal cavity. And the descent of the testis in mammals is actually under the control of two major hormones. So in fetal life, the testis and the epididymis is attached to the diaphragm by a cranial suspensory ligament. And then you see this long-thin gubernacular ligament here as well. Under the effects of insulin light factor three, you start to see movement of the testis down from up near the kidneys towards the inguinal ring. The gubernacular starts to develop and it really drags it down towards the inguinal ring. And then it's under androgen control that final testis descent occurs through the ring and into the scrotum. So it's a two-stage process in mammals. And we know, for example, in mice that have been knocked out for INSL3, they're 100% cryptocortid and they're very high up near the kidneys. We also see hypospadias. And this is probably one of the worst cases, I think, that we saw of DVP. So here's a normal penis and here's one that's been treated with a 500 milligrams per kilogram of DVP. And you can see that the failure of the penile folds diffuse here. So this is totally open. The opening of the urethra is at the base of the thalus and you can even see the ospinus here that is exposed through here. So this is a fairly major hypospadias. You can see them much less severe than that from the treatment. But it's not just those. You also see effects in the accessory sex glands. So in this particular instance, again, an animal taken from 500 milligrams per kilogram per day, there is no ventral prostate in these animals. It's totally missing. And you can see that one of the horns at the seminal vesicle here is also much, much smaller than it should be. And there is no anterior prostate or calculating glands here at all. So we see major effects on the normal development of the accessory sex organs that are normally under androgen control for their normal development. So what I've explained to you is absolutely typical of what you would expect to see for an androgen receptor antagonist like flutemite. Exactly the same kinds of lesions that you see. Exactly the same targets. Although what you do see, I think, with thalus is they don't occur at the same prevalence as that you would see with an androgen receptor antagonist. So we obviously assumed that the thalate was likely to be an androgen receptor antagonist and it wasn't. And so here's the... Here's an androgen receptor reporter gene assay basically showing that dibutyl thalate has no effect on its own. Here's dihydrotestosterone, the endogenous ligand. And here's testosterone in the presence of dibutyl thalate. Didn't do a thing. So we thought, well, clearly it needed metabolism. So we did the same experiment with monobutyl thalate and we didn't get any effects there either. So what we have are the classic effects on phenotype of an androgen receptor antagonist, but no mechanistic evidence that actually it binds or inhibits the androgen receptor. But what we did see was a dramatic decrease in fetal testosterone levels. And so here's one of our studies where we looked at different ages in gestation and compared dibutyl thalate with flutamite. So the classic anti-androgen will not reduce androgen levels in the testis. And you can see here it really doesn't. But dibutyl thalate can knock it down by 50% or more. In other words, it differentiates the effect in terms of testosterone concentration in the fetal animal. You don't see the same thing as you see with a pure AR antagonist. And this was another study that wasn't done for this purpose but shows you some of the dose-response relationships. And at 500 milligrams per kilogram you're down at 5% of control levels. And you can even see there's a, maybe a 20-25% decrease at around 13 milligrams per kilogram per day. So there is a dose-dependent decrease in the level of fetal testicular testosterone. But really some of the interesting things are what's going on with the morphology in normal fetal development. And so here's a control. And you can see at this time there are no seminiferous tubules. We have the seminiferous cords. This is just prior to birth. And normally one sees small pockets of latex cells in the interstitium here. Maybe half a dozen cells at most. And here's a blow-up of one of these cords. So these would be the primitive germ cells, organocytes, and these would be the satoli cells around the outside. And as you can see in the phthalate-treated animal, we see these huge rafts of interstitial cells, much, much more than what we see in the controls. And within the seminiferous cords, you actually also start to see many of these multi-nuclear gonocytes. So these are primordial germ cells. And this one I think we've got in one cell. This is a problem with the normal differentiation of the gonocytes into the stem spermatogonium. Okay, so what explained that? So we went ahead and did a whole range of different gene expression experiments in fetal life, mainly around gestation 19, mainly because they're pretty small when you need a fair amount of tissue to do these kinds of work. And what this slide really shows here is effects on steroidogenic gene expression. This is a P450 C11A1 or Cy-Chang cleavage enzyme, one of the rate-limiting steps. This is 3-beta HSD, hydroxy-steroid dehydrogenase, down by about 50%. P450 SIP17, down again by 50%. But not all of the steroidogenic enzymes were down-regulated. So this is 17-beta hydroxy-steroid dehydrogenase. It actually, with anything, was slightly elevated, but not significantly so. And by and large, these were also found when one looked at protein expression. That was, we did that by RT-PCR. And so again, so this is star, steroid-acute regulatory protein, present in the mitochondria of latex cells. And the brown stain here shows the presence of star, and you can see it in these fetal testes lighting up the latex cells of the interstitium. And this is what you see in the phthalate-treated animals. You don't need to, again, to be the world's greatest pathologist to spot what you get a much reduced expression of the protein as well as the RNA. And this kind of summarizes a whole range of things that we looked at on gestation day 19 that I didn't have time to go into today. So we see effects on the receptor that transports cholesterol into the latex cell. We see effects on star, on side-chain cleavage, on HSD, on SIP17. And testosterone could be down to 10% of the control. And what I haven't showed you is that INSL3, remember that's the hormone responsible for the first-part testicular descent, is really only about 25% of control. We didn't have a protein for that, but that's just on... by RT-PCR. But to me, some of the classic effects that we saw really were on epididymis. So this is what you see at gestation day 19 in a DBP animal. So this is the wolfie induct here that will become the epididymis. Here's the field. Test this. This is what you see in the control. It's absolutely no different. But by gestation day 21, hopefully you can see that this has become now a very highly convoluted structure in the wolfie induct that will be the epididymis. Hopefully you can see those coils that are occurring there. But you don't get it in the phthalate-tree animals. And in fact, these then start to go and go apoptosis. So we know that the normal development of the epididymis is under androgen control. So immediately we went to look at androgen receptor levels. And we did a whole range of RT-PCR and we also did microarray on the fetal epididymis, looked at androgen receptor levels, and actually they weren't changed. Which came as a big surprise to us. Until we actually then went and looked at the protein levels. And what you see is actually a change in the expression of the protein in different parts of the epididymis. So here's the control where you can see AR, which is again the brown stain, is present both in the epithelium of the developing duct and also in the parenchyma. But when you look in the phthalate-treated animals it's almost absent in the epithelium but still present in the parenchyma. And so when you take a little sample of fetal epididymis and just look for message level, you can never differentiate that cell-specific activation. But it's not all down-regulation. So you can actually look at IGF receptor 1 and you can see it's almost the opposite. So this is weakly expressed in the control animal in the epithelium of the duct but when you give DVP you see over-expression in the parenchyma. So this isn't just because these are dying, it's a true differential expression. So to try and summarise that for you we know that malaria inhibiting substance from the Satole cell is responsible for malaria and duct regression. The removal of that female plumbing. INSL3 from the latex cell is responsible for trans-abdominal descent of the testis down towards the inguinal region. But testosterone from the latex cell is responsible for the inguinal descent of the testis into the scrotum and also for Wolfian duct differentiation. So you need testosterone for the epididymis and the vas seminal vesicles. But testosterone needs to undergo 5-alpha reduction to dihydrotestosterone and that can take place in the urogenital sinus and the genital tubercle. And that's why you see changes in differentiation in these particular fetal tissues. And then what you end up seeing is DVP can actually affect many of these processes and the size of the X is kind of roughly the prevalence of the lesions. So we get effects on genital tubercle, urogenital sinus, Wolfian duct, inguinal and both parts of testicular descent that result in a whole sweep of malformations that you can see which we term the phthalate syndrome. Now what this means is not every animal has every effect but because they are so closely related to normal effects from latex cell products what we generally do when one's doing developmental toxicity is to look at a syndrome of response that can plausibly be related together. And so that's what we ended up doing when we started looking at these things is that you can get animals that have cryptocortism and epidermal malformations but they might not have a hypospadius or they may have a hypospadius but they're not having cryptocortism. But I think essentially what you can say is there's a syndrome of response there. So then we went on to actually look to see okay we've gone throughout the period of sexual differentiation from 12 to 21 can we actually narrow down that window and we actually undertook a range of different studies and I think this is sort of like the take home message here. So we did it from about gestation day 14 to 20 and we did it in windows. Some of them were just single day windows and it's absolutely true. You can see a low incidence of malformations after a single dose. That is possible. So one dose is enough to produce a malformation. However, if you want to see the complete syndrome where you're going to get cryptocortism and you're going to get hypospadius and effects on seminal vesicles, etc. what we found was you needed to have three daily doses and that the critical period actually was before any of the genomic work that we've done. So I think we probably have to say that those might not necessarily be causal in the production of these effects but they are clearly related to the adverse effects and this window actually corresponds to the recently published malprogramming window that Richard Sharp published after we did. I don't think we realized that it actually was the malprogramming window and this really just shows you that the window itself is from gestation day 15 to 17 and as you increase the dose you can see this high prevalence of different reproductive malformations and you can get them all. They can all occur and you can get it just with that three-day dosing window. Okay, so what do we know about phthalates and malreproductive development? Well actually it's not one mechanism I think is the thing to take home. Is that we do see effects on testosterone production and clearly that's going to be related to the androgen dependent reproductive tract development particularly things like the epididymis and hypospadius which is really DHT. We see inhibition of INSL3 production and that's going to be related to malformations of the gubernaculum which we do see and obviously cryptocortism that's where we see the abdominal testes in these animals. But we've also noted and I really had a chance to go into it in any more detail changes in gonocyte development and differentiation we see these multi-nucleated cells and you can also see inappropriate age dependent gene expression that occurs here. So there's three things that are going on all at the same time and I thought this was quite interesting because there's been a whole range of different metabolism type studies that have been done and they haven't always been done in development at the right time. So many of them were done around gestation day 20 or 21 because you've got more tissue to work with. But I think this was an interesting study from Earl and from Antonio Calle perhaps grouped down at CDC where they looked for the level of free monobutal phthalate at GD18 so this is where you have the peak of testosterone occurs. And I think what they tried to show here is the comparison between looking at amniotic fluid monobutal phthalate concentrations compared to maternal urine monobutal phthalate concentrations and what they found was in amniotic fluid the vast majority of this is actually 90 to 100 percent is actually unconjugated and I think one would normally tend to think that the conjugation is actually a detoxifying step. And the other thing you have to remember is amniotic fluid is essentially fetal urine and so there's the potential there even for a recirculation that you can excrete into amniotic fluid and the fetus can then swallow it. And so again looking at the right time becomes very important in understanding what the active entity might be to which the fetus is actually exposed. And so the critical window for induction of reproductive tract malformations is around this time. And the other thing to note as I said is you get age dependent fetal differences in the glucuronidating enzymes. So at 18 the UDPGTs for phthalates are really not there for the phthalate monobutal phthalates. They occur later. You can see them by GD 20. So the time of exposure makes a difference. And so if you give 100 milligrams per kilogram per day of DVP which is an effect dose you see an amniotic fluid mean concentration in the rat of about 1400 nanograms of monoester per ML. Antonia Calafat looked at a very small sample of pregnant women. I think it's 54 which were from the general population. They didn't really measure the difference between conjugated and free levels. And I think it's interesting that the highest level that they found was about 264 nanograms per ML of amniotic fluid which actually is a margin of exposure only of about 5 from an effect dose of diputal phthalate in a rat. So to conclude diputal phthalate and other phthalate esters are anti-androgens that disrupt reproductive development via disturbance in androgen signaling in the fetal testers and reproductive tract. They don't work they also inhibits INSL3 and I think that's also been found for DEHP and a number of the others and I'm sure Earl will go into that as well. I didn't show this data but you can also see effects on stem cell factor and its receptor C kit which may be related to the mononutrient gonocytes that we see. We can see structure activity relationships for reproductive toxicity that seem to be parallel by changes in testosterone production and gene expression we know that the critical development or window is in the rat is GD 15 to 17 and that seems to coincide with the male programming window and I'd say it's unlikely that P par alpha has a significant contribution to this role and really the effects that you see in rats are very much parallel the testicular dysgenesis syndrome hypothesis. So why do I say we don't think it's related? Well there's not really been a huge number of studies. I just tried to pull together some of the pieces of evidence that I think support that it's probably not driven. So some of the active phthalates both moronastas and diastas are actually really I'm sure Geoff you're going to talk about this are really weak P par alpha ligands they are not potent by any stretch of the imagination but what's really interesting is where you do see differential binding so for example MEHP is a much better binder of P par alpha than is monobutal phthalate that's not what you see in the potency for the effects on mal reproductive development. There are user about equipotent or molar terms or over other stronger P par alpha agonist have not been reported to produce reproductive tract malformations so that's not necessarily the drugs but if you look at something like PFOA where there are good multigenes available you don't see reproductive tract malformations so if this really was a critical pathway wouldn't you have expected to see a bigger response in the more potent ligands and I think the other thing that's quite interesting is about the guinea pig so this is actually a non responsive species for the induction of proxazone proliferation by phthalates you don't get it you don't get the proxazone proliferation and yet when you give an equimolar dose of DEHP to the juvenile guinea pig it's every bit as potent in producing testicular toxicity as is DEHP so there seems to be again a disconnect here between what the potential mechanisms are through the production of adverse effects I really wanted to acknowledge a lot of the people that did this work it couldn't have been done without a lot of really talented postdoctoral fellows and I also want to acknowledge Kevin Guido who's now with the FDA Richard Sharp who's in Edinburgh and the good Dr. Gray who's just around the road there and be happy to take any of your questions or you might want to wait to get Earl first thank you Paul I think we should go ahead with questions any questions on the committee thank you Paul that was very informative two questions based on what you know now about motive action what do you think some animal species are insensitive and the second one is what do you assume about the relevancy of these findings for humans so I'll try and answer that so many moons ago we actually tried to look at the reason for some of the species differences that we saw in testicular toxicity in the pubertal animal and so for example the hamster is really quite resistant to the effects of dipental phthalate and we actually looked at that and the major difference that we found actually was in roots and rates of metabolism that the testis was not seeing as much of the material in the hamster as it was in the rat now that's only just one so there clearly could be pharmacodynamic reasons as well as pharmacokinic we honestly do not know but you know that we did publish a study back in the 80s looking at species difference and found that the guinea pig was as good as the rat the hamster was pretty resistant the mouse is pretty resistant as well you can see some testicular effects in the mouse but you've got to give huge dose levels to see it and in all honesty we don't know what the true reason is for those species differences we know that some of the factors that might contribute but certainly not all of them and I think that if you're dealing with androgen if we go flip now to the reproductive development side if you're really dealing with androgen dependent development we know that's a highly conserved mode of action that's present in all species you know we know that if you lower androgen levels in humans you can get crypt orchidism we know that if you have a a mutated androgen receptor you can get humans that are totally feminized so it just strikes me that it's quite plausible that you might see such effects in humans based on what we know about various syndromes because of the conserved mode of action can we elaborate on this a little more because that's a point of major importance which cropped up yesterday so we had yesterday evidence presented to us or that was evidence but presented to us data suggesting that rather than the rat the mouse and the marmoset are more like the human the people concerned actually under questioning didn't quite want to go that far saying it explicitly but we felt that some of the slides presented to us suggested this conclusion to us but so we got a little confused can you elaborate on that a little to help us out here okay I'll try and I suppose it depends again on which life stage you're talking about so for example I already explained to you in the pubertal animal the mouse is incredibly resistant to effects we really don't know why but I think it's also worth bearing in mind that of the known environmental agents that we know produce testicular toxic in humans there are a large number of these that are inactive in the mouse so for example dibromochloropropane which was probably the archetypal testicular toxic and doesn't work in a mouse it works in the rat it clearly works in the human gossiphole which was used as a potential contraceptive agent in China clearly works human it works in the rat it doesn't work in the mouse for the effects on reproductive development I'm not sure that I've ever seen a real publication that looks at the adult offspring following a neutero exposure in the mouse I don't believe I've ever seen that published well the good doctor Sharp in a recent publication from his group McKinnell at 2009 I quote says humans are not good models for human fetal germ cell development this may do you think that's a comment relevant to the issue at hand I mean fetal germ cell development is just one fragment of the processes we're discussing here and I think you could honestly say so for example for a testicular germ cell cancer there is not a good rodent model I think there's maybe the horse that you can get we're going to do toxicity studies in horses anytime soon so none of our the dog maybe it's got to be like 7 or 8 years of age we don't have good experimental models for testicular germ cell cancer and if we believe the hypothesis that this is due to a change in normal differentiation then you would have to agree that no rodent would be as good as a human but then again human primates might not be as good either this statement which we find in this paper may be correct but if I understand you correctly it doesn't invalidate the experimental evidence which we have from the rat model and which you produced in order to make extrapolations to the human I don't think so correct me if I'm wrong on this Paul but there are a lot of assessments done by the national toxicology program in the reproductive assessment by continuous breeding protocol in the mouse many of those had adverse reproductive effects in the mouse with phthalate exposures in both male and female offspring so I think that they're negative in the mouse is not entirely accurate and I think that's what I meant to say is that you need to give much higher dose levels to produce effects in the mouse so they're less sensitive but they're not what's the right word they're not intransigent to any effects you can see it if you push the dose high enough I might add that some of those studies were done at 4 grams per kilo so I was struck by the absence of any discussion of AGD could you talk about that a little bit so analgenital distance is the length of the perineum and it's actually under the control of dihydrotestosterone so I think that it's the way we normally sex rodents visually and newborns and so what it is is a non-invasive method that's a very good if you like phenotypic marker of the androgen status of that pup in utero so in other words animals that have lower androgen levels tend to have shorter AGDs and males becoming more female and would you include that as part of a short AGD as part of the phthalate syndrome oh yeah, yeah as is retaining nipples but I think that there's always been some question marks about is that an adverse response and I think and from my point of view is if it's a permanent change then it's an adverse response and I think I tried to show you that if you do these nipples in adult animals you have to shave them to find them but they're still there in those animals and AGD is a permanent change and then conversely I don't know if you have any data on this but would you expect then that increased viralization of the female would create a longer age of general distance I think Dr. Gray did that experiment if I remember with androgens right, well and you do see that can I continue inquiring a little about DINP we had yesterday presentations from ExxonMobil about DINP one issue was there were studies presented Niko Forov at hour 95 Waterman at hour 99 Helvick at hour 1997 where I think we sorted out that the dosing period was up until gestational day 15 and responses were not seen what's the response I mean you wouldn't expect to see an antiandrogenic response because you're not dosing during the period of male sexual differentiation but I mean you could see other responses potentially on development yeah I think the responses on male sexual differentiation well I mean it's an inappropriate dosing regime we learned yesterday that the responses normally so if dosing continues beyond GD 15 some effects of DINP etc were observed but would you therefore class DINP as a salae that causes the salae syndrome and would you call that chemical and endocrine disruptor thank you for those wonderful questions so is DINP active in producing antiandrogenic effects yes I think that there are several studies that have shown that now you can see effects on AGD well I'm sure Dr Gray will be talking about that in his presentation it might be as well show the data or at least present that to you first what was the other question endocrine disruptor I hate that term to be honest with you I suppose we have to live with it now is it a reproductive and developmental toxicant yeah and it does it do it via an endocrine pathway probably yes you use the wealth health organization definition of an endocrine disruptor which is producing an adverse effect in an intact animal answer is yes that definition doesn't take into account dose or potency at all thank you for these clarifications because yesterday we had in a different submission from ExxonMobil that using the same definition by WHO IPCA that DINP is not an endocrine disruptor and this line of argumentation was then mainly based on the fact that DINP so we were told wouldn't show up in OECD guidelines studies levels one through to five would this contradict anything we heard from you today or in other words is DINP likely to show up in these studies well depending on the dose level I would have said yes but I think you would have to do the study to the current I'm trying to remember OECD the EPA in 1998 OECD I think was a year or two later before they incorporated a lot of the endocrine endpoints and a lot of the data on some of these satellites is really quite old as I have in the last century I happen to say I was responsible for some of it myself so you know there was an element that you know you can't criticize people for using the guidelines that were current at the time that they did the work and those are the data that they found I think the issue is if you don't look for some of these effects you wouldn't expect to find it because those kinds of endpoints were not part of the normal guideline approach so you didn't do AGD as a triggered response you didn't do pre-putial separation for measuring puberty and as I also mentioned one of the issues that we have with some of our standard OECD guidelines is you only look at one male one female pup per litter adulthood and so your power to detect is really quite low for less active agents so if I understand you correctly you're saying in defence of these older studies age is a sign of quality is it not that the that it needs carefully designed studies which to crystallise to recapitulate these effects and that the OECD guidelines in general there are exceptions are not designed to capture that effect. Do I understand you correctly? I think what I was trying to say is that when these studies were done the OECD guidelines did not incorporate many of the endocrine dependent endpoints which they now do so you're not really giving yourself a good chance to find it if you don't look for it and on the other hand even under the current guidelines I think that I think I had a publication that just came out in Toxide really illustrating that we have very little power when we only have one male and one female per litter. So for example I think that paper said that if you only had one male one female and you had a rare event like these reproductive trap malformations to get a statistically significant change you're only going to see that 4.5% of the time so you have very little power to actually detect those effects and increase that to 3 or 4 pups per litter that goes up drastically to 75 to 80% chance of detecting those kind of effects. A different direction of questions but you had mentioned about the amniotic fluid study and in humans we're really limited to collecting urine samples so is there data or suggesting in the rat what urine to amniotic fluid ratios would be and likewise in the human that you're aware of so that we can get a better handle on what fetal exposure would be relative to traditional biomarkers that we use. I'm trying to think if they've been done at the same time and I'm not so sure they have Russ I don't think that study has really been done but I think it's something where just looking at the dose level doesn't necessarily equate to what the fetus is exposed to and I must say I've been burnt with that several times in a former existence that you know looking at maternal urine is not necessarily reflective of what you're seeing in the fetus. I think there are tissue banks with amniotic fluid in hospitals that collected for the amniocentesis. I think that there was some discussion about measuring contaminants and I just don't think at the time when we talked about that a few years ago that funding was available I think you have to also be careful that you know whether or not it has cells or it doesn't have cells that are in obviously the cells are what's being used for amniocentesis and the fluid is not but if you have cells there there's a good chance that they can metabolize away what it is you're really trying to look for and so you have to be fairly careful about how you would preserve those samples before you really did your analytical chemistry. Just to add that obviously the samples for amniotic fluid are based on a particular subset of women who are older or perhaps have indications for problem pregnancy so in terms of a population you know it's an elective population but there was I think one of the Korean studies did look at the correlation between amniotic fluid and urine levels. I can send that to you. Paul, thank you of course for a very nice presentation. One comment you said was that you think it's important to keep pups through adulthood to evaluate them later on. Do you ever do any cognitive assessment? I know that's a huge problem. No, I did not. So do you have a sense of neurological effects on these pups? I think all we can say in terms of I suppose they must undergo normal reproductive behavior because you can actually they can mate but the females do undergo lordosis you know you can get pregnancies at lower doses where you haven't got huge numbers of reproductive translegions but that's really a very much like a two steps removed from really doing a formal evaluation of sexually dimorphic behavior. What happened to you? I've only done very little behavioral observations with a rat mounting behavior after high dose exposure has not been rigorously evaluated. Not like hyperactivity or behavior that's whatever. Sexually dimorphic or non-sexually dimorphic behaviors or the standard developmental neurotox I don't know, I'm not sure about that. Sexually dimorphic behaviors. Okay. Is there any reason to suspect that routes of exposure make a difference? I mean your studies how are the dams? They're dosed orally by Gavage but we've also done dietary studies and found exactly, in fact those oral Gavage studies were based on an NTP multi-generation study that gave the dose in the diet and produced it pretty well exactly the same effects at the same equivalent dose level. You're not going to get the parent chemical, I mean but the real thing is that there are lots even in red blood cells. So even if you gave it IV you get fairly effective deesterification. And the chemicals in terms of low molecular weight versus high molecular weight do some tend to be more problematic than others? Yeah so we've never been able to show any effects of propyl, methyl and diethyl phthalates. You have to have chain links longer than that. We've done studies in the Pupil animal looking at what happens if you have them in different so we're talking about ortho phthalates but what happens if you have you know, terephthalates where you have one four and one three and it has to be in the ortho configuration to produce the effects. It has to be in the ortho phthalate. Yeah so it has to be an ortho phthalate and it has to be of a certain chain link for branching. So if you have too much branching like so for example you can take an isomer diambutyl phthalate produces the effects diantersary butyl phthalate does not produce the effects. So that's an isomer but it just has a different degree of branching in the side chain. And lastly one thing I was interested in you saying that you see in the same litter you can have different effects. How often does that happen that you have the same litter of different effects? All the time. So you can have the same litter and you could see nearly all the pups affected but they won't be affected by the same things. Any other questions? That will move on to our next speaker Dr. Gray. Escape? It's too late. No. Yes of course you can. So I'm going to follow on Paul's talk somewhat but I'm going to talk more in this about what I think some of the critical endpoints might be in a risk assessment and following from postnatal studies and then I'm going to talk about the potency of some of the different phthalates in these postnatal studies and compare that to the potencies that we find for inhibition of fetal androgen levels from studies we're doing now. So and then following that I'm going to talk about our mixture studies and sort of propose the framework that we think is appropriate for designing cumulative risk assessments and so most of these studies are published, not all of them and this is as that says not EPA opinion I'm not sure whether they agree with me or not and I reserve the right to change my opinion based on new data. Over the last 25 years or so we've studied the in-utero effects of exposure to a variety of different chemicals and I put them on the list here and they're grouped sort of by the mode of action for disruption and these have looked at male and female offspring as adults and for this particular talk I'm going to the discussion will include some of the androgen receptor antagonists that we used in mixture studies and then several phthalates that were we've studied in the fetal animal or in postnatal studies that have been shown to interfere with fetal androgen synthesis and we've also started some preliminary studies with dioxin interacting with phthalates and I hope to make it clear why we would do that so I sit in our studies the animals are exposed in utero and we measure a variety of endpoints in the males after birth throughout the infant at the neonatal and infant period and as adults we look for the malformation shown there and we weigh the reproductive organs and then all these data are conducted analyzed by logistic regressions to see what the ed50s are and the slopes and we use that information in our mixture studies to make predictions about how these different chemicals especially the phthalates will interact with one another what I wanted to show here is is from a study of ours that was recently published on diaxylethylphthal eHP and it had the highest dose was 300 milligrams per kilogram and I think that this gives an idea of what the phthalate syndrome is in the rat and what the most sensitive effects are that we see. How to Paul? Paul how did you get this to I'll just so this is pointing it the wrong way flip it around third training for this there we go thank you what this shows is the sensitivity of different endpoints and the first thing here is the red line is the fetal androgen levels and this is exposure gestational at 18 and the fetal androgen was measured at 18 and then all the other endpoints are from the postnatal males and you can see that the percentage of animals displaying some malformation in the phthalate syndrome closely parallels the fetal androgen levels. Analgenal distance is less sensitive it's not the most sensitive endpoint the percentage of retained nipples in the males is similar and sensitive to the androgen disruption if you look at something like hypospatias for phthalates at these particular doses we didn't see any hypospatias and if you look in the literature for phthalates some of the studies even with high doses don't report it because it's rare so this is not the most sensitive endpoints you would want to use as a critical effect in a risk assessment the organ weights are less sensitive than these neonatal and infant parameters and generally we find that in our studies the most precise and sensitive endpoint is fetal androgen levels and we feel like we're developing and others are developing sufficient information to that actually be used as a an adverse effect in a risk assessment as EPA proposed for diabetes phthalate several years ago. Reductions and testosterone during sex differentiation are causally related to many of these malformations and as you can see as Paul said that if you lump together the different individual malformations of the phthalate syndrome that gives you a lower more sensitive dose response curve than if you consider them individually because one animal has one lesion and another has another now what I tried to do here was go through the literature on several of the chemicals and I feel like these are the postnatal studies from several phthalates and I think they show that these chemicals when you examine the animals after utero exposure these phthalates are of similar potency in inducing changes in the male offspring and so this is testicular or epididymal malformations which we find as a single malformation to usually be the most sensitive and these studies have fairly similar ED50s and this is testis weight this is epididymal weight reduction and seminal vesicle weight reduction so we felt like this was given these are different studies in different labs and some even in different centuries that this was pretty good evidence that these had common potencies as opposed to dipental phthalate which we have found in our study that's ongoing and Paul found in 1983 that this was more potent than the other phthalate in effecting the testis of the pubertal animal and DINP is positive in these but it is considerably weaker than this group and I think the data we're collecting on fetal androgen levels shows the same sort of profile as the DPP is more potent the others are pretty much the same and the preliminary data we have on DINP dose response and fetal androgen levels is that it's less potent I know what I did took the data from the previous slide and just lumped all of the data together to give you a single logistic regression because I assumed that they were all the same they weren't really different and then this is the for the different effects and I would add that when we compare ED50s from studies where the fetal testosterone to these even the most sensitive malformation singly taken the fetal testosterone is more sensitive the way the literature exists is it's not possible to go back from a publication and reconstruct the percentage of animals that display the phthalate syndrome because the lesions are usually just presented individually in a postnatal study and not as the percentage of animals that are affected so this is what we're doing now in collaboration with Paul at the NTP under interagency agreement is we're exposing dams during the critical period of sex differentiation and looking at the ability yes or no to inhibit fetal testosterone and some genes we've selected three genes in the testis to look at STAR, INSL3 and SIP 11A because these are consistently found by multiple investigators to be reduced message levels in the testis during this period and then having determined whether something's positive or negative we're now running those response curves for the individual phthalates we're going to use the potency factors from these studies to conduct mixture studies in the fetus and for the important phthalates with data gaps we'll run small post-neal studies and then suggest that the NTP run a more rigorous one and we'll also use this, we'll try to run some of these mixture studies not just in the fetus but also with a post-needle assessment but as I said I think that the changes in fetal testosterone are likely causally related to the malformations and useful as a consideration as a critical effect and a risk assessment so what we've run so far in this ongoing study is these are the chemicals that we've run through the fetal phthalate screen looking at androgen levels in three genes these are positive the color denotes the potency for reducing fetal androgen levels so DINP appears to be less potent these are positive and equipotent and dipentyl phthalate is more potent than the others the chemicals with asterisks are the ones that we're now running dose response studies in this protocol looking for reductions and androgens in the three genes so we can make predictions these are negative so that's the TIRT phthalate this is DINCH and the diethyl phthalate there's some that we would like to run dimethyl dipropyl DIDP and the propylhexyl phthalate which is becoming more widespread we'd like to run these and possibly some others if you have some suggestions we don't take formal nominations but we take suggestions of all of the chemicals that Paul ran back in the last century the only real difference we found in these the sort of structure activity of the chemicals that he ran then the heptyl phthalate as you mentioned in his talk and we found that to be as positive as the others and we were surprised so we repeated that and so that was sort of the you know what I think what we and others have used as the structure activity was three to six carbons or four to six carbons were active and others were not seven straight chain seven and we thought maybe that this is a propylhexyl might also be active but we ran this one twice one with a sample we purchased and Paul said how do you know what it is and I said I read the label and then the other with some chemical that Paul provided that his chemist certified was actually dihexyl phthalate and they were the same there are advantages to GLP like studies to be sure that what the what's in the jar is really what you think it is this is a generic example because of the kind of results we're seeing when we look at dose response studies in the fetal phthalate syndrome for T production extracted testosterone to three genes in terms of the order of sensitivity so when EPA proposed this dibutyl phthalate risk assessment there were some concerns that EPA should have used gene expression instead of testosterone and I would argue that our data says that you would not want to do that because they are less sensitive consistently we've done this now with three or four phthalates and they are also more considerably less precise so that you get a much higher NOEL or benchmark dose and then what I've plotted on here also is the dose response curve for normal epididymal development in a parallel post needle study for this particular chemical again fetal testosterone is more sensitive I believe it's causally related and can be accurately measured so I'd like to talk briefly about the mixture studies just to throw out our ideas about a framework for cumulative assessment there are we've done a series of studies with chemicals that have similar molecular mechanisms of actions so this is androgen receptor antagonists and phthalates that inhibit fetal testosterone synthesis and we've done some studies where they're just binary mixtures of two chemicals and we've done a study with a mixture of five phthalates examining the effects on fetal testosterone synthesis and some of the genes and then for chemicals where we've mixed chemicals with different precise mechanisms of toxicity we've done some binary studies also we've done a seven chemical study which include pesticides and phthalates we've done a ten chemical study bigger and better than seven chemicals and I think the take home message from all of those is that whether independent of the molecular mechanism of toxicity the responses in the mixture are following a dose additive cumulative response rather than independent action or response addition which is not generally the way cumulative risk assessments are conducted and in the US EPA the current guidelines or that you would include chemicals in a common mechanism group for a cumulative risk assessment and that's chemicals that induce a common toxic effect by a common mechanism of toxicity the assumption of that approach is that chemicals that disrupt differentiation of the same tissue like the epididymis by a different mechanism of action are going to act independently and not cumulative and would not be included in a cumulative mechanism group and so this is response addition so it's assumed if you mix a phthalate with an androgen receptor antagonism those are different mechanisms of toxicity a dose is where you have nothing with the phthalate and nothing with the pesticide you'll have no adverse effects that's response addition well if that's correct and these act by a dose additive manner you can add a phthalate and a pesticide a dose is where they produce no effects and you can see 100% of the animals are malformed so that has significant consequences in terms of the so the objectives of our researcher determine how these chemicals with similar and dissimilar mechanisms of toxicity interact during sex differentiation to provide a framework for how chemicals should be included in a cumulative risk assessment and our working hypothesis is that chemicals that disrupt the development of a common reproductive system during sex differentiation will produce dose additive responses regardless of the molecular mechanism or signaling pathway that's disrupted and I'll talk more about why I believe that after I go through just some of the mixture data so these are some of the studies common mechanisms of toxicity even close on a prosimidon or two androgen receptor antagonists they block the effect of androgen at the level of the target tissue so these are working in the epididymis and in the in the phallus and they alter the same genes in the tissues so they have a common mechanism of action so in this particular study this is a binary study with just um we took a dose of inclosalin and gave it to the mother and a dose of prosimidon and gave it to the mother and then in the combination we mix this and this so if this is a response addition you'd have 10 plus 0 or 10% of the animals would have hypospatias and you'd see animals with a vaginal pouch and so this is what you see so you see 10 plus 0 equals 96% 0 plus 0 equals 54% so this is, we called it the new math I think Andreas called it something from nothing what it is is I always like to get Andreas upset and say it's synergy but we don't believe it's synergy we believe that this is dose addition is that correct Andreas? yeah these were simple studies just to demonstrate this sort of response is not response additive as you would expect for making chemicals with a common mechanism so we've mixed a couple phthalates so we've mixed benzobutyl and dibutyl phthalate they have one active metabolite in common and again you see 0 plus 0 equals 50% incidence of hypospatias and epididymalagenesis is 9 plus 23 equals 100% these are not low dose studies this is a limited exposure during short window of sexual differentiation and we're focusing on some of the higher dose endpoints one because these are things that people actually consider adverse without debating whether AGD changes are adverse or not and they also show a very steep threshold so it's easy to distinguish the difference in the predictions of the model from response and dose addition for some of the lower dose endpoints that are sensitive phthalates they appear more linear and you can't really distinguish response and dose addition and the purpose of these is not to set no wells or necessarily to be used in risk assessment but to establish ultimately which is the best model overall for beginning to do this with broader dose ranges and at lower doses so this is another pair of phthalates with different active metabolites and again you see malformations with the individual chemicals but they're much more at a higher incidence in the mixture group so we're seeing greater than response addition with mixing two phthalates together and those phthalates we consider to be equipotent so in this five phthalates study we had dose response studies on the fetal androgen levels of each of these phthalates and based on those predictions we made a fixed ratio dilution study at a to see if we could predict how these would behave with the expectation that they would be dose additive and so if you look at that outcomes of the androgen production this is the observed effect and this is the prediction from the dose addition model and if you looked at the individual responses of the phthalate at what it contribute to here they'd be here now we're in the process of completing a postnatal study with similar exposures to these five phthalates and what you see there is that this is the response addition prediction and this is the dose addition prediction for the incidence of epididymal malformation so clearly these are not response additive these five phthalates did appear to act equipotent manner in producing lesions in a dose additive cumulative fashion and the same is true for seminal vesicle agenesis response addition is not a particularly useful model these are not acting independently we have found in some of these studies the mixture studies at high doses we find things that we did not expect we don't have enough individual data on the chemicals to talk about whether it's synergy or dose addition but I think it's important to point out that and with this what we call the mega phthalate study when we looked at the female offspring we saw a very high incidence well 60% of the females had agenesis of the lower vagina and uterus unicornis agenesis or partial agenesis of one uterine horn and this had been seen in some of the individual phthalate studies in our laboratory or in Paul's lab in Delcris paper and a very low incidence just two or three animals so we were quite surprised to see this in 60% of the animals whether or not the critical period for development of this is the same whether the potency of the phthalates in the mixture for contributing this is the same we don't really know yet it's clear though that this is not related to a reduction in testosterone in cell three or any of those and it just points out that there's a lot about the upstream events in the genes and what's happening in sex differentiation with the phthalates that we don't understand and we think that what's going on in male rat sex differentiation is in the changes in testosterone in genes or downstream events from what's really there and so we are working with Paul to try to figure out what's happening earlier in the genes and things but I think people have been working on the mechanisms of phthalate toxicity since 1982 so we my career may not last that long but that's what we're trying to do so what I've talked about is chemicals with diverse mechanisms of toxicity but they disrupt the same signaling pathway they're disrupting androgen signaling in the fetal tissues and so these so these are different mechanisms of toxicity we've mixed the phthalate ester with linearon, a pesticide that has some weak androgen receptor antagonism and also inhibits fetal testosterone since it's a little bit different with Paul about that and so we put these together in this combination because that's what we thought the relative potencies were like and this is a binary study so this is the effect of the benzalbutyl phthalate alone and the effect of the linearon alone and when you put these two chemicals together you see 0 plus 0 equals 56% so this is not a response or independent action, these are acting in a cumulative presumably dose additive manner even though there are different classes and some different pathways we've also mixed phthalate with prosimidone and I think this is probably the clearest example in a binary study this is a androgen receptor antagonist that has no effect on fetal testosterone levels like flutamide and this is the dibutyl phthalate that doesn't bind the androgen receptor but does lower fetal testosterone dibutyl phthalate is working on the fetal testis prosimidone is working in the tissue so they're not even affecting directly the same fetal tissues and when you combine these in this simple study you see 1.5% hypospatias plus 0% equals 49% so we decided this study was interesting enough to expand it and so we took we did a fixed ratio dilution study with this which Andrew just published and so we have a variety of dilutions of the top dose and we used dose and response addition equations to predict what the outcome should be and you know when we did the study which is harder than actually just doing predictions so what we found was as indicated from the earlier study dose addition provides a better prediction of the observed effects than response addition so these two chemicals with very distinct mechanisms of toxicity acted jointly in a cumulative manner now as I said in the phthalates sometimes you find things that you hadn't anticipated probably just because you don't have all dose and dose response data and what we observed in this study was a very nice dose related incidence in testicular tumors like the ones that Paul showed earlier with Diabutyl Phthalate so these have been observed that are low incidence in a few animals with Diabutyl Phthalate they have not ever been observed with Prosimidon but when we combined these two we saw a dose related increase I think it was up about 25 or 30% in the highest dose group so that was just unanticipated but not necessarily represented of synergy so we've done, I'm going to mention two other mixture studies of chemicals that disrupt the androgen signaling pathway by a different mechanism so there's seven pesticides administered that we examined the offspring we tried to construct this mixture study so that each of the seven chemicals contributed equally to the mixture and we did a dose dilution with a limited number of dose groups and this is the result of that again this is the observed, this is dose addition this is independent action and response addition models in blue and green and you can see for the endpoints presented here dose addition is consistently a better model for these seven chemicals in response addition or independent action models for each of these oh that's pretty interesting I don't think I typed that this is 10 chemical mixture study Nina did you do that to my slide last night in this this is bigger and better, this is 10 not 7 we tried to have larger sample sizes and more dilutions but the intent was the same to compare the responses of the models and the high dose group we call the ED 100 so we have the same four pesticides and six phthalates the last one had fewer phthalates in the seven so it was four pesticides and three phthalates and we have more dilutions so we have better dose response curves Cynthia Ryder who worked with us before was always critical of our simple two by two studies as we really only had one mixture dose group to compare with our models she was very critical she wanted more dilutions in this study than the last one so we're always responsible and trying to improve especially for dear Cynthia and so we did this one and we would like to build one of these with more and more chemicals with the idea of being able to show that they behaved in a dose additive manner where each chemical is really administered at a dose where you get below the no effect adverse effect level for each chemical so we're getting there with 10 and we now have more phthalates that we've identified we need to conclude in here and maybe more pesticides so these are just again the results here are the same dose dose addition is a good predictor or a better predictor for these two end points than integrated addition or response addition and then for undescended test this these are sort of equivocal so it's response addition is about as good as dose addition so what we often see in these is that dose addition is better or equivalent to response addition in all of these end points and all of these studies we have not seen that independent action or response addition models are better than dose addition models so my recommendation would be if you have to pick one to start with you'd start with a dose addition model and have to have a reason why we're not doing that and then this is just more the same data showing the dose addition is a better predictor of the black line the observed than the others so what I've said I think this is the it's the most logical model for the data response addition often fails to explain the results it's consistent actually with the biology of hormone action on this tissue and that all of these chemicals are disrupting a common pathway the androgen signaling pathway what the tissue sees is a reduction in the androgen receptor bound to testosterone or dihydrotestosterone so in both cases androgen dependent gene expression is attenuated the androgen receptor antagonists do this by preventing testosterone from binding the androgen receptor desynthesis inhibited by the phthalates and so it's reducing it through it's causing the same effect at the gene level through these two mechanisms the tissue doesn't know the difference and why the response is I believe dose additive what we're also trying to do now is expand the studies beyond a common pathway and look more at a common tissue so there are dynamic interconnected pathways in development and so what we're trying to do here is look at ethalate and dioxin these disrupt some of the same androgen dependent tissues through apparently very different pathways how will these interact and we only have preliminary data so far but the preliminary data indicates that many of the effects in the mixture groups exceed response addition so we're repeating this study with a larger number of animals with a larger number of dose groups to see if if we go for different pathways not just androgen signaling pathway how do those behave and again we in this study we saw effects on the liver of the offspring in a small percentage of the animal that we've never seen before and those are out now for histopath evaluation so I think that's going to happen at the high end of the dose response curve but we haven't really seen unanticipated effects at the low end of the dose response curve which you would really be more interested in so we believe that the framework for these tissues should be based on disruption of common reproductive tissues differentiation of these tissues is requires critical interactions of dynamic interconnected pathways as I said they are dynamic interconnected pathways so what as Paul said what's happening on day 15 is not what's happening on day 18 is not what's happening on day 20 if you look at the genes at day 4 postnatal or day 18 or 19 that's not telling you what happened to cause the problem it's telling you it's it's an effect and I don't know that there's any expectation that the there's no reason to expect that the pathways are the same and I think that all the chemicals that affect the same tissue during the same critical period could be included in a single cumulative risk assessment and so the effects of a mixture would be predicted from the relative potencies on a tissue by tissue basis and then these are the folks that have collaborated on this project I should update Dr. Reiter going to Duke University is now working with Dr. Foster recently and they are developing a fairly active mixture program to sort of expand this idea so thank you thank you questions great Chris thank you for a very good presentation couple of small questions first so when you do your mixing ratios you look like you've used equipotent mixing ratios is there any reason to expect that if you use different ratios the effect of being mostly dose addition wouldn't hold we started the seven chemical study and the others were equipotent when we did the 10 chemical study we fudged that a little bit because we thought well we have four pesticides and three phthalates and we put that together so that they were supposed to be equipotent but if you look at it in the integrated addition model I thought if we put six phthalates in the four pesticides in the equipotent that the phthalate effect would predominate so what we did in that one was had the same percentage of pesticides but cut the phthalate contributions in half but it wasn't exactly equipotent but I think the question is if you go to a protocol where you're looking at the raise lower contribution would it deviate from dose addition some of the chemicals are more dominant than others and that's something that might be more relevant to actual human exposure where it's based on our problem in doing those kinds of studies is if the difference between response and dose addition models would be much smaller and therefore we'd have to have huge sample sizes but in terms of just expecting dose addition at the end I would expect dose addition and we've done some of these we've started to do some things in vitro to look at that with estrogen but there isn't anything we can do with the phthalates in vitro we might in the fetal phthalate studies be able to do that sort of redesign I think you're talking about where there's different mixture ratios because that's a higher throughput five day eight day study as opposed to the postnatal studies take us a year a small study has 150 animals but I'm not asking so much about studies you do as much as just what you would expect I don't see any reason dose addition should hold to see that it would not be dose additive in some of your plots you had IA and RA integrated addition that's where you cluster that's a LeBlanc cluster the phthalates together as dose additive and then the androgen receptor antagonist would be dose additive and you'd combine the prediction based on independent action right so I guess the final question I'm interested in when you're putting chemicals together that are very diverse mechanistically and you're starting to show things that are dose addition and in terms of thinking about risk assessment if we're thinking about a risk assessment of just phthalates I mean is that going to be underestimating risk if now we should be concerned about phthalates mixing with dioxins or mixing with pesticides or mixing with other things what's your thinking about that in terms of how to choose chemicals for inclusion in a risk assessment yeah I'm I think I mean I do think that the data indicate that these will interact in accumulative fashion and so that but I do think at the point where you want to do a real risk assessment you're going to want to know something about exposures is what to include or not so I so in Closelin may be useful in this model but there may not be much of in Closelin if any in the general human population and so for the general human population you wouldn't include that in the model on the other hand most people have some body burden of PCBs and dioxins so you might want to consider that but I think these are sort of to establish the behavior of the so they're not necessarily relevant doses of relevant mixture ratios but I do think ultimately you'd want to go back and if there was a typical mixture ratio of there's people had generally this much dioxin and the highly exposed people had this much phthalates that those would be good studies to repeat but I think I think it's a long range question because sort of the beginning of cumulative risk assessments more work may I carry on in this vein you pointed out to us quite convincingly that the most sensitive end point for phthalates is fetal testosterone synthesis reduction suppression and you made some comments that you would recommend that as an end point to be used as basis for risk assessment and I see the logic for that now considering the phthalates first are there studies available to your knowledge that would enable us to define points of departure either no observed effect levels or benchmark doses for these end points for phthalates you're interested in so we've run some benchmark dose models and some of our the dose response studies we're running are really probably more appropriate for a benchmark dose model and then no well because we have more dose groups in the lower dose range not large sample sizes and I know that the data that we're providing to the EPA and CEA that's beginning to do the risk assessment on six phthalates is their intention is to run benchmark dose models on these for their risk assessment is this data published in the peer reviewed literature or publicly available there's the only data on the fetal androgen levels and dose responses from the how to shell paper and we're working on several other papers and as I said we're doing several other studies can I just about the how to shell paper the how to shell paper is a fantastically well designed mixture study but in terms of animals used for dose group I wouldn't say that that is a study suitable for deriving any points of departure would you agree or whether I think the point of departure question I guess is it would be nice to know how much of a reduction in testosterone produced an adverse effect as opposed to using just a benchmark dose say a standard deviation or a no effect and we are doing studies to look at that so we have fetal androgen levels from these studies and then we have a postnatal study and so how do they compare and I don't think we can really say that we've done that in three studies and adults weren't in terms of the percent reduction that led to a change they weren't identical it just needs more work but I think it can be done and determined it just worked so just for a point we just published our DEHP multigen with benchmark doses for the phthalate syndrome and in that particular study we actually kept back more malpups to improve our power and I think it was like a seven dose level study so it was a huge amount of work so that's in like the current addition of toxin with the BMDs continuing the line of questioning from Chris just a minute ago so we would be able for a cumulative risk assessment of phthalates to integrate the cumulative effects using suppression of testosterone synthesis as the end point now the issue is that many of the other androgen receptor antagonists which you also investigated in your mixtures are not affecting that end point because they operate by a different mechanism so it's not easy to integrate these two end points. Do you see a way out of this dilemma or VRSA making allowance for background exposures to other antioxidants to correct the dose responses for phthalates in some way? Do you have any thoughts? Yeah, you're absolutely right we could use the fetal androgen level for the phthalates or other chemicals that reduce fetal androgen levels but the androgen receptor antagonists don't do that so that's not a useful approach if you're going to mix chemicals with dissimilar mechanisms and so there I don't know now of any other way to establish potencies then from the postnatal studies there's not a lot of good markers in the fetus of what's happening with androgen receptor antagonism in terms of looking at gene expression in the tissues and stuff. I think we've gone through five or six postdocs asking them to look at gene expression in the phthalates after high dose finclosal and exposure and saying have we changed the genes in a female like direction and the problem is that a very small percentage of the cells are affected by the androgen receptor antagonist that lead to failure of closure of the midline groove of the penis and it's just it might be 0.5% of the cells so you have a complete dilution effect and it was an incredible waste of time of five postdocs so when we have a new postdoc that's sort of the dirty trick is to say do you want to do this and they go oh yeah so I don't know what the answer to that is just a last question about dinge you presented in your one slide well you didn't present data but you said dinge doesn't work we yesterday heard from Dr Otter from BASF who also tested dinge that there were changes downward changes in anodental medicines both in the males and the females so your work would that contradict what Dr Otter told us yesterday what we we have our study was in this fetal phthalate screen which used a minimal number of animals at a single dose it would be possible if you gave higher doses than we used it's also possible that the effect on anal genital different distances I don't know if the animal's body weight was drastically reduced or anything like that so Dr Otter would have to share the data with us well that is the problem Dr Otter is not willing to share those data only a robust summary so it is very difficult to investigate or evaluate what actually is going on I'm not even sure I correctly summarized what Dr Otter said I think we have indication that it's negative and you can never prove a negative and we haven't had my feeling was we were going to run positives through a dose response to develop potency factors but some of these may be potent important enough it would run dose response studies anyway at higher doses like we did with the INP is it possible Dr Otter is dying to say something would this be a pick-out without giving him a chance I think you are perfectly right that it's negative and also I said it's negative but we have reported that it's a minimal effect in both sexes and this is related most probably to body weight changes and we have seen not any other parameter so this is sorry a misquotation and that's what I wanted to say but still it's very it's very difficult to evaluate this we're stuck there a little I think it's an important chemical as are many of the phthalate there are other chemicals that are being used as replacements that we need more data on thank you Earl for sharing the information and the wisdom question about metabolism of phthalates the effect of phthalates on itself and other phthalates when you do these mixture studies is there an effect of over repeated dose time does one phthalate affect its own metabolism and if you have a mixture does one phthalate affect the metabolism of other phthalates and if there is an effect does any of that hold up at levels of human exposure well the let me take that we've done some of the dose response studies we've done like with the EHP we're 0 to 300 milligrams per kilogram we don't see the leveling off of the dose response curves that might indicate limited uptake when we do the mixture studies Paul always warned us that we might at some point saturate metabolism more interfere with one he keeps trying to get us to do a DEP DEHP mixture study with the idea that high doses of DEP might interfere with the metabolism of DEHP we haven't done that one yet but he's so that's possible we haven't seen anything like that and our dose response curves are the mixtures that the doses we've used some of which are going quite quite high haven't we haven't seen the dose response curves really flatten off but I think that I think when you go down to the levels of human exposure at the top end of human exposures those are at the lower end of our dose response curves I think the studies we did with Antonio Calafat were the I think it was 11 milligrams per kilogram of DEHP and 100 milligrams per kilogram of DVP which just produced a little effect in the animal so we're human the general human population even the highest exposed don't seem to be at high doses we're using given the exposure information we have which is I'll let you deal with that but it's it's hard to get it's dynamic it changes what tenfold and day with an individual and from day to day and I'm not sure what human exposure really is they can probably he can probably tell me better Dr. I guess just taking that a little further you know apart from phthalates or other chemicals that may work through altered androgen signaling there are chemicals that may alter glucuronidation or metabolism of phthalates that don't work at all through androgen signaling and if those lead to longer half lives or retention of phthalates potentially they could potentiate responses Mike did you have a yet your light on one point in the so the work that you're doing for NC you're doing dose responses on the six individual phthalates or is it mixtures know that right now they're doing the six individual risk assessments and then they want to do the maybe do the mixture they're going to evaluate at that point so I mean we're trying to see where are the data gaps so there are some of these chemicals they don't have the data they'd like or some of them like the dipental they realize there's almost no data other than Paul's paper they said well we said we do a risk assessment by a certain date and we can't find any data so we're doing the individual chemicals in some cases like that and then there are some that like so I guess when in my conversations with NC scientists including Andrew Hachkes who was supposed to come with us but you know they say well you know we really would like to have dose response data on the DINP in the fetal phthalate screen and we they wanted the DBP data because they're working on that one so that we're trying to at least provide them the short term data they can use I think that for some of the other phthalates we already have the data okay I'd like to come back to the exposure data we had some discussions about it yesterday on the validity of the exposure data let's say the best before date on the studies currently available and we have to be aware that the fourth report just published in 2009 is based on urinary data from 2003 and 2004 so literally we are working in the black box for the last five years now regarding exposure data but I have another point regarding the testicular tumors you mentioned my understanding was that the testicular tumors are part of the testicular dyskinesis syndrome but I haven't brought them into connection with the phthalate syndrome right now so are these testicular tumors in effect you found in the mixture studies or would you count this point also to the phthalate syndrome now we we and paul include the light excel tumors and light excel hyperplasia that we see in the phthalate syndrome I think in the testicular dyskinesis syndrome they are primarily talking about germ cell tumors which we haven't seen in the rat there are paul can tell you more about this but human pathologists use different criteria for testis tumor categorization and rat pathologists and so if you use similar criteria there's actually what light excel micro adenomas and human testis that are often associated with subfertility or infertility it's more common than is referred to as light excel tumors in men just because of the way they score them so but it's not the germ cell semenoma that skankybeck is talking about I have a few questions Earl so you very elegantly showed that the decrease in fetal androgen is the most sensitive endpoint but unfortunately we can't look at that in humans at least I don't know anyway to look at that in the population study of humans the next most sensitive was nipple retention but as I understand it this is not a human endpoint so I think the next one is then the AGD and so would you agree that for a human study then for a population based study the most sensitive human endpoint would be AGD that's my first question well I don't know if you can extrapolate one to one across species like that okay but we also saw that more sensitive than AGD was just the percentage of animals that displayed some malformation consistent with the valley stream more sensitive than AGD but actually most of those endpoints unfortunately are not accessible to us in terms of our human infants in our new study we will be doing a sensitive ultrasound and I would love to hear from others you and what endpoints we might hope to visualize but at this point we're limited to the external genitalia so my other question was if you could comment on the Christiansen study that came out a month or two ago from Ulachas' lab in which they reported decreased end genital distance and increased nipple retention at 10 milligrams per kilogram per day 10 milligrams per kilogram of what DHP well we have we have effects at 11 and 10 milligrams per kilogram but it wasn't anal genital distance ours were in Paul's study the NTP was malformations of the testis epididymis and in our study it was it was 11 milligrams per kilogram we had a 10% incidence of some animal with the phthalate syndrome so it's plausible then fine actually it's not wildly radical right you mentioned a couple of times the equipotency of the phthalates in question at your let's say high dose experiments now when I look the low dose TDI data or the reference doses I can see a difference of factor of 50 in the phthalates you mentioned as equipotent so do you think this is an effect of different slopes those response or is it an effect that the data basis is different what's your yeah I think I think if you go back and look at the individual risk assessments on some of these phthalates as Paul said they are very old I don't think any of the studies include all of the end points that we include in our studies and none of them have put together how many animals have any malformation consistent with this syndrome so I think that that's some of the difference in what we see but the studies are done in different labs and people have different skills and expertise and they're done over a long time frame so as I said more than half of the studies don't even talk about hypospatias and that's seen more frequently in newer studies so I think that people are becoming more aware of what to look for I don't think anybody looks at the gubernacular ligament in these studies and scores whether it's there or normal or not I think there are a lot of factors that could contribute to those and my expectation was that if the NTP were taken to redo all those studies in a similar protocol using all the sensitive end points that they would come out very similar and that would probably exhaust their budget and might not be the best use of their funds so in other words you're saying a non-observed adverse effect level would be around let's say 15 for these five I think that's the other point as Paul made is in his study in our study he has 70 to 80 animals per dose group and we had 60 to 80 and if you're doing a no effect level determination it's dependent upon the statistical power so if you do if you have 20 animals 20 males per dose group you can pick up a 25% incidence of malformation and not lower and so if there's a 10% incidence of malformation in there it's not going to be significant just because of the sample size so I think that if you really want to establish the some of these chemicals that needs a special study design not just the standard which is designed for lots of different chemicals and not just reproductive toxicants but you certainly wouldn't expect a difference in with a factor of 50 between these stellates? No that would be my expectation if not let's take a short break and reassemble at 10 after 11 to accommodate the travel plans of one of our speakers because we've gotten behind on our schedule we're going to have a change in the order which Juan will now make her presentation Dr. Peters this afternoon early. Do I need to stand here? That's your only mic okay okay so I have really an impossible task which is to tell you about all the studies that looked at health effects in humans in 30 minutes so obviously I cannot go critically into any of these studies so what I'm going to try to do is to go through them quickly you'll all have the presentation and I'd be happy to make available bibliography or any of the studies PDFs if you want them and what I'm trying to try to do is to give an overview and then summarize at the end what I think they add up to okay so the first place to start I think is prenatal phallid exposure and the studies that have looked at alterations of infant genitalia and those have been the endpoints that have been looked at have been AGD penile size and hypospatias and I should have put their testicular adjacent because we also looked at that there are studies on length of gestation and then there are some endpoints that it's a little hard to unscramble whether the associations are with prenatal or postnatal exposure because the children are examined sometime later and they can have been affected by both prenatal and postnatal exposure so the studies that have looked at alterations of infant genitalia there's R2 05 and 08 there is an abstract presented at IC by Bustamante there is a study by Huang and study by Orman and to my knowledge those are the only ones that have looked at this and by the way if I have omissions I would love to know about them later because it's hard to cover the whole literature so just to remind you what Paul and Earl have been talking about the phthalate syndrome and the point I want to make here is that our studies were designed to specifically examine this syndrome in humans unlike a lot of epi studies where the endpoints are classical epidemiological endpoints I designed this study after talking to toxicologists and pediatricians about what they might expect to see in male infants who are under androgenized so the other thing I want to point out is that yes is that the dark indicates what we could look at in principle in humans and nipple retention we could but it's not relevant so we really have very few endpoints visible externally and we can't do surgery on these infants obviously unless there are special cases like surgical patients and or as I think I mentioned in the discussion that we're starting in our next study we're going to be using ultrasound of the abdomen and the testes of these children so we'll have possibly other endpoints to examine so just briefly our studies the study for future families included women and their partners recruited at prenatal clinics around the United States and the women were eligible if they were over 18 and their pregnancy was naturally conceived and we obtained questionnaire serum and urine samples and the second study was a follow-up of the children born to these parents now the original study was not designed as a pregnancy a cohort follow-up study we had not planned to follow the children when we designed the study it was actually designed to look at semen quality in four cities in the United States so there was a gap between we actually did the second study which is why we lost sample size because we were not funded to do this follow-up until later the first study was NIEHS funded, the second one was EPA funded so the design was simple we had the mid-pregnancy urine samples which we analyzed for phthalite metabolites it was 28.6 weeks on average the urine was collected obviously not ideal and then we measured we had an exam of the infant genitalia which included a measure of the penile size the analgenital distance by several measures scrotal size and the scored degree of descent of each of the testicles and I want to show you this diagram because it's not actually clear what we should be measuring and this has come up again and because we are funded to do another study which is going into the field next month and so we are doing a lot of work on trying to figure out what are various markers landmarks that we can measure in these boys and also in the girls so in the literature on human analgenital distance the anal scrotal distance the shorter of these measures has been used more often actually if you look at the say images from pictures from Richard Sharp and others it's actually neither of these but I believe and Erlen and Paul can correct me if I'm wrong and why can't I see this there it is it's this place just below the penis the lower insertion of the penis to the center of the anus but it's a little hard to say with a newborn pup so we are probably going to measure all three of these in our next study and try to see which ones are most sensitive but in our first studies it was the longer of these that was more associated with phthalates although the shorter distance was also associated with some phthalates and so this is just a very brief summary and I think you've all seen at least the O5 paper in the O5 paper we used anal genital distance divided by weight because of the work of John Vandenberg and Andrew Hodgkes this was a very common thing to do in rodent studies it actually statistically is not a great thing to do because it doesn't remove the effect of weight and so in order to appropriately control for weight you have to put it in the regression model as a covariate but then if you put age in which we had to do because our boys were on average 12 months of age but there was a variation in age you can't put age and weight in the same model because they're highly correlated and so what we put in was weight for age using CDC standardized curves and age and those two variables are quite independent and this is a very good way to control for both weight and age in our next studies we'll be examining other markers of body size and exploring which ones are optimal but what you see here is that and this is only a summary using these as dichotomized or trichotomized variables of course we also had to use these as continuous measures but just to simplify things for example if we just take oh I'm sorry if we just take let's just take MEOHP well that's the biggest one you'll say I'm picking that because it's biggest how about MEHHP and you look at high versus low that means that if the mother exposure was in the highest quartile compared to the lowest quartile of MEHHP in our distribution her son was 13 times more likely of having a shorter in a general distance that is in the lowest 25th percentile controlling for weight and age so this is a very quick summary of a lot of data and you can look at our papers to see more details we also looked at other endpoints and we found associated only with DEHP were a penile width and a degree of testicular descent so in this study it looks like DEHP is associated with several markers of the phthalate syndrome but not other phthalates the paper by Wang is on Taiwanese women undergoing amniocentesis so it's a different and this is what this question came up recently about amniotic fluid they did correlate that with urine they detected several phthalates in the amniotic fluid and they did use the shorter measure of in a general distance in both males and females and they found an association with AGD which is actually anis for set distance in the females of all the studies that I'm going to show you this is the only one that found this an association in females it was based on 32 females they found no association in males there is a study out of Mexico which unfortunately I've only seen an abstract form 73 Mexican women and they found no association for MEHP but MBP and MEP were inversely associated with some marker of the phthalate syndrome and finally I want to mention Ormond which is a different kind of study this is a case control study of hypospatias in the UK this is a very large study for a study of hypospatias which is so rare almost 500 cases in controls and they didn't have biomarker of exposure they had report questionnaire which they used to define exposure using an exposure matrix and they found significant associations with exposure to phthalates not broken down further they couldn't do that or use of hairspray so this is interesting it's the only study I know that looks at phthalates and hypospatias ok so now I'd like to talk about another class of endpoints and those are the endpoints of brain and behavior so if you think about effects of testosterone and development of course it's not just the general development that's relevant but the brain is also affected and this is of course well known and so we also have data not from phthalates but from other anti-androgens that have been identified that rodents who are exposed prenatally have changes in both their brains which have been measured the sexually dimorphic nucleus of the preoptic areas altered but also they play in a less male typical manner so we decided to look at this question and so we use something called the preschool activities inventory which is a maternally completed questionnaire that's used and validated thousands of children and it's been shown to show excess more masculine behavior in girls with congenital adrenal hyperplasia which is a genital defect associated in girls with a longer intergenital distance and more masculine behavior and PCBs in one Dutch study showed differences in play behavior so this questionnaire is pretty simple it's it's a Likert scale there's 24 questions how often does your child play with dolls, play with guns play dress up etc etc and we mailed it to 334 mothers whose children were old enough this was for age 4 to 7 at preschool and many of the mothers had moved out of their children and we had a completion of 72% of those who were delivered and we ended up with about 150 children with usable data and we hypothesized that again as in the genital studies we made these hypothesize these ahead of time we hypothesized that we would not see phthalate related differences in girls and that is what we found we found that the masculinity score which is a score constructed from the PSAI there are three scores masculine, feminine, composite excess more masculine is a higher masculine score it's also a higher composite score which adjusts for the feminine side of things and what we found was that two DEHP metabolize but not MEHP were significantly related to more excuse me less masculine behavior and there was no effect on the composite and feminine scores and interestingly that for we saw a borderline significance for MBZP for the masculine score and for the composite score this was significantly decreased for MIBP and borderline for MBP and again this is less masculine behavior so this was a preliminary study on a relatively small number of children which suggests that there are alterations in play behavior linked to prenatal exposure to phthalates and in a Korean study of 261 children they found just looking at metabolize of DEHP and DVP they found that there were associations with DEHP on ADHD and MBP on certain errors of commission and omission which are related to impulsivity and attention I wrote to Dr. Kim and I asked him for whether we could get this information by sex of the child and he said that would be interesting but I never got it so I don't know whether this is a finding in boys or girls or both that's what we know so the study of Cho in Korea is children at nine years and this was on IQ they did get the mother's IQ and they found that MEHP again and MEOHP were inversely associated with vocabulary score and they said in the text that the associations were in boys only but this was not shown in the table so again I'm not certain about the sex dimorphism of this association and I think that's something that's really critical to the plausibility of these studies oh I'm sorry I didn't summarize the Engel studies and I can't do that on the cuff and I just realized that's an omission two studies out of Mount Sinai by Stephanie Engel on newborns in relation to prenatal exposures and I'm gonna have to send you a slide on that I'm sorry so then there are studies on the length of gestation which is really interesting and complicated and still I think unresolved so the first study was a long time ago it was by Latini in Italy and it was a fairly that was early on and there wasn't a lot of we didn't know as much then so what he did was measure MEHP and DHP in cord blood just wasn't measurable or was it not measurable plus minus and he found that there was a shorter gestation when there was a positive finding you know when MEHP was detectable but it was quite short and it wasn't he did both DEHP and MEHP and DEHP itself was not associated with gestational length Mary Wolf in Mount Sinai in the same cohort that Stephanie Engle examined found that gestational day was actually increased by about one day for each one log unit increase in MEHP and typo there I'm sorry also associated with the sum of the low molecular weight valid so in this study it looked like increased exposure was associated with a longer as opposed to the Latini was a shorter gestational length in Jennifer Adibi study in our populations we also found a longer gestational age associated with MEHP metabolites and they increases were statistically significant again about one day so consistent with the Wolf studies and one thing that's different about this is that Jennifer found a significant increase in the rate of unscheduled C-section and the risk of delivering at more than 41 weeks conversely in the Wyatt paper in New York and Columbia there was a significant shortening of gestational length and an interquartile increase in metabolite concentration for all the DHP metabolites was associated with a five days shorter gestation and finally we have a study in Mexico from John Meeker and this is a small study looking at preemies and controls and all the metabolite metabolites were higher in the cases and the strongest associations after adjustment for urine dilution which they did both by specific gravity and creatinine were with MbP and MCPP which is interesting because MCPP doesn't usually show up here but I have to tell you that we have a finding on MCPP related to growth of the child after birth which is significant and will be published this year so MCPP may turn out it's a non-specific metabolite of DHP and also of MbP of DbP so it's interesting I think we may want to look further at MCPP so these studies are hard to summarize there were differences in ethnicity socioeconomic status for example one population was all black and Dominican Republic one was primarily white and so on methods of controlling for urinary concentration the timing of the samples were different and it's not clear what's the appropriate time to collect the sample related to timing of labor and gestational age and the phthalate exposure levels actually varied quite a bit so most of the strongest associations were with DHP we saw considerable geographic variation even within the study for future families and so we need to repeat this and we will be doing that in our new pregnancy cohort study so postnatally almost all the exposures almost all the papers have found associations in males and these are associations with serum hormone levels premature breast development female one male that's an exception behavior and neurodevelopment and liver function and allergy and asthma and I just wanted to show you this if you haven't seen it this is on our children we actually got urine samples from our children at their approximately 12 month visit and we measured phthalate metabolites in their urine and this is the percent of infants that had a number of phthalates that were detectable in their urine just to point out that there is considerable exposure to children postnatally and so the Katrina main study in Denmark collected breast milk over the first three months postnatally and related it to serum hormones in the children's blood serum hormones at three months of age and this is in boys and so they found a decrease in free testosterone and increases in the gonadotropins there are two studies only on premature breast development and they're quite different the early study by Cologne in Puerto Rico looked at girls and found associations with I'll go into that a little more detail and then Dermaz in Turkey looked at related question in boys so the Dermaz study looked at male gynecomastia at 13 years of age 40 boys with this condition and 21 controls they measured serum DHP and MEHP and found these to be higher in the boys with premature breast development and these were not related to the boys serum hormone levels and the Cologne study were girls with premature thalerky and they were quite young mean age of 31 months in Puerto Rico and this was a little tricky study to interpret they just said whether these were levels were these phthalates were detectable or not and the largest differences between the cases and controls were for DEHP but there were higher levels pretty much all of the phthalate esters and also MEHP in the cases in fact 68% of the cases had one or more of these phthalates compared to only one in five of the control subjects there was a big difference but it's not clear what their methods were there's been a lot of criticism of this study there are a large number of studies primarily out of Scandinavia in Bulgaria now looking at the question of asthma and allergy and the history of these studies are interesting Bornehag actually was an engineer who was studying mold in relation to allergy and asthma and found this kind of serendipitously the fact that polyvinyl chloride in the home and particularly in the environment of the children to the prevalence of allergy and asthma he did the initial study in 2004 and then a colleague he repeated this in Bulgaria and found pretty similar findings in Bulgaria and there's a lot of studies now that look at PVC not phthalates but PVC which of course is where a lot of DHP and DVP are used to so if you want to look at that review I couldn't summarize them all but that's just a very recent review by Bornehag but most of them are associating indoor air exposure to PVC in relation to these symptoms and they are now conducting a large study in Sweden called the Selma study which is actually getting biomarkers so that they can follow up on this now I just want to talk briefly about phthalate exposure in adults and these associations are all in males so there are the semen quality studies and there are studies of adult male serum hormones there's also study on pulmonary function metabolic syndrome and thyroid function so the semen quality studies initially there was the Russ Housers and groups study at MGH and those are men at an infertility clinic and they found that there were inverse associations with monobutal phthalate and butyl benzel mbzp I'm sorry I've repeated that and sperm concentration and then mbp was inversely associated with sperm motility conversely Johnson study in Swedish conscripts it's his young men unselected but they're being seen for military service he didn't find associations with mbp or mbzp but he did find association with mbp and reduced sperm motility so these published data are inconsistent and I have to just add that and I will make these available to the committee that there are two new large studies coming out and one is infertile man that finds no association with semen quality and one in a young man who actually have poor semen quality who have significant associations with several of the phthalates and semen parameters so it may be that if you put these together that it's men with poor semen quality that are more sensitive to effects of phthalates on their sperm parameters so that's not unusual for many studies of semen quality many risk factors affect only those on the low end of the semen quality distribution as far as male serum hormones there are two studies that have been published one is the Russ Hauser's group the same men that were examined for semen quality were also examined for serum hormones and in the study for future families we also looked at serum hormones in relation to phthalate levels so there are some differences in the serum hormones as you would expect in a partially infertile population at Harvard they have poor hormone profiles and better among fertile men and as far as the metabolite levels they are also quite different and that was kind of surprising to us that DHP levels are higher in the infertile or possibly infertile population whereas the other phthalates tend to be higher in the fertile men and so in both of the populations one or more of the metabolites that DHP are associated with one or more measures of decreased free testosterone that's the biggest finding across the two studies that there is a decrease in free testosterone and it's only with DHP metabolites no other phthalates and MEHP is associated with also with decreased estradiol and total testosterone at MGH but not in the future family studies and the gonadotropins are not associated with any of the phthalate metabolites so we'll be publishing a joint analysis of those two populations shortly so turning to metabolic syndrome there is a paper by a fellow of mine Rick Stallhut published in 2007 where he analyzed NHANES data very large population of course and he found that MBZP and two of the DHP metabolites and MEHP were significantly associated with increased WASER conference and that MBP and MBZP were associated with increased insulin actually using a measure called HOMA but you might consider this study when you talk about the P-PAR I'm sure you will talk about the question of metabolic syndrome in relation to phthalates and only one study on thyroid function by John Meeker again the Harvard group and men's infertility workup and T3, T4 and TSH were measured and an inverse association was found between MEHP urinary metabolites and free T4 and total T3 serum levels and no other phthalates were associated with any marker of thyroid function I want to tell you a little bit about what's going on now if you plot the number of studies of phthalates and human health effects in the literature from 2000 when the first one came out until 2010 you will see exponential growth of those numbers it's just huge the increase in studies I think one in 2000 a few by 2005 and everything I've shown you up until 2010 and there are many many in the pipeline some I know about some I don't know about there are large ongoing studies now so our study tides the infant development and environment study will go in the field in August we're going to have 880 pregnant women enrolled the Selma study in Sweden has several thousand women there's a Canadian study called MIRAC that has I think 8,000 women and so on so there are a large number of large studies going on right now that I think within five years will have much more conclusive data and we are just going to get to the sample size and start looking at mixtures and identifying more precise exposure windows and more precise outcome measures so I just want to talk about this whole body of studies that I've thrown by you really quickly and ask could this be chance could this all be chance could this have happened from tossing a coin and I think not and first of all I want to say that exposure misclassification in these studies is undoubtedly present we don't have the exact right window we certainly don't have the mixtures but by and large the exposure misclassification will lead to an underestimate of the effect measures that we're estimating so that's not going to make exposure misclassification is not going to explain any positive findings we have how about bias well unlike a study of smoking or pharmaceutical where you can say well the mother remembered better when she had a bad outcome there's no memory involved here none of us know our phthalate exposure there is no way that recall bias can affect these studi