ATDEPARTMENT OF HEALTH AND HUMAN SERVICES

> FOOD AND DRUG ADMINISTRATION
>
> CENTER FOR FOOD SAFETY AND APPLIED NUTRITION
>
> FOOD ADVISORY COMMITTEE
>
> ACRYLAMIDE
>
> Monday, February 24, 2003
>
> 8:30 a.m.
>
> Sheraton College Park Hotel
>
> 4095 Powder Mill Road
>
> Beltsville, Maryland 20705
>
> PARTICIPANTS
>
> Sanford A. Miller, Ph.D., Chairman
>
> Catherine DeRoever, Executive Secretary
>
> PARTICIPATING FOOD ADVISORY COMMITTEE MEMBERS:
>
> Francis Fredrick Busta, Ph.D.
>
> Annette Dickinson, Ph.D.
>
> Johanna Dwyer, D. Sc., RD
>
> Brandon Scholz
>
> TEMPORARY VOTING MEMBERS:
>
> Jean Halloran
>
> Ken Lee, Ph.D.
>
> Harihara Mehendale, Ph.D.
>
> Robert Russell, M.D.
>
> Clifford W. Scherer, Ph.D.
>
> J. Antonio Torres, Ph.D.
>
> GUEST SPEAKERS:
>
> Robert Brown, Ph.D.
>
> Tim Fennell, Ph.D.
>
> Stephen S. Olin, Ph.D.
>
> Sorell Schwartz, Ph.D.
>
> David Zyzak, Ph.D.
>
> [C O N T E N T S]{.underline}
>
> Welcome and Introductions,
>
> Dr. Sanford Miller 4
>
> Conflict of Interest Statement,
>
> Ms. Catherine DeRoever 7
>
> Opening Comments,
>
> Mr. Joseph Levitt 9
>
> Dr. Lester Crawford 12
>
> Development of the Acrylamide Action Plan,
>
> Dr. Terry Troxell 18
>
> Report of the Contaminants and Natural Toxicants
>
> Subcommittee (CNTS) Findings,
>
> Dr. Henry Kim 29
>
> Questions of Clarification 39
>
> Revised Action Plan,
>
> Dr. Terry Troxell 46
>
> Questions of Clarification 68
>
> Mechanisms of Formation,
>
> Dr. David Zyzak 99
>
> Questions of Clarification 116
>
> Reduction Strategies,
>
> Dr. Robert Brown 127
>
> Questions of Clarification 151
>
> Exposure Assessment,
>
> Dr. Donna Robie 152
>
> Questions of Clarification 185
>
> Adduct Studies,
>
> Dr. Tim Fennell 195
>
> Questions of Clarification 221
>
> [P]{.underline} [R]{.underline} [O]{.underline} [C]{.underline}
> [E]{.underline} [E]{.underline} [D]{.underline} [I]{.underline}
> [N]{.underline} [G]{.underline} [S]{.underline}
>
> **Call to Order and Opening Remarks**
>
> DR. MILLER: Good morning. I am Sandy Miller and I am Chairman of the
> Food Advisory Committee. I want to take this opportunity to welcome
> the members of the committee and the subcommittee that are with us
> today, and our guests and staff members, to deal with the issues that
> are concerned with acrylamide and food safety.
>
> Before we go any further, let me take this opportunity and ask each of
> the members of the committee to introduce themselves briefly and then
> we can go on to the next agenda item.
>
> DR. MEHENDALE: I am Dr. Mehendale from the University of Louisiana and
> the Monroe School of Pharmacy.
>
> DR. RUSSELL: Rob Russell. I am Director of the USDA Human Nutrition
> Center in Boston at Tufts.
>
> DR. TORRES: Antonio Torres, Oregon State University Food Science.
>
> DR. BUSTA: I am Frank Busta, Professor Emeritus at the University of
> Minnesota.
>
> MS. DEROEVER: Catherine DeRoever, the executive secretary for the Food
> Advisory Committee, FDA.
>
> DR. MILLER: I am Sandy Miller, as you all know.
>
> DR. DWYER: I am Johanna Dwyer at Tufts New England Medical Center in
> Boston.
>
> DR. DICKINSON: Annette Dickinson, Council For Responsible Nutrition.
>
> DR. LEE: Ken Lee, Ohio State University Food Science and Technology.
>
> MR. SCHOLZ: I am Brandon Scholz, Wisconsin Grocers Association.
>
> DR. SCHERER: Cliff Scherer, Cornell University, with an interest in
> risk communication.
>
> DR. MILLER: Thank you. The committee has been organized for the
> purposes of reviewing an Action Plan prepared by the staff in order to
> deal with the issues of acrylamide in food.
>
> I am not going to provide the background to this because this will be
> done by some of our speakers in time, but I want to remind the
> committee that, as you will see, what the committee is being asked to
> do is to review the Action Plan, not to make a decision concerning the
> safety of acrylamide in food. As we continue our activities I want to
> focus on that issue rather than the ancillary issue, although there
> are questions concerning safety that are going to have to be resolved.
>
> Let me also remind all of the speakers that we have a number of people
> to speak and to make sure that everybody gets their proper time, we
> are going to have to stick very close to the time schedule. What I
> will do, I will interrupt you when your time begins to run out and
> then as you continue to talk, continue to interrupt you and, if
> necessary, I will adjourn the committee until you sit down. But it is
> really important. Quite seriously, it is very important that people
> stick to their time.
>
> I will also at periodic times ask the committee if they have questions
> and, hopefully, these will be questions for clarification rather than
> comment. We will come to comments and opinions at the end of the
> meeting.
>
> Before we move on to our opening comments, Kathy DeRoever has some
> comments she needs to make concerning conflict of interest.
>
> **Conflict of Interest Statement**
>
> MS. DEROEVER: Good morning. As you have heard, I am Catherine
> DeRoever, executive secretary for the Food and Drug Advisory
> Committee. First, I would like to welcome all of you to the meeting,
> particularly the members of the committee, Dr. Miller, Dr. Busta, Dr.
> Dickinson, Dr. Dwyer and Mr. Scholz; the members who serve on our
> subcommittees who graciously agreed to be here today, Dr. Lee, Dr.
> Mehendale, Dr. Torres; and also our temporary voting members\--Jean
> Halloran, as you can see, has not arrived yet but Ms. Halloran, Dr.
> Russell and Dr. Scherer have been appointed as temporary voting
> members for the purpose of this meeting by the authority granted to
> the Center Director, Mr. Josepph Levitt, in the Food Advisory
> Committee Charter.
>
> Second, the following announcement addresses the issue of conflict of
> interest with respect to this meeting, and is made part of the record
> to preclude even the appearance of a conflict of interest. The issues
> to be discussed at this meeting are issues of broad applicability.
> Unlike issues in which a particular sponsor\'s product is discussed,
> the matters at issue do not have a unique impact on any particular
> product or manufacturer but, rather, may have widespread implications
> with respect to foods and their manufacturers.
>
> To determine if any conflict of interest exists, the committee has
> been screened for interests in the food industry. As a result of this
> review, in accordance with 18 USC, Section 208(b)(3), Dr. Busta, Dr.
> Dwyer and Dr. Miller have been granted a particular matter of general
> applicability waiver that permits them to participate fully in the
> matters at issue. Copies of the waiver statements may be obtained by
> submitting a written Freedom of Information Act request.
>
> Our invited guest speakers have also been screened for conflicts of
> interest. Dr. Saunders, who I understand is being replaced by Dr.
> Brown, and Dr. Zyzak are employed by the regulated industry. No other
> conflicts have been reported.
>
> I now turn the meeting back to Dr. Miller.
>
> DR. MILLER: Thank you, Cathy. For opening comments, Mr. Joseph Levitt,
> Director of the Center for Food Safety and Applied Nutrition, will
> make some remarks.
>
> **Opening Comments**
>
> MR. LEVITT: Good morning. Let me again welcome you to our wonderful
> Washington, DC area in the middle of winter. I last saw this committee
> in the heat of summer. So, I guess, we have a way of playing these
> things around the weather. At least the subcommittee that Dr. Busta so
> ably chaired in December was in the middle of a snow storm so here we
> at least waited till some of the dig-out occurred.
>
> Today\'s subject is acrylamide. We are at what I think of as phase two
> of this process. Phase one I would describe as discovery, where we all
> heard less than a year ago, last spring, of new findings reported from
> Sweden on acrylamide levels in food. That created a lot of activity
> that many of you know about and you will hear about in more detail.
> Phase two is what I call formulating a course of action. That is what
> we have been doing for the last six months or so. We presented a Draft
> Action Plan at a public meeting at the end of September, presented
> that to the Subcommittee of Contaminants and Natural Toxicants in
> December and are trying to finalize that today. Phase three,
> therefore, would be the implementation and finding solutions.
>
> This has been such an important issue that actually phase three
> started before phase two was over. We are happy also to be sharing
> continued results of research that has been ongoing.
>
> So, what you will hear today in summary is as follows: Number one, you
> will be hearing a summary of our plan, including the subcommittee\'s
> review and suggested improvements to that that have been incorporated.
> You will be hearing continued findings of research that FDA has
> conducted and, for the first time, our overall exposure assessments on
> acrylamide. You will be hearing a presentation from industry research
> later this afternoon, preliminary research on how to reduce the
> levels. That is really a major goal here. The results, though
> preliminary, look very encouraging from what we have heard, and we are
> pleased to be able to present that to you today.
>
> So, in less than a year I think you will find, as all of us involved,
> that an awful lot has been done in less than a year though, clearly, a
> lot more needs to be done to see this important issue through to its
> conclusion.
>
> As always, what we want from you is your best advice. How can we best
> crystallize, direct and target this plan to accomplishing what we need
> to do in the shortest time possible, recognizing that research does
> take time, that it does take work by a lot of people in order to try
> and solve a problem of this degree of complexity?
>
> With that, it is my pleasure to make way for the FDA Deputy
> Commissioner, Dr. Lester Crawford. Thank you very much.
>
> **Opening Comments**
>
> DR. CRAWFORD: Thank you, Joe and thank you Dr. Miller. I would like to
> share a few thoughts with you with respect to the agency\'s current
> position on acrylamide, but before doing that I want to recall a
> couple of points of nostalgia to be sure that I run over my time, Dr.
> Miller.
>
> \[Laughter\]
>
> One is that I used to be on the Food Advisory Committee back in the
> golden days when we successfully solved the problem of food
> biotechnology\--
>
> \[Laughter\]
>
> \--and also somatotropin and then finally Ephedra. But we didn\'t have
> a chairman like Dr. Miller. That is for sure. Even earlier, about half
> a mile from here, my first stint at FDA was out in Beltsville,
> vaccinating pigs. So, I am happy to be here.
>
> \[Laughter\]
>
> Somebody is out there vaccinating pigs right now.
>
> First of all, let me just say that we are fully committed to
> developing better knowledge of acrylamide in foods and passing this
> information along to the public as quickly as possible. We have had a
> number of issuances on acrylamide, and we are about to have another as
> a result of this meeting.
>
> At this point we simply don\'t know what the actual human health risk
> of acrylamide might be at the low levels found in food. We know it
> causes cancer and reproductive problems in animals in high doses and
> is a neurotoxin in humans at high doses. That is why the FDA created
> an Action Plan that Joe just talked about and that Terry Troxell will
> talk about a bit later to understand the risk that might be associated
> with acrylamide in foods, and to reduce levels of acrylamide in foods,
> including active involvement of this committee.
>
> As a result, we are learning more about acrylamide levels in a broader
> range of foods than has been previously analyzed. We are also learning
> more about acrylamide forms in foods and steps that may help reduce
> acrylamide formation.
>
> Based on our current understanding of the science, FDA continues to
> advise consumers to eat a balanced diet, choosing a variety of foods
> that are low in trans and saturated fat and rich in high fiber grains,
> fruits and vegetables.
>
> The purpose of the meeting, as Joe has said, is to seek input from the
> committee to assist FDA in analyzing and finalizing the revised Draft
> Action Plan. The revised Action Plan includes greater detail and
> reflects new research activities and comments from the subcommittee
> which we received on December 4 and 5 of last year.
>
> We are releasing 110 additional test results in the spirit of openness
> and transparency. The findings released today are generally similar to
> the preliminary results FDA released previously.
>
> FDA exploratory survey findings on levels in foods have shown
> substantial variability among a wide variety of foods, as well as
> substantial variability within foods. The initial exposure assessment
> has found results similar to those conducted by organizations
> worldwide, such as the World Health Organization and the Food and
> Agriculture Organization.
>
> The exposure assessment has found that many foods contribute to
> acrylamide exposure. No single food accounts for the majority of
> acrylamide exposure for the U.S. population. Some foods that have
> lower levels of acrylamide contribute appreciably to the overall
> exposure because they are commonly consumed. Researchers are seeking
> ways to get acrylamide levels down. Preliminary data are encouraging
> and we are convinced that there will be some techniques and some
> technologies developed that might help and we are eager to learn more
> about that from presentations that will be made at this meeting.
>
> With that, I will conclude and will officially declare, from my
> position as Deputy Commissioner, that I was on time, Dr. Miller. Thank
> you.
>
> DR. MILLER: Thank you. I am always happy to be present at rare events.
>
> \[Laughter\]
>
> I wanted to remind Dr. Crawford that the function of this committee is
> to provide advice, not to make decisions on implementation, and if a
> problem still exists that is not because the committee didn\'t give
> good advice.
>
> \[Laughter\]
>
> That brings me to the next issue. As I said before, the function of
> the committee is to review the Action Plan and to review the
> conclusions made by the Subcommittee on Contaminants and Natural
> Toxicants and to add or make recommendations concerning any changes in
> this plan.
>
> There are three basic issues we have to think about as far as the
> Action Plan is concerned. One, as mentioned, is the toxicology. How
> strong is the data? What does the data tell us in terms of calculating
> risk? And, what else needs to be done in order to provide a stronger
> base for an agency Action Plan?
>
> Secondly, exposure and, as Dr. Crawford mentioned, some additional
> data is being released today. Is that right? It is going to be
> released today, some additional exposure data?
>
> DR. CRAWFORD: Yes, 110 additional analyses.
>
> DR. MILLER: All right, I wanted to be sure I heard that right.
> Thirdly, as was pointed out, there are available technologies for
> reducing exposure to acrylamide and to determine whether or not these
> are important in order to reduce the potential risk from exposure from
> this material.
>
> In order to accomplish these tasks we have a number of speakers who
> will talk about the different subjects, but I think the committee
> ought to focus on these issues, and we will come back to them at the
> end of the meeting when we have our general discussion concerning
> recommendations that we need to make to the agency.
>
> The first speaker this morning to talk about the Action Plan is Dr.
> Terry Troxell, who is director of the Office of Plant and Dairy Foods
> and Beverages, CFSAN. Terry?
>
> **Development of the Acrylamide Action Plan**
>
> DR. TROXELL: Good morning. We bring you another simple problem to
> solve, like biotech and Ephedra.
>
> What I would like to do first thing is to go over the charge. You all
> have the charge and questions in your packets. I think we should focus
> on that for a minute so we orient the meeting. The charge is to
> evaluate the revised Action Plan as a tool for providing the
> scientific basis from which to assess the significance of acrylamide
> in foods and potential public health consequences.
>
> The first question of the committee is does the revised Action Plan
> meet its intended goal of serving as a tool for providing a scientific
> basis to assess the significance of acrylamide in foods and its
> potential public health consequences?
>
> The second question is new data on acrylamide levels exposure and
> potential interventions have become available in recent months. Does
> the Action Plan accommodate these new data? Please comment on the new
> data, including the exposure assessment of potential interventions.
>
> The final question is FDA\'s consumer message stresses the importance
> of eating a balanced diet. Given the uncertainties associated with the
> current state of the scientific knowledge, FDA has concluded that
> there is insufficient data to revise this message. Please comment.
>
> \[Slide\]
>
> For this talk I want to go over the development of the plan and the
> content, FDA\'s overall goal and ongoing work on the Action Plan.
>
> \[Slide\]
>
> This all began back on April 24 of 2002 when the Swedish national food
> agency surprised the world with a report that acrylamide was in
> numerous foods, surprised the world partly because cooking process had
> been thoroughly studied and the mutagenicity, and so on, had been
> worked on for many years and this one was missed apparently because it
> wasn\'t positive in the Ames test.
>
> Basically, what happened then was that many, many countries began
> developing methodology. The methodology was not initially available.
> We knew we had an LC/MS/MS method but we basically had to go from
> scratch, pretty near scratch, to develop a method. Then, as I said, we
> started thinking through the problem to try to clear up what in the
> world could be underlying the formation.
>
> We posted our first version of our LC/MS/MS method on June 10th and we
> have updated it two times. It is either being updated on the web or
> was just recently updated, in the last couple of days. It is an
> excellent method of linear quantitation for 10 ppb.
>
> Due to the intense interest worldwide in the subject, the WHO and FAO
> put on a consultation only two months after the announcement by the
> Swedish authorities. FDA sent three scientists to this meeting and the
> consultation concluded that acrylamide was a major concern.
>
> We posted our original Action Plan on September 20th so it didn\'t
> have the benefit of the next meeting, which was our interagency round
> table for which FDA brought together various components of CDC, NIOSH,
> National Institute of Occupational Safety and Health, and the National
> Center for Environmental Health. Of course, our National Center for
> Tox. Research was there and NIEHS of NIH was also at the meeting to
> exchange information on what was going on with acrylamide research and
> pathways and work of interest to various groups. So, we began our
> process of trying to organize the work to deal with the problem.
> Finally, we had our public meeting on September 30th and that was the
> first time we presented the plan.
>
> \[Slide\]
>
> The next event was the JIFSAN/NCFST workshop on October 28th to 30th.
> JIFSAN is our Joint Institute for Food Safety and Applied Nutrition.
> It is a consortium with the University of Maryland. Our National
> Center for Food Safety and Technology is our consortium with the
> Illinois Institute of Technology. This workshop brought together
> researchers from many countries, four months after the original
> consultation, to thoroughly review the status of methods, mechanisms
> and formation for reduction of exposure, toxicology and risk
> communication. The workshop led to a list of priority research for
> each category. In addition to this, JIFSAN is operating the WHO/FAO
> acrylamide in food information network on the web to try to bring
> together research from around the world.
>
> The next event was our subcommittee meeting in the snow storm of
> December 4th and 5th. At that time, of course, we presented the
> original Action Plan and got very valuable comments from the group.
>
> \[Slide\]
>
> The Action Plan outlines FDA\'s goals and planned activities on
> acrylamide in food over the next several years. The plan discusses
> intentions to work with other federal agencies and participate in
> international efforts. We saw from the very beginning that there was a
> large amount of work to do, and work that would cost a lot.
>
> It was our belief that we could get the answers that we needed as
> quickly as possible by leveraging our efforts and as important was a
> means to coordinate work with researchers and communicate throughout
> the process in order to accelerate the solutions. For example, several
> labs discovered concurrently that asparagine and glucose through their
> reaction were the primary cause of acrylamide formation.
>
> It would be useful if there was a way of limiting redundancy in order
> to optimize our efforts. However, the desire of academics and other
> researchers to publish gets in the way of that because they want to
> hold their work until the publication occurs. In any event, the Action
> Plan and coordination we are trying to foster, we believe, will move
> this effort in the right direction.
>
> \[Slide\]
>
> The original Action Plan included sections on testing foods,
> toxicology formation, methodologies, meetings and collaborative
> projects, consumer messages and regulatory options. The revised plan,
> summarized in the next talk, covers the same areas pretty much in more
> depth, with the addition of a couple of new sections.
>
> \[Slide\]
>
> The scientific review at FDA and WHO indicated cause for concern, as I
> indicated. Actually, although we note neurotoxicity here, the
> consultation did not expect neurotoxic effects from acrylamide levels
> present in foods. But discussions since then, for example at JIFSAN
> meetings, suggested that more work was needed to be done to
> characterize potential neural development of effects in chronic
> exposure.
>
> Another factor that was involved in developing the Action Plan was
> that the consumption was in the tens of micrograms range. In contrast
> to that, food additives and water purification effects of exposures
> were 100 to 1000 times lower. Also, the scientific review, as I have
> said, indicated that there were quite a few gaps and a lot of work
> needed to be done.
>
> \[Slide\]
>
> The results were intense worldwide interest, as indicated by the
> desire to have the consultation very shortly. Our optimal desire, of
> course, would be to harness that intense interest and at least try to
> influence it toward a better coordination and less redundancy.
> Research indicates acrylamide is formed through traditional cooking
> practices. As I said, it appears to be the product of one of the
> standard cooking reactions that is essential to cooking foods at
> processors and in the home. The reaction produces desirable flavors
> and browning. Thus, acrylamide is formed in a wide number of foods and
> is going to be a challenging problem for chemists.
>
> \[Slide\]
>
> We developed an overall goal for this project, and that is, through
> scientific investigation and risk management decision-making, to
> prevent and/or reduce the risk of acrylamide in foods to the greatest
> extent feasible.
>
> \[Slide\]
>
> As far as ongoing work, we revised the plan based on the
> subcommittee\'s input as well as other developments along the way. We
> will present that plan today and will look forward to your assistance
> in finalizing the plan.
>
> \[Slide\]
>
> What is going on in the future here? Of course, our work on the
> projects is outlined in the plan. Then, there is the CCFAC meeting
> coming up shortly, March 17th. Of course, as we have discussed, there
> is intense national interest and we expect that the CCFAC will
> undertake new work at this meeting on acrylamide. As part of our
> effort to coordinate and share information on acrylamide, we are
> proposing a formal workshop on acrylamide in conjunction with the
> CCFAC meeting. The WHO and FAO have adopted that proposal and we will
> be conducting a workshop at the meeting. Finally, the WHO and FAO
> JECFA, the Joint Expert Committee on Food Additives, which also
> evaluates contaminants, will be looking at acrylamide sometime in
> 2004. Whether it is February or June is not clear but they will be
> evaluating, and that is the group that does the risk assessment that
> flows into the CCFAC committee which is the risk management group.
>
> There are tens of meetings on acrylamide going on all over the world,
> as well as in the U.S., and we have just highlighted a few of those
> but there is just very intense interest and it is hard to know which
> ones to highlight and which ones not to highlight.
>
> \[Slide\]
>
> Anyway, in the next talks we will be betting a report from Dr. Kim on
> that subject and then I will come back and summarize the revised
> Action Plan. Thank you for your attention. Any questions?
>
> DR. MILLER: We have some time if there are any questions of
> clarification. Johanna?
>
> DR. DWYER: Thank you for an interesting introduction. I have one
> question on one of your early slides where you mentioned that the
> Swedish report came out in April, and then you developed a method
> which you released on June 20th. Why was the Swedish method not
> available?
>
> DR. TROXELL: Again, they released a method but they were waiting to
> publish the work.
>
> DR. DWYER: And they would not release it to the authorities?
>
> DR. TROXELL: Right, they released a sketch of the methodology which
> helped accelerate our development of the method pretty quickly and
> observe it in an appropriate LC Mass Spec. method.
>
> DR. MILLER: Any other questions? If not, thank you. As you know, the
> organization of the Food Advisory Committee consists of the committee
> itself and several subcommittees. This issue was referred to the
> Contaminants and Natural Toxicants Subcommittee, which you have heard
> about already. Dr. Henry Kim is the executive secretary and he will
> present the report of the committee. Henry?
>
> **Report of the Contaminants and Natural Toxicants**
>
> **Subcommittee (CNTS) Findings**
>
> DR. KIM: Thank you, Dr. Miller. As the executive secretary for the
> Contaminants and Natural Toxicants Subcommittee, I was asked by Dr.
> Busta, who is the chairman of that subcommittee, to present a report
> of the subcommittee meeting\'s findings and it is my pleasure to do so
> this morning.
>
> \[Slide\]
>
> For my presentation what I would like to talk about is briefly on the
> purpose of the subcommittee meeting, as well as the presentation that
> was made at this meeting, and then talk a little more in detail about
> the subcommittee\'s discussions and recommendations that were made.
>
> \[Slide\]
>
> The purpose of this subcommittee meeting, which was held on December
> 4th and 5th of 2002 was to present the major components of the FDA\'s
> Draft Action Plan and then to seek advice and recommendations from the
> subcommittee.
>
> \[Slide\]
>
> In presenting the FDA\'s Draft Action Plan, the subcommittee was asked
> to evaluate whether the research steps outlined in FDA\'s Action Plan
> are scientifically adequate to describe and address the public health
> significance of acrylamide in food.
>
> \[Slide\]
>
> In addition, the subcommittee was posed with three questions. That is,
> given what we know of acrylamide, that is, the toxicology, the
> occurrence, formation, exposure and risk, one, are the research steps
> appropriate to describe and address the public health significance of
> acrylamide in food? Two, are there gaps in the research plan or areas
> where emphasis should be increased? Three, are there priority research
> needs that should be addressed first?
>
> In order to facilitate our responses from the subcommittee on the
> Action Plan, FDA representatives presented five major components of
> the Draft Action Plan, that is, the toxicology, the occurrence,
> formation, exposure and risk.
>
> \[Slide\]
>
> First, Dr. Canady talked about information that we already know in
> addition to data that we need, and what we are planning to do to
> obtain those additional data with regard to the five toxicology
> elements that are shown on this slide. That is toxicokinetics, animal
> carcinogens and human neurotoxicants, reproductive/development effects
> and safety and risk assessment.
>
> \[Slide\]
>
> For the occurrence component of the Draft Action Plan, Dr. Musser
> discussed the development and performance of the LC/MS/MS method for
> the quantitation of acrylamide in a wide variety of foods, and also
> presented results of the exploratory survey data that was collected
> through November 15th, 2002.
>
> \[Slide\]
>
> Then Dr. Jackson provided an extensive summary on the current state of
> knowledge about mechanisms, the precursors and factors affecting
> acrylamide formation, and identified additional research needs in this
> area, and then discussed two major elements regarding acrylamide
> formation in the Draft Action Plan, that is, understanding the
> conditions leading for acrylamide formation and developing methods to
> prevent or reduce acrylamide formation.
>
> \[Slide\]
>
> For the exposure component of the plan, Dr. DiNovi talked about the
> FDA approach for conducting exposure assessment using the occurrence
> and consumption data to estimate exposure, and then talked about the
> two exposure studies that were conducted by Sweden and FAO/WHO and
> indicated that the FDA was currently conducting an initial exposure
> assessment which is to be completed shortly.
>
> \[Slide\]
>
> Finally, Dr. Acheson talked about achieving a balance with respect to
> the importance of balanced diet, risk from exposure to acrylamide in
> food and potential dangers of inadequate cooking for addressing the
> complex issue of reducing risk to consumers from the presence of
> acrylamide in food.
>
> \[Slide\]
>
> Some of the other presentations that were made just briefly, Mr.
> Levitt provided an opening remark sort of to kick off the meeting, if
> you will. Dr. Schwetz talked about scientific overview of acrylamide
> in food, focusing mainly on the work that was done at the FAO/WHO
> consultation in June. Then, Dr. Lineback, the Director of Joint
> Institute of Food Safety and Applied Nutrition, provided an overview
> of the JIFSAN workshop on acrylamide that was conducted last October.
> Finally, public comments were made by a representatives from National
> Food Processors Association, SNF and the American Council on Science
> and Health.
>
> That is what happened on the first day of the meeting so now I would
> like to move on to the second day of the meeting, which was devoted
> mainly to discussions by the subcommittee members about the various
> components of the Draft Action Plan, as well as responding to the
> three questions that were posed to them. So, I would like to first
> talk about the responses that were made by the subcommittee members
> with respect to the three questions and then highlight some of the
> major recommendations that were made on five components of the Draft
> Action Plan.
>
> \[Slide\]
>
> In response to question one, are the research steps appropriate to
> describe and address the public significance of acrylamide in food,
> the subcommittee generally supported the overall Draft Action Plan as
> well as the research plan that was outlined in the plan.
>
> \[Slide\]
>
> In response to question two, are there gaps in the research plan or
> areas where emphasis should be increased, the subcommittee felt that
> more detailed information was needed in the plan in discussing the
> risk assessment, human toxicology and epidemiology studies, as well as
> animal tox. studies and sampling and analytical variability and food
> consumption data by various population groups.
>
> \[Slide\]
>
> In response to question three, are there priority research needs that
> should be addressed first, the subcommittee generally felt that the
> priority as outlined in the draft plan, that is, the methodology,
> occurrence, formation, exposure and then risk\--they felt that this
> outline was appropriate but there were suggestions made that perhaps
> an explicit statement about the priority in the plan may also be
> appropriate.
>
> The subcommittee also recommended that the toxicology and risk
> assessment studies should move forward quickly because these types of
> studies take a long time to conduct, as well as focusing on developing
> a rapid and inexpensive method to analyze a wide variety of foods and
> to provide science-based risk communication messages.
>
> Now I would like to move on to highlighting some of the major
> recommendations that were made by the subcommittee on various
> components of the plan.
>
> \[Slide\]
>
> In the area of toxicology the subcommittee recommended that more human
> studies should be conducted, such as physiological studies looking at
> absorption, metabolism, distribution and excretion, as well as
> toxicokentic studies of ingested acrylamide in humans. They also
> recommended that more detailed discussion about studies with respect
> to animal neurotoxicity and genotoxicity should be made; as well as
> animal bioassay studies, particularly the short-term studies; as well
> as looking at the dose-response relationships at lower levels, that
> is, between the no observed hazardous effect level and the levels at
> which there is tumor formation; and also looking at the mechanism of
> action in these bioassay studies.
>
> Finally, they also suggested that the Action Plan should look at
> discussing biomarker-exposure relationship in smokers. They felt that
> data from those type of studies may be useful in conducting biomarker
> studies for acrylamide exposure from foods.
>
> \[Slide\]
>
> In the area of epidemiology the subcommittee recommended that a
> separate section on epidemiology should be included in the Draft
> Action Plan, talking more about the human epidemiology studies such as
> identifying the study populations with higher or lower exposures to
> acrylamide from various diets, as well as investigating epidemiology
> studies of workers exposed to acrylamide to determine whether those
> types of studies may be applicable to food exposure.
>
> \[Slide\]
>
> With regard to the exposure assessment component of the plan, the
> subcommittee recommended that the Action Plan should highlight the
> exposure assessment element of the plan more prominently by providing
> information about data bases that would be used to conduct the
> exposure assessment; as well as looking at improving the ability to
> blind the data that will be released to the public, such that that
> will facilitate more data sharing; as well as obtaining quality data,
> and to statistically determine when enough samples have been
> collected, such that the exposure assessment can be conducted; as well
> as obtaining inputs from the exposure assessors in the type of foods
> that should be sampled; and, finally, to use food consumption data by
> various population groups.
>
> \[Slide\]
>
> With respect to risk assessment, the subcommittee recommended that
> more information should be incorporated into the Draft Action Plan,
> particularly in discussing the importance of conducting a risk
> assessment, as well as the methods for conducting that risk assessment
> and on how to incorporate new data as they become available.
>
> \[Slide\]
>
> Finally, this is the last recommendation section within the area of
> risk communication. The subcommittee recommended that more emphasis
> should be made on the risk communication activities by FDA and to
> provide, as I mentioned previously, science-based information for
> dietary choices, as well as involving dietetic and nutrition
> associations in communication efforts, such as the American Dietetic
> Association and the American College of Nutrition and, finally, to
> disseminate consumer and cooking messages through extension services.
>
> \[Slide\]
>
> In summary, the subcommittee spoke positively about the FDA\'s Draft
> Action Plan and generally agreed with the approach and the planned
> research activities that were outlined in the plan, and then provided
> some very valuable recommendations with respect to toxicology,
> epidemiology, exposure and risk assessments and risk communication.
>
> With that, I turn the meeting back to Dr. Miller.
>
> DR. MILLER: Thank you, Henry. Are there any questions or comments?
>
> **Questions of Clarification**
>
> DR. BUSTA: I just want to compliment Henry on doing a very succinct
> presentation of our two-day meeting. There are other members who were
> at the subcommittee meeting if there are additions, but I think you
> summarized in a very succinct manner a lot of comments.
>
> DR. KIM: Thank you.
>
> DR. MILLER: Dr. Torres?
>
> DR. TORRES: We heard a lot about variability within food, within food
> batches and then I see that the plan does include studies on the
> formation of acrylamide in foods. But when I look at the subcommittee
> recommendations there seems to be nothing in that area of formation
> and I am a little bit surprised about that. I don\'t see any specific
> recommendations about the formation kinetics of acrylamide. We see so
> much variability within food, between foods, between batches of food,
> however, I don\'t see any recommendations for further studies on
> formation.
>
> DR. KIM: Yes, I did mention in the formation slides that Dr. Jackson
> presented formation information and did go into some of the additional
> research needed, such as looking at factors like pH, temperature,
> moisture content and those types of research activities. But I did not
> kind of highlight that in this presentation because of lack of time to
> present everything that was discussed at the meeting. There was a
> whole lot of discussion on all the major topics and I just wanted to
> focus on the major components of the plan.
>
> DR. MILLER: I think the point is that this issue which is, of course,
> vitally important wasn\'t highlighted as one recommendation for the
> Action Plan in your presentation.
>
> DR. KIM: Well, I can add that to the recommendation list.
>
> DR. MILLER: That is the point that Dr. Torres was trying to make. It
> is a vital issue and is certainly deserving of the same emphasis, at
> least in my view, as other aspects such as toxicology.
>
> DR. KIM: Yes, I would agree with that.
>
> DR. MILLER: Do you want to say something, Terry? You look anxious.
>
> DR. TROXELL: I always look that way. Our original Action Plan did
> highlight research on mechanism and formation. So, I think the
> subcommittee recognized that and I know that no recommendations came
> out on that, to change that. They just agreed that we should keep
> pushing on those aspects because they are vitally important.
>
> DR. MILLER: I think the report ought to reflect that. It is an area
> that obviously got missed because in reading the report, it doesn\'t
> come across. Johanna?
>
> DR. DWYER: Thanks for a good report. One thing that troubles me about
> this whole area is the database that one is using to get at the foods.
> Just a cursory reading of the report we were given this morning
> doesn\'t seem to highlight that for further attention. My concern is
> this, I know that on page two or three of the Action Plan you talk
> about FDA doing some analyses and then we were told that today more
> values will be released, but the question is how to capture all the
> values that are there, not just these but others. We went through this
> with methyl mercury where we found out that there is a lot more data
> out there than we had in the database. I wondered what efforts would
> be focused on all the different government agencies that may have data
> on this.
>
> DR. KIM: I was told that this will be addressed in the revised Action
> Plan. There were discussions about data collection. I think with
> respect to FDA\--we obtained data from surveys\--I don\'t know, maybe
> Dr. Busta or Dr. Lee may have some additional thoughts.
>
> DR. DWYER: I am talking about the presence of this compound or
> compounds in food, not the further step of taking those data and
> trying to get exposure estimates for food. I am talking about the
> database that is used to say the French fries have this much, the
> broccoli has that much.
>
> DR. BUSTA: I thought Henry covered that under the exposure assessment
> in improving the ability to blind the data and facilitate the data
> sharing, and maybe that should be more explicit but when I see that I
> think that there a lot of data in the food industry that, if they were
> made blinded, would be made available but might not be offered freely
> if it said somebody\'s brand.
>
> DR. LEE: Just to amplify on the subcommittee\'s recommendation, a lot
> of time was spent on the JIFSAN website and ways to share acrylamide
> data, and there was also a lot of discussion about the need to at
> least single-blind acrylamide levels in food so an industry that has a
> lot of data on acrylamide content of its products would not be in
> danger of being fingered as a source in the diet. So, I think that
> thought is there and, in all fairness, Henry gave a great summary but
> couldn\'t get into that level of detail in his presentation.
>
> DR. MILLER: Other comments? This is not a clarification, Henry or
> Terry, but how much emphasis\--excuse me, let me go back, I think it
> was you, Terry, who said something about acrylamide not being positive
> in the Ames test? Did I hear that correctly?
>
> DR. TROXELL: That is my understanding, and when they did research on
> cooking carcinogens that form in foods, and so on, apparently that is
> probably the reason acrylamide was missed among the other chemicals
> that are formed.
>
> DR. MILLER: But it is genotoxic?
>
> DR. TROXELL: Yes.
>
> DR. MILLER: That is what I thought. That is why I didn\'t understand
> why it didn\'t show any results the Ames test.
>
> DR. TROXELL: If you want further clarification, Dr. Canady probably
> can provide some.
>
> DR. MILLER: It is a matter of curiosity, but thank you. Any other
> comments? If not, we are actually ahead of time. I think what we are
> going to do is\--Terry, why don\'t we move on?
>
> MS. DEROEVER: We have a technical difficulty.
>
> DR. MILLER: We have a technical difficulty? All right, we will take a
> break and return in about 30 minutes.
>
> \[Brief recess\]
>
> DR. MILLER: Our next speaker is Dr. Terry Troxell, who will describe
> the revised Action Plan. Terry? I assume these are revisions that were
> made in response to the recommendations of the subcommittee.
>
> **Revised Action Plan**
>
> DR. TROXELL: Right. Thank you. You should have the revised Action Plan
> before you and also those 110 new data points should be in your
> packet. They are kind of at the bottom of the packet, under
> \"exploratory survey.\"
>
> \[Slide\]
>
> I am going to go very briefly over the revised Action Plan that we
> updated based on the input from the subcommittee, as well as the
> public, and also all the things that have happened in the meantime.
> Like I said, we put out the first Action Plan before the interagency
> meeting that we had, also before the JIFSAN meeting, and so on. Again,
> what we are looking for today is we are seeking your input to assist
> us in finalizing this.
>
> \[Slide\]
>
> What I am going to do is summarize key changes; review the major goals
> of the plan; walk through the action sections; and then highlight the
> changes section by section.
>
> \[Slide\]
>
> The major change, of course, is that we reorganized the plan. It now
> has a more logical flow, at least from my viewpoint. We have added new
> sections on exposure assessment, epidemiology and risk assessment and
> this is in response to subcommittee recommendations. We added more
> details and updated the information. The plan is about double the size
> of the previous plan and this also was in response to the
> subcommittee.
>
> \[Slide\]
>
> There are seven sub-goals to our work. I already mentioned our overall
> goal before. The first sub-goal is to develop rapid and inexpensive
> screening methods and validate confirmatory methods of analysis. The
> second is to identify mechanisms responsible for the formation of
> acrylamide in foods and identify means to reduce acrylamide exposure.
> Again, we know at this point that asparagine and glucose with high
> heat are responsible for most of the formation of acrylamide. What we
> don\'t know at this point is how to quench that reaction.
>
> \[Slide\]
>
> The next goal is to assess the dietary exposure of U.S. consumers to
> acrylamide by measuring the levels in various foods and estimating the
> dietary exposure.
>
> \[Slide\]
>
> The next goal is a rather long one and I am not going to read this but
> basically what it says is that we are going to explore the toxicology
> and epidemiology, and we are going to use quantitative risk assessment
> to determine the risk and the uncertainty associated with those risk
> calculations.
>
> \[Slide\]
>
> The next goal is to develop and foster public/private partnerships to
> gather scientific and technological information and data for assessing
> the human risk. We believe this is really essential and at the core of
> getting this work done and also it will harness that interest around
> the world by all parties, academia, industry and the governments.
>
> \[Slide\]
>
> The next goal is to inform and educate consumers and processors about
> the potential risk associated with acrylamide throughout the
> assessment process and as knowledge is gained.
>
> \[Slide\]
>
> The next goal is to provide all the essential elements for risk
> analysis, that is, risk assessment, risk communication and risk
> management. That is kind of a sum-up goal because that kind of covers
> the waterfront of what we need to do on this process and pretty near
> in any project we have on food safety.
>
> \[Slide\]
>
> There are nine sections detailing the actions toward accomplishing
> these goals. They cover the methodologies, research on formation,
> measuring exposure, toxicology and health effects, epidemiology, risk
> assessment, meetings, inform and educate the public, and then further
> actions. I will now look at each of those sections.
>
> \[Slide\]
>
> As I said, our LC/MS/MS method was posted on the web back in June. We
> have recently updated it. Dr. Musser of CFSAN and his group have done
> an enormous job of developing the method and analyzing around 400
> samples to date. This is in addition to the numerous analyses they
> have done in determining the characteristics of the method.
>
> I am going to diverge a second to make a point. Acrylamide has been a
> real team effort here, at FDA. It is not only my office, Office of
> Plant and Dietary Foods and Beverages, that is involved, but the
> Office of Food Safety is doing the exposure assessment, the Office of
> Systems and Support, the office in which Dr. Musser has done the
> analyses, and there are others in CFSAN like the food safety staff and
> the Office of Science. In the FDA in a broader sense, our National
> Center for Tox. Research is doing an enormous amount of work on
> toxicology and our Office of Regulatory Affairs will be running total
> diet study samples. This gives a sense of the energy going into
> developing the science around this issue, and is being repeated in
> many countries by many different kinds of parties.
>
> With respect to validation, while there are certain excellent methods
> for determining acrylamide, Dr. Musser\'s method is an excellent
> definitive method that we believe should go through the full AOAC
> validation process.
>
> Then, there are screening alternatives. We want to explore, and are
> encouraging others to explore the screening methods. Dr. Diachenko\'s
> lab in CFSAN is presently looking at use of LC/UV as a screening
> method. We believe the equipment, the LC/UV detectors are available in
> many labs around the world and this could provide a simple means for
> people in many countries to get a handle on the screening levels.
>
> \[Slide\]
>
> So, what is new in methodologies? Our second update is being posted
> for our LC/MS/MS method. We are explicitly talking about AOAC
> validation rather than just validation and we are currently exploring
> LC/UV alternatives.
>
> \[Slide\]
>
> Research on formation, CFSAN\'s Division of Food Processing and
> Packaging is located in Chicago at the National Center for Food Safety
> and Technology. NCFST has the capability of doing pilot process
> research so we can take the bench-top research on reduction, whether
> done in academia or done in industry labs, and test it under pilot
> processing conditions. Currently, NCFST is planning research on the
> formation of acrylamide during home cooking of toast, French fries and
> other foods. This seems to be a gap that we don\'t believe others are
> pursuing. The laboratory out there has the capability of measuring
> surface temperatures of food and also looking at the degree of
> browning so we can actually bring some science to bear on what would
> be happening in the home with toasting and frying of foods.
>
> Also, the interaction of academia and industry, again, we expect that
> reduction strategies will vary with many types of food in which
> acrylamide is formed. Therefore, it will take the efforts of the food
> science departments like Mike Pariza\'s Food Research Institute at the
> University of Wisconsin and the food industry to do the heavy lifting
> on research in this area. You will hear from Frito Lay and Procter and
> Gamble this afternoon on their progress on reduction strategies.
> Procter and Gamble will talk about the mechanism, Frito Lay will talk
> about reduction strategies.
>
> \[Slide\]
>
> So, what is new in the research on formation? NCFST is going to
> investigate scientifically home cooking.
>
> \[Slide\]
>
> Now to get at some of the questions we had this morning of measuring
> exposure, as I think I said earlier, Dr. Musser\'s group has done
> about 400 locally collected food samples thus far with this additional
> 110. These are the analyses we have used in our initial exposure
> assessment that you will be hearing about later, and we have handed
> out that data. I hope you each have a copy of that.
>
> We are contracting through JIFSAN, our consortium with the University
> of Maryland, for 400 to 500 more foods to be analyzed, and these will
> be collected across the country, so we will get a national scope now,
> by our field forces.
>
> In addition, we have begun testing total diet study foods. The total
> diet study look at foods as eaten and serves as a good indicator of
> exposure to contaminants\--I mean, that study looks at contaminants
> and pesticides, and looks at nutritional minerals, and so on. So, it
> will be a means for us to understand what the kind of gross exposure
> is and then what our progress is on reduction.
>
> As far as other testing goes, we plan to continue testing in
> subsequent years in part to fill in the distributions. You will see in
> our exposure assessment presentation that there are too few samples in
> many categories so we need to fill those in. As was discussed earlier,
> JIFSAN is compiling data from industry and academia which will fill
> gaps in distribution and help solve this data problem.
>
> We also understand that Europe has compiled a large amount of data.
> The U.K. apparently has a database that may run up to 4000 samples but
> their processing conditions may be somewhat different than processing
> conditions in the U.S., therefore, they may not always be directly
> applicable to the U.S. situation. But we think it is really important
> for us to tap into the data that is out there from all parties to
> basically fill out the distributions for each of the food categories
> and understand the variability on exposure.
>
> \[Slide\]
>
> Then there is exposure assessment and I am going to pretty much skip
> over this part because Dr. Robie will be thoroughly discussing our
> methodology but, again, we will be periodically updating that exposure
> assessment as more data is compiled and, hopefully, we will be
> bringing to bear this blinded data that industry can make available to
> us.
>
> \[Slide\]
>
> So, what is new in exposure? Well, it is a new section and we put in a
> lot more details in the revised Action Plan on what we are going to
> do, and those details include information on how we are going to model
> the database we are going to use.
>
> \[Slide\]
>
> Now we move on to toxicology and health effects. Toxicology will
> provide information for risk assessment and consist of two general
> areas, animal toxicology and human toxicology.
>
> \[Slide\]
>
> Now I am going to pretty much summarize the next three or four slides
> with the following: There are basically four things we want out of
> this work. One, we need a better understanding of the carcinogenicity,
> neurotoxicity and germ cell toxicology in rodents.
>
> Two, we really need to understand the bioavailability of acrylamide
> from food versus water in which most of the studies have been done.
>
> Three, we need to understand the difference between high and low dose
> metabolism at high doses and, again some others may need to correct me
> because, as they always say, I am not a toxicologist, I am only a
> \"Troxell-cologist.\" So, at high doses the p450 pathway produces
> glycidamide which is thought to be the penultimate carcinogen, but at
> low doses a smaller fraction of acrylamide may go through the p450
> pathway. So, it is really important for us to understand the high
> versus the low dose differences.
>
> Fourth, we need to understand the differences in metabolism and
> processing of acrylamide between animals and humans.
>
> For carcinogenicity we need to understand the levels of adducts that
> form on ingestion of acrylamide. There is a very convenient biomarker
> adduct that is formed with hemoglobin. Once the relationship is
> established between acrylamide intake and adduct formation we will be
> able to use the adduct levels in red blood cells as a convenient means
> of understanding population exposure to acrylamide. Of course, that
> will integrate everything. That will integrate the acrylamide from
> smoking and occupational and all possible sources.
>
> We don\'t really believe that this adduct with hemoglobin leads to
> adverse effects. In fact, it probably prevents acrylamide from
> ultimately reacting with the DNA. It is the DNA adducts after enough
> insults to the DNA that are thought to lead to the tumor production.
> So, we need to also understand the correlation between acrylamide
> intakes, the hemoglobin adducts to the DNA adducts, and we need these
> studies for rodents where we can study cancer production at the
> highest doses and we need these studies on humans who are exposed to
> very low levels from foods. Such studies should significantly reduce
> the uncertainty about the risk to humans but, so everybody is clear,
> we never are going to be totally precise in this. There are still
> going to be fair uncertainty bars but we can reduce those uncertainty
> bars by quite a bit.
>
> The challenge is going to be to measure adducts in situations where
> there are low levels of exposure because, obviously, at low level
> exposure you are forming very few adducts and, therefore, you are
> having difficulty pulling those out in your analysis. Many of the
> studies that we list on the next several slides can contribute to
> these key needs in a relatively short time for toxicology. To me, a
> relatively short time for toxicology is one to two years. So, with
> that having been said, I think I have summarized the short-term
> studies at the National Center for Tox. Research on bioavailability
> and adducts.
>
> \[Slide\]
>
> Then, they are going to be doing a gold standard, you know, NTP
> bioassay. The subchronic and mechanistic components of that will be
> done fairly fast and will contribute to our understanding of the
> toxicokinetics and can be pumped into physiological-based
> pharmacokinetic modeling. But obviously definitive results on number
> of tumors that are produced in the rodents won\'t be available for
> many years because that takes a lifetime of study in the rodent.
>
> The NCTR will be looking at neurodevelopmental effects and also there
> will be mechanistic research on germ cell toxicology and neurotoxicity
> by the National Institute for Environmental Health studies.
>
> \[Slide\]
>
> Then there will be the adduct studies that CDC is going to be party
> to, as I mentioned.
>
> \[Slide\]
>
> We are looking at using the NHANES study and measuring adducts to
> understand population exposure to acrylamide. As well, NIOSH is
> looking at this area and the acrylamide manufacturers are doing a
> variety of toxicokentic studies. So, we are trying to bring all
> parties to bear in the process.
>
> \[Slide\]
>
> Before I leave this area, again, there are many groups that are going
> to be expected to contribute to this work, and this work costs
> millions. We are trying to facilitate the research that needs to be
> done in all areas, but outside the agency it will be up to the
> researchers, their priorities, and how much they can invest to see how
> far they go. This will be particularly true for doing epidemiology
> studies which I will discuss a little later.
>
> \[Slide\]
>
> So, what is new in the tox. area? We have listed a series of new
> toxicology studies, new areas, neurotox. and germ cell tox. and
> industry working on toxicokinetics. We have talked about doing PBPK
> modeling and so on. Since the early report, we now know that the NTP
> is going to be doing a chronic bioassay.
>
> \[Slide\]
>
> Let\'s move on to the epidemiology area. Some of the work on adducts
> is viewed as epidemiology so it is kind of cross-cutting. Of course,
> we are going to be exploring the feasibility of prospective studies
> and also we will be using all the studies that are reported in the
> literature, whether they are going to be on worker exposure or food
> exposure.
>
> Now I will go to a little description of my view of the epi. area
> here. There are basically two avenues of epi. studies in my view, the
> occupational and the food exposure. If I understand it right,
> occupational studies are most likely to be able to tell us that
> acrylamide at high levels can cause cancer in humans. I think
> occupational exposures may be the highest. Thus far they have not
> shown an effect. This may be due to the fact that not enough years of
> exposure have been studied.
>
> The other avenue, of course, is intake from foods. Can epi. studies
> make a difference? Dr. Acheson will discuss the Mucci study, which was
> published several weeks ago, in his talk. I believe that is tomorrow.
>
> This was a case control food intake study and no effect was seen. The
> basic problem, as I see it, is that we should have an epi. study that
> can detect a lifetime risk of 1/10,000 and we don\'t want to wait 50
> years for the result. If the study can only detect 1/1000 risk then we
> are not any better off than what the rodent studies can give us. So, I
> have asked our experts if it would be possible to detect such low
> risk. I don\'t have the answer yet but the initial reaction is that it
> would be very hard, at best.
>
> The kind of study we have been discussing at the FDA is to do a case
> controlled study within one of the ongoing prospective studies, like
> the Nurses Health Study. They have collected diets as well as blood
> samples for the study. Many pieces would need to come together in
> order to do such a study but, if they did come together, it could be
> done in one to two years.
>
> But consider the complications. How accurate are the diets?
> Misclassification would reduce the power of the study. Further,
> because acrylamide is in so many foods, it will not be possible to
> find a population that is exposed versus not exposed. What we are
> talking about is groups that may vary in levels of exposure by maybe
> 10-20 microgram differences. Then, there is the further complication
> with food in that an array of chemicals make up the composition of any
> foods. So, if one sees an effect or doesn\'t see an effect, is it
> because of acrylamide or is it because of other chemicals that are
> also present in the foods that are being looked at which have
> carcinogenic and also anti-carcinogenic effects?
>
> So, there are a lot of difficulties in looking at these food studies
> because of the confounding factors and many other factors that are
> also involved concurrently in causing various effects. So, in sum, we
> are exploring epidemiology but we need to carefully consider any study
> to determine if it will likely provide added value once it is
> completed.
>
> \[Slide\]
>
> So, what is new in epidemiology? Well, it is a new section. It
> explains how the epi. data could benefit FDA work on acrylamide, and a
> number of studies and collaborations are under consideration.
>
> \[Slide\]
>
> The next action section is risk assessment. Clearly, when we have
> adequate information we want to characterize potential risk, including
> the uncertainty analysis. We think that key data needs are
> bioavailability, biomarkers, metabolism and toxicokinetics.
>
> \[Slide\]
>
> We would revise the risks assessment when significant developments
> materially change the assessment.
>
> \[Slide\]
>
> So, what is new in risk assessment? We have given an explicit section
> to risk assessment and it outlines our goals, data needs and expected
> output of the risk assessment.
>
> \[Slide\]
>
> The next section is meetings which we will continue to participate in
> and convene meetings as appropriate. As I said earlier, we convened
> the interagency meeting and we participated in the JIFSAN meeting, and
> so on. I am not repeating the meetings here because I went over the
> key things that have been going on in the first presentation.
>
> \[Slide\]
>
> The next action section is to inform and educate the public. We are,
> and have been, committed to communicating with the public throughout
> this process. This is the third public meeting. So, that is one form
> of our communication and transparency on the acrylamide issue. Also,
> our website has a lot of information on our Action Plan, on levels and
> so on. Also, there has been an [FDA Consumer]{.underline} article
> published in the January/February issue.
>
> \[Slide\]
>
> We have a message and do not believe that we have enough science at
> this point to change it. That message is eat a balanced diet; choose a
> variety of foods that are low in trans fat and saturated fat and rich
> in high fiber grains, fruits and vegetables. We may recruit diet and
> nutrition organizations to help spread that message and changes to the
> message.
>
> \[Slide\]
>
> What is new in this section? We have expanded it to indicate that we
> have a continued process of risk communication going on and
> participation of outside organizations.
>
> \[Slide\]
>
> The last section is further actions. One of our further actions, of
> course, is to develop and revise regulatory options as additional
> knowledge is gained on acrylamide in food. Another action is to
> encourage industry to adopt feasible, practical and safe processes
> that are successful at reducing acrylamide, as needed.
>
> \[Slide\]
>
> Another action is to develop and revise the consumer message about
> dietary choices and cooking, as additional knowledge is gained to
> assist consumers in making informed choices. Finally, any adjustments
> to messages would be made as dictated by the totality of the science.
>
> \[Slide\]
>
> In conclusion, the Action Plan outlines goals and planned actions. It
> was revised to reflect comments of the subcommittee and new
> information from many sources. It was reorganized, with new sections
> added and more details. Finally, we are here seeking your input to
> assist us to finalize the plan. Thank you for your attention.
>
> **Questions of Clarification**
>
> DR. MILLER: Thank you. Before we begin the questions, let me just
> introduce Jean Halloran. Comments or questions?
>
> DR. RUSSELL: Yes, I was wondering if it is known which p450 enzymes
> are involved in creating the ultimate carcinogen. The reason I am
> asking that is that you might be able to target certain populations
> whose p450 enzymes, for example, are turned on or induced for a
> particular effect of this carcinogen because more carcinogen would
> have been formed. One example would be people who drink alcohol where
> certain p450 enzyme profiles are turned on, and if those enzymes are
> the same ones that are involved in forming the carcinogen you may be
> able to target more supopulations that may be at particular risk. So,
> the question is do we know that or is that a part of the puzzle also?
>
> DR. TROXELL: I don\'t know that. Dr. Canady may be able to comment on
> that.
>
> DR. CANADY: Rick Canady, FDA. 2E1 is the enzyme and it is pretty
> clearly implicated in the conversion of glycidamide and it is one of
> the things that we are looking at in designing studies.
>
> DR. RUSSELL: Another comment is that you mentioned in the
> epidemiology, perhaps just as an example, that the Nurses Health Study
> might be a population to look at. My only caution there would be that
> they are not a particularly representative group because they have
> other health habits. They may be more health conscious, for example,
> than the normal population that you are dealing with. So, it may be
> that a longitudinal population that has been followed that is more
> representative, perhaps a Framingham study for example, might be a
> better population group to look at.
>
> DR. TROXELL: Thank you.
>
> DR. SCHERER: I am just wondering whether any consideration has been
> given, or discussion, in terms of expanding a bit the risk
> communication section. For example, it seems to me that the idea of
> communicating all of this to the public is certainly a sound one but
> it seems to me that maybe a part that is missing is more of the
> preparation for how to do that. This particular risk, it seems to me,
> is in some ways potentially, if the science bears it out, what might
> be described as the perfect risk, risk challenge. It has all of the
> elements of being one of the more difficult ones to communicate. So,
> the idea would be that to some extent there needs to be some work done
> on understanding how to change human behavior in this case because
> that seems to me to be the real challenge.
>
> DR. TROXELL: I think we would agree with you that it is always a
> challenge to provide messages and education that consumers will
> respond to. That is the challenge of any message and in this case
> probably more so because consumers hear many negative messages and
> they are puzzled many times about which ones are really important to
> listen to. So, yes, a study of what would work and what won\'t work is
> important. You know, if we are going to go out with a targeted
> message, for example relating to cooking, we certainly want to make
> sure that what we do has scientific underpinnings like the toasting
> issue. We clearly don\'t want consumers to hear cook food less
> generally because then we would end up having more problems from
> pathogens. So, we would be reducing a risk from this a little bit but
> we could be creating an actual health hazard from people not cooking
> their hamburgers well enough.
>
> DR. TORRES: When I look at where most of the studies will be done on
> formation, it seems that the focus is on the National Center of Food
> Safety. So, I have two questions. One is that most of the research at
> that center is fee-based access. So, if you pay a fee you have access
> to the research. So, the question is how accessible would that
> research be to any industry?
>
> Second, you mentioned that the focus should be on home use study so
> where would the non-home use study be done?
>
> DR. TROXELL: Actually, most of the FDA research will be at the
> National Center for Toxicology Research. They are undertaking the NTP
> bioassay and all the mechanistic work. As to the studies relating to
> reduction, the National Center will do some of that work, and has the
> capability to do some additional work on pilot process testing, and
> that work will be fully available to everybody as early as possible
> and generally is presented as early as possible.
>
> Yes, I think the Center does some private work but our Division is not
> involved in that kind of work out there. That is work that is between
> the researchers at IIT and particular industry members. We will only
> do a portion of the formation research. We don\'t have the capacity to
> do a large portion. We really need to depend on other organizations,
> other governments, industry and so on to push the research on
> reduction forward. We also expect that is necessary because food is so
> complex we will probably need different strategies for different
> foods. While we can hope for a magic bullet to quench the reaction,
> that is something that I am not aware of anybody having a handle on at
> this point.
>
> DR. TORRES: My question specifically is will we hear more details
> about the research done at the National Center for Food Safety. I
> agree with you that they do have the capability to do pilot studies
> and they have the equipment to simulate industrial processes, but I
> don\'t see that anywhere in this report.
>
> DR. TROXELL: Well, part of our effort is to make information available
> as early as possible to help fuel the process of discovery. Obviously,
> the FDA is committed to that so we will make any discoveries available
> as soon as possible. Dr. Jackson, at the subcommittee meeting,
> presented on the mechanism and also showed some initial results on
> potato chip work. So, we are making our research available as soon as
> possible.
>
> DR. MILLER: Johanna?
>
> DR. DWYER: Thanks for a good presentation. Just a couple of points,
> the first is I was delighted to see that you are using HANES, or are
> hoping to use HANES, but the number of subjects above the 95th
> percentile will be very small, as you well know, because the sample
> size is only 5000. But the notion of using blood and connecting it
> with diet and other exposures is very appealing.
>
> I was taken by Dr. Russell\'s comment about longitudinal studies and
> Framingham is one example of a place where other agencies in the
> public health service have invested considerable money in maintaining
> sample sizes. The other one is the Women\'s Health Initiative which,
> not surprisingly, eliminates men but is very large, about 35,000
> people I think in the randomized study and I think the observational
> study is even larger. That, again, is heavily supported by the NIH. It
> is one of the largest studies in the country. It would seem like you
> would also want to explore that particular study when you look at
> feasibility of prospective studies. The advantage of that particular
> study is that the data and the assumptions that are used in the food
> frequencies are totally transparent because it is all federally
> funded, and basically the forms they use are things that were
> developed I think at NCI.
>
> The second point is simply this issue again of getting all the data
> that are available on acrylamide levels in food into your database. It
> sounds to me like you are spending at least a million dollars, if not
> more, to try to do these assays of food within the agency. I haven\'t
> looked at the FDA budget lately but it strikes me that it is a lot of
> money, and if there are others who also have data on this we have to
> find ways\--and food composition in general\--to get this data into
> national databases for risk assessment.
>
> DR. TROXELL: Thank you for those comments. Getting the data together
> on levels was a major discussion point at the JIFSAN meeting. It looks
> to me like they are going to be able to compile, hopefully, an
> enormous amount of data that is going to fill in the gaps for us
> substantially.
>
> Clearly, we are going to have to look at individual foods to see what
> kind of data we have for those distributions and where we need to fill
> them out. We will be consulting with our statisticians to make sure
> that we have adequate information. But, as you know, you can do an
> exposure assessment with one data point and it is just how good is
> that exposure assessment. So, we ultimately want to understand the
> distributions from the major foods.
>
> DR. MILLER: More comments? I have just a couple of points. Clearly,
> some of this work is already in progress. Can you give us some idea of
> what the timeline is for your priorities about what is going to be
> done first?
>
> DR. TROXELL: I am sorry, I don\'t have a timeline on each of these
> studies. Much of the tox. work is either in the planning stage or has
> already begun. There is short-term work from which within a year or
> two we should be reaping benefits. Of course, the bioassay is going to
> be very long. We have just put in a proposal for the home cooking work
> for the National Center. It is going to take some time, I am sure, for
> JIFSAN to compile the database and so on but that work was begun a
> while ago to try to get the different players to submit some of their
> analyses.
>
> We are\--what?\--ten months into this project. We started without a
> method and, obviously, the first thing we need is to be able to detect
> and to find out what we are dealing with so we are pushing forward as
> readily as we can. I don\'t know if that is enough. Dr. Canady may
> have some additional information on timelines.
>
> DR. MILLER: Well, it just occurred to me that we are looking at an
> action plan that may already be in progress. Unless there is something
> seriously wrong, other than beating a dead horse, there isn\'t much
> you can do about changing it. The committee made a decision that they
> would like to see you modify some of the activities but if a study is
> already under way it would be difficult to change.
>
> DR. TROXELL: Well, I think any recommendations coming out of the
> committee would be carefully examined to see if something should be
> modified in approach to a short-term study or other studies. Things
> are not that far down the road, I believe, that they cannot be altered
> and, I mean, we are here to receive this input. It is important to us,
> very important to us that we get it as right as possible at this point
> so we have what we think is a good framework, a good template for
> proceeding, not that we won\'t need to evolve that template in another
> year.
>
> DR. MILLER: The other question I had concerned funding. How are you,
> or are you, attempting to get other groups, particularly university
> research groups, involved in some of this research in order to expand
> the research base?
>
> DR. TROXELL: We have tried to do some extramural proposals. I don\'t
> know that we have been successful in getting those through. It is not
> clear to me how much we are going to be able to accomplish as, you
> know, the budgets are not growing at this point; they are decreasing.
> So we are hoping to tap into I guess other people\'s budgets.
>
> DR. MILLER: That is what I meant, like NIHS.
>
> DR. TROXELL: Right, we will work on that but, again, that isn\'t in
> our control. We can control the agency\'s dollars and where they go.
> We can work through the Department to try to get additional funds
> focused on this and, hopefully, we will be successful in that.
>
> DR. MILLER: Have you had discussions with NIHS on this issue, or any
> of the NIH institutes?
>
> DR. TROXELL: Well, they are definitely interested in looking at the
> neurotox. and the germ cell part of the work so that is in progress.
>
> DR. MILLER: Dr. Lee, yes?
>
> DR. LEE: Terry, since we are on this subject of \"Troxell-cology\" I
> would just like to ask perhaps philosophically what is the long-term
> outcome? Do you think we will ever get to a point with acrylamide that
> no more information is needed?
>
> \[Laughter\]
>
> DR. TROXELL: Well, in the sense of good \"Troxell-cology\" as with all
> toxicology, we always need one more study.
>
> DR. LEE: Right.
>
> DR. TROXELL: If we can nail down those essential needs of
> bioavailability, the high dose/low dose and so on, that is going to
> really help us get a handle on what the potential human impact is.
> Again, trying to understand high dose/low dose is going to depend on
> some pretty sophisticated chemistry trying to measure adducts. This is
> science and, as you know, you never know exactly where science is
> going to take you and how far you are going to go with it, and we are
> going to push it as well as we can and certainly some of the best
> researchers in the country are measuring adducts and you will hear
> from one of them later, Dr. Fennell, on adducts and we will see how
> far they can go with this work.
>
> DR. MILLER: Johanna?
>
> DR. DWYER: I am concerned a little bit though about singing from the
> same hymn issue of all the government agencies working on this. I know
> you are trying very hard to coordinate the work, and so forth, but I
> am just concerned that we don\'t sit here in a year or two and hear
> three different agencies, all telling us different things about the
> exposure assessments they have done, and so forth. And, what kind of
> guarantee is there that this isn\'t going to happen? I am also
> concerned about Dr. Miller\'s question of the time frame. How do we
> know if the time frame has been revised if we don\'t know the time
> frame to start out with?
>
> DR. TROXELL: Well, as far as the exposure assessment, the FDA is
> responsible for the exposure assessment I believe so we will be doing
> that. We are doing our best to coordinate the work and all I can say
> is we are working very hard at it. We had an interagency meeting. We
> are going to go back and have further meetings to try to ensure
> coordination. There have been meetings along the way on the
> neurotoxicity. Dr. Canady attended a meeting that had a session on
> acrylamide in November. We will be doing a meeting at some point this
> spring, I believe, on the germ cell toxicity, and so on. So, we are
> making every effort to coordinate the actions and we hope, you know,
> that it pulls together. I don\'t want to provide any absolute
> guarantees because obviously it is not all under the control of FDA
> but we will work our best to keep it coordinated and on track.
>
> DR. MILLER: Have you made an attempt to develop some kind of a
> consortium of those people who are working in this area? Because if
> you just have meetings periodically there is always a delay in this
> thing but a consortium with different groups given the responsibility
> of coordinating information on different aspects of the problem might
> be a way of getting the data distributed much more rapidly.
>
> DR. TROXELL: The closest thing to a consortium is the work of JIFSAN
> to coordinate the efforts.
>
> DR. MILLER: Coordinate which efforts?
>
> DR. TROXELL: They are basically coordinating all aspects and gathering
> the information together on all aspects internationally to make sure
> that all information is available to everyone. That is more of an
> information exchange kind of system. So, I guess I would have to say,
> no, we don\'t have an explicit consortium to work on this. The FDA,
> because this is a problem in FDA regulated foods, is basically taking
> the leadership to coordinate the efforts among the agencies and to do
> what we can to leverage the academia and industry. So, in a sense, we
> are the head of the consortium to explore acrylamide.
>
> DR. MILLER: Yes, Dr. Mehendale?
>
> DR. MEHENDALE: I was pleased to see that you mentioned the high
> dose/low dose bioavailability studies as being very critical. I was
> hoping that there was a timeline on those studies. But it seems like
> the results of those studies would help us to at least get the
> direction of the larger issues I think.
>
> The second point I wanted to make is I didn\'t see anything in what
> you mentioned, if you are considering any special populations. A
> couple of them I can think of, You know, just last week we had an
> issue of [Science]{.underline} devoted to obesity and I know lots of
> people are considering diet restriction or caloric restriction. One of
> the things that happens with caloric restriction is induction of Cyp
> 2E1. That is a special population or special physiological condition.
> Diabetes is also thought to be associated with some induction of Cyp
> 2E1, and there may be other special populations. I wonder if you have
> any comments on that aspect. I think considering these populations
> might also be helpful when you are looking at more sensitive
> populations and, therefore, it might be helpful in arriving at safe
> levels.
>
> DR. TROXELL: Number one, I don\'t have any further information
> personally on timelines, other than to expect that the toxicology on
> the short-term studies, which can give us some insight on high/low
> dose bioavailability, are going to take one to two years. Do you have
> anything further, Rick?
>
> DR. CANADY: With regard to the toxicology studies, we have already got
> some results. For example, work on adduct formation, DNA adduct
> formation has already been developed. Bioavailability and studies
> along those lines with regard to toxicokinetics, some of the initial
> findings I guess we will have within the next six months to a year. We
> have proposals that we are currently reviewing on the specific studies
> but some of the studies as you can imagine, for example
> bioavailability, are relatively short-term studies comparing area
> under the curve for different routes of exposures and that is
> something that can be done fairly quickly. So, those studies with
> regard to toxicokinetics we can expect to see, as Terry was saying, in
> the next year or two but probably some of the initial results will be
> in the next six months to a year. Again, some adduct studies are
> already available.
>
> I am not sure how else to give a more specific deadline or specific
> timeline, other than to say we have proposals and we have specific
> information on modes of action we are evaluating within those
> timelines. Is there more detail you would like with regard to that? It
> is not like we can give you that the bioavailability studies will be
> done by June or\--
>
> DR. MILLER: I think the question was in terms of priorities, what is
> being done and what is going on. I think you have done that.
>
> DR. CANADY: Good. With regard to the special populations, clearly in
> considering the prospective studies that is a very important
> determination, very important source of confounding or a way of
> designing the studies. Glutathione transferase, Cyp 2E1, polymorphism
> with regard to those inductions are things that are clearly important
> to consider when thinking about how you might go about doing those
> studies, and we are well aware of that.
>
> With regard to specific animal studies, NIEHS has already initiated
> some studies with Cyp 2E1 knock-out mice to look at the conversion of
> acrylamide to glycidamide and looking at the mechanistic
> interpretation of that information with regard to neurotoxicity, germ
> cell toxicity and also DNA adduct interactions. NCTR and NHANES are
> working together to develop that line of mechanistic research.
> Specifically, NCTR will do the DNA adduct work for that set of
> experiments.
>
> The NHANES studies, as you know, sampled some specific populations but
> they weren\'t designed to specifically look at acrylamide issues so
> maybe others can comment more specifically with regard to how NHANES
> would be used to look at specific populations. You would have
> information that would be relevant to intake but whether it is
> specifically relevant to caloric restriction, probably not. Whether it
> is specifically relevant to foods that are high in acrylamide, that is
> something we need to evaluate still. But the bloods have been set
> aside to do specific adduct work, the bloods that were taken earlier
> this year, in fact, for NHANES so that information would be worked
> into the study design to the degree it could.
>
> Results of separate studies being considered by CDC and CEH are
> intended to look at sort of add-on acrylamide dosing or foods that
> have acrylamide and through that we could evaluate some of the
> questions that you are talking about.
>
> DR. MILLER: I think this issue of stress populations, particularly for
> something as ubiquitous as this is, would seem to me important enough
> to mention as part of the Action Plan. I am not exactly sure how it
> would be done but it seems to me it needs to be considered, and more
> general stress situations where we know there are metabolic changes
> and inductions that occur in response to stress.
>
> DR. TROXELL: See, it makes a difference when you have a real
> toxicologist instead of a \"Troxell-cologist\" to provide the answers!
>
> DR. MILLER: Any other comments or questions? Yes?
>
> DR. RUSSELL: Jumping ahead to risk communication because I don\'t see
> any other time to really comment on this, I think it is a good idea to
> have the dietetic nutrition associations in concert with you in
> communication efforts, but you will have to cast the net pretty wide
> for these nutritional organizations. It is not that you can just pick
> one or two or them. There is a whole group of them that have different
> kinds of missions, if you will, but they have very important audiences
> and very important journals, a couple of them, even though the
> societies are pretty small. So, you need to really cast the net pretty
> wide there. You have mentioned one very big one, the American Dietetic
> Association, and one very small one but you need to go much beyond
> that.
>
> DR. TROXELL: Thank you.
>
> DR. MILLER: Yes, Johanna?
>
> DR. DWYER: Specifically, I would suggest that you go to the American
> Society for Clinical Nutrition, the American Society for Nutrition
> Sciences and certainly IFT. I believe Dr. Russell is currently the
> president of the ASCN.
>
> DR. TROXELL: Thank you.
>
> DR. MILLER: Have you made a commitment, Robert?
>
> DR. RUSSELL: Well, that journal reaches a large audience.
>
> DR. MILLER: Yes?
>
> DR. SCHERER: I wonder whether there has been any thought or discussion
> about the potential problems that the agency faces in terms of release
> of information? I certainly support the idea of open communication but
> given the nature of the wide range of studies that are being done, it
> is likely, it seems to me, that at least one of those studies at some
> point will have implications for serious health concerns, whether that
> is eventually supported or not but the possibility exists. So, the
> challenge, it seems to me, for the agency is how do you respond to
> that information that is likely to be picked up by the media and, as
> we all know, perhaps be exaggerated? But the idea of trying to put it
> in perspective of the wide range of studies that are going on, whether
> there has been any thought given to that process?
>
> DR. TROXELL: Well, for every one of our meetings we do a lot of
> thinking about how appropriate the message is about acrylamide, and we
> will clearly bring a lot of energy and information to bear from our
> people who do consumer messages, and so on, in the chain who
> understand risk communication we bring that kind of to bear.
>
> I think also, as you suggested, it is important for us to think about
> how we are going to communicate what would reach consumers, and I
> think that is a valuable comment.
>
> DR. MILLER: Actually, that is an issue that really needs special
> emphasis because I have been trying to think if there were any other
> materials that I could remember that are so ubiquitous in diets
> everywhere in the world so any culture that produces a dough baked
> product is going to be exposed to some level of this material and how
> do you deal with that? That is going to be a big problem, it seems to
> me, as a research issue and it needs to be emphasized. I think maybe
> it requires going back and looking at it again to reemphasize it
> again.
>
> DR. TROXELL: Thank you. I think these are valuable points.
>
> DR. MILLER: Any other comments or questions? We are caught between a
> rock and a hard place in terms of timing. We are substantially ahead
> of time and I was thinking of going on to our next speaker.
>
> DR. TORRES: Maybe I could ask one more question.
>
> DR. MILLER: Please, do.
>
> DR. TORRES: On the subject of cooperation with different groups, one
> thing that really caught my attention was the message that the U.K.
> had done analysis on 4000 food items. That seems to be a lot of work.
> Could you explain to me why there is a difference between 100 in the
> U.S. and thousands in the U.K.?
>
> DR. TROXELL: Well, I don\'t think the Central Science Lab of the U.K.
> has done 4000. They have compiled 4000 from industry and around
> Europe, as well as their own analyses. It is just the level of
> progress we are at in different areas.
>
> DR. TORRES: Thanks.
>
> DR. MILLER: Terry, there is another question that I meant to ask
> before, given the different methods\--well, let me ask you this
> question, how many different methods are being used by the different
> laboratories?
>
> DR. TROXELL: That is a good thing to dwell on for a minute because
> even with the most elegant methods, like the LC/MS/MS method, it is
> pretty easy to mess up and get erroneous results. But there is
> LC/MS/MS; there is GC/MS; there is GC after bromination. Well, if you
> brominate your extract and then run it through GC with electron
> capture detection you get pretty good results. We are trying to look
> at this LC/UV as more of a screening method. I mean, we are looking
> for something that people can use as quick and dirty to screen their
> products, to help fuel their research, for developing countries to be
> able to use, and so on, and also maybe as a quick screen for us
> although we will probably end up as an agency, when we get into the
> screening mode, using something like the GC/MS.
>
> Surprisingly, when you look at the cost, and they did this at the
> JIFSAN meeting, the costs are\--what?\--\$200 or something per sample
> for the LC/MS/MS and you are still talking about \$100 or so per
> sample for some of the GC/MS. So, this is very expensive work and you
> can imagine that developing countries wouldn\'t have the capability to
> be doing much of this work.
>
> Of course, some countries have quite different foods, different fried
> products and so on that can reach the high temperatures. So, for the
> world to get its arms around the distribution of acrylamide in foods
> is an interesting point, and also for us to understand if the analyses
> that are being put into the database are valid is also another
> interesting question. We are going to have to be very careful, based
> on the information they provide, to understand if the results are
> good. I mean, there are many methods that give good results but,
> again, you have to perform the analyses correctly and it is very easy,
> initially particularly, to have problems. Therefore, people are
> circulating proficiency samples to try to make sure that the results
> that other labs are getting are going to be accurate. That is one of
> the efforts that is going on, to get proficiency samples in different
> types of foods to help improve the credibility of the results.
>
> DR. MILLER: Looking at the developing world, is FAO doing anything to
> try to collect some samples?
>
> DR. TROXELL: Not that I am aware. As I said, we have proposed an
> informal workshop and FAO/WHO were behind getting that together. Of
> course, FAO and WHO were behind the consultation and also will be
> working on gathering information for the next meeting.
>
> DR. MILLER: Someone has to do those samples.
>
> DR. TROXELL: Right, and if you look at the JIFSAN info. net you will
> find, I think, there are samples in there from South Africa and I
> think there might be some samples from Egypt. People are sending data.
> That is not the JIFSAN info. net; it is the WHO/FAO information
> network that JIFSAN is operating for them. Anyway, these data are
> coming in; the databases are growing and there are criteria for
> methods. There are many fields to fill out\--what is your methodology,
> and so on\--so that in the end, hopefully, we will be able to
> understand what these results look like and how valid they might be in
> different foods.
>
> DR. MILLER: I imagine a lot of these populations that consume flat
> breads might get a little higher exposure.
>
> DR. TROXELL: Yes.
>
> DR. MILLER: Johanna?
>
> DR. DWYER: Back to the issue of the quality ratings of the values that
> are coming in, I don\'t have any knowledge of this particular compound
> but certainly with flavonoid in food, where there are a lot of
> advantages for industry and others to try and find out what is in
> there because there are possibly positive health effects, there are
> quality rating systems already in place that can be used and adapted
> for determining whether a value is adequate or not adequate. I believe
> Dr. Beecher and others at the Ag. Research Service, right over in
> Beltsville, developed those methods some time ago. I wonder whether
> you are using those methods, adapted for acrylamide, to screen your
> values so that the database is as inclusive as possible, at the same
> time meeting quality criteria for analysis, sampling and so forth.
>
> DR. TROXELL: Clearly there are performance criteria one can apply to
> analytical methods, and the GEMS database at WHO has many data fields
> which list out what is your method, what is your limit of quantitation
> and how you establish it, and so on. I believe an awful lot of
> information is requested of submitters so probably it is more than
> adequate and, in fact, if anything, there is so much information
> requested that it kind of inhibits people from submitting data because
> they have to put so much energy into providing the data. So, yes, I
> think we should be able to distinguish between the good data and the
> questionable data. Clearly, when it is questionable we won\'t use it.
>
> DR. MILLER: Any other comments or questions? If not, we are going to
> adjourn for lunch and return at 12:45 for the afternoon session. Thank
> you.
>
> \[Whereupon, at 11:15 a.m., the proceedings were recessed, to resume
> at 12:45 p.m.\]
>
> [A F T E R N O O N P R O C E E D I N G S]{.underline}
>
> **Mechanisms of Formation**
>
> DR. ZYZAK: April 24, 2002, this is a shocking date to many people in
> the food industry as Stockholm University, in connection with the
> Swedish NFA, revealed acrylamide presence in a variety of foods. As
> many organizations began to develop analytical techniques with a
> capability of analysis of acrylamide in foods it came out that the
> data released at this time was accurate.
>
> \[Slide\]
>
> We, at P&G, did our own product survey and here is a sample list of
> that. As you can see, we also found acrylamide present in a variety of
> foods such as toasted bread products, roasted asparagus, corn chips
> and potato chips.
>
> \[Slide\]
>
> What is acrylamide? Acrylamide is a conjugating e-amine molecule. It
> has a high boiling point and it is also very hydrophilic or water
> loving. I think this is probably why it took a while for us to detect
> it in the food system. Typical analytical techniques in the food
> industry involve head space which is dependent on the boiling point or
> extraction of organic solvents and in this case acrylamide would tend
> to stay more in the aqueous phase.
>
> \[Slide\]
>
> Now that people have accepted the fact that acrylamide is present in
> foods, the next issue is how is it formed. Some initial mechanisms
> that were proposed from the Food Research Institute were based on
> equivalence formed in the frying process so there are lipid oxidation
> products which could be precursors, and some of these have similar
> structures to acrylamide. You can see acrylamide here and this is
> acrylic acid. The only difference is we need an amide bond here and
> acrolein, another lipid oxidation product.
>
> However, this formation of the amide bond is not a very favorable
> reaction in typical food conditions and subsequent research in this
> area has shown that this is not occurring under typical cooking
> conditions. Again, the trouble came in the formation of this amide
> bond.
>
> Well, if you look at the amino acids in the food system, there are
> already a couple that have amide bonds present in the side chain. One
> of these is asparagine and the other is glutamine. Now, amino acids
> during typical cooking conditions undergo a process of decarboxylation
> followed by deamination. If you look at this asparagine side chain, it
> looks very similar to acrylamide. What is known as the structured
> aldehyde of the amino acids would form this aldehyde here, and there
> is the possibility that there is a side reaction going on where we can
> form acrylamide from asparagine. You will see, as I show further on,
> that this is a small part of the reaction. Glutamine here just has an
> extra methylene group but there is the possibility that under typical
> cooking of food where you get decarboxylation followed by deamination
> it might form some type of rearrangement of products which could lead
> to a conceivable small portion of acrylamide formation.
>
> \[Slide\]
>
> To address this we developed a model system. In this case we tried to
> make it like a potato chip. We took potato starch and water. We were
> able to heat this kind of like a dough sheet, and to this system we
> could add a variety of amino acids, reducing sugars and a variety of
> other ingredients which could include inhibitors. After this we could
> take it to a frying process and then measure acrylamide in the
> finished product. This could also be baked and we showed in our
> studies that baking can result in the formation of acrylamide.
>
> So, the elegance of this model system\--we have to make sure that our
> system is inert itself. Here we looked at a potato starch system and
> we went through the frying process and got less than 50 ppb
> acrylamide. To our potato starch we started adding reducing sugars
> such as dextrose. We take our potato starch, we add in asparagine
> alone and we start to detect acrylamide formation. However, the
> combination of dextrose and asparagine gives us significant amounts of
> acrylamide formation here.
>
> We also looked at other amino acids, such a alanine, aspartic acid,
> lysine, threonine and glutamine. We can actually detect a small level
> in glutamine, 156 ppb versus asparagine with 9000. So, about one
> percent the level is formed in glutamine compared to asparagine.
> However, arginine, cysteine, all these other amino acids do not form
> detectable levels of acrylamide. You can see from this that we kind of
> felt that asparagine is really the source of acrylamide.
>
> \[Slide\]
>
> How does this relate to food systems? Well, what about amino acid
> composition of potatoes? We looked at that and approximately 50
> percent of the amino acids in potatoes are in the free state, which
> means it is not incorporated into the protein. Out of that, asparagine
> is roughly half of the free amino acid content. So, it is conceivable
> that potatoes have such a high level of free asparagine that that
> could be the source of acrylamide formation in potato products.
>
> \[Slide\]
>
> Other reactions were carried out with using free amino acids such as
> asparagine. We wanted to find out whether protein-bound asparagine
> could also participate in the formation of acrylamide. So, as an
> analog, you can purchase N-acetyl asparagine where the alpha-amine
> group is tied up to this bond, here, mimicking a protein analog. We
> reacted this with dextrose to see if it formed acrylamide. The results
> were that no acrylamide formation was observed. So, from our
> understanding, we felt that all we need to be concerned with is free
> asparagine because that is what is the precursor to acrylamide
> formation during heat in the food system.
>
> \[Slide\]
>
> We know that asparagine is required for homolysis and dextrose is also
> required. So, we looked at a dose-response curve from dextrose. If you
> look at a potato, to get a rough estimate, asparagine is actually 1.25
> percent in a potato. The dextrose or reducing sugars is about 0.5
> percent in a fresh product when harvested, but as product is
> harvested, usually in the late summer early fall, it may go into
> storage and be stored for quite a period of time and the level of
> reducing sugars will actually increase in potatoes. So, you can see
> that as you increase your level of free reducing sugars you actually
> increase your level of acrylamide. So, we know that you need not only
> asparagine but a level of reducing sugars in potatoes.
>
> \[Slide\]
>
> Are there other carbonyl sources that can form acrylamide? Some recent
> work speculated that the formation of acrylamide from asparagine, the
> structured degradation reaction\--structured degradation reaction is
> implicitly explained, actually a di-carbonyl such as, in this case,
> glyoxal reacting with the amino acid causes the reaction to proceed.
> We also showed that glyceraldehyde, 2-deoxyglucose and ribose are also
> efficient at forming acrylamide in food systems.
>
> People who are familiar with the Maillard reaction understand that the
> typical browning reaction involves first a reaction of a carbonyl
> amino acid. If you use a molecule such as 2-deoxyglucose where it is
> C2 here, you do not have a hydroxyl group. This prevents the molecule
> from undergoing the rearrangement. So, this actually lets us know that
> all we need is to Schiff base the formation for the formation of
> acrylamide. This is also verified by reactions we did, lipid aldehyde
> such as decanel, and Dr. Adam Bakowsky at Health Canada also published
> about octynal, another lipid aldehyde that can react with asparagine
> to form acrylamide.
>
> However, people may ask is lipid oxidation contributing to acrylamide
> formation? I would think not because if you look in the food system,
> typically the reducing sugars are probably on the order of about one
> to two magnitudes higher than the lipid aldehydes. So, I think what we
> need to be concerned with is level of reducing sugars.
>
> \[Slide\]
>
> To prove that, initially we are thinking that the asparagine actually
> going into acrylamide is the side chain here. Just to make sure we can
> prove that this is going on, you can purchase isotopes which can be
> incorporated and enriched in either ^15^nitrogen or ^13^carbon. So, we
> carried out experiments just to confirm that this is where the source
> of carbon nitrogen is coming from in asparagine.
>
> \[Slide\]
>
> For the initial experiment we used amide-labeled asparagine so it is
> ^15^N here. Reacting with dextrose, we should form acrylamide where we
> have a ^15^nitrogen at the amide bond. Acrylamide has a molecular
> weight of 71. We are going to be monitoring and any unlabeled
> acrylamide would show a mass at 72. However, since we are
> incorporating ^15^N here, we think we should be detecting this at a
> mass of 73. And, this is what we see. We do not detect any unlabeled
> acrylamide. Really 97 percent of the total acrylamide response is at
> the monolabel, suggesting that this amide nitrogen is being
> incorporated into the acrylamide.
>
> \[Slide\]
>
> We did further studies where we labeled the alpha-amine nitrogen.
> Again, this is the source of the carbon nitrogen so when we add
> dextrose to form acrylamide we should get a mass at 72 here so any
> detectable levels of acrylamide were the unlabeled acrylamide, again
> suggesting that this nitrogen is not being incorporated into the
> acrylamide formation.
>
> \[Slide\]
>
> Next was to verify where the carbons were coming from. So, we
> purchased a uniformly labeled asparagine where all the nitrogen and
> carbons are labeled. In this case we got this nitrogen label and these
> three carbons and we should see an increase of four mass units and we
> should be detecting acrylamide at a mass of 76. Indeed, from our
> analysis all we could detect was the acrylamide at 76. You see in this
> chromatogram that we also monitored at 75, 74, 73 and 72 for any other
> molecules. So, from these experiments we concluded that this side
> chain of asparagine is what is being incorporated into the acrylamide.
>
> \[Slide\]
>
> From these studies we were able to form this following mechanism of
> acrylamide formation. The alpha amine group of asparagine here is a
> mucophilic attack on the carbonyl source, forming glycocyamine. As you
> are driving away the water, you get the formation of Schiff base. So,
> actually this process is favored under reduction of water in your
> cooking system. After this, as heat is applied we get decarboxylation
> that forms as intermediate which rapidly degrades to form acrylamide,
> os hydrolyzed to form beta alanine amide, and beta alanine amide
> itself can undergo elimination of acrylamide. We showed that we can
> heat this under typical frying conditions and it will be able to
> decompose even in the absence of sugars to form acrylamide.
>
> So, this is kind of a proposed mechanism. How can we prove that? Well,
> utilizing LC/M mass we are able to do that. What I will show you on
> the next slide is where we are going to be monitoring our carbonyl
> source. In this case we use dextrose so we can monitor that at a
> molecular weight of 180. We are going to be monitoring asparagine.
> And, as we heat a product out we are going to be looking at the
> formation of Schiff base, the beta alanine amide and the acrylamide to
> prove our mechanism.
>
> \[Slide\]
>
> Here are the first monitoring intermediates in acrylamide formation.
> In this case, we use just regular asparagine reacted with dextrose.
> You can see that at our initial time, zero seconds, we get a response
> for dextrose and a response for asparagine. At intermediate time, 180
> seconds, we actually start to see our first intermediate Schiff base
> being formed. As we heat this on to 270 seconds we have actually
> depleted all our source of asparagine, dextrose. The Schiff base is
> gone. We get an extra intermediate beta alanine amide and also
> acrylamide. So, we can monitor these intermediates in this reaction
> system.
>
> Just to confirm that they were there we actually used isotope labeled
> ^13^C and ^15^N molecules and we can see the corresponding shift to
> mass units. In this case we are monitoring asparagine at 133, with
> these all being labeled, and incorporation of six mass unit
> difference. We monitor at 139 and we can see the increase so the
> acrylamide has gone from 72 to 76. The beta alanine amide has gone
> from 89 to 94. So, we have confirmed that these intermediates are
> actually formed during the reaction process.
>
> \[Slide\]
>
> Next is understanding acrylamide formation in food products. All these
> studies I have been showing you right now are a model system so we
> need to prove in a real food system that asparagine is the source of
> acrylamide. Questions arising\--is asparagine the only precursor to
> acrylamide in heated foods? What about other potential sources of
> acrylamide, methionine, glutamine, cysteine or acrolein? These have
> been postulated by people to maybe provide a minor amount of
> acrylamide. In our model system, we think we have disproved this fact
> and have shown that they are not sources.
>
> But another way to do this, we decided to do selective removal of
> asparagine from a real food product with the enzyme asparaginase to
> address these questions because we felt like if we could have
> asparaginase in this real food system degrade all the asparagine and
> look at acrylamide formation we would show that that reduces
> acrylamide formation and asparagine is the source of acrylamide in a
> real food product.
>
> \[Slide\]
>
> Asparaginase, this enzyme, will hydrolyze the amide bond of asparagine
> utilizing water and will form aspartic acid. If you remember our
> initial model system that I showed you, we analyzed aspartic acid\'s
> ability to form acrylamide and it formed undetectable levels. So, we
> feel like if we do convert asparagine to aspartic acid it should
> result in reduction of acrylamide formation.
>
> \[Slide\]
>
> So, here is our real food system. We took washed, Russet bake potatoes
> purchased from the local grocery store, boiled for one hour, and then
> we blended the flesh on a one to three ratio with distilled water. We
> have two plots here, one as a control and the other one we did with
> enzyme asparagine-treated, carried out for 45 minutes at room
> temperature. Then we microwaved this at two minute intervals for a
> total of ten minutes. This is a highly cooked product to maximize
> acrylamide formation. Both the control and asparaginase treated
> products were dry and brown after the step.
>
> To make sure that the enzyme was working correctly we analyzed for
> change in asparagine and aspartic acid. This is our control sample
> and, as mentioned earlier, free asparagine is high in potatoes so you
> have a nice peak response here for asparagine and a smaller response
> for aspartic acid. In our asparagine-treated sample you can see where
> the asparagine has been depleted. We depleted about 88 percent here
> and the aspartic acid is subsequently increased. So, we know that in
> our system here the reaction was carrying out the way we expected.
>
> Next was to monitor for acrylamide. We can see in our control product
> we have 20,000 ppb acrylamide. Asparagine is treated down to 164 so we
> actually got greater than 99 percent reduction in acrylamide by using
> asparaginase. So, we feel that this experiment is able to prove that
> asparagine is the mechanism for acrylamide formation in a real heated
> food system, in this case being the potato.
>
> How does this relate to other foods? In our studies we looked at the
> yield of acrylamide from asparagine. We deduced that the yield was
> less than 0.5 percent. Dr. Adam Bakowsky at Health Canada also showed
> in his work that the yield was about 0.1 percent. So, we have taken
> these numbers and looked at a variety of food products. If you look at
> the amount of free asparagine in the starting food products and you
> correspond to the yield of about 0.1 to 0.5 percent, I think that will
> compensate for all the level of acrylamide that has been detected out
> there. So, we feel that asparagine is the source of acrylamide
> formation in all food products.
>
> \[Slide\]
>
> Acrylamide precursors are ready to intervene. We know that asparagine
> is important for this formation and also reducing sugars. Typically in
> food systems reducing sugars are glucose and fructose. Some foods will
> contain sucrose and in the cooking process will undergo hydrolysis to
> form glucose and fructose.
>
> How is this affected in potatoes? Well, the level of asparagine and
> reducing sugars actually varies by the source of potato. I think there
> are many people out there in potato processing areas who are
> monitoring asparagine in a variety of potatoes and also looking at
> reducing sugars, and we know that in storage conditions, as potatoes
> are stored for periods of time, the level of reducing sugars will
> increase. If you look at a product in the early fall, it will probably
> have a low level of acrylamide but as people start to use more
> potatoes that have been stored for a longer period of time the
> acrylamide will potentially increase.
>
> \[Slide\]
>
> In conclusion, asparagine is the major source of acrylamide formation
> in foods. Carbonyl source typically in food systems is going to be
> reducing sugars as required in the reaction. Oil oxidation products
> and starch do not appear to be significant factors in acrylamide
> formation.
>
> One thing I forgot to mention when I was showing you our model system
> where we took our potato starch and added amino acids and fried it, we
> also looked at fresh oil versus an oxidized, aged oil to see if
> acrylamide formation was affected and there was no difference. So, we
> were able to conclude that oil quality such as oxidized oil did not
> significantly affect the level of acrylamide formation.
>
> So, that is the conclusion of my talk. Thank you.
>
> **Questions of Clarification**
>
> DR. MILLER: Thank you. Comments or questions?
>
> DR. BUSTA: You were generating acrylamide in a microwave in a water
> system which wouldn\'t be over 100 C. Right?
>
> DR. ZYZAK: Yes.
>
> DR. BUSTA: I thought we required a higher temperature than that to
> generate acrylamide.
>
> DR. ZYZAK: I mentioned that in the microwave system it was dry and
> brown, and there are results out there that you can form acrylamide
> during microwave conditions. What we found is a big factor in the
> level of acrylamide formation is the moisture of the product. So, if
> you do microwave something and you still have a higher moisture
> content, it is probably okay. It is when you get down to low moisture
> content that you drive that reaction. As I showed the mechanism, as we
> remove water from our Schiff base you get the decarboxylation step.
> So, I think finer moisture content is a critical factor in acrylamide
> formation in food products.
>
> DR. BUSTA: Are you saying temperature is not?
>
> DR. ZYZAK: Temperature is. I think it is a combination of both. You
> need to have a low moisture environment to get that Schiff base and
> you need to get heat involved to get the decarboxylation step going
> on. You do need both of those going on, but we do see that at 100
> degree C you can form acrylamide.
>
> DR. MILLER: Johanna?
>
> DR. DWYER: I think I am right that ascorbic acid is a reducing sugar
> and is present in some foods.
>
> DR. ZYZAK: Correct.
>
> DR. DWYER: Is that a significant factor? For instance, my ancestors
> ate a lot of potatoes and I want to know if I have gene damage.
>
> \[Laughter\]
>
> DR. ZYZAK: I think you were talking about ascorbic acid and four
> carbon sugars can participate in this reaction, but I think if you
> look at the level of reducing sugars, such as the glucose and
> fructose, they are about half a percent and can range up to two
> percent in a potato. So, that far outweighs the level of ascorbic acid
> in there that is enough to facilitate the reaction.
>
> DR. MILLER: Yes?
>
> DR. MEHENDALE: I was wondering if you have tried any carbonyl blocking
> mechanisms in your reactions.
>
> DR. ZYZAK: Yes, the typical anti-browning reason are sulfites. All
> this would be simple if we could add sulfites to solve the problem but
> it didn\'t work and you can only add a pretty low percentage of
> sulfites in a food product, like if you buy dehydrated potato products
> I think it is less than a percent or something like that, the level of
> sulfite you can add in there. Since you already have a couple of
> percent of reducing sugars we didn\'t see any benefit to adding
> sulfites.
>
> However, we also looked at another amino acid like lysine. We added
> lysine in there to block the carbonyl source, dextrose in this case,
> that was ineffective. However, if you add the amino acid cysteine, you
> can actually decrease the level of acrylamide formation. The question
> is, is the cysteine reacting with the dextrose tied to the carbonyl,
> or is it reacting with the acrylamide once the acrylamide forms? It is
> actually a later part of the reaction so it is complexing with the
> acrylamide.
>
> DR. MEHENDALE: I have a follow-up question. You know, in some old
> literature a gentleman by the name of Serami has done a lot of work on
> di-cosylated end products for aging.
>
> DR. ZYZAK: Yes.
>
> DR. MEHENDALE: It seems to be that it was carbonyl groups of dextrose
> that are involved.
>
> DR. ZYZAK: Yes.
>
> DR. MEHENDALE: So, it seems to me like there may be some potential for
> either blocking or reacting the carbonyl groups with other things.
>
> DR. ZYZAK: Yes, you bring up Dr. Anthony Serami and I did my graduate
> studies with Dr. John Baines, who were kind of competing with each
> other so I am very familiar with his work. We also did studies by
> adding one protein and other things in our model reactions. Maybe we
> can add a protein source that will either react or just tie up the
> reducing sugars. We weren\'t very successful at that.
>
> We did try adding a protein source to our model system and didn\'t
> seem to have a significant increase. But, again, in that case we were
> using something off the shelf like a relatively inexpensive source. I
> mean, you can go out and buy some yeast products which may have a high
> content of glutathiamine. It is a very pricy product. So, if you want
> to try to reduce the level of acrylamides, you could probably
> incorporate a source of protein which may have a high concentration of
> thiol groups in there which are known to be very active with
> acrylamide once it is formed. People have done studies looking at
> amino acids. Amine groups will react with acrylamide but not very
> readily where there are high thiols.
>
> DR. MEHENDALE: So, how long can we keep potatoes?
>
> \[Laughter\]
>
> DR. ZYZAK: I think many people in the industry are also looking at
> that, you know, how is acrylamide going to be affected as later in the
> season we are using older potatoes? I think we are all looking at that
> now and I think many people are addressing that so it will come up in
> the future I believe.
>
> DR. MILLER: It depends on how you cook the potato.
>
> DR. ZYZAK: Sure, yes.
>
> DR. MILLER: Any other comments? Yes?
>
> DR. TORRES: Are there any other food systems where there would be a
> lot of free asparagine?
>
> DR. ZYZAK: Yes, asparagus has a high level of free asparagine I
> believe. I believe at the subcommittee meeting Dr. Lauren Jackson
> showed some data that the level of free asparagine is high in almonds.
> I think it is high in legumes, beans, bean products. Asparagine
> actually is used by plants as a source of nitrogen storage system. So,
> most plants utilize asparagine to store the nitrogen for further use
> as energy or convert it into protein. So, I think we are kind of stuck
> with this because, you know, that is the way plants are going to grow
> so they are going to use nitrogen as a fixation source until we can
> use some biotech and utilize some other source of nitrogen.
>
> DR. RUSSELL: I was just wondering is there much of a difference
> between white potato and sweet potato in the acrylamide formed under
> similar conditions?
>
> DR. ZYZAK: You know, I don\'t have that data. We haven\'t done that
> experiment but I think definitely there is activity in that. We,
> ourselves, know that the variety of potato will affect the level of
> acrylamide because different varieties of potatoes will have a varying
> factor of asparagine in them and reduced sugars so we are also looking
> at that.
>
> DR. MILLER: It seems to me that most root vegetables cooked at high
> enough temperatures should be excellent sources of acrylamide.
>
> DR. ZYZAK: Yes. We actually talked with kind of a potato professor in
> industry just to understand more about how it is using asparagine so
> we can get potatoes with a lower source of asparagine. There doesn\'t
> seem to be a lot of information out there about that. You know, I was
> specifically told that roots are different from tubers so I don\'t
> know all the botanical aspects of that but it can be different whether
> it is a potato versus a carrot.
>
> DR. MILLER: Well, if it stores asparagine as a nitrogen source then,
> depending on how it is cooked, it will have a high level of
> acrylamide.
>
> DR. ZYZAK: Exactly, yes. I think any source of product out there that
> has free asparagine, if you cook it under conditions where you are
> going to drive off the moisture and heat it up, you are going to get
> acrylamide formation.
>
> DR. MILLER: Certainly the big concern would be legumes as well.
>
> DR. ZYZAK: Yes.
>
> DR. BUSTA: Is this information readily available to anyone who wants
> it now?
>
> DR. ZYZAK: Which information? What I just presented? I think it is
> going to be up on the website so anybody can download it.
>
> DR. BUSTA: How about before this?
>
> DR. ZYZAK: Yes, actually the JIFSAN\--you know, you have heard the
> struggle between people whether you are in academia and there is a
> need to publish\--I think Procter & Gamble is a great company to work
> with. Actually, at the JIFSAN meeting back at the end of October I
> presented the mechanism and I told people we identified these
> intermediates and I informed people we used the enzyme to confirm
> that. At AIOC we showed mechanism formation, which was in late
> September. So, once we felt confident and we knew this was the
> mechanism we have been trying to be forthcoming to the industry and
> the academic people, releasing the information.
>
> DR. MILLER: Terry, do you have a comment you want to make?
>
> DR. TROXELL: Thank you. If you look at the spectrum of foods in which
> we find acrylamide, you are tracking foods that contain enough
> asparagine and glucose to form acrylamide so we are talking about
> wheat products, corn products. So, it is not just tubers and so on.
>
> DR. MILLER: No, no, that is the point I am trying to make.
>
> DR. TROXELL: Exactly. Might I ask a question of the speaker?
>
> DR. MILLER: Not a good idea! Johanna?
>
> DR. DWYER: I was just wondering, I think I followed your chemistry but
> I wasn\'t sure about instant mashed potatoes. Would those be high
> because of the extrusion product?
>
> DR. ZYZAK: You may have small levels.
>
> DR. DWYER: I am talking about the instantizing process.
>
> DR. ZYZAK: Yes. In industry I think most of the mashed potatoes you
> buy from the shelf actually have sulfites in there but there is still
> a level of acrylamide. We are even monitoring our flakes, our starting
> material, and during the flaking process of potatoes they go through
> cooking and they are mashed and they undergo a spray-drying process
> and, yes, there is actually a small amount of acrylamide. I think it
> is probably around 100 ppb but you also have some precursors there too
> which are formed, such as the Schiff base. So, during the formation of
> these dried potato products you do have a small amount of acrylamide
> and probably some precursors.
>
> DR. MILLER: Depends on how they are cooked again.
>
> DR. ZYZAK: Yes.
>
> DR. MILLER: Other questions or comments? Thank you.
>
> DR. ZYZAK: Thank you.
>
> DR. MILLER: Our next speaker is Dr. Robert Brown, substituting for Dr.
> Steve Saunders, from Frito Lay.
>
> **Reduction Strategies**
>
> DR. BROWN: I feel very fortunate to be here today. There have been
> some very good presentations this morning and it has been nice. I
> don\'t have a handout. We will have to print one off. Anyhow, I feel
> fortunate to have heard the particulars this morning. There is some
> interesting science going on and it is amazing how far we have moved
> forward in a short period of time on this. FDA and other groups have
> really moved forward quickly.
>
> \[Slide\]
>
> I also feel very fortunate to be standing in here for Dr. Steve
> Saunders because Steve is not only my mentor and my colleague but he
> is also a good friend of mine. Unfortunately, Steve was not able to be
> here today due to unforeseen circumstances and I know that he wishes
> he could be here to be making this presentation today, and he wanted
> me to convey to all of you his sincerest regret for not being able to
> be here in person to make this presentation. I am a nutritionist and I
> was attending a nutrition meeting in town, and he asked me if I could
> step in and present this information for him. I am sure I can\'t
> really substitute for Steve but I am going to give it a shot. That
> said, I want to present the slides that Steve sent to me and the notes
> that he provided for me.
>
> \[Slide\]
>
> That was an excellent presentation from Procter & Gamble today, very
> compelling information on the formation of the acrylamide passing
> through a Maillard reaction product and that is what we have here so I
> can just skip past this.
>
> \[Slide\]
>
> If we look at the first intermediate of the reaction between
> asparagine and glucose or reducing sugar, we see this intermediate.
> When the typical Maillard reaction product is formed that typically
> has an energy activation level of about 25050 kilo calories per mole.
> These products that are formed are the typical browning colors and the
> flavor compounds that are formed in the typical Maillard reaction
> product.
>
> As David mentioned, there is a second pathway, a minor pathway
> proceeding through the Schiff base and going to decarboxylation and
> beta elimination and proceeding to acrylamide. We have done some work
> in our laboratory on a model system similar to what P&G has done, and
> we have estimated that the energy of activation of acrylamide
> formation is on the order of 70 kilo calories per mole. So, you see,
> it takes more heat energy to form this compound and it is more of a
> minor pathway as compared to that going to the Maillard reaction
> products.
>
> \[Slide\]
>
> Clearly, our first insight then is that in a chemical pathway leading
> to acrylamide is a low yield pathway with a higher activation energy.
> This will be demonstrated in a couple of slides I have coming up to
> show a difference in concentration between reactants and the products
> in this reaction.
>
> \[Slide\]
>
> If we look at a summary of the data on acrylamide values in food, you
> are all very familiar with this data but I want to make a couple of
> points about the different concentrations of acrylamide in foods.
> First of all, if you look at the foods on this table you will see that
> there is a wide variety of foods that contain acrylamide, and across
> these different foods there is a huge range of concentrations of
> acrylamide found in these food products.
>
> Additionally, even across and within a category of food there is a
> very, very wide range of acrylamide formation. You will see in some
> foods that the range of the acrylamide can be as much as two orders of
> magnitude. So that is quite a bit.
>
> The third thing is that undoubtedly we are going to uncover more food
> products that are going to contain acrylamide, and I think the data
> that was presented by Procter & Gamble makes it clear that we probably
> can find those foods quickly by determining the concentration of
> asparagine in those foods and looking at potentially the cooking
> process, and then looking at the level of reducing sugar in those
> foods.
>
> \[Slide\]
>
> This slide looks at those different foods that were on that list. We
> did a food consumption survey and we looked at this information to
> look at all foods that contain acrylamide to get an idea of what the
> impact on the American diet would be. If we look at this list, this
> list shows that approximately 38 percent of total calories consumed in
> the American diet are foods that contain acrylamide. As you look down
> the list you see that, of course, many of the nutrients at about that
> same level also are coming from foods which contain acrylamide. The
> variation in micronutrients is dependent on the type of food, some of
> which is fortified. For instance, many of the bread products are
> fortified with iron, and such. So, that level of iron would be higher
> than you would expect as a percentage of the calories.
>
> \[Slide\]
>
> Clearly, this insight tells us that the acrylamide question really is
> affecting a large fraction of the American food supply and is
> something that we have to be concerned to understand.
>
> \[Slide\]
>
> When we began our thinking at Frito Lay about means to look at the
> issue of acrylamide formation and how one might begin the
> investigation of reduction of acrylamide in food we organized our
> thinking in these three areas: One could look at removing the
> reactants, either the glucose or the asparagine; one could look at
> disrupting the reaction to form acrylamide by a number of means; or
> one could look at removal of acrylamide once it is formed in food
> products.
>
> Finally, at the end of the talk today I just want to talk a little bit
> about the significance of the study of exposure to acrylamide, which
> should be an interesting endeavor that we are looking forward to.
>
> \[Slide\]
>
> If we start with looking at ways to reduce the reactants, first of
> all, this data is coming out of Dr. Mottram\'s lab at the University
> of Reading in the U.K., and if we look at this, this was mentioned
> just a few minutes ago, the difference in acrylamide that is
> potentially found in different potato products and, in this case, what
> we see is that if we look at the baking potatoes as compared to the
> King Edwards potatoes we will notice that in the raw state we find
> essentially no acrylamide. In both potatoes, when they are boiled, in
> other words the heat stays below 100 degrees C, we see no formation of
> acrylamide. But once the products are fried we see the formation of
> acrylamide. In this case we see a ten-fold difference in the formation
> of acrylamide between the typical baking potatoes and the King Edwards
> potatoes.
>
> Clearly, what we would anticipate from the information we just saw
> from Procter & Gamble is that the level of reactants is probably
> different between these two, whether it be more reducing sugars in
> King Edward\'s potatoes, though those were not measured in this
> particular study, but that would be a good assumption that we might
> make.
>
> DR. MILLER: Just a matter of clarifying something, which is the potato
> generally used to make chips?
>
> DR. BROWN: There is a variety of potatoes used in all industries.
> Frito Lay uses a proprietary potato and I am not sure of the exact
> variety name of that one. We call it the Frito Lay variety and it is
> called a chipping potato, and they tend to be lower in reducing sugars
> but, as was mentioned, as potatoes are stored over time the levels of
> reducing sugars rise.
>
> \[Slide\]
>
> In this slide if we look at the asparagine in various crops, as was
> mentioned just a couple of minutes ago, in fact the level of
> asparagine does vary quite significantly across crops. As you look at
> this, there are many crops and food products that have quite a high
> level of asparagine. In this case, you see in some of the products
> that there is a huge range of asparagine level in potatoes, looking at
> 0.5 to 10 milligrams/gram. In other products such as asparagus there
> is a huge range in the level of asparagine. Then, if you look at wheat
> the range can be 100-fold difference between low level wheat and high
> level wheat. So, what we have is a system that has a lot of
> complexities in it and you can\'t look at one solution that is going
> to work for everything.
>
> Just to mention too, this table was compiled by the Food Research
> Institute and they have a list that goes quite a bit longer than this,
> but just to know that we have a very complex problem involving the
> entire food supply.
>
> \[Slide\]
>
> In our laboratory we did a model system and we looked at the substrate
> concentration and applied the reactants, both glucose and asparagine,
> and looked at the change in concentration with the formation of
> acrylamide. What we see here is a second order reaction where the
> maximum level of acrylamide is formed when both the substrates are in
> fairly equal concentration. Whenever one of the substrates is at a
> reduced concentration you see that there is quite a large reduction in
> the level of acrylamide formation. This is an interesting insight for
> us because at this point we were able to understand that you need to
> look at your specific food product and make the determination which is
> the reactant that is highest in that food product and that is the one
> that is probably going to be the one reactant that you want to go
> after in that particular food product. The equation on the bottom of
> the slide describes the fit of the surface plot.
>
> \[Slide\]
>
> Then, the insight is that the reaction is a second order reaction and
> that the concentrations of the two reactants need to be in fairly
> equal concentrations to get maximum acrylamide formation. The reaction
> becomes very limiting for that reactant that is at lower
> concentration. So, in the case of the example that Procter & Gamble
> brought up when the reducing sugars are at much lower concentration
> than the asparagine in potatoes the reducing sugar is, in fact, the
> rate limiting step on the formation of acrylamide.
>
> \[Slide\]
>
> The next area that we want to look at is what is the possibility of
> disrupting the reaction of acrylamide formation. If we go back to the
> data from the U.K. and we look at the bottom there and we look at what
> is the effect of cooking on acrylamide formation, if we look at the
> data on the bottom there, you look at frozen frying potatoes, French
> fries, and you look at a cooked product having approximately 3500 ppb
> acrylamide, then overcooking the product increases the level of
> acrylamide by four-fold. This demonstrates very clearly the
> time-temperature relationship to the formation of acrylamide and is
> something that gives us insight into some of the things that we may
> begin looking at to reduce the formation of acrylamide.
>
> \[Slide\]
>
> In our laboratory we wanted to validate our model system and this is
> similar to data that was done by Mottram et al., in their lab in
> Reading. In Reading, they showed that there was an inflection point of
> formation of acrylamide at 120 degrees C.
>
> I would be interested in the data that was presented on microwave
> cooking, looking at the moisture level of the products that were done
> in the microwave, what the real temperature was at the surface of the
> product in formation of acrylamide and looking at the browning
> reaction. If there was a browning reaction that was actually taking
> place in the microwave, I think that the surface temperature may have
> been higher than 100 degrees, but that may not be the case. I don\'t
> know if they had measurements of the surface temperature but that
> would be interesting to know.
>
> But in our model system in the laboratory, if we look at this in the
> model system we have no formation of acrylamide at temperatures under
> 110 degrees C. The reaction really starts going as you go above 120
> degrees C and then is exponential in the increase in rate of
> acrylamide formation. I think this data is important because, as Terry
> mentioned earlier today, the study to look at surface temperatures and
> being able to probe, temperature at the surface is going to be very
> critical. And, the information from Procter & Gamble regarding
> moisture content\--in most food systems, as you know, if the moisture
> content is high you cannot drive temperatures beyond 100 degrees C. As
> you drive off the moisture, that is when your surface temperatures can
> actually reach above 100 degrees C and that is where you will see the
> formation of acrylamide beginning.
>
> \[Slide\]
>
> We plotted a kinetic model of the formation of acrylamide over
> temperature in Kelvin here, and if you look at the formation of
> acrylamide in this kinetic plot what you see is that the inflection
> point appears to be around 120 degrees C. From that point the rate of
> acrylamide formation is very rapid. The rate on the bottom of the
> chart, on the right-hand side there, is approximately 175 degrees C
> and that is the typical baking temperature for most food products. As
> you can see by that temperature, at 175 degrees that is well above the
> temperature to drive the formation of acrylamide to a maximal rate.
>
> \[Slide\]
>
> The insight, therefore, is that acrylamide formation is extremely
> temperature dependent and occurs well below temperatures needed in
> typical baking or frying operations. It is probably not possible to
> cook products without any formation of acrylamide. The surface
> temperature studies that will be undertaken I think will be very
> interesting, looking at whether one could modify the final surface
> temperature of products by alternate cooking methods.
>
> \[Slide\]
>
> We have also investigated the pH dependency of acrylamide formation in
> foods and we found that at pH under five acrylamide formation is
> severely inhibited. Even at the pH of six there is some significant
> inhibition of acrylamide formation and as you get towards neutral pH
> you see that the acrylamide formation is maximized at a pH of around
> seven.
>
> \[Slide\]
>
> A very interesting idea in the whole disruption of acrylamide
> formation is can you come up with an inhibitor of acrylamide formation
> similar to the case of vitamin C added to reaction to inhibit the
> formation of nitrosamines. It is a very exciting idea to think that we
> could come up with something like that, but in this case the whole
> Maillard reaction process is one of amino acids forming the color and
> flavor compounds that we expect in our cooked foods and in this case
> we are trying to inhibit a single amino acid reaction rather than the
> whole cascade of free amino acids that would react.
>
> However, one other point on that is that the inhibitor would have to
> be food safe and it would have to be approved for use in foods. I
> think we are all hoping that a similar simple story can be developed,
> and we have seen published reports and we have heard from Procter &
> Gamble today about some of the potential ingredients that could be
> added to foods to inhibit this reaction. Rosemary flavonoids have been
> reported to inhibit the reaction of asparagine going to acrylamide but
> we haven\'t seen data on that.
>
> There was, of course, the interesting JIFSAN conference that we just
> heard about, and the use of the amino acid cysteine to inhibit this
> reaction. It would be interesting to look at what levels of cysteine
> could inhibit the formation of acrylamide and how could it be added to
> foods. Obviously the wide number of foods we have would require
> different mechanisms to incorporate cysteine into a surface of a
> product that would try to inhibit this reaction because there would be
> no need to have the inhibitor throughout a product if, in fact, the
> formation of the acrylamide was occurring only on the surface of the
> product where the browning is taking place.
>
> In our model system we have also studied other inhibitors that might
> be functional in this area, and we have looked at divalent and
> trivalent cations and found that they also inhibit the formation of
> acrylamide. However, it takes a large amount of the divalent or
> trivalent cation to have this inhibition come into place. It takes
> approximately one equivalent of cation per mole of reactant to inhibit
> the acrylamide formation. We are not sure how these could be
> practically applied to foods. It may not be successful in all types of
> food because, again, of trying to get the cation in the area to
> inhibit the reaction.
>
> \[Slide\]
>
> If we turn to the kinetic model of Wedzicha and Mottram we have a
> pretty interesting array of areas that we might look at to try to
> develop a mechanism to inhibit the formation of acrylamide. As we see
> here, if glucose is in fact in a particular reaction system, the rate
> limiting step, there would be means to force the reaction of glucose,
> to deplete the glucose by other reactants that might react with
> glucose such as other free amino acids that would compete against
> asparagine for the formation of this reaction. Also, pH, temperature
> and time variables are other potential things that we might look at.
> If we can look in the cooking process at some way to control the final
> surface temperature of the product as another means of looking to
> inhibit this rate of formation. As we learn more about these reactions
> and these kinetic constants out of the lab, we hope to learn much more
> where the best place to attack this issue will be.
>
> \[Slide\]
>
> Finally, I want to talk just a couple of minutes about our attempts to
> actually remove acrylamide after formation in food products. In this
> case we have tried a couple of things. The supercritical CO-2 is very
> effective in removing acrylamide. Of course, it removes everything and
> completely destroys the product.
>
> \[Laughter\]
>
> UV light is something that we had high hopes for to be an effective
> use, a fairly simple technique to cause the acrylamide to polymerize
> and effectively eliminate the hazard. In this case we take ground
> product. We expose it to all levels of light. I think we came up with
> this, remembering back when we used to make polyacrylamide gels and we
> thought this was something that, you know, was really going to work.
> We exposed to product to wavelengths of light ranging from UV to red
> and essentially we have found no effect at any wavelength that we have
> tried to date. Potentially the level of acrylamide is too low in food
> products for this to be effective.
>
> \[Slide\]
>
> Finally, I am going to talk just a little bit about the toxicology of
> acrylamide and its presence in the food supply.
>
> \[Slide\]
>
> On this first slide is data that was also developed for us looking at
> the food consumption survey and mirroring that to the data on
> acrylamide content of foods consumed in the American diet. If we look
> at the total here and then if we look at the red line on the bottom,
> which is essentially drawn on the X axis, that red line on the X axis
> would be the standard risk assessment line that would be developed
> under standard techniques of developing risk assessment for average
> daily exposure. As you look at that line you can see that it is orders
> of magnitude, at least two orders of magnitude lower than the exposure
> of acrylamide in the U.S. food supply. You can see that no one food is
> going to be the bulk of that. Assuming that we can eliminate the
> content of all potato products, fried potatoes, other kind of cooked
> potatoes, mashed potatoes and potato chips and we remove the
> acrylamide from all those products, we are still in the range of two
> orders of magnitude too high with the total acrylamide concentration
> in the diet using standard risk assessment techniques to form this
> line.
>
> This is where I think the research on toxicology, looking at the low
> concentration versus high concentration p450, 2E1 metabolism of
> acrylamide is going to be very important to understand the difference
> in low and high concentration of acrylamide in the diet.
>
> \[Slide\]
>
> Steve gave me some food for thought here. Everybody is probably not
> that hungry right now but I didn\'t have any lunch so I still have
> some room for food. The whole idea of carcinogen in food is not a new
> one to us, and is something we have dealt with before if we look at
> cooked meats and such. We also have the NAS report and the Ames/Gold
> as other references to talk about that.
>
> Also, as we evaluate acrylamide we have to understand that humans have
> been cooking foods for millennia and we have been exposed to
> acrylamide for all those years. So, we need to understand what is
> going on and what is happening or not happening with acrylamide
> exposure low dose versus high dose. We need to understand the
> toxicology there because it is going to be very important to us.
>
> As Terry said this morning, there are going to be no quick fixes of
> this issue because acrylamide is going to be so widespread in the
> entire food supply. We are going to have to look at different bullets.
> Instead of one magic bullet for all foods, there are going to have to
> be different bullets for different food products as we move forward to
> look for means to address this issue.
>
> \[Slide\]
>
> As we begin doing feasibility analyses of what kind of intervention we
> can have in the food supply and what we are going to do, we really
> need to understand the whole kinetics and the removal of substrates
> from food products. We need to understand each individual food,
> determining whether in this case the asparagine or the reducing sugar
> would be the rate limiting step for the formation of acrylamide.
>
> Also, if we look at low temperature intervention what would be
> required in the development of new cooking techniques that could
> potentially reduce the level of acrylamide in foods. For some foods it
> will be impossible to develop these low temperature techniques. In
> other foods it may be possible to use two-step cooking where you heat
> at higher temperatures before moisture levels are driven down and then
> lower temperatures as the food product is drying out and temperatures
> on the surface can increase.
>
> Again, there are no magic bullets here. We have to look at individual
> potential solutions to each of the problems, and there is absolutely
> no precedent to the kind of ordered magnitude of intervention we have
> to have in the food supply from the processing to the cooking to the
> growing of foods in our food system. It is dependent on what we find
> in the toxicology and how far we have to drive the acrylamide level
> down.
>
> \[Slide\]
>
> Then some final thoughts here, the issue affects a large portion of
> the food supply. Lowering acrylamide clearly in one food is not going
> to do much to lower the overall level of acrylamide exposure in the
> population. We need to understand the toxicology here so that we know
> what our target is in this case.
>
> Clearly, as was mentioned this morning, foods cooked at home and foods
> cooked at restaurants are going to be a significant source of
> acrylamide so foods that are processed by the manufacturer are taken
> into the home and then baked out, for instance as apple pie. Those
> foods are going to have a large potential exposure to acrylamide so we
> need to look at methods that would also address those.
>
> If we look at what does the future look like, given the magnitude of
> the change in the food supply that could be represented by this, we
> need to really understand the nature of the low dose hazard of
> acrylamide to humans, and we need to really look at the impact of any
> proposed interventions and the consequences, if there are any
> unintended consequences to the public health. In going forward, as we
> begin to study the toxicology of acrylamide, we need to be
> simultaneously looking for all the interventions that are possible to
> be driving the acrylamide level down so that we are working on both
> ends of the spectrum, to both lower acrylamide concentration in the
> food supply and to understand the real significance and the real
> health effects of acrylamide levels in the food supply and on human
> health. Thanks very much.
>
> **Questions of Clarification**
>
> DR. MILLER: Thank you. Comments or questions?
>
> DR. DWYER: Thanks, Bob, for a very, very interesting talk. I just have
> a question that is sort of silly but I was wondering does free
> asparagine have a taste in food so if you took it out it would taste
> different?
>
> DR. BROWN: I don\'t know. I am not a flavor chemist so I am really not
> too sure. Thanks very much.
>
> DR. MILLER: Thank you. We are ready to take a break. We are a little
> early again. I will tell you what, why don\'t we go ahead with the
> exposure assessment, Dr. Robie? We will go ahead and have that and
> then we will take a break after she finishes.
>
> **Exposure Assessment**
>
> DR. ROBIE: I am going to be referring to the handout you have in your
> package. I am going to be presenting the exposure assessment for
> acrylamide for FDA as prepared by Dr. Michael DiNovi and myself.
>
> \[Slide\]
>
> I am going to start out the presentation by going through some
> history, then I am going to move right into our exposure estimates and
> the model that we used, the assumptions that we made and the future
> work on it, and then the results and the interpretation.
>
> \[Slide\]
>
> You have already seen this a bunch of times. Less than a year ago a
> group of Swedish scientists reported the occurrence of acrylamide in
> food, and in their report they included a preliminary exposure
> estimate. This was based on acrylamide data in about 100 food samples.
> They split that into eight food categories. They may some assumptions
> about the foods for which they didn\'t have acrylamide data, and they
> calculated a preliminary exposure estimate of about 0.7 mcg/kg body
> weight per day for a 60 kg individual.
>
> \[Slide\]
>
> Then, about two months later, two months after the Swedish scientists
> publicized their findings, there was an FAO/WHO consultation held to
> discuss the issue of acrylamide exposure through the consumption of
> food. They also performed some exposure estimates. They used the same
> residue data as the Swedish scientists used in conjunction with some
> food consumption data from several national food consumption
> databases. They used a couple of different approaches to exposure
> estimates but the bottom line they reported was a range of about 0.3
> to about 0.8 mcg/kg body weight per day, which is in agreement with
> the Swedish scientists.
>
> \[Slide\]
>
> That is all the history I am going to bore you with. Now I am going to
> go on to how we approached calculating the exposure to acrylamide.
> Really we approached it in the same way that we would approach the
> exposure to any additive or contaminant or naturally occurring
> substance in food, following this general equation.
>
> Basically, we need information on the concentration of the substance
> that we are interested in, in the food. Then, we need information on
> the food, and we need to know how frequently it is consumed, and we
> need to know how much is consumed and when it is consumed. We get the
> frequency and portion size from the food consumption databases and the
> concentration data for acrylamide we get from the laboratory. This
> expression, this multiplication takes place for every food, each
> individual food, the concentration of a food, times the intake of the
> food. This is summed over all of the foods that would contain the
> substance to get estimated daily intake of the substance for an
> individual. Then, this information is summed over individuals to get
> an EDI for the population. Again, this is the way we do it for every
> food additive, everything in food. So, there is nothing different
> there.
>
> \[Slide\]
>
> Food consumption surveys\--I mentioned that we are going to be getting
> a lot of our data from them. We use three food consumption surveys.
> Let me explain them, the differences between them and why we use
> three.
>
> The first two that we use are CSFII surveys. We use a three-day
> consumption survey and a two-day, and each has about 20,000
> participants. We just wanted to see the comparison between these two.
> This is going to help us evaluate the robustness of our model. Of
> course, in making this comparison we have to have knowledge as to how
> the number of days of the surveys is going to affect the outcome, the
> final answer that we are going to get. So, that is what I have in this
> last bullet. I was just going to leave this up here and then it was
> explained to me last week that this might not be intuitive so I am
> going to spend a couple of minutes explaining what this means.
>
> The percent eaters, the percentage eaters value that we get from our
> food consumption survey is the percentage of eaters that report eating
> or consuming food on any one day of the survey. So, the longer the
> survey, the better chance we have to get higher percentages. We
> capture more of the eaters the longer the survey duration is.
>
> The intake for the eaters is considered overestimated for a shorter
> survey because the intake for the eaters as reported from the survey
> is reported as an average over the days of the survey. So, the more
> days of the survey, the lower the intake for the eaters, the mean
> intake for the eaters.
>
> \[Slide\]
>
> We also use an MRCA 14-day survey. Again, we had the data; we wanted
> to make the comparison and test the robustness of our model. After
> saying what I just said about how the difference in the number of days
> is going to affect what we expect to see from the survey, I need to
> point out that this is a 14-day survey but it is 14 days of reported
> frequency. So, the percent eaters is going to be higher. The
> participants in the survey, every time that they consume the food,
> they report it as a consumption occasion. So, we are going to get
> percent eaters but they don\'t report the amount of food. The amount
> of food we get from the USDA Nationwide Food Consumption Survey so the
> frequency data from MRCA is linked to this food consumption survey and
> this is a three-day survey.
>
> So, I am going to talk about the differences between the CSFII surveys
> and the MRCA surveys because, again, we are going to be comparing them
> and we have to have knowledge as to what the differences are between
> them to make valid comparisons. There is a lot of difference in the
> food groupings between the two surveys. The CSFII food coding system
> allows us to be a lot more specific about the foods that we choose.
> The MRCA survey has big, broad categories. For example, for the CSFII
> survey we were able to separate crisp breads from crackers and other
> salty snacks, whereas for MRCA we weren\'t able to do that and they
> were all grouped together. When we get to the data and you see the
> tables you will see how the categories are changed slightly.
>
> Another difference in the surveys is the time periods over which they
> were carried out. For the MRCA we are talking about early to
> mid-\'80s, and the two-day CSFII survey was carried in the mid to late
> \'90s. Certainly, food consumption patterns and habits have changed
> over that time. Whether or not it is significant enough to affect the
> model is debatable but it is a difference that merits mention.
>
> \[Slide\]
>
> I am not going to spend a lot of time talking about this because our
> office is not actually doing this. This is work that is being
> performed by Dr. Clark Kerrington with the Office of Plants, Dairy
> Foods and Beverages Risk Assessment Division. He is looking at taking
> the two-day CSFII data, our most recent data, and expanding it to
> longer than two days. We really want to model product exposure and
> using a two-day, three-day or even a 14-day survey may not be that
> appropriate. I mean, it is data that we have so that is what we are
> using right now but Clark is going to be working on adjusting this
> two-day CSFII survey data and that is work that is in progress right
> now.
>
> \[Slide\]
>
> So, we are back to our exposure equation. This is basically the same
> equation that we had a few slides ago. The food frequency and portion
> size are combined here from the information that we get from the food
> consumption survey. Again, we multiply that by the concentration of
> the substance that is in each individual food. This is summed over
> food and summed over individuals. This is typically done at the mean
> so we arrive at a point estimate. The result we get is one point. This
> approach is useful for substances only in a few foods or when the EDI
> and the ADI to TDI in this case are very different from each other. In
> the Frito Lay presentation we saw that the ADI and TDI are not that
> different from each other so these two things don\'t really fit for
> acrylamide. Acrylamide is in a lot of foods so we decided to use
> probabilistic modeling.
>
> So, instead of just using the means and getting point estimates we are
> able to use the entire distributions for the food consumption and also
> for concentration data. I am going to talk about this in a lot more
> detail in the next few slides.
>
> \[Slide\]
>
> Probabilistic modeling is an iterative process and for each iteration,
> as I said, we use the whole food consumption distribution so each
> iteration is a random sampling of the food consumption distribution
> for an individual food. In a similar way, the computer will randomly
> sample the distribution for the acrylamide level that we have so we
> are not just going to be using an average acrylamide value that we
> have for each food. We are going to actually use distribution and then
> we are going to again apply the percentage of eaters to the result we
> get from multiplying the food consumption to the acrylamide level.
>
> \[Slide\]
>
> I mentioned random sampling. I am going to show an illustration of
> what that is. This is known as Monte Carlo sampling. To be strictly
> correct, Dr. DiNovi and I used a variation of Monte Carlo sampling
> called Latin hypercube sampling. But for purposes of what we are
> talking about today, which is illustrating how the food consumption
> distribution is sampled, this sampling is certainly appropriate.
>
> What we have is the cumulative probability distribution for a given
> food from zero to 100 percent. So, from zero to one the computer
> generates a random number for each iteration. It generates a random
> number from zero to one. So, in this case, this is where the computer
> is generating a random number for this iteration. This random number
> is used to sample the food consumption distribution. This is the
> generated value for food consumption that we got for this iteration.
> The acrylamide concentration distribution is sampled in a similar way.
> Again, those two numbers for each iteration are multiplied together to
> arrive at the acrylamide intake that we will get from consuming that
> much of this particular food.
>
> \[Slide\]
>
> For this expression I am going to be using AA for acrylamide
> throughout this presentation. I believe this is probably the first
> time you have seen this. This is just the same exposure equation that
> we have seen, just reworked a little bit. The food amount, the
> information that we get from the food consumption surveys is here. We
> are going to view this as one iteration. This is what is happening for
> each iteration for each food. The acrylamide level that is randomly
> sampled from the acrylamide concentration distribution is put there.
> Then, these two things are multiplied together to give us the amount
> of acrylamide that this iteration or virtual consumer will be exposed
> to by eating this amount of the food containing this amount of
> acrylamide.
>
> Now, I have been saying that we are applying the percent eaters to
> this, and the way that we do that is also in each iteration and either
> a zero or a one is multiplied by the amount of acrylamide that we have
> decided we get from eating that food in this expression, here. One is
> in proportion to the percent eaters. Let\'s say a certain food is
> reported as having 80 percent eaters, for 80 out of 100 iterations
> this number is going to be one, and this food will be considered to be
> eaten by this iteration, this virtual consumer. The other 20 times
> this number will be zero and there will be no contribution to the
> total acrylamide for this iteration or this consumer from this
> particular food.
>
> Again, this expression is for a food for an iteration. The values are
> summed over all the foods and then we have combined all of the
> iterations to arrive at distributions for acrylamide intake, which I
> am going to be showing later on in the presentation.
>
> \[Slide\]
>
> This is what we will refer to as the first page of the handout,
> virtual consumer number one. I just want to illustrate the point that
> we showed on the last slide a little bit better by actually showing
> you the output that we see on the computer screen when we run an
> iteration.
>
> I just have the first seven rows reproduced up here. You have the
> whole table. The food consumption of the eater is the result that we
> get from sampling the food consumption distribution for this iteration
> for each individual food and then the acrylamide concentration, again,
> is sampled from the acrylamide level distribution for this particular
> food. These are multiplied together and, again, they are multiplied by
> either zero or one depending on whether or not this food is considered
> to be eaten by this consumer for this iteration. So, we either have a
> zero here or we have a number for acrylamide contribution to this
> particular eater. These are all summed then to arrive at the value of
> 0.49 for this particular eater or this particular iteration.
>
> We have provided you a couple of additional virtual consumers in your
> packet. I think there are three more virtual consumers just to give
> you an idea and make sure everybody understands the point of what we
> are doing here and the results that we are getting.
>
> \[Slide\]
>
> We did 25,000 iterations. I am going to explain some of the
> assumptions that we made in the model. The first one is that there has
> been no accounting for correlations between food choices, either
> positive or negative. Examples of that could be if you look at virtual
> consumer number four, virtual consumer number four is shown as
> consuming both oven browned and restaurant French fries on the same
> day. All of this is entirely possible as an example of a negative
> correlation. A positive correlation could possibly be between peanut
> butter and bread or coffee and toast. These are things that we
> haven\'t included in the model. It is not that it can\'t be done; it
> can be done but historical knowledge has shown us that really it
> won\'t make that much difference and won\'t have that much of an
> effect on the bottom line result that we are going to get from running
> the model.
>
> Another assumption that we made, or what we wanted to make you aware
> of at least, is that we have used the food consumption distributions
> only from zero to the 99th percentile. The 100th percentile we saw
> what we considered to be some pretty irrationally high values. I know
> that is a strong word but 13 liters of coffee for one person in one
> day not only seems like a lot of coffee but it is also 11 liters
> higher than what is reported at the 99th percentile. So, we didn\'t
> want these numbers, which we considered irrationally high and possibly
> reporting errors or calculation errors of some kind, to interfere with
> our model. Another example of that is cookies, 620 grams of coolies
> per day was reported at the 100th percentile by the CSFII two-day
> survey and the recommended portion of cookies is 30 grams, and also at
> the 99th percentile this value of cookies was 130 grams. So, you know,
> it is a five times difference between the 99th and 100th percentile.
> So, we just went ahead and took off the 100th percentiles and used
> from zero to 99.
>
> DR. BUSTA: Why did you use zero?
>
> DR. ROBIE: It is possible that people consumed zero. I see what you
> are saying, to just cut off both extremes of the distribution. There
> is no zero percentile? Good point.
>
> \[Slide\]
>
> Some of the limitations that we see in the model include the surveys
> and the laboratory data which are two inputs for getting information
> from the surveys about the food consumption. We have already talked
> about these. We have talked about the duration of the surveys. Two or
> three days, even 14 days to model product intake is not an ideal
> situation. And, the food classifications, I have already talked about,
> especially for the MRCA. They have very broad groupings. It would be
> better if we could separate the groups as much as possible.
>
> A primary limitation of the model is laboratory data. You have heard
> people talk about the laboratory data all day and I am going to do it
> again. Some of the samples that we have of food types are represented
> by fewer than five data points from our laboratory and we see that as
> a limitation. Most of these are the samples that are lower consumption
> and don\'t contain a lot of acrylamide. We have tried to focus on the
> ones that we think are going to have a big impact on the total overall
> intake for the population.
>
> Another model limitation that we see is variability in acrylamide
> levels in different foods. Again, you have already heard people
> talking about this all day today. What we do see is that as you go
> down this list we see greater variability. There is a significant
> amount of lot-to-lot variability, even more brand-to-brand
> variability. Different products can be included in the same food
> category. I don\'t want to pick on potato chips here but we have a lot
> of potato chip data so we have definitely seen this. We have seen
> differences in lot-to-lot, brand-to-brand and product-to-product. If
> you have a baked potato chip versus a fried potato chip, they both
> have acrylamide. The differences between the two are pretty drastic.
>
> Then, there is always the problem with the foods prepared at home and,
> again, you have heard about this. We do have data on toast and we have
> data on oven browned French fries but these are samples that were
> prepared in our laboratory following package instructions and I don\'t
> really think we have probably captured the variability that we are
> going to see in people making it at home to their color and taste
> preference.
>
> \[Slide\]
>
> Just one more slide and then we will go to data. We wanted to make you
> aware that we did apply some factors to some of the food types,
> typically the foods that are consumed as liquids. The analytical
> technique that we are using makes it a lot easier to test these as
> solids than as liquids as they are consumed but, of course, they are
> reported in food consumption surveys by the survey participants as
> liquids as they drink them or eat them.
>
> So, for the last three on this list, instant coffee, dry soup and dry
> cocoa powder, the calculating effect was pretty straightforward in
> that we are taking the amount of the dry powder that we are going to
> then add a known amount of water to and dilute and consume the entire
> amount. So, whatever is in the package, you are consuming it all.
>
> For ground coffee it is not so straightforward because when you make
> coffee you don\'t eat the grounds. We know how much acrylamide is in
> ground coffee. What we need to know is how much acrylamide is in
> coffee as consumed so we can compare it with the food consumption data
> that we have. We use a value of 24 for this. This has been
> experimentally derived by Dr. Musser\'s lab. They measured the
> acrylamide in the ground coffee. They made coffee from it and then
> they measured the acrylamide in the coffee as consumed. They are still
> working on fine-tuning this number, if it is 25 or 23, but we are
> right there with the preliminary value that they have given us, I am
> sure.
>
> \[Slide\]
>
> As I said, we used three food consumption surveys and data from those
> and we did it for two populations. So, really you are going to have
> six slides for these. Instead of tabulating all 30 foods for all three
> surveys for both populations, I am going to go ahead and just show two
> slides just to show you all the foods that we are considering, and
> then we are going to go on to the survey-specific information.
> Actually, these are results from the CSFII two-day survey for the two
> years and older population. The data that you have here is the mean
> population for acrylamide intake for this food. This is restaurant
> fried French fries; this is oven browned French fries. These are
> sorted in order of their contribution for acrylamide, the contribution
> that they are going to have on the total acrylamide intake for the
> population. And, we have cumulative percentiles too.
>
> So, restaurant fried French fries will contribute 15 percent
> acrylamide to the total population. Oven browned French fries will
> contribute another 13 percent. So, the cumulative percentile there is
> 27 percent, and so on down the slide to soft bread. We pick it back up
> again at corn snacks and then down to soup mix. Here are the last,
> cocoa to breaded fish and then donuts to multi-drinks, with
> multi-drinks having the least contribution to the total population
> acrylamide intake of 0.37 mcg/kg body weight per day. I know that
> these look like they have no contribution. I decided to only go out to
> three decimal places on the slide but they do contribute, just not
> very much.
>
> \[Slide\]
>
> Now we are going to look at data specific to the populations in food
> surveys. Let me refer you to page six of the handout. The way that
> these are ordered is just from oldest data to most recent data. There
> is no other significance to the order that I will be showing you.
>
> These are the results of the MRCA 14-day survey two years old and
> older population. I have up here the foods contributing five percent
> or more individually to the total acrylamide population of 0.48. These
> are eight foods. You have the rest in your handouts. For each food we
> have the percent eaters, the mean food consumption for the eaters, the
> acrylamide concentration for that food. If we multiply the mean food
> consumption of the eaters by the mean acrylamide concentration for
> each food we get an eaters only acrylamide intake, which we then
> weight using the percent eaters to get a mean population acrylamide
> intake for that food. Then we sum all these to arrive at a value of
> 0.48 mcg/kg body weight per day as the mean acrylamide intake for the
> population using this survey data.
>
> \[Slide\]
>
> This is the same table for the CSFII three-day survey. Again, the
> foods that we have chosen to tabulate on the slide are the ones that
> contribute five percent or more individually to the total mean
> acrylamide intake of the population which, for this survey, is about
> 0.32. It is a little bit less than what we saw for the 14-day survey
> but, again, over 14 days we are going to have a lot greater percent
> eaters. That is why this value is lower here than for the MRCA 14-day
> survey.
>
> The other thing of significance to look at on this slide\--actually, I
> was going to show you the percent eaters difference. Look at the
> percent eaters for potato chips here. It is 18 percent for this survey
> and it is 76 percent for MRCA 14-day survey. So, the difference in the
> percent eaters can be very significant for some foods.
>
> The other thing to note on this slide as compared to the slide for the
> MRCA data is that these top foods that each contribute five percent or
> more are the same for both surveys. The order is changed a little bit
> but the top contributors are the same for both surveys.
>
> \[Slide\]
>
> Then I have the same slide, which is the next page in your handout,
> for the CSFII two-day survey. The bottom line here is 0.37 and we saw
> 0.32 for the three-day survey. It is a little bit higher, which is
> expected, but everything is pretty much in the same ball park. Again,
> the top eight foods are the same. The order may be a little bit
> different but the top contributors are the same no matter which survey
> you look at, which shows us that our model is very robust.
>
> Another thing I just want to point out is that all these values are
> very consistent with each other that we have gotten from these three
> surveys, and also they are consistent with previous exposure estimates
> which I talked about on the first couple of slides of this
> presentation.
>
> \[Slide\]
>
> We are going to go in the same order of surveys for two- to five-year
> old population. That is on the next page of the handout. The first
> things that are probably pretty obvious to you are that we no longer
> have eight foods; it is seven foods because the coffee dropped off.
> Two- to five-year olds aren\'t drinking a lot of coffee, which is
> good. Each of these foods, these tops seven foods, are again
> contributing five percent or more individually to the mean acrylamide
> intake for this population and it is the same seven foods as we saw on
> all the other slides, except for coffee.
>
> Another thing that I am sure you have noticed is the fact that the
> mean acrylamide intake for the population is about twice what it was
> for the two years old and older population. This is expected when we
> are talking about data on a kilogram body weight basis. Children tend
> to eat about half of what adults eat but they weigh about a fourth of
> what adults weigh. So, the factor of two is an expected result for
> what we see here.
>
> \[Slide\]
>
> Then, for the CSFII three-day survey data for the two- to five-year
> old population, again the coffee is gone and the top seven foods are
> the sane. The order is moved around a little bit. Again, the value
> that we have here is lower than we had for the 14-day. Again, we are
> capturing fewer eaters, and it is also about twice what we saw for the
> CSFII three-day survey for the two years old and older population.
>
> \[Slide\]
>
> One more table of data, the CSFII two-day survey, and we see the same
> trends as we saw for the two years old and older population. Again,
> this mean acrylamide intake for the population is a little bit higher
> than we saw for the three-day survey but with a shorter survey that is
> expected. Again, the top seven foods are the same.
>
> \[Slide\]
>
> I have shown you tables of data, lots of tables of data. Now I am
> going to show you a lot of distributions to go along with those tables
> of data. On the data tables that you saw we showed you mean acrylamide
> concentrations and mean food consumption and a mean result but we
> really don\'t get out just a mean; we get distribution of acrylamide
> intakes. This is for the MRCA 14-day survey two years old and older
> population. Acrylamide intake is on the X axis in units of mcg/kg body
> weight per day. The mean, again, is about 4.8 mcg/kg body weight per
> day. This occurs about the 70, 75th percentile and that is the case
> for all of the survey results, all the distributions that I am going
> to show.
>
> DR. MILLER: Just to clarify, we are talking here about population
> means; we are not talking about eaters?
>
> DR. ROBIE: Right, we are talking about population means. I am sorry if
> I misspoke.
>
> Another thing of note, the 99th percentile we have shown here is 0.91
> mcg/kg body weight per day, just about twice the mean for the
> population, which is an expected result.
>
> \[Slide\]
>
> This is the same looking type distribution for the acrylamide intake
> from the CSFII three-day survey data for the two years old and older
> population.
>
> \[Slide\]
>
> For the CSFII two-day survey for the two years old and older
> population.
>
> \[Slide\]
>
> Then we will go on to the two- to five-year old population. One
> difference of note here is that we have expanded the scale to six. We
> cut the scale off at three for the two years old and older population.
> We have already discussed how we expect to see a doubling in the
> population mean intake when we are talking about two- to five-year old
> population when comparing to a two years old and older population on a
> kilogram body weight basis.
>
> \[Slide\]
>
> Here is the acrylamide intake distribution for CSFII three-day survey
> two- to five-year old population.
>
> \[Slide\]
>
> And the two-day survey. So, we haven\'t seen anything unexpected.
> Again, all the surveys agree with each other and the previous exposure
> estimates, and that is all I am going to say about that.
>
> \[Slide\]
>
> The table that you have in your handout to go along with this slide I
> believe is on page five. What we have done here, just to round out the
> whole picture and put things in perspective, is to show you how much
> acrylamide an eater would get from consuming one recommended portion
> of any of these foods. We are applying the mean acrylamide
> concentration. We are multiplying these by recommended portion sizes.
> These are not portion sizes that we got from any survey; these are
> portion sizes from 21 CFR 101. This is the labeling section of CFR,
> the food labeling section. These are the portion sizes that you are
> going to see on labels. These are recommended portion sizes.
>
> What I have tabulated here are the top eight foods that you kept
> seeing consistently in the previous tabulated data. You have the full
> list in front of you, and these are also in alphabetical order.
>
> I guess the thing I want to point out here first of all is that this
> is wrong in the copies of the slides. This is 3.2 and the actual
> number is 2.0. It is correct on page five of your handouts. Something
> else to note is that certain foods that we saw having large
> contributions appreciably contributing to the total acrylamide intake
> for the population, notably breakfast cereal and soft bread, there is
> really not a lot of acrylamide per portion for these foods. These are
> frequently consumed foods and highly consumed foods.
>
> \[Slide\]
>
> We also ran some \"what if\" scenarios. Probabilistic modeling lends
> itself very well to carrying out these types of scenarios. What we are
> talking about here is looking at the effect of a chosen mitigation
> measure on the population acrylamide mean, final result. So, we have
> chosen some foods and food groups and set the acrylamide level of
> these foods and food groups to be zero and we ran the model. I am
> going to show you the results for several food groups.
>
> It is important to note, however, that the foods are still included in
> the model. We are just assuming that the acrylamide can be removed
> from the foods. To remove the food from the model and consider it not
> being in the diet anymore we would have to consider what it would be
> replaced by and the implications behind that. So, that is not anything
> that we did here. We just set the levels to zero in the foods.
>
> \[Slide\]
>
> These are the results for several different food groups for the CSFII
> two-day survey two years old and older population. Remember, the
> population mean result for the acrylamide intake was 0.37. We have
> assumed zero concentration of acrylamide in French fries, both types
> of French fries, both the restaurant fried and oven browned French
> fries, and recalculated the mean to be 0.26 mcg/kg body weight per
> day.
>
> We also ran it assuming that acrylamide would be removed from snack
> foods, and in snack foods we included potato chips, corn snacks,
> popcorn and pretzels. The mean was reduced to 0.31 mcg/kg body weight
> per day.
>
> We did the same thing for breakfast cereal and coffee. The bottom line
> here, and Terry mentioned this in the morning, is that no one food is
> contributing the majority of the acrylamide to the total population of
> acrylamide intake that we are seeing in any of the surveys.
>
> \[Slide\]
>
> For our future work I have already mentioned the modeling of
> longer-term food consumption that Clark Kerrington is working on for
> us to more accurately model chronic intake. We will continue to run
> \"what if\" scenarios based on the technological capabilities in
> industry. Also, we are going to analyze the sensitivity analysis of
> our model. Sensitivity analysis will allow us to determine the
> sensitive inputs for the model which, in turn, allows us to identify
> important uncertainties and that is going to help direct our future
> efforts in our exposure estimate.
>
> \[Slide\]
>
> In summary, we have seen that the mean population acrylamide intakes
> that we got from three survey are consistent with previous exposure
> estimates. The greatest contributors to the mean population acrylamide
> intake, the top eight or the top seven for the children, is the same
> for all surveys. We see that some of the foods that have lower
> acrylamide levels do contribute appreciably to the overall mean
> population but, again, this is because they are commonly consumed
> foods. On the \"what if\" slide I just showed no one food or food
> group accounts for the majority of the mean population acrylamide
> intake.
>
> Thank you very much for your attention.
>
> **Questions of Clarification**
>
> DR. MILLER: Thank you, Donna. Comments or questions? Yes?
>
> DR. BUSTA: If you ran the \"what if\" scenario on the two- to
> five-year olds that had a mean of 1.0, knowing that the coffee
> wouldn\'t be there, would it be a similar kind of reduction or would
> it be more dramatic? These range somewhere between 35 percent
> reduction and less than that.
>
> DR. ROBIE: Let me find the data.
>
> DR. BUSTA: I can look for it. Down to 0.84, so go down from 1.0 to
> 0.84. You are talking about removing French fries?
>
> DR. BUSTA: Breakfast cereal.
>
> DR. ROBIE: Breakfast cereal.
>
> DR. MILLER: Johanna?
>
> DR. DWYER: Thanks for a very good presentation. I am sort of hung up
> on one thing, and that is this business of using food consumption
> surveys of different lengths of time, two, three 14 days, and the
> issue of a person reporting they didn\'t eat it because they didn\'t
> eat it on that day when what you are really trying to model is chronic
> consumption. It could be that they do eat it but they don\'t eat it
> the day that you observe them or they report on.
>
> It is my understanding that the group at Iowa State has been doing
> some statistics, as they always do, and have developed a method that
> may help to adjust distributions in that respect. Now, I understand
> that, particularly when you get up to the 90 percentiles, they can be
> up to the 95th percentiles. It is also my understanding that a group
> at the National Cancer Institute, Dr. Dowd and his colleagues, are
> working on something called propensity to consume that basically is
> just\--I hope he is not here\--a guess as to how often you consume
> something that you are telling the observer, and that has now been
> approved for use in NHANES. Could you tell me in your modeling if you
> are taking that into account, adjusting in any way for the artifact
> because of those issues?
>
> DR. ROBIE: The longer-term food consumption modeling that we are
> talking about that another scientist in our office is working on, I
> believe that is about the same as the IOC. I don\'t understand the
> intricacies and I didn\'t talk about it too much about it today, but
> this is something that has been done before and been published. I
> believe this is along the same lines as what you are talking about.
> This is just taking a little bit more time. We went ahead and did what
> we could with the two- and three-day surveys to see what results we
> could get. Of course, we are not stopping there. We are considering
> this preliminary. But I believe that what we are doing with these data
> here, these two-day data to expand over 365-day period is similar or
> the same as what you are talking about.
>
> DR. DWYER: Well, if you are eventually going to be using the NHANES
> survey for looking at hemoglobins and all these other things and
> trying to relate it to consumption it might be worth talking to the
> people at NCHS to find out if, on the instrument they have, they have
> 120 foods I think where they are asking for propensity to consume, if
> those eight foods that you care a lot about from the standpoint of
> acrylamide are included in that list. If they are not, perhaps it is
> worth talking to them and seeing if there is a way to get them.
>
> DR. ROBIE: Thank you.
>
> DR. MILLER: If you did the same analysis you did on the population of
> eaters only, would the top seven foods change any or would the
> distribution change any?
>
> DR. ROBIE: Well, we can look at one of the tables because on your
> tables we do also have a column of eaters only. I am just going to put
> up one of the tables for anybody who doesn\'t have the handouts. If we
> were to sort on the eaters only column, certainly the foods that you
> are seeing right now are not going to be the same ones if you sort if
> by that column. We have looked at that. The breakfast cereal is where
> it is because the percent eaters is high, especially for the 14-day
> surveys, 37 percent. So, I guess the answer to the question is yes,
> they would sort differently.
>
> DR. MILLER: It just seemed to me that there were enough differences
> between the two, just quickly glancing at these curves, that you would
> get a different pattern of what the top seven or eight might be for
> people who are eaters and also, of course, the total intake is going
> to be considerably higher or at least would be significantly higher.
>
> DR. ROBIE: For eaters only, yes, that is true.
>
> DR. MILLER: It would be interesting to know how much greater the
> intake is for eaters only.
>
> DR. ROBIE: Well, it is not really appropriate to add the eaters only
> values. If each of these categories was 100 percent eaters then that
> would be appropriate, or if we knew that they were the same eaters.
> That is why we have added for the mean population.
>
> DR. MILLER: Right, I understand. Anybody else?
>
> DR. DWYER: I have one more.
>
> DR. MILLER: Yes, please.
>
> DR. DWYER: I guess the first thing I would ask if I were coming off
> the street is the Swedes came up with an estimate of about 0.7 and
> then FAO came out with an estimate of 0.3 to 0.8. You are coming out
> with estimates that are toward the lower end. Is this because you are
> from a fast food nation or is it because of some defect in the model,
> or is our model better and were those early estimates simply
> imprecisely high?
>
> DR. ROBIE: Well, I\'d like to think our model is better but I can\'t
> necessarily say. I can say for the Swedish data, again, this is a very
> limited data set, only 100 food samples for only eight food
> categories. They made a lot of assumptions and predictions for the
> foods that they didn\'t have data for, but they assumed that they had
> acrylamide, things like meats and vegetables and fruits. They went
> ahead and assumed that they had the same average level of acrylamide
> as the food they did test. I believe that is why their value is so
> high.
>
> As for the results from the FAO/WHO, it is hard to know reading the
> report. Again, they used the same residue data so we are talking about
> a limited data set to start with. It is not really clear from reading
> this report what they have done about the foods that haven\'t been
> tested at this point, if they also made assumptions or if they assumed
> those to be zero and ran the model. Again, there are several different
> food consumption databases so it is possible that the value is higher
> for Sweden and lower for another country and that is how they came up
> with this range. Their lowest number in the range is 0.3; our lowest
> number is about 0.3, and we go from about 0.3 to about 0.5.
>
> DR. MILLER: Dr. Torres?
>
> DR. TORRES: What is the body weight difference between the U.S.
> population and the Swedish population? I imagine it must be
> significant.
>
> DR. ROBIE: I can\'t say I know the answer to that question.
>
> DR. MILLER: It really isn\'t that much different. Anybody else?
>
> DR. TORRES: One last question, if you were to run the computer model a
> couple of times what would be your position about the value obtained?
> Is it going to be pretty reproducible? The question I want to ask you
> is how confident are you of the numbers that you are getting?
>
> DR. ROBIE: We are very confident in the numbers. The number of
> iterations we have chosen is 25,000 and Dr. DiNovi and I ran it once
> just to see how long it took to converge. It was 5000 iterations and
> we have run it several times and get the same results.
>
> DR. MILLER: We will take a 20-minute break and be back here at three
> o\'clock.
>
> \[Brief recess\]
>
> DR. MILLER: Given the nature of the product and how widely is used,
> why don\'t we see pizza on this list?
>
> DR. ROBIE: Somebody else asked me the pizza question. We don\'t have
> laboratory data on pizza. We have some data on Boboli pizza crusts
> where they detected above the limit of detection. I can\'t remember
> the actual residue levels. Forgive me, I don\'t have the data right in
> front of me and there are quite a few data points. But I know that
> Steve Musser\'s laboratory has tested the Boboli pizza crust. I don\'t
> think we have tested any like take-out pizza from any pizza chain or
> anything like that.
>
> DR. MILLER: Well, what kind of data did they get?
>
> DR. ROBIE: For the Boboli pizza?
>
> DR. MILLER: Yes.
>
> DR. ROBIE: I think it was non-detected. It might have been detected
> but under the limit of quantitation for the method. We deemed that the
> values weren\'t high enough to really have enough data. We are talking
> about a Monte Carlo method so we don\'t want to add something that has
> just a few levels that non-detects or are under the limit of
> quantitation.
>
> DR. MILLER: But you have large numbers of eaters.
>
> DR. ROBIE: Well, we would assume we have large number of eaters. I
> can\'t necessarily say that without looking at the survey data. You
> would assume I guess that there would be a large number of eaters but
> I don\'t want to say that unequivocally without looking at the data.
>
> DR. MEHENDALE: To follow up, you know, crust is one aspect and also
> cheese and the crust, of course, is relatively low in moisture and so
> on.
>
> DR. BUSTA: In one table it said 33, the crust. It is really low.
>
> DR. MILLER: That is interesting. Given that it is basically a flat
> bread, you would expect to find a high concentration. Thank you.
>
> DR. ROBIE: While I am up here, I just want to point out an error on
> the last two tables of the handout that you have. It says two-plus
> population and it should be two- to five-years population. That is one
> of the hazards of cutting and pasting. I apologize for that error.
>
> DR. MILLER: Thank you for your patience. Dr. Tim Fennell, RTI
> International, will talk about adduct studies.
>
> **Adduct Studies**
>
> DR. FENNELL: Thank you very much. I am very pleased to be here. I
> would like to thank the organizers for inviting me, and I would like
> to thank you all for your time.
>
> \[Slide\]
>
> What I am going to talk about, what I was actually asked to talk about
> are adducts of acrylamide, and metabolism is one of those things that
> is inextricably thrown in there so I am going to talk about metabolism
> also.
>
> I am going to review some of the general concepts of metabolism and
> pharmacokinetics, hemoglobin adducts and DNA adducts, and then get
> into a little bit about the history of metabolism of acrylamide,
> hemoglobin adducts and DNA adducts, and some of the recent studies
> that we have been doing and where we are going.
>
> \[Slide\]
>
> I would like to acknowledge a number of my collaborators. I used to be
> at CIIT and while I was there I worked extensively with my wife, Dr.
> Susan Sumner. I would like to particularly call attention to a couple
> of people. One is Rodney Snyder who has moved to RTI with me, and
> Burham Ghanayem at NIEHS who has collaborated with me on Cyp 2E1 null
> mice. I would also like to call attention to the various sources of
> support I have had from CIIT and from the acrylamide industry, and in
> particular most recently SNF who is currently funding some of my
> studies.
>
> \[Slide\]
>
> Going back just to give you a little bit of history, I started work on
> acrylamide in 1989. I had never been to a meeting on acrylamide until
> last year.
>
> \[Laughter\]
>
> It just shows you how much interest has been generated since April of
> last year. Since August of last year I have been to five specific
> meetings on acrylamide.
>
> \[Laughter\]
>
> That is what it is like to be in vogue.
>
> Acrylamide is a reactive chemical. It undergoes Michael additions. As
> we have heard, it is very reactive with sulfinyl groups. It also
> reacts with amino groups. It is extremely reactive with proteins and
> reacts very slowly with DNA.
>
> \[Slide\]
>
> I am going to be talking about a number of different kinds of labeled
> acrylamide and I just wanted to go through and review for everybody
> what I am talking about. For unlabeled acrylamide, we usually use that
> for pharmacokinetic studies for measurement of chemicals. For chemical
> measurements with things like mass. spec. For radio-labeled acrylamide
> we use 2,3, ^14^C acrylamide. The labels are in the vinyl carbons.
> Usually we have a small percentage labeled used for metabolism
> disposition, pharmacokinetics, adducts\--you name it, we can do it.
>
> The limitation here is how hot you can make this, how high a degree of
> radioactivity. It has a tendency to polymerize. So, that is always one
> of the big concerns. You can\'t make it tremendously high.
>
> What we have done a lot of studies with is uniformly labeled 1,2,3
> ^13^C, ^3^C acrylamide which is essentially 100 percent labeled at
> each site. We use this for metabolism disposition studies and also for
> adducts. We developed at CIT a method for analyzing metabolites by
> taking this material, giving it to animals, collecting the urine and
> looking at metabolism in the urine using ^13^C NMR spectroscopy and we
> can use that to find and characterize specific metabolites even if we
> don\'t know they are there until we go looking for them and we can
> measure them.
>
> \[Slide\]
>
> One of the other things we need to talk about when it comes to
> metabolism is glycidamide. This is the epoxide metabolite of
> acrylamide. It is formed by oxidation. It is a reactive epoxide. It
> reacts with protein and it also reacts with DNA. This is the big
> concern from the standpoint of carcinogenesis. This reacts with DNA
> and can cause mutations.
>
> \[Slide\]
>
> So, when we are dealing with metabolism and pharmacokinetics,
> generally most chemicals undergo metabolism to things that are more
> water soluble and less toxic. While most of the metabolites are
> unreactive and heavily excreted, some are more reactive. The problem
> with reactive chemicals or metabolites is that they can react with
> micromolecules, with glutathione, and they can disrupt all kinds of
> cellular processes that can lead to toxicity or carcinogenicity.
>
> When you have metabolism by more than one route, and we actually do
> have that with acrylamide, you may have one route that will give you
> the reactive metabolites and the other that will give you stable
> metabolites. Then, you can have those reactive metabolites undergo
> further metabolism so that life gets complicated.
>
> What we really want to know is the balance between the various
> metabolic processes, the relative rates, and these can be an important
> determinant in toxicity and they can differ between species and
> between high and low doses.
>
> \[Slide\]
>
> So, when it comes to risk assessment, we want to know about metabolism
> and adduct formation so we can understand relationships between
> exposure and internal dose. Here, internal dose is something that
> pharmacokineticists think of as area under the curve in blood. It is
> the amount integrated over time at a particular site.
>
> For dose response\--we have already heard about dose response and
> linearity. Do we have linearity in range of effects? Can we compare
> our internal dose measures with effects that are generated in
> bioassays? Do we have differences between species? Can we use measures
> of dose for reactive chemicals or metabolites to improve other studies
> such as epidemiology studies, interpretation of bioassays? Can we
> start looking at susceptible subpopulations?
>
> \[Slide\]
>
> Really what we are interested in here is how much acrylamide gets in
> the body, how much is metabolized, what is our area under the curve in
> blood and the tissues? Do we have adducts in proteins, and do we have
> adducts in DNA? And, the same thing for glycidamide. And, what is the
> balance between the various processes?
>
> \[Slide\]
>
> To go back to a little history, one of the more comprehensive studies
> on pharmacokinetics was conducted by Miller et al., in 1982. They
> administered radioactive acrylamide to rats. I just draw your
> attention to the very last bullet. The half-life of acrylamide, they
> found, was about two hours and about 12 percent of the radioactivity
> remained associated with red blood cells. That is something that is
> actually specific to the rat. They have a very high level of binding
> in red blood cells and it is because of the presence of a highly
> reactive cysteine in rat hemoglobin that does not occur in mouse or in
> human red cells.
>
> \[Slide\]
>
> I have already alluded to this study that was conducted in 1992. This
> was the first study that I was involved in with acrylamide, looking at
> urinary metabolites following administration of this 1,2,3 ^14^C
> acrylamide in rats and mice. We used a dose of 50 mg/kg in male rats
> and mice and we were able to characterize five urinary metabolites
> including four derived from glycidamide. Interestingly, we actually
> found glycidamide itself in the urine. We find that there was a big
> species difference, with rats metabolizing most of the acrylamide
> directly by glutathione conjugation and about 33 percent by
> glycidamide, whereas with mice it was the opposite, much more was
> oxidized by glycidamide than was conjugated directly with glutathione.
>
> \[Slide\]
>
> On this slide I just have a summary of the various metabolic pathways.
> The acrylamide undergoes direct conjugation with glutathione to form
> this mercapturic acid that we see in urine. This is the epoxide
> glycidamide and this is actually detected in urine and we see it is
> ring opened form, the dihydroxy form, and we also see two glutathione
> conjugates at each of the epoxide carbons to form also mercapturic
> acids.
>
> \[Slide\]
>
> Now I am going to switch to hemoglobin adducts. These are one of the
> great ways of detecting exposure long after the exposure may have
> happened. So, what do we know about hemoglobin adducts and how do they
> behave? Well, they are proportional to area under the curve for
> reactive chemicals or metabolites. You can get a lot of hemoglobin
> rapidly from a blood sample so it is easy to get and easy to measure.
> It has a number of reactive amino acid residues that can react with
> chemicals and their metabolites, cysteine, histidine, the internal
> valine and carboxyl groups. The internal valine is particularly useful
> in performing adducts because it is the same in both the alpha and
> beta chain of hemoglobin and it is the same in rats, mice and humans.
> So, that gives you a lot of simplification. You don\'t need to have
> different assays for different species.
>
> The red cell has a long lifetime in circulation of about 120 days, and
> it is removed with zero order kinetics. That also is a big advantage.
> It is not a first order process so it is very predictable and we can
> really nicely model what is going on with adduct accumulation. What
> happens is if you have continuous exposure over the life span of the
> red cell, in humans, if you have an adduct formed per day of exposure
> of X, you would expect that the life span of the red cell, if you had
> continuous exposure, is that your adduct level would reach 60 times X.
> So, that is very useful for long lived exposures and long experiments
> but it makes it very difficult to actually do a short-term study
> because you need to have a fairly high level of exposure if you are
> trying to do a calibration study.
>
> With repeated exposures the adduct accumulate and reach a steady state
> when the duration of exposure exceeds the red cell life span. So, it
> is good for things like dietary exposure, smoking, exposures in the
> workplace, those kind of endpoints.
>
> \[Slide\]
>
> This is just a very crude sketch of what happens if you have a
> reactive chemical in the blood. This is the concentration with time
> and, as your chemical would go up and down maybe in one or two
> exposures on consecutive days the adduct concentration would increase
> and would reach plateau when your chemical has disappeared from
> circulation, and then would increase again on the second exposure. So,
> that is sort of an illustration of the integration. You are looking at
> accumulation over time and the dose or the area under the curve equals
> the adduct concentration that you measure divided by the reaction rate
> constant for basically a second order reaction, a rate constant that
> you can determine chemically [in vitro]{.underline}. So, that is one
> way that we can actually go from an adduct measurement to an
> indication of what the area under the curve was and can give us a way
> of tying back into pharmacokinetics and pharmacokinetic modeling.
>
> \[Slide\]
>
> Again to review just a little bit of the history, the first
> measurements were done with cysteine adducts in the rat. There has
> been some indication of non-linearity, first in a study by Bailey and
> then in two studies by Calleman. These are probably the more
> authoritative ones. They measured adducts from acrylamide and
> glycidamide produced by reaction with cysteine. They detected the
> adduct from glycidamide as well as acrylamide. Then they did a very
> elaborate compartmental pharmacokinetic model and it ended up with a
> description of a saturable metabolic process, oxidation of acrylamide
> to glycidamide. what it implies is that there is a greater risk per
> unit exposure of low doses if glycidamide is the metabolite that
> generates adverse effects.
>
> \[Slide\]
>
> This just shows acrylamide internal dose that was calculated by
> Calleman. This is the administered dose in milligrams per kilogram
> body weight. You can see this is a straight line extrapolated from
> these points, and then this is the actual curve of the model with
> individual data points of each dose. So, that is for acrylamide.
>
> \[Slide\]
>
> For glycidamide it shows this evidence of saturation where, as you
> increase the dose up to 100 mg/kg, the amount of glycidamide produced
> is falling off.
>
> \[Slide\]
>
> Then, the last slide from this paper shows that they calculated the
> percent of acrylamide that would be metabolized through glycidamide at
> 15 mg/kg, which was actually the concentration we used or the dose
> that we used in our studies, and find about 30 percent or so
> metabolized through glycidamide in the rat. We got pretty good
> agreement. As you go down to lower doses the percentage that gets
> metabolized through glycidamide appears to go up.
>
> \[Slide\]
>
> Now I would like to switch gears to valine adducts. I am sure this is
> a name that is familiar to you all. Before Margarita was working on
> acrylamide in food she did a lot of work on hemoglobin adducts and
> actually developed one of the major methods that we use for acrylamide
> adduct analysis. That is when valine is involved in the adduct
> formation. It is the internal residue in both the alpha and beta
> chains, as I indicated before.
>
> She developed a modified Edman degradation that selectively cleaves
> the N-terminus with its adduct, using a variant of the classic Edman
> degradation for peptide sequencing. This has been used widely with gas
> chromatography with negative chemical ionization mass spectrometry,
> and more recently with GS-MS. It is, however, a real pain to do this
> assay. It is tremendously labor intensive, although as assays go it is
> pretty good.
>
> \[Slide\]
>
> This method was applied in a number of studies. One was by Bergman et
> al. They looked at analysis of acrylamide and glycidamide adducts in
> China. Just to go through these really quickly, they looked at
> acrylamide and glycidamide adducts in 41 workers exposed to
> acrylamide. They only looked at glycidamide adducts in six samples and
> they find that there were about 30-100 percent of the acrylamide
> adduct levels. This methodology though left something to be desired.
> It was a completely different method for the AAVal or acrylamide
> valine versus the glycidamide valine.
>
> There were other measurements reported by Emma Bergmark on acrylamide
> valine in smokers and non-smokers in laboratory workers. Then Perez et
> al. developed a method for acrylamide valine and glycidamide valine
> that was applied to workers in a production plant, and they recorded
> glycidamide valine levels that were about 3-12 percent of the
> acrylamide valine levels. These are probably a reasonably good
> measurement.
>
> Then we have the Hagmar study, in 2001, where acrylamide valine was
> measured in 210 tunnel workers. This is the one that precipitated a
> lot of the measurements in food. In the unexposed workers it measured
> from 0.02 to 0.07 nanomoles/gram for acrylamide valine and in the
> exposed workers it was found in considerably elevated levels.
>
> \[Slide\]
>
> One of the things that I have been doing over the last few years has
> been to work on a number of different aspects. These are metabolic
> data on acrylamide and adduct data. One of these has been how is the
> internal dose related to exposure with different routes of exposure
> and different species. Is the GSH conjugation to oxidation ratio
> altered by exposure route and by dose?
>
> We have looked at comparing route of exposure, dermal and inhalation
> and IP, and we have looked at comparing inhalation exposure in rats
> and mice, and looked at Cyp 2E1 metabolism and its role in acrylamide
> metabolism.
>
> \[Slide\]
>
> This just shows sort of a summary plot of glutathione conjugation
> versus oxidation. The first bar is the glutathione conjugation. The
> second bar is all of the metabolites of glycidamide. The last one is
> glycidamide itself in urine. When we look at dermal in the rat, it
> actually has the lowest amount of glutathione conjugation, the highest
> amount of oxidation, and compare that with inhalation IP and gavage
> and we see about 70 percent here at the peak. Whereas, in mice when we
> look at inhalation and gavage, we have a much greater percent of
> oxidation in the mouse compared with the rat.
>
> \[Slide\]
>
> We looked at metabolism of acrylamide via glutathione conjugation,
> oxidation to glycidamide and further metabolites of glycidamide in
> wild type mice and Cyp 2E1 null mice. We administered acrylamide at a
> dose of 50 mg/kg. In the wild type mice we find basically what we have
> seen before. About 50 percent or so was metabolized by glutathione
> conjugation, the remainder by glycidamide and glycidamide further
> metabolites. Whereas, in the 2E1 null we saw absolutely nothing
> derived from glycidamide or glycidamide itself. So, it argues that Cyp
> 2E1, at least in the mice, is the major or the only form of p450 that
> catalyzes the reaction and there doesn\'t appear to be any other
> oxidation pathway.
>
> \[Slide\]
>
> In my lab recently we have been working on a new method for adduct
> analysis, basically using the Edman degradation. We have adapted this
> for using LC/MS/MS for analysis. We get a higher throughput, a greater
> sensitivity with a smaller amount of globin and we can distinguish
> adducts from natural abundance acrylamide and ^13^C-labeled
> acrylamide. We have just recently published this, actually this month.
>
> \[Slide\]
>
> The method essentially works on taking our acrylamide valine
> hemoglobin, adding Edmund reagent and we selectively cleave our adduct
> and we form this kind of an adduct derivative, where this part, here,
> is derived from the acrylamide and this part, here, is derived from
> the valine, and this part, here, is derived from our derivatizing
> agent. We add an internal standard and we monitor by LC/MS/MS. We have
> three different ion reactions that we monitor. One is for our analyte;
> the second is for our ^13^C-labeled analyte where we have acrylamide
> administered to animals. The third one is our internal standard which
> is derived from labeling the valine with ^13^C.
>
> \[Slide\]
>
> We have a similar kind of procedure for analyzing glycidamide valine.
> This basically is the adduct. I won\'t bore you with the details here.
>
> \[Slide\]
>
> Our chromatograms look something like this. This is acrylamide valine.
> I think it is in the mouse. Here is our natural abundance channel, our
> ^13^C channel where we have administered ^13^C-labeled acrylamide, and
> this is our internal standard. From this we can understand our curves,
> we can quantitate and we can make measurements.
>
> \[Slide\]
>
> This is what we get with glycidamide valine. We actually have two
> isomers of our analyte and here is our internal standard.
>
> \[Slide\]
>
> We have basically looked at the same kind of studies as I mentioned
> with our metabolism studies so I won\'t run through all this.
>
> \[Slide\]
>
> We have looked at acrylamide valine and glycidamide valine in rats
> administered acrylamide. We have used a number of different routes and
> exposure scenarios. What I wanted to draw your attention to, since we
> are talking about food, primarily gavage as a route of exposure. We
> have done two studies here, at 50 mg/kg and at 3 mg/kg. These are
> actually exposed to ^13^C so the channel to really focus on is this
> one, AAVal and GAVal, and we see high levels at 50 mg/kg and much
> lower levels at 3 mg/kg.
>
> The important thing is that the ratio of glycidamide valine to
> acrylamide valine changes in going from high dose to low dose. So, we
> do seem to have a dose-response difference in the percent going to
> acrylamide valine versus glycidamide valine, and that fits with the
> Calleman data.
>
> \[Slide\]
>
> I am just going to bypass this.
>
> \[Slide\]
>
> To look at inhalation exposure we used rats and mice actually during
> the same exposure event. Again, I am going to call your attention to
> these two sets of data and just look at the numbers. These got the
> same exposure and we have similar amounts of acrylamide valine from
> the acrylamide in blood, whereas in the glycidamide valine we have
> very considerably higher levels in the mouse than in the rat. Our
> ratio here is completely different. It is about 1.0 for the rat and
> about 3.5 for the mouse. So, we have big species differences and mice
> seem to make a whole lot more glycidamide valine than do rats.
>
> \[Slide\]
>
> So, what kind of range of adduct concentrations have we seen and what
> kind of ranges have been reported? There have been a number of studies
> by Bergmark, by Hagmar and by Perez. The background is sort of in
> general agreement in humans from these studies. Some are in the 20-70
> range. In smokers that may be elevated. In acrylamide exposed workers
> we can have fairly high levels that have been reported and that vary
> from 300-34,000 in the Bergmark study and in the 3-12 percent range in
> the Perez study for glycidamide valine compared to acrylamide valine.
>
> In rats, what is in the literature, apart from what I summarized on
> cysteine adducts, we have a background and in rats administered a
> fried diet there is an increase from about 20 up to about\--well, an
> increase of the order of I guess somewhere around seven-fold. So,
> there has been reported an increase in rats fed a fried diet.
>
> \[Slide\]
>
> So I would like to conclude from some of our published work that we
> have seen route differences in internal dose in metabolism to
> glycidamide. We see that dermal administration results in a lower
> percentage of absorption. I didn\'t really go into that but I think
> dermal is one of the significant worker place exposure rates. We do
> see a species difference in metabolism to glycidamide.
>
> \[Slide\]
>
> We can readily measure acrylamide valine and glycidamide valine by
> LC/MS/MS. We do see a background for both of our adducts. The ratio of
> acrylamide valine to glycidamide valine depends on dose, rate of
> exposure and species. We do see a higher amount of glycidamide valine
> in mice compared to rats. Although I didn\'t really go into it, we do
> see a good correlation between metabolism data and hemoglobin adduct
> data.
>
> \[Slide\]
>
> I think one of the things we are all concerned about is where are the
> data gaps. I have my opinions on where these are. One of the big ones
> is when are we concerned about glycidamide and when are we concerned
> about acrylamide, and how metabolism fits into mode of action when you
> are dealing with risk assessment. How is acrylamide taken up and
> metabolized in people and what is the relationship between exposure
> and hemoglobin adduct levels in humans. Then, a final point is how
> good are the data on DNA adducts.
>
> \[Slide\]
>
> We have a number of studies in progress and I just wanted to summarize
> those very quickly.
>
> \[Slide\]
>
> The first one is a human study that has been funded by SNF. We are
> actually just doing the measurement of the adduct concentrations. That
> is to evaluate uptake of acrylamide, metabolism to glycidamide and to
> calibrate hemoglobin adducts with the known exposure and to compare
> uptake on dermal and oral administration. This was an exposure of
> sterile male volunteers to three different dose levels of
> ^13^C-labeled acrylamide, a dermal exposure to three times three doses
> of acrylamide, and then collection of urine for analysis of
> metabolites by ^13^C NMR, collection of blood for analysis of
> hemoglobin adducts before and after administration. We are almost at
> the conclusion of this study and we hope to have this finished
> probably by the end of next month.
>
> \[Slide\]
>
> There has been one paper published on acrylamide DNA adducts, and that
> is this paper by Segerback. They had synthesized an adduct,
> characterized an adduct standard from the reaction of glycidamide with
> guanine and then administered ^14^C-labeled acrylamide to rats and
> mice. They had identified DNA adduct based on comigration of ^14^C
> with adduct standard on HPLC. They quantitated the adducts.
>
> These kind of approaches are limited by the specific activity of the
> acrylamide you can prepare and we do have more sophisticated methods
> for this. We have been working on one. I know that at NCTR there has
> been work going on, on this also and I think they are further ahead
> than we are. But I think it is something that needs to be done and we
> need to have a better understanding of is this, for example, the only
> DNA adduct and how is it repaired. There are quite a number of issues
> of how one deals with an adduct if it is formed.
>
> \[Slide\]
>
> I just wanted to finish up with this slide which is basically a
> summary of some of the points from the FAO/WHO consultation
> recommendations. At the time when these came out we were actually
> actively involved in pursuing some of these. One is evaluating and
> calibrating biomarkers of exposure. We need data on absorption,
> metabolism and distribution and excretion in humans by the oral route.
> We need information on glycidamide and binding to the DNA as a marker
> of toxicity and carcinogenicity. We need dose-response characteristics
> of acrylamide and glycidamide and the relationship between adducts
> with hemoglobin and adducts with DNA in different organs.
>
> With that, I would like to stop and thank you for your attention.
>
> **Questions of Clarification**
>
> DR. MILLER: Comments or questions? Yes, Frank?
>
> DR. BUSTA: I your human study that you are just concluding, the dose
> of 0.5\--
>
> DR. FENNELL: That was the lowest dose.
>
> DR. BUSTA: Was it given orally?
>
> DR. FENNELL: Yes.
>
> DR. BUSTA: I am just guessing, I tried to calculate that, that is
> about 100 and 200 times above what you might get with a high
> concentration in food?
>
> DR. FENNELL: Yes, I think we are talking about ball park. There are a
> couple of things that we were interested in, in this study. One is
> that when we initially developed our exposure protocol the driver was
> not acrylamide in food. The driver was acrylamide in the workplace.
> These are not, you know, outrageous levels that could be achieved in
> the workplace. I think that is issue number one.
>
> The other is that when you come to measuring DNA adducts or hemoglobin
> adducts, or any of these things that have a background, and you are
> accumulating a background from the dietary exposure it is difficult to
> actually see a significant increment above that background without
> going to a higher dose than you would see ordinarily for example in
> food. So, in order to see, for example, a two-fold or just a doubling
> of a background in hemoglobin adducts you would probably need to
> administer something like 60 times the daily dose to see an increase
> statistically, and the levels that we operate for determining
> background that is quite a challenge. So, one needs to actually get to
> much higher levels in order to make that happen.
>
> One thing that would did was to use ^13^C-labeled acrylamide so we are
> actually looking at a different species of acrylamide adduct and
> glycidamide adduct than we see in our indigenous background but even
> then we were concerned that it was going to be too big a challenge for
> us to go much lower than 0.5.
>
> DR. MILLER: How do you deal with that when you have a substance that
> appears to be metabolized differently at high concentrations than low
> concentrations?
>
> DR. FENNELL: I think the range that we are in\--well, one of the
> things that we did want to find out was over that small concentration
> range did we see a difference. Certainly, what has been shown in the
> rat has been 0.5 to 100 mg/kg. So, in the range of 0.5 to 3.0 there is
> probably not going to be as wide a difference. You know, that big
> difference has been in the rat for the two dose points that showed the
> maximum or that showed the best evidence of saturation were at 50
> mg/kg and 100 mg/kg. So, it is quite possible we won\'t be anywhere
> close to that at this point.
>
> DR. MILLER: You said you calculated it was about 60 times?
>
> DR. BUSTA: Well, if you have 1000 ppb in a food, I calculated 1 mg/kg
> of food and feeding at 0.5, for an average person that is 35 mg that
> you have to feed. So, you would have to have 35 kg of French fries to
> get that average load. Maybe my numbers are a little off.
>
> DR. FENNELL: No, I think your numbers are about right. I think this is
> one of the issues. You know, this study is not going to be the answer
> to what happens to acrylamide in food but I think it is going to go a
> long way to providing a calibration for hemoglobin adducts which in
> the end will give us some idea of what is happening in food.
>
> DR. BUSTA: Where is the background coming from?
>
> DR. FENNELL: The background is coming presumably from food. One of the
> things that we have had difficulty with is as you get down to the low
> end of the calibration curve your matrix blank always has a
> measurement, there is always something in that, so how do you deal
> with that if you are going to use the appropriate matrix? We are still
> wrestling with some of those issues on our assay at the moment for
> dealing with our pre-exposure measurements. We clearly do see adducts.
> You know, it is quite easy to find them. I guess the question becomes
> when you do a calibration curve how you deal with them, and we are
> wrestling with some of those issues at the moment.
>
> DR. BUSTA: I would think that there are groups of people that are
> consuming very, very low levels of acrylamide in the kinds of foods
> they are eating. So, you should be able to run an assay on their blood
> and find the minimum amount of adducts as a result of their
> consumption of that food. But you say you still get a baseline.
>
> DR. FENNELL: We haven\'t done a huge number but we get a baseline even
> when we set up with rat hemoglobin where we are dealing with rats on
> defined diets, none of which we believe are fried but at least may be
> heated, and we do see a background. At the time we first observed it,
> it was more of an extreme annoyance and we thought, you know, when are
> we ever going to get a decent batch with a low background but now we
> know how to interpret this and it is more of an interesting
> observation.
>
> DR. MILLER: Other comments?
>
> DR. RUSSELL: With regard to the consultation recommendations that are
> up here now on the data on absorption, distribution and excretion in
> humans by the oral route, were those recommendations both for
> acrylamide in the food matrix as well as pure acrylamide? I guess
> another way of asking is not so much about the recommendations but is
> the work envisioned to go on in both realms, both looking at the pure
> acrylamide and in a food matrix?
>
> DR. FENNELL: I wasn\'t at the consultation and I know there is at
> least one person in the room that was so.
>
> DR. MILLER: Go ahead.
>
> DR. CANADY: Yes, our intention was just for food but information from
> other routes would be informative to interpreting existing tox.
> studies.
>
> DR. MILLER: Just one question, was there any suggestion made about the
> matrix itself, what the matrix should consist of? If everyone uses a
> different diet you may end up with different results.
>
> DR. CANADY: Right. No, that level of detail was not gone into. It was
> more just the observation that we don\'t have information for
> absorption of acrylamide through the food matrix and so we need
> information that can help us interpret how we receive acrylamide
> through food versus what the animal studies tell us. That was a simple
> observation.
>
> DR. MILLER: I have to admit that I am just uneasy about the apparent
> lack sometimes of agreement on fundamental standards. For example, if
> you are going to argue that there is a possibility that absorption or
> metabolism is going to have some impact on the outcome of the
> experiment, it would also be probably clear that different matrices
> are going to cause different effects, or at least it is possible. So,
> it seems to me there has to be some venue where people can agree on
> what that matrix is going to be. That equally applies to the analysis
> too. I mean, if everybody is using different analyses you have to show
> it doesn\'t make any difference, the differences between the different
> analytical methods, or you are going to have to agree on which method
> you are going to use.
>
> DR. DWYER: I am just curious. It seems to me there are a lot of
> feeding studies around the country where people are fed defined diets
> or some kind of diet, kept samples of what they are fed or at least
> records of what they were fed, where they have blood samples. Have
> people looked at the similarities or has there been any work done to
> get an estimated dose and then looking at the hemoglobin adducts and
> so forth? Rather than starting from scratch and doing new studies, it
> would seem like this would be far less expensive and might give some
> first approximations.
>
> DR. FENNELL: That is a good point. I think we are at a level of
> complexity in analysis that is much, much higher than acrylamide
> analysis in food when we are dealing with hemoglobin adducts, and the
> cost reflects that difference.
>
> The way that we have collected samples in general has involved a
> fairly extensive sample preparation right at the point where the
> samples are collected. We wash the red blood cells is isotonic saline
> and then we store them frozen. We do that in order to decrease the
> amount of albumin binding or serum protein contamination. Once we have
> the red blood cell frozen it is pretty stable and we can do a lot of
> things with it at a later time.
>
> So, I think if there are banked samples that are stored as red blood
> cells we think that we can analyze them. We are not quite there yet in
> having a definitive answer. You know, we can take an unwashed red cell
> where somebody has spun off the plasma or serum and see what we can
> get out of that. I think that is one important question. It has been
> raised to us. We have actually done one study and we think it was
> successful but we would like to do it a few more times before we make
> any definitive statements on it.
>
> DR. MILLER: If you are going to use NHANES samples you are going to
> have to determine whether the storage conditions allow you to develop
> any worthwhile conclusions.
>
> DR. FENNELL: To address just one point about NHANES, the procedure
> that is planned by CDC for NHANES is different from the one that I
> presented. I attended a meeting at CDC to discuss that and I think
> CDC\'s plan is that we will have some standards that can be passed
> around the labs to make sure that we are all in agreement as to what
> the measurement is, but they are planning to set up a new method which
> will involve HPLC mass spec. with tryptic digests and whether they can
> do that on banked samples or not I don\'t know.
>
> DR. MILLER: If what you say is true, the sample storage might make a
> difference in the results.
>
> DR. FENNELL: I think the way the sample is stored can make a
> difference depending on whether you want to store it to go
> prospectively or whether you are looking at retrospectively stored
> bank samples and I think they are looking at it from the standpoint of
> going forward from here on.
>
> DR. MILLER: I see.
>
> DR. DWYER: Would you say that again? Why are they developing another
> method if you already have a method?
>
> DR. FENNELL: I am not sure I can give you an answer for that.
>
> DR. DWYER: Aren\'t you all being funded by the Department of Health
> and Human Services?
>
> DR. FENNELL: I am not. I am funded entirely by either by grant or
> contract and I am not funded by CDC at all.
>
> DR. CANADY: One of the reasons the CDC has given for developing a new
> methodology is that the same methodology, if used for acrylamide and
> glycidamide adducts, could also then be used for other adducts to
> other chemicals. So, they are trying to develop a more generalized
> approach that they can use for other chemicals that they would asses
> through NHANES.
>
> Having said that, they are in the process of determining what the
> method will be, whether it works and how well it correlates with other
> methods. They haven\'t decided on the method they will use, rather,
> they are developing a method that is appropriate. So, it is still in a
> method development stage although they are taking another approach, as
> Dr. Fennell indicated. They are trying to take another approach.
>
> DR. DWYER: What are they doing with the blood they are collecting each
> day while they are waiting since the survey is in the field right now?
>
> DR. CANADY: Right. The red blood cells are being stored for this
> purpose. There are samples being set aside of red blood cells for
> future analysis including acrylamide adducts and glycidamide adducts.
>
> DR. MILLER: That is bothersome, frankly. I can understand why they
> would want to develop a more multi-purpose analysis given the things
> they have to deal with, but this is an issue that needs some
> resolution, and why wait until they have a multi-purpose analysis when
> it might be easier just to produce a more specific assay to begin
> with?
>
> Any other comments or questions? Since there is no one registered for
> public comment, which is required by the rules of the committee, then
> we have come to the end of our first day\'s activities and, unless
> there is some further question or comment from the committee, I am
> going to adjourn the committee until tomorrow morning at 8:30.
>
> \[Whereupon, at 4:00 p.m., the proceedings were adjourned, to resume
> on Tuesday, February 25, 2003 at 8:30 a.m.\]

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