FOOD ADVISORY COMMITTEE AND DIETARY


                                    SUPPLEMENTS SUBCOMMITTEE











                                         FURAN MEETING



















                                     Tuesday, June 8, 2004


                                           1:55 p.m.









                                       Bethesda Marriott

                                         Grand Ballroom

                                      5150 Pooks Hill Road

                                       Bethesda, Maryland














                  Food Advisory Committee


                  Sanford A. Miller, Ph.D., Chairman

                  Linda Reed, Acting Executive Secretary


                  Douglas L. Archer, Ph.D.

                  Patrick S. Callery, Ph.D.

                  Goulda A. Downer, Ph.D.

                  Johanna Dwyer, D.Sc, RD

                  Jean M. Halloran

                  Norman I. Krinsky, Ph.D.

                  Daryl B. Lund, Ph.D.

                  Margaret C. McBride, M.D.

                  Mark F. Nelson, Ph.D.

                  Robert M. Russell, M.D.

                  Carolyn I. Waslien, Ph.D., R.D.


                  Contaminants and Natural Toxicants Subcommittee


                  Alex D.W. Acholonu, Ph.D.

                  Marion F. Aller, D.V.M., DABT

                  George M. Gray, Ph.D.

                  Ken Lee, Ph.D.

                  Henry B. Chin, Ph.D.


                  Temporary Voting Member


                  P. Joan Chesney, M.D.




                  Dr. Henry Kim











                                        C O N T E N T S




                  Welcome and Introductions

                  Sanford A. Miller, Ph.D., Chair                            4


                  Conflict of Interest Statement

                  Linda Reed, Acting Executive Secretary, FAC                6


                  Opening Remarks

                  Nega Beru, Ph.D.                                          10


                  Scientific Overview of Furan in Foods


                    Analytical Methods/Occurrence

                    Dr. Kim Morehouse                                       22



                    Jeremy Mihalov                                          36



                    Dr. Don Forsyth                                         49


                  Questions of Clarification                                58


                  Scientific Overview of Furan in Foods

                  Dr. Glenda Moser                                          69


                  Questions of Clarification                                86


                  Public Comment                                            98


                  Summary and Charge to the Committee

                  Dr. Terry Troxell                                         98


                  Questions of Clarification                               105


                  Committee Discussion and Recommendations                 115











                                     P R O C E E D I N G S


                                   Welcome and Introductions


                            DR. MILLER:  I think I would like to get


                  started to enable us to finish on time and give



                  people a chance to make their planes, and so on.


                            First of all, let me welcome the new


                  members of Food Advisory Committee meeting for this


                  afternoon's session, which will deal with furans


                  and the data necessary in order to estimate the



                  risk of furans in food.


                            For the record, when I call your name, I


                  going to introduce the new members of the


                  committee.  This meeting is being held in


                  conjunction with the Contaminants and Natural



                  Toxicants Subcommittee of the Food Advisory


                  Committee, and there several members of that


                  committee that will be sitting with us in our




                            When I call your name, will you please



                  just repeat your name and the institution with


                  which you are associated.


                            First, Dr. Acholonu.











                            DR. ACHOLONU:  My name is Alex Acholonu,


                  Alcorn State University, Mississippi.


                            DR. MILLER:  Dr. Aller.


                            DR. ALLER:  Marion Aller with the Florida



                  Department of Agriculture and Consumer Services.


                            DR. MILLER:  Dr. Gray.


                            DR. GRAY:  George Gray with the Harvard


                  School of Public Health.


                            DR. MILLER:  Dr. Lee.



                            DR. LEE:  Ken Lee with Ohio State




                            DR. MILLER:  Dr. Chin.


                            DR. CHIN:  Henry Chin with the National


                  Food Processors Association.



                            DR. MILLER:  Dr. Chesney.


                            DR. CHESNEY:  I am Joan Chesney.  I am


                  Professor of Pediatrics and Infectious Diseases at


                  the University of Tennessee and also the title you


                  see on the roster at St. Jude.  I am also here



                  representing the FDA Pediatric Drug Subcommittee.


                            DR. MILLER:  Thank you.


                            Since we have some new members, we are











                  required to repeat the discussion of conflict of


                  interest for this particular issue on furans.


                            Linda Reed, who is Acting Executive


                  Secretary of the Food Advisory Committee, will read





                                 Conflict of Interest Statement


                            MS. REED:  Good afternoon, everyone.  As


                  Chairman Miller indicated, I am Linda Reed, the


                  Acting Executive Secretary of the Food Advisory



                  Committee meeting.  I would like to welcome


                  everyone and particularly our member from CDER.


                            I need to read the conflict of interest


                  statement into the record again.


                            The authority to grant permission to



                  borrow Special Government Employees currently


                  serving on an advisory committee in a sister


                  center, in this case, the Center for Drug


                  Evaluation and Research, is granted to the


                  Associate Commissioner for External Relations, Mr.



                  Peter Pitts.


                            Relying on that authority, Mr. Pitts has


                  signed a memorandum granting permission for Dr. P.











                  Joan Chesney to serve as a temporary voting member


                  for this portion of the meeting concerning furan on


                  June 8, 2004.  Dr. Chesney will represent, as she


                  just indicated, the Pediatrics Advisory



                  Subcommittee of the Anti-Infective Drugs Advisory




                            Because of the breadth of topics to be


                  discussed at this meeting, all of the members and


                  temporary voting member have been screened for any



                  and all financial interests associated with


                  regulated industry.


                            Based on this review, FDA has determined


                  in accordance with 18 U.S.C. Section 208(b)(3) to


                  grant general matters waivers to Dr. Marion Aller,



                  Dr. Douglas Archer, Dr. Johanna Dwyer, Dr. George


                  Gray, Dr. Norman Krinsky, Dr. Margaret McBride, Dr.


                  Sanford Miller, Dr. Robert Russell, and Dr. Carolyn




                            The granting of these waivers permits



                  these individuals to participate fully in the


                  matters before the committee.  Copies of the waiver


                  statements may be obtained by submitting a written











                  request to the agency's Freedom of Information


                  Office, Room 12A-30 of the Parklawn Building.


                            In an effort to enhance consistency within


                  FDA, the agency has recently adopted a policy



                  whereby all public commenters will be asked to


                  report any personal financial interests that could


                  be affected by the committee's deliberations.  A


                  copy of the policy was provided to any individual


                  who registered to make comments at this meeting.



                  Additional copies of the policy may be obtained


                  from the registration desk.


                            Similarly, we have asked all of our guest


                  speakers to complete a financial interest and


                  professional relationship certification for guests



                  and guest speakers to identify any potential


                  conflicts of interest.


                            Dr. Don Forsyth and Dr. Glenda Moser will


                  be the guest speakers at this portion of the


                  meeting.  Both have indicated they have no



                  financial interests in the food industry.


                            I would like to thank for your attention


                  and I will turn the meeting back over to Dr.













                            Thank you.


                            DR. MILLER:  Thank you, Linda.


                            As a matter of procedure, each of the



                  speakers have been assigned a time for their


                  presentation, and in order for us to make certain


                  we get through the presentations, and most


                  important of all, the discussion, I intend to be as


                  ruthless as I can in keeping the time.



                            We have several limitations on our time.


                  For one thing, we have to be out of here by 6


                  o'clock at the very latest.  Otherwise, as I


                  indicated this morning, we may be involved in


                  somebody else's wedding.



                            Also, there are some of you who have


                  planes to catch, and in order for the committee to


                  complete its business, which will be explained in


                  just a moment, it is important that we stick to the


                  time schedule.



                            The first presenter is Dr. Nega Beru of


                  the FDA, who will provide the background and


                  discuss the charge to the committee.











                                        Opening Remarks


                            DR. BERU:  Thank you, Dr. Miller, and good


                  afternoon.  My name is Nega Beru.  I am the


                  Director of the Division of Plant Product Safety in



                  CFSAN's Office of Plant and Dairy Foods.


                            My purpose here today is to provide you


                  with some of the background on furan in foods to


                  set the stage for the scientific overviews that


                  will follow immediately.



                            I will also lay out what input we are


                  seeking from the committee.


                            The structure of furan is depicted on this


                  slide.  It is a 5-member O-ring with two double


                  bonds.  It goes by a number of names as shown on



                  this slide also, has a molecular weight of 68, a


                  melting point of -85.6 degrees Celsius, and a


                  boiling point of 31 degrees Celsius.


                            This last property, it is fairly volatile,


                  may be important with respect to how much furan



                  consumers are exposed to in foods as consumed.


                            Furan is a colorless liquid that is used


                  in some segments of the chemical manufacturing











                  industry.  It is used, for example, as a solvent


                  for resins and in the manufacture of lacquers.


                            It was the subject of a 2-year bioassay by


                  the National Toxicology Program in 1993.  As a



                  result, it is listed in the Department of Health


                  and Human Services report on carcinogens, because


                  it was found to cause cancer in rodents in the NTP




                            Furan is formed in food during traditional



                  heat processing techniques, such as cooking and


                  canning.  Its mechanisms of formation are beginning


                  to be elucidated, and there appear to be a number


                  of them.


                            Later in this session, Dr. Don Forsyth



                  from Health Canada will present to you their


                  studies on mechanisms of formation of furan in




                            The discovery of furan in foods is not


                  new.  Furan has been reported in a small number of



                  foods starting as early as the 1960s, although very


                  little quantitative data exists in the literature.


                            Furan was found in coffee, canned meat,











                  baked bread, cooked chicken, sodium caseinate,


                  hazel nuts, soy protein isolates, hydrolyzed soy


                  proteins, rapeseed protein, fish protein


                  concentrates, and caramel.



                            What is new here is that FDA has developed


                  a quantitative method to measure low levels in food


                  and has found that furan forms in a wider variety


                  of foods than previously thought including in some


                  baby foods.



                            In addition to FDA, Health Canada and


                  NFPA, together with some of its members, are


                  investigating furan levels in foods, and, in fact,


                  FDA, Health Canada, and NFPA are also currently


                  collaborating In a round robin evaluation of the



                  method that was developed by FDA.


                            FDA's finding was made during


                  investigations aimed at confirming a report in the


                  scientific literature that furan forms when apple


                  juice is irradiated.  As part of that



                  investigation, a number of non-irradiated, but


                  processed foods were also evaluated using a


                  semi-quantitative method.











                            In the exploratory survey we posted on the


                  web on May 7, we used a more refined quantitative


                  method.  FDA initially concentrated on foods that


                  appeared to have high levels during the initial



                  screen using the semi-quantitative method.  FDA


                  also analyzed foods that didn't necessarily have


                  high levels in the initial survey, but could


                  potentially result in high exposures based on


                  consumption data.



                            For each type of food, foods were obtained


                  from two to three manufacturers, and, in addition,


                  to get at the lot-to-lot variation, two lots were


                  examined per food.


                            Foods that were tested include baby foods,



                  such as apple juice, applesauce, sweet potatoes,


                  carrots, and green beans, infant formulas, both


                  liquid and powder, and adult foods, such baked


                  beans, soups, chilis, spaghetti sauce, tuna,


                  coffee, and chicken broth.



                            Over 160 samples were tested in the


                  exploratory survey including replicas of the same


                  brand or product, and the results ranged from











                  nondetectable to approximately 100 parts per


                  billion furan.


                            Right after my presentation, Drs. Kim


                  Morehouse and Jeremy Mihalov will present more



                  detailed results of the survey, as well as the


                  exposure assessment that was based on the results.


                            FDA made the data collected in this


                  exploratory survey public on May 7 by posting them


                  on the FDA's web site.  At the same time, we posted



                  on the web a detailed description of the method


                  used to analyze the food samples, as well as a set


                  of questions and answers on the issue of furan in




                            FDA also issued two notices in the Federal



                  Register on May 7.  One was to announce a call for


                  data on various aspects of furan in foods, which I


                  will go into a bit later.  The other was to


                  announce this very meeting of the Food Advisory


                  Committee and the Contaminants and Natural



                  Toxicants Subcommittee.


                            When we announced the data to the public,


                  we did so with a number of message points.  Of











                  course, we said that finding furan in foods is a


                  concern because based on studies in rodents, furan


                  is a potential carcinogen in humans.


                            At the same time we made it clear that



                  furan certainly did not appear suddenly in food,


                  its occurrence in food has been reported before.


                  What is new here is the discovery in a broader


                  variety of foods than previously thought including


                  some baby foods.



                            We also said that this discovery is not an


                  immediate public health concern.  This was based on


                  our preliminary exposure assessment and a National


                  Academy of Science's review of the toxicology of


                  furan done for NASA, and this review is in your



                  briefing books, which concluded that, one, the


                  weight of the evidence suggests that furan is an


                  indirect carcinogen, and, two, calculated and no


                  observable adverse effect level of 80 mcg/kg body


                  weight per day.



                            Nonetheless, we said that there are many


                  questions that must be answered to improve the risk


                  analysis.  Thus, we said that we intend to conduct











                  an expanded survey including foods as eaten in


                  order to determine exposure and risk to consumers


                  more accurately.


                            We also said that we will look at what



                  additional studies are needed to determine furan's


                  potential risk to human health, as well as studies


                  on mechanisms of formation and reduction methods if


                  the risk assessment warrants such studies.


                            Finally, we said that we will seek input



                  from our Food Advisory Committee and Contaminants


                  and Natural Toxicants Subcommittee on what data are


                  needed to fully assess the risk posed to consumers


                  by furan in foods, hence, this meeting.


                            We intend to evaluate all the available



                  data including input from this meeting, and develop


                  an action plan to address the issue of furan in


                  food.  The action plan will certainly include an


                  expanded survey of foods, but may also include


                  mechanisms of formation/reduction in foods, as well



                  as toxicity studies to address mechanism and dose




                            In the call for data we issued on May 7,











                  we asked for data in several areas.  With respect


                  to occurrence of furan in foods, we asked for data


                  on the particular foods in which furan occurs and


                  the levels in these foods, the formation and



                  occurrence of furan in home-prepared foods as


                  opposed to, say, manufactured foods, and on


                  environmental sources of furan in which a typical


                  consumer is likely to be exposed.


                            With respect to mechanisms of formation,



                  we asked for data on possible mechanisms of


                  formation, as I mentioned earlier, we wrote a


                  letter here about studies that Health Canada


                  conducted on mechanisms on formation.


                            We also asked for data on variables that



                  enhance or mitigate furan formation in foods, on


                  the stability or dissipation of furan in foods, and


                  on the effect of post-production practices, such as


                  consumer heating of canned foods on the furan


                  levels in foods.



                            With respect to toxicology of furan, we


                  requested data on mechanism of furan toxicity,


                  mutagenicity, and carcinogenesis, on reproductive











                  and developmental toxicology, and on metabolism of


                  furan in vivo including characterization of any


                  reactive metabolites, and the role of such


                  metabolites in producing furans adverse effects



                  including carcinogenesis.


                            We also asked for data on the diversity of


                  furan pharmacokinetics in humans or the alteration


                  of furan metabolism as a result of dietary,


                  medical, or environmental interactions, and data on



                  whether sub-cytotoxic doses of furan produce any


                  adverse effects, such as a change in enzyme


                  activities or ATP levels.


                            Importantly, we asked for data on the


                  effects of furan at doses lower than those used in



                  the 1993 NTP study in order to accomplish the


                  following objectives:


                            1.  To establish a dose-response curve for


                  the various toxicological endpoints.


                            2.  To determine whether furan toxicity,



                  including carcinogenesis, is a threshold dependent




                            3.  To determine whether the carcinogenic











                  activity of furan is secondary to its hepatotoxic




                            Last, FDA is also asking for data on the


                  mutagenicity of furan in the TA100 strain in the



                  Ames test, and the behavior of furan in other in


                  vivo assays for mutagenicity or toxicity.


                            In the Federal Register notice call for


                  data, we asked that data and comments be submitted


                  to FDA by July 9, 2004.  We also said that we would



                  share with the committee and the subcommittee any


                  data or comments we received by June 1.


                            To date, we have not received any data on


                  any of the areas we specified in the Federal


                  Register.  We did, however, receive one comment.



                  That comment was from Dr. James Coglin [ph],


                  president of Coglin & Associates, a consulting firm


                  on food, chemical, and environmental toxicology and




                            The comment describes work done on various



                  heat-induced heterocyclic compounds including furan


                  as antioxidants and urged the committee and


                  subcommittee to consider the beneficial health











                  protective effects of such compounds in evaluating


                  the safety of furan in foods.


                            This brings me to the charge and the


                  question we are posing to the committee.  This, by



                  the way, are found in Tab 2 of your briefing




                            The Food Advisory Committee and


                  Contaminants and Natural Toxicants Subcommittee are


                  being asked to provide input on data that would be



                  helpful for further evaluation of the potential


                  risks posed by the presence of furan in foods.


                            Essentially, this is the question we are


                  asking the committee.  Taking into consideration


                  the data needs already identified by FDA in the



                  Federal Register notice requesting data on furan,


                  and the presentations you are about to hear at this


                  meeting, are there any additional data that are


                  needed to fully assess the risk of furan in foods?


                            With that I will end my presentation.  I



                  trust this will provide an adequate background for


                  the more detailed presentations that follow, and I


                  thank you for your attention.











                            DR. MILLER:  Are there any questions for


                  clarification?  Dr. Dwyer.


                            DR. DWYER:  I wasn't clear from the data


                  needs if you are also considering doing home-cooked



                  foods, for example, if I made a sweet potato pie at


                  home, are you planning on doing those, as well?


                            DR. BERU:  I think in the long run, we


                  want to do that, and perhaps even consider adding


                  furan to the total diet study.  Certainly, we have



                  done some preliminary work on home cooking in terms


                  of what dissipation of furan may take place during


                  normal home preparation of meals of canned or


                  jarred foods, and Dr. Morehouse will present some


                  of those data later.



                            DR. MILLER:  Dr. Callery.


                            DR. CALLERY:  Are you planning to also do


                  the Ames test on metabolites of furan, especially


                  metabolites that may have some predicted





                            DR. BERU:  Well, at this point we are sort


                  of in a data collection mode.  We want to see what


                  work has been done out there, and certainly we











                  intend to do what we can to fill the data gaps


                  including those studies.


                            DR. MILLER:  Thank you.


                            We next have three papers dealing with



                  overview of furan in foods, the first presented by


                  Dr. Kim Morehouse from FDA.  Ten minutes.


                             Scientific Overview of Furan in Foods


                                 Analytical Methods/Occurrence


                            DR. MOREHOUSE:  Hello.  My name is Kim



                  Morehouse and I am a research chemist with the


                  Office of Food Additive Safety, Division of


                  Chemistry Research and Environmental Review.  My


                  collaborators on this project have been Ms.


                  Patricia Nyman, Mr. Timothy McNeal, and Dr. Gracia





                            Today, I am going to present some data


                  that we have obtained on furan in foods and sort of


                  explain to you why we got into this in the first


                  place, even a little bit more than what Dr. Beru



                  has presented already.


                            As was noted earlier, during our


                  investigation of the possible formation of furan by











                  ionizing radiation, we noted that heating the


                  sample caused an increase in the amount of furan


                  that was detected.


                            This increase was not due in an increase



                  in the volatility of the furan, but rather was


                  indeed due to generation of furan.


                            We also noted the presence of furan in


                  pasteurized apple juice that we had purchased


                  locally at a store, but that furan was not present



                  in apple juice that we prepared fresh in our




                            This led us to investigate the presence of


                  furan in heat-processed foods, and we started


                  looking at various foods.  Originally, we were just



                  looking at it from the standpoint of comparing


                  radiation treatment to heat treatment of foods, so


                  we were doing a very random sampling of products.


                  Basically, I just went through the store, picked up


                  samples off the shelf that were canned and



                  pasteurized products, and this was a quick


                  semi-quantitative determination.  We weren't as


                  determined that we had to have exact numbers, but











                  rather an order of magnitude because we were just


                  trying to say was the radiation going to


                  significantly increase the amount of furan that


                  would be present in the total diet at that time.



                            However, as we got further into this


                  project, we began to realize that there was a large


                  number of foods for which furan was present and in


                  substantial amounts, and it became clear that we


                  needed to look at it further, as well as needed to



                  know the quantitative numbers that were there, not


                  just from a qualitative standpoint.


                            So, we modified our procedure.  In order


                  to do this, we were using static, headspace


                  sampling with gas chromatograph determination with



                  mass spec detection.  Our quantitation was based on


                  stable isotope dilution, as well as standard


                  addition with known amounts of furan to each food




                            It is important to note that we were doing



                  it on each food product because each food product


                  had a different partitioning coefficient of the


                  furan between the headspace and the sample.











                            This method has been peer verified within


                  our lab group itself by three different scientists,


                  as  I mentioned earlier, and we are currently


                  participating in a round robin study of the method.



                            Basically, what we did was we took for


                  what I call liquid samples, we took 10 grams of the


                  sample from the food container and placed it into a


                  headspace vial.  For solids and semi-solids, we


                  took 5 grams of the sample, added 5 grams of water



                  in the headspace vial.  The headspace vial was then


                  sealed and analyzed.


                            For some products, it was necessary to


                  homogenize the sample, and for those products they


                  were homogenized on ice either using a blender or a



                  tissue homogenizer.  After the samples were sealed


                  upon the addition of either D4 furan or furan if


                  necessary.  They were vortexed to ensure adequate


                  mixing of the samples.


                            It was important to make sure that we did



                  have adequate mixing because we noted that when we


                  did not, we retained rather spurious results, but


                  upon proper control of our samples with proper











                  mixing and everything, we were able to obtain


                  extremely good quantitation.


                            For our studies, we listed limits of


                  quantitation on the data tables that were presented



                  on the web.  We used rather conservative estimates


                  of those limits, and for liquid samples, we


                  determined that was about 2 ng/g, and for solids,


                  it was about 5 ng/g.


                            Like I said, these values are fairly



                  conservative, however, we know that our limits of


                  detection are much lower than that.  For liquid


                  samples, we estimate those to be about 0.7 parts


                  per billion, and for the solid matrices, about 1.5


                  parts per billion.



                            As Dr. Beru mentioned earlier, we selected


                  foods based on that initial survey that we were


                  doing during our radiation studies, as well as from


                  the literature reports of foods that were known to


                  contain furan, and using the FDA database to



                  determine which ones were higher consumption foods.


                            For each food analyzed, we analyzed from


                  either two or three brands, and usually from two











                  different lots per brand.  Using this data, we


                  undertook a systematic manner to obtain


                  quantitative data.


                            I am going to go through classes of some



                  of the foods that we looked at.  From the infant


                  formulas, we looked at powders, concentrates, and


                  what are called ready to feed foods.  The


                  concentrates and powders were prepared according to


                  label directions, placed in the vials and analyzed.



                  The ready to feed, of course, are already ready to


                  feed, so they were just simply transferred into the




                            You can see that we have a range for the


                  powders of non-detected to 2 parts per billion, for



                  concentrates of non-detected to 15, and for the


                  ready to feed, non-detected to 13.


                            For the powder and concentrate, they are


                  based on what would have been consumed.


                            The ranges I am listing here is because



                  you still see in the next presentation on the


                  exposure estimates, the range is what is used for


                  doing that calculation.











                            For some of the baby foods that we have


                  analyzed, you can see the apple juice range from 2


                  to 8, and you can go on down the list up to the


                  sweet potatoes and garden vegetables, which were up



                  to 100 part per billion.  Again, you can see that


                  we do have a fairly large range.  Again, the garden


                  vegetables, we are talking about three


                  manufacturers and two lots per sample.


                            For some of the adult foods, we have done



                  a lot more work.  You can see that we range from


                  bread, where it is non-detected to below our


                  quantitation level.  When we have less than 2


                  there, that means we can detect it, but it was


                  below our quantitation level, and in the cases of



                  the tuna and the canned meats, we listed as less


                  than 5.  That means it was within our detection


                  limits, but below our quantitation level again.


                            Again, you can see the spread of the


                  numbers that we are seeing and the various



                  different types of products that we have been able


                  to analyze so far.  Just so you don't think it is


                  all so bad, from our original survey, we do know











                  that many foods do not contain furan, some of those


                  listed here, and you will notice that man of these


                  foods are fairly high consumption products, such as


                  milk and margarine  and yogurt nowadays type of



                  thing.  We also included pasteurized eggs and


                  potato chips in our original survey, as well.


                            I was asked the question about the heating


                  the products.  We haven't gotten to the point yet


                  where we are actually cooking unprocessed foods to



                  look at that, but it is something we do intend to


                  do eventually, but what we did look at was what


                  about the foods from the can and if you heat them.


                            For the foods we looked at here, a very


                  limited preliminary study, we did chicken broth,



                  two different pastas, and the infant food sweet


                  potatoes.  The pasta No. 2 and the sweet potatoes


                  were only treated one way, that is why there is no


                  second bar there, but you can see from the pasta


                  sauces and the sweet potatoes, there is not what I



                  call a significant change upon heating, whereas,


                  with chicken broth where you basically have water,


                  and not much lipids or proteins to be holding back











                  the furan, it does substantially decrease.


                            So, depending upon what the food would be,


                  you would either lose the furan or not, and this


                  gives us a little bit of idea that we may have less



                  furan actually in the consumption than what would


                  actually be in the food as we are opening up the




                            For the heated samples, they were heated


                  basically on a hot plate in an open environment



                  until they boiled for about 10 minutes.  In the


                  microwave, they were heated to boiling, usually for


                  about a minute for the chicken and pasta.  The


                  sweet potatoes, they were heated what I call until


                  they were tepid, similar to what a consumer would



                  have done.


                            What is ongoing?  We are obviously


                  analyzing more foods.  This was set as just a


                  preliminary survey so far, we are doing a lot more.


                  We are now looking at foods based on using the USDA



                  consumption database to say what are some of the


                  other high use foods that we should go ahead and


                  analyze that we haven't already done before.











                            Again, still looking at foods that have


                  been reported in the literature that contain furan


                  for which no quantitation is available in the


                  literature.  It should be noted that in most



                  literature they would state that they found furan,


                  but would not state what the amount was, they


                  didn't quantitate the amount there.


                            Of course, we are going to continue to


                  investigate the effects of heating on the



                  concentrations of furan.


                            For those who would like to see the full


                  tables, of course, the entire method that we used


                  is available on the web site as was stated earlier,


                  as well as all the foods that have been analyzed.



                            Thank you.


                            DR. MILLER:  Questions?


                            DR. ARCHER:  A question, just curiosity.


                  What do you make of the potato chip data?


                            DR. MOREHOUSE:  There was no furan in



                  potato chips.


                            DR. ARCHER:  Any hypotheses?


                            DR. MOREHOUSE:  Nope.  Again, you are hear











                  later on some of the mechanisms, and some foods


                  that we saw high amounts of furan in, we look at


                  some of the mechanisms that have been proposed for


                  where furan is coming from, and they don't



                  correlate with the products, so obviously, there is


                  multiple mechanisms, multiple pathways, and potato


                  chips was one of the things that we thought would


                  contain furan, and did not.


                            DR. DOWNER:  Thank you very much.



                            It seems to me that the higher fat foods


                  tended not to have furan detected.  I want to ask a


                  little bit about the milk, though.  Were you able


                  to look at fat-free milk, 1 percent, 2 percent,


                  regular milk to see if there were any detectable



                  differences in those grades of fat content in the


                  milk with respect to furan?


                            DR. MOREHOUSE:  That was back from the


                  survey work, and I believe all we did was whole


                  milk, and we didn't see any furan in the whole



                  milk, so we didn't bother with looking at any of


                  the others.  We figured if it wasn't in whole milk,


                  why would it be in the others.











                            DR. MILLER:  Dr. Waslien.


                            DR. WASLIEN:  I was particularly concerned


                  with the furan content of formula, maybe


                  non-detectable, the 13 sounds low when you are



                  looking at a gram quantity, but if an infant


                  consumes a liter a day, you are up there in the




                            I went and looked at the l.d., the least


                  dose for mice or rats, and the calculated based on



                  that, of course, we don't have any data for doses


                  for humans, would indicate that the amount of furan


                  taken in is 13, and the dose that is least


                  detectable or least risk is something like 12, so


                  you are getting close for some of those infant





                            Now, my calculation might be wrong, I just


                  sat and did it right now, and we are encouraging


                  infants to drink less than a liter of milk a day,


                  but it is a concern, and that was my major worry.



                            DR. MILLER:  That's true, but the issue


                  that we are concerned with here is what work would


                  we suggest to the agency in order to get enough











                  data in order to be able to come to that




                            DR. WASLIEN:  Well, partly I would think


                  one of the things you might want to look at is



                  age-related differences in metabolism since a


                  newborn infant has all kinds of other metabolic




                            DR. MILLER:  Hold that thought.


                            DR. WASLIEN:  Okay.



                            DR. MILLER:  Dr. Chesney.


                            DR. CHESNEY:  I also have many, many


                  thoughts as you do, but for the moment, I wondered


                  if you could clarify the infant formula slide for


                  me.  I didn't quite understand



                  non-detectable-2-15-13, and you also said based on


                  consumed, and I may have heard wrong.  I wanted to


                  be sure I understood the slide.


                            DR. MOREHOUSE:  The slide, that is the


                  range that we found for the products that we have



                  analyzed.  From non-detectable to 2 for the powers,


                  from non-detectable to 13 for the concentrates, I


                  think it was, and the powders and concentrates are











                  based on as it would have been prepared by the


                  consumer for consumption.


                            In other words, we took the powder and


                  made up the solution was it was by label, so it is



                  based on the prepared formula, not the powder




                            DR. CHESNEY:  I understand.  Thank you.


                            DR. MILLER:  Dr. Chin.


                            DR. CHIN:  Going back to your table or



                  figure that showed the effect of cooking on furan


                  levels in various foods, there were I guess a


                  couple of bars where either the value was zero or


                  there were no values.


                            DR. MOREHOUSE:  Those were because for the



                  second pasta sauce and for the baby food, we did


                  not do the second treatment, so the pasta sauce No.


                  2 was only heated, and the baby food was only




                            DR. CHIN:  Thank you.



                            DR. MILLER:  Dr. Aller.


                            DR. ALLER:  A question again on the infant


                  formula.  I know you mixed that.  Was it heated













                            DR. MOREHOUSE:  No, just mixed.


                            DR. DWYER:  Just a question.  Could you


                  explain the difference between limit of



                  quantitation and limit of detection?  It is just


                  that you can't above the limit of detection, you


                  can't quantify until you get to 2 parts per


                  billion, is that right?


                            DR. MOREHOUSE:  Right.  Because of the



                  mass spectroscopy's sensitivity, we can detect it


                  or we put very stringent requirements on


                  quantitation right now because the method has not


                  been totally peer verified, we felt that we didn't


                  want to say that we could do 1 part per billion,



                  even though we can see it, but we don't want to


                  take the quantitation level there yet.


                            DR. MILLER:  Thank you.


                            The next speaker is Mr. Jeremy Mihalov,


                  FDA, will talk about exposures.





                            MR. MIHALOV:  My name is Jeremy Mihalov,


                  Office of Food Additive Safety.  This also was done











                  with Dr. Michael DiNovi.  I am going to give you an


                  overview of our exposure assessment for furan from


                  the consumption of adult and baby foods.


                            I will start off, give you an idea for the



                  model that we used to estimate exposure, and this


                  is fairly similar to most exposure assessments,


                  simply that the total exposure for a person to


                  furan is the sum of the exposures from each food,


                  overall foods that contain furan, and exposure from



                  each of those foods is simply the product of the


                  intake of that food modified by the concentration


                  of furan modified by the concentration of furan in


                  that food.


                            We looked at adult foods, baby foods and



                  also the infant formula, and they were considered




                            The sources of our data.  For intake data,


                  we used the USDA 1994 to 1996 and 1998 USDA


                  Continuing Survey of Food Intake by individuals.



                  This was a two-day survey, two nonconsecutive days.


                  For each of the years, there was about 5,000


                  people, so we have data for basically 15,000











                  individuals, and we know what they ate and how much


                  for each of those days.


                            We then looked at the furan concentration


                  data which you just heard about, and we looked at



                  those lists of foods, and looked at the survey


                  data, how much of those foods did those people eat


                  multiplied by the concentrations, and you can get


                  an exposure for each individual.


                            This may be somewhat of an iteration of



                  what you have already heard.  By looking at the


                  infant foods, we group them into juices, fruit


                  purees, vegetables, mixed chicken meals, had a


                  separate for infant formula.


                            For the adult foods, we grouped them into



                  brewed coffee, instant coffee, broths, soups that


                  contain meats, spaghetti sauces, chili, pasta,


                  ravioli--they were both canned--juices, pork and


                  beans, canned string beans, canned tuna, canned





                            Just to go over some of the levels again,


                  within each food type, the ones I just listed,


                  within the food types, there wasn't a lot of











                  variability.  Overall, the range, looking at all


                  the food types, went from limited detection up to


                  about 125 mcg/kg.


                            Specifically, looking at the infant food



                  groups, the highest were the sweet potatoes and the


                  garden vegetables, juices were generally low, below


                  10 mcg/kg.  The fruits and mixed meals were below


                  30.  Other vegetables ranged between 30 and 60.


                  With the formula samples, about half were below



                  limit of detection, and we used the mean, which was


                  about 7 mcg/kg.


                            With the adult foods, the coffee had the


                  greatest variability, between limit of detection up


                  to 80.  The juices, tuna, broth, sauces were all



                  generally low, below 15.  The soups and the pork


                  and beans had a fairly wide variation, the soups


                  being the highest.  The chili, beef ravioli, and


                  spaghetti, the canned pastas were between 30 and





                            Going back to discussing the model,


                  generally, when you do an exposure assessment,


                  there is a certain amount of uncertainty, and we











                  compensate the uncertainty with making certain


                  assumptions.  Whenever we make an assumption, we


                  tend to make it conservative, and this is typical


                  for agency exposure assessments.



                            The first assumption is that the


                  concentration of furan and all the furan-containing


                  foods will be at the mean within the food type, and


                  as I said there is generally little variability


                  within the food types, so we use the mean.  When we



                  are looking at chronic exposure, that is generally


                  how we do it.


                            Second assumption, for all foods within a


                  food type that are shown to contain furan, we


                  assume that it does contain furan.  In other words,



                  they have seen it in canned chili, so when we did


                  the exposure assessment, we assume anytime anybody


                  eats chili, it also contains furan, and as there is


                  more data collected in the future, those


                  uncertainties could be reduced.



                            The last assumption is that the two-day


                  survey intake data that we used reflects a lifetime













                            So, getting to the final numbers, we used


                  the published April 20th concentration data that is


                  on the internet.  Using that, for the adult foods


                  for people ages 2 and older, that ate those foods,



                  the mean consumption was 0.3 mcg/kg-body




                            The 90th percentile, which is what we


                  consider to be the heavy consumer, on the upper end


                  of the distribution, is at 0.6 mcg/kg/day.



                            When we looked at the infant foods, and


                  these are age 1 or less, that ate those foods, the


                  mean was 0.4 mcg/kg/body weight, and the 90th


                  percentile was 1 mcg/kg.


                            We ran the exposure assessment looking at



                  the individual foods just to get a sort of profile


                  of how those different food types contribute to


                  that overall mean, and this is just a table of how


                  those foods contribute, coffee being the highest


                  out of the groups that were tested, going down to



                  broths being negligible.


                            For the infant formula, we took a slight


                  different approach, a more simple approach.  There











                  is sort of standard numbers for infant formula.  In


                  order for an infant to thrive, they need to consume


                  between 100 and 120 kilocalories per kilogram per


                  day, and infant formula is usually formulated to



                  contain 0.8 Kcal/gram when it is prepared, and I


                  used the mean furan concentration of 7 mcg/kg, and


                  if you do the arithmetic, you can come out at a


                  mean exposure of 0.9 mcg/kg/day for an infant


                  consuming infant formula at the level needed to





                            To sort of sum up overall, the variability


                  of the furan levels within a food type is generally


                  small, so we can pretty much assume that additional


                  measurements within food types won't have much



                  effect on the overall exposure, however, because


                  the number of food types that have been tested is


                  generally limited, additional measurements in other


                  types of foods could have an overall effect on the


                  exposure, especially with foods that are consumed



                  in high quantities or also foods that have high


                  concentrations could affect the exposure.


                            Thank you.











                            DR. MILLER:  Dr. Waslien.


                            DR. WASLIEN:  I did a quick recalculation


                  of my numbers, and I am off by 1,000, so I skipped


                  nanograms in there.  I just wanted to make that





                            But even so, I think when you look at


                  infant formula, I hesitate to take the mean of


                  values, because the likelihood of a person changing


                  from one formula to another is not that high, so I



                  think you are looking at the individual risk from


                  formula, so the child who is consuming a formula


                  with 13, if it is a ready to consume formula, is


                  probably going to be consuming that reasonably


                  every day.



                            DR. NELSON:  I guess a similar question.


                  On the other food products, did you use the mean in


                  your conservative estimate, or did you use the


                  highest value?


                            MR. MIHALOV:  We used the mean of all the



                  concentrations for all the food types.  Generally,


                  when you are looking at a lifetime exposure, you


                  can pretty much assume that if there is a











                  distribution over time as you consume that food,


                  one day you might consume the minimum, the next day


                  you may consume the max, but over the course of


                  time, you are going to consume at the mean.



                            Of course, if there is additional data to


                  demonstrate that there is some reason to why there


                  is a distribution, you know, that could change, but


                  generally, for now we use the mean.


                            DR. RUSSELL:  Just a question of



                  information.  With so many adult Americans eating


                  out, particularly in fast food type restaurants, do


                  you have any data on fast foods that have been


                  prepared under high heat conditions?


                            MR. MIHALOV:  Well, the survey data



                  includes restaurants and home cooking.  It is


                  essentially the survey is given out and whatever


                  was eaten by those individuals on those two days,


                  that is what they report.


                            DR. RUSSELL:  But in your analysis of



                  foods that FDA has analyzed, how many foods come


                  from that type of an environment that were analyzed


                  actually?  I noticed a lot of canned and jarred











                  things, very important for infants particularly,


                  but I was just concerned about the adult exposure.


                            MR. MIHALOV:  Just looking at the list, I


                  would say a few of them are probably restaurant.



                  Like I had said, if they found it in a food, we


                  assume that it is in all foods of that type, so,


                  for instance, the chili was a canned chili, but we


                  assume that all chili contained furan when we did


                  the exposure assessment, so if they had chili at a



                  restaurant or if they made it at home, that was


                  taken into account.  If there is further data to


                  show that canned is higher than home-cooked or


                  restaurant, then, we can make that change.


                            DR. MILLER:  Dr. Lee.



                            DR. LEE:  To continue that thread, I


                  assume that there is a fair amount of looking at


                  canned and jarred foods because the furan is fairly


                  volatile, so the packaging method itself keeps the


                  furan present in the food, is that a fair





                            MR. MIHALOV:  I couldn't say.


                            DR. MILLER:  Dr. Nelson.











                            DR. NELSON:  That would fit with the


                  infant formula data because the powdered stuff is


                  typically spray dried where you have a lot of


                  opportunity for dissipation of furan as opposed to



                  the canned concentrate or ready to drink formula.


                            DR. MILLER:  Do you want to respond to




                            DR. LEE:  I just want to continue along


                  that line of thinking.  Have you ever considered or



                  does anyone have any data on animal exposure,


                  particularly pets consumption, because you


                  basically have a pretty monotonous diet, and there


                  are pet foods that do come in cans, so one would


                  expect that there would be a fairly good exposure



                  there that you can model, is there any interest in


                  looking at that?


                            MR. MIHALOV:  That could probably be done


                  if we had concentration data.  I am sure that there


                  is some information on how much food a typical



                  animal eats per day, but it would be pretty much as


                  simple as that, because a pet would consume one can


                  or two cans, or something along those lines, but











                  that could be one.


                            DR. MILLER:  Dr. Chin.


                            DR. CHIN:  I just wanted to comment a


                  little bit on the thought about foods purchased at



                  restaurants.  I think one of the other


                  considerations in terms of foods that are purchased


                  at restaurants is that not only do you consumer the


                  food at the restaurant, but there are situations


                  where you have takeout food and you take it home.



                            You might reheat it in the microwave.  We


                  have seen some limited data where you take a food


                  from a restaurant, put it in the microwave, and


                  under those circumstances, at home, you would


                  produce some more furan.



                            DR. MILLER:  Dr. Dwyer.


                            DR. DWYER:  Just a question about the


                  exposure assessment.  I am a nutritionist and so


                  when we use these kind of data, we use the Iowa


                  State method for adjusting the nutrients to pull in



                  the tails of the distribution.


                            Do you do that in exposure assessments, as


                  well? In other words, you have two days worth of











                  data, and so you are able to get an estimate of


                  usual intake from that, and I wondered if you


                  adjust for that.  The effect would be to change the


                  exposure, i believe.



                            MR. MIHALOV:  It doesn't sound familiar.


                  Basically, we take the distribution of all the


                  consumers and pull a mean in 90th percentile right


                  from the distribution, but not adjusting it.


                            DR. MILLER:  Dr. McBride.



                            DR. McBRIDE:  In answer to Dr. Nelson's


                  point, I looked at that data of the prepared


                  formula and the powdered formula and thought maybe


                  it was a difference in processing, might be heating


                  it more when it is packaged in liquid form.



                            I also did the calculations for the worst


                  case scenario because that is something I think you


                  were getting at, and if you have a chubby


                  8-month-old who consumes a liter of formula and 5


                  jar of sweet potatoes a day, I assumed it had to be



                  at least 8 kilos to do that, I got a worst case


                  scenario of 8 mcg/kg.


                            DR. MILLER:  How much?











                            DR. McBRIDE:  Eight.


                            DR. MILLER:  Dr. Chesney.


                            DR. CHESNEY:  Not why I am here, but the


                  fast food issue is intriguing.  I wonder if the



                  packaging contains furan.  Most fast food, you get


                  plastic containers to put it in, and most people


                  reheat it in the container.  Just a thought.


                            DR. MILLER:  Any more comments?  If not,


                  thank you.



                            The next speaker is Dr. Don Forsyth from


                  Health Canada, who will take about the formation of






                            DR. FORSYTH:  First of all, I would like



                  to thank the committee for the invitation to appear


                  here today on behalf of Health Canada.


                            My name is Don Forsyth.  I am a research


                  scientist with the Food Research Division of the


                  Bureau of Chemical Safety with Health Canada in





                            I would like to take you through the


                  background as far as Health Canada is concerned on











                  this issue.  In late March 2004, we became aware of


                  U.S. FDA's investigation of furan in canned and


                  bottle food commodities.  Upon learning that furan


                  has been shown to be carcinogenic in rodent models



                  and has been classified as possibly carcinogenic to


                  humans, we commenced method development as of April


                  of this year for support of the study of mechanisms


                  of formation, as well as a preliminary survey of


                  Canadian food products.



                            Although furan is used in industrial


                  processes, as has been discussed this afternoon, we


                  considered that the likely source would be


                  formation during food processing during the initial


                  start-up of our investigations.



                            One thing we should mention about furans


                  in foods, however, is that furan derivatives not


                  only have been reported in a wide variety of foods


                  previously, but they are also a significant flavor


                  and odor component in coffee, cocoa, and various



                  cooked meat products.


                            So, these are products or compounds, I


                  should say, which arise naturally during the











                  processing and cooking of various food commodities.


                            Furan itself, the parent compound, has


                  been previously isolated in coffee, canned beef,


                  sodium caseinate, soy and rapeseed protein, as well



                  as caramel.


                            Looking through the literature, you can


                  find a variety of possibilities or comments from


                  previous authors working in flavor and odor studies


                  about how these compounds are formed.



                            The three that we chose to look are the


                  thermal degradation of carbohydrates or the


                  Maillard reaction, thermal oxidation of lipid, and


                  decomposition of ascorbic acid and its derivatives.


                            Just to take a look at an older study



                  conducted by Persson and von Sydow back in 1974,


                  one of the first studies that you find where they


                  are able to determine that certain components in a


                  processed food could increase the levels of furan


                  produced within that food commodity under typical



                  canning conditions.


                            Using a beef, water, and sodium chloride


                  formulation fairly typical of the day for canned











                  beef products, they found that even with just these


                  basic components, there was fairly large levels of


                  furan produced, however, with the addition of the


                  fat, as you see in the second formulation shown



                  here, the levels increased dramatically above the


                  formulation without the fat.


                            Then, when they looked at the formulation


                  with a small amount of carbohydrate added, they


                  found essentially no increase over the basic



                  formulation of beef, water, and sodium chloride,


                  and then finally with the fourth different


                  formulation shown here, with the fat and the


                  carbohydrate added in addition to the other


                  constituents, you get levels similar to the beef,



                  fat, water, and sodium chloride formulation.


                            So, in this particular study, the authors


                  determined that the fat was a precursor for the


                  formation of the furan.


                            Briefly looking at our own analytical



                  methods that we developed to support these studies,


                  one was a headspace analysis which we used for the


                  mechanisms of formation and for the food survey,











                  and also the microextraction technique, which is a


                  SPME related method developed at Health Canada


                  which we applied to the food survey results, which


                  we will be showing later on in this presentation.



                            Both methods are based on isotope dilution


                  using a d4 furan surrogate, measurement by gas


                  chromatography/mass spectroscopy.


                            Formation studies.  We took some of the


                  test compound or precursor to a small vial



                  containing 0.5 ml of water.  The vials were then


                  heated for 30 minutes at 118 degrees, conditions


                  not too dissimilar to commercial canning


                  procedures, allowed to cool, and then force cooled


                  to 4 degrees when the D4 furan surrogate was added,



                  so that we could analyze the resulting anilides


                  which may have formed during this study.


                            The first table is on the level of furan


                  which were formed with the addition of ascorbic


                  acid and ascorbic acid derivatives.  Virtually all



                  of these compounds are commercial antioxidants


                  which are used in foods, and you can see that the


                  ascorbic acid with or without the iron present,











                  iron is a known promotor of oxidation and


                  therefore, would be expected to, at least in some


                  cases, increase the amount of furan which would be





                            The sodium ascorbate, again relatively low


                  levels.  The dehydroascorbic acid, however, with


                  either the iron present or absent gave higher


                  levels, almost 10 times higher than the ascorbic





                            Isoascorbic acid, again similar in this


                  case to the dehydroascorbic acid, and the sodium


                  isoascorbate in the presence of the ferric iron


                  produced almost again 10 times as much as the


                  sodium isoascorbate by itself, but again, both half



                  the levels that we found with the addition of


                  dehydroascorbic acid.


                            Finally, the ascorbyl palmitate compound


                  produced fairly low levels of furan as well.


                            Then, when we looked at fatty acids and



                  oils, we found that the degree of unsaturation in


                  the compound had an effect with an increase in the


                  levels of furan formed increasing as you go from











                  linoleic up to the linolenic with an increase of


                  about 4 times in this case.


                            Now, in these two fatty acid series, we


                  did see an increase in the production of furan with



                  the addition of the ferric iron, and in the case of


                  the oils, what you see in the last four rows of the


                  table, again, a similar increase as you go from the


                  trilinoleate up to the trilinolenate, approximately


                  again roughly 4 times.



                            In this particular case, with the oils,


                  the ferric iron had an increase in the production


                  of the furan for the trilinoleate, but not for the




                            Comparisons were made between the reaction



                  products and the furan standard, and as you can see


                  in this particular case, the comparison between the


                  linolenic acid reaction mixture and the furan


                  standard, you get a very similar pattern both for


                  the total iron chromatogram as well as the



                  fragmentation pattern for these two.


                            So, what we have determined so far is that


                  at least in the model systems that we have tested











                  so far, we found that the polyunsaturated fatty


                  acids, such as the linoleic and the linolenic, did


                  produce furans likely through a free radical


                  formation mechanism with ring closure resulting in



                  the formation of the furan, and also decomposition


                  of ascorbic acid derivatives particularly the


                  dehydroascorbic acid and the isoascorbic acid also


                  led to the formation of furan.


                            Some of our survey results in baby foods.



                  Here, we have a comparison between our two


                  methodologies, the microextraction technique in the


                  first column, and the static headspace in the




                            Levels varied as low as 6 parts per



                  billion, and went as high as approximately 154


                  parts per billion in the mixed vegetable.  Each one


                  of these values that you see is the average of two


                  individual analytical determination for each





                            When you look at adult foods, we found


                  that the chili products had the highest levels


                  amongst those that we analyzed with levels ranging











                  up to as high 227, 236 depending upon the method


                  value, as well as 152, soups there was a broader


                  range ranging from as low as 35 ppb up to


                  approximately 115, 117 ppb.



                            We have looked at one stew product so far


                  with a value of approximately 80 parts per billion,


                  one bean product with relatively low value, 14


                  parts per billion.


                            The luncheon meats that we looked, I



                  believe were both beef or pork based, and they were


                  all relatively low with levels down to 4 parts per


                  billion, and no higher than approximately 30 parts


                  per billion.


                            Fresh brewed coffee, as would be typically



                  served, would range between 14 to approximately 50


                  parts per billion.


                            Next steps for our work at Health Canada


                  include further studies on the mechanisms of


                  formation using additional model systems, as well



                  as precursor fortified food matrices.


                            Examining losses of furan during food


                  processing and cooking operations, as well as











                  further examinations of canned and bottled


                  products.  We also have a round robin method


                  validation study to complete, and that is ongoing


                  as we speak, and we should be reporting back on



                  that in just a few weeks.


                            Then, finally, to continue updating our


                  health risk assessment as new data becomes




                            With that, I would just like to thank



                  everyone for their kind attention.


                            DR. MILLER:  Thank you.


                            Any comments or questions?


                                   Questions of Clarification


                            DR. ACHOLONU:  I was wondering, is it



                  advisable to check the concentration of furan in


                  mixed vegetables?  Could you justify using that?


                  Mixed vegetables, which has different kinds of


                  vegetables put together, what do you do?


                            DR. FORSYTH:  The premise of that, of



                  course, is for health risk assessment, in which


                  case we are interested in consumption of food


                  commodities that are related to a typical diet, so











                  this is one particular food product that we


                  happened to analyze, and that is essentially the


                  extent of our interest in it at that point.


                            DR. ACHOLONU:  But does it have any



                  scientific basis?


                            DR. FORSYTH:  It has a scientific basis in


                  the sense that with that particular food matrix,


                  those are the levels that you are reaching.  It


                  also brings to mind what is causing that formation,



                  which is something that we are certainly interested


                  in, because it doesn't fit into the existing models


                  that we have pursued so far.  So, yes, I think it


                  has a lot of scientific interest.


                            DR. MILLER:  Dr. Krinsky.



                            DR. KRINSKY:  Could you just describe the


                  conditions for generating the furan from the


                  linolenic acid?  Was this heated, cooked, baked, or


                  was it just linolenic acid out of a jar?


                            DR. FORSYTH:  I didn't actually conduct



                  this study myself.  My understanding is that the


                  compound, which I believe was 10 mg of the


                  precursor would have been added to the vial











                  containing 0.5 ml of water, and then that would


                  have been heated to the 118 degrees for 30 minutes.


                            DR. KRINSKY:  Thank you.


                            DR. MILLER:  Dr. Lund.



                            DR. LUND:  Looking at the Persson and von


                  Sydow data, I wondered if you have had any comments


                  with regard to the degradation of furan upon


                  prolonged heating.  Some of their data, at least on


                  the surface, would suggest that upon prolonged



                  heating, you probably get formation rates equal to


                  degradation rates because the concentration is not




                            DR. FORSYTH:  First of all, I am not sure


                  if that is what they were alluding to or not.  I



                  thought that data was to look at probably losses of


                  furan due to revolatilization during heating and


                  processing in the kitchen.


                            I know that there is some concern that you


                  may actually be creating more furan with



                  post-processing sample manipulation, but I don't


                  know if anybody has actually really looked at that













                            DR. MILLER:  Dr. Callery.


                            DR. CALLERY:  We addressed part of this


                  already, but it's an impressive amount of work that


                  you have done since March.  I have been looking at



                  this, and I admire you for being able to get so


                  much data so rapidly.  I have a couple of little


                  questions, though.


                            The ascorbic acid one in particular, from


                  what I remember the structure of ascorbic acid,



                  it's a highly oxidized species and it gets even


                  more oxidized readily, and that you are actually


                  asking iron to participate in this reaction to


                  facilitate an oxidation.


                            I think the point I am trying to make is



                  that the furan is more like a reduction or


                  elimination of water, a couple water molecules, and


                  more a reduction.  If you looked at the oxidation


                  states of the various carbons, they are not at a


                  higher oxidation state than ascorbic acid.



                            So, it may be something very different


                  going on here that is involving the metal in the


                  process of making furan, if that is what you are











                  actually doing.  I think the question was also the


                  yield that you are addressing here, maybe there is


                  10 mg of ascorbic acid or I am sure 10 mg of fat,


                  but that nanograms per gram is incredibly small



                  yield in the process of cooking, so I am wondering


                  a little about that, too, if you aren't just making


                  some furan this way out of this particular




                            DR. FORSYTH:  I have no doubt that the



                  yields, particularly with the ascorbic acid tend to


                  be quite low, but typically, levels used in food


                  are reasonably high, and this wouldn't necessarily


                  be the only way that furan would be formed, and it,


                  of course, had been alluded to earlier by one of



                  the other speakers, that we undoubtedly will find


                  that there is multiple pathways contributing to the


                  overall levels of furan present in the food.


                            These are, I can't stress strongly enough,


                  preliminary investigations into possible means that



                  furan could be formed.  There had been previous


                  work with some of the ascorbic acid related


                  derivatives that had indicated that a variety of











                  furans were formed during thermal degradation, and


                  this is what we were attempting to follow up on


                  with this study.


                            DR. MILLER:  Dr. Chin.



                            DR. CHIN:  I would also like to compliment


                  you on doing such an impressive amount of work in


                  such a short period of time.


                            Just a question in terms of your thoughts


                  on other possible precursors.  Are you planning to



                  look at the possibility that perhaps carotenoids


                  and similar types of materials might be a precursor




                            DR. FORSYTH:  Our immediate plans, and we


                  are doing this as we speak, looking at Meyer type



                  reactions at present.


                            DR. CHIN:  Just a follow-up, and the


                  reason I am asking is because in products like the


                  sweet potatoes where there are amount of furan have


                  been detected, I mean those materials are high in



                  carotenoids, whereas, they are generally low in


                  fat, and I don't think the ascorbic acid levels are


                  particularly high, so just a possibility in terms











                  of another possible precursor.


                            DR. FORSYTH:  It sounds like we will be


                  following up with you shortly.


                            DR. MILLER:  Dr. Dwyer.



                            DR. DWYER:  Just a question more from


                  ignorance than anything else.  Are the methods that


                  you are using in Health Canada and the Food and


                  Drug Administration's methods the same?  I just


                  looked at chili, and it looked like the Canadian



                  chili was much more potent than the American chili,


                  and just wondered if there were some way if they


                  are not the same method, if there is some way to


                  get some comparable methods in both countries or to


                  divide up the work and do some round robin studies,



                  so that we are not overduplicating things that are


                  basically the same trading area.


                            DR. FORSYTH:  Round robin testing is


                  underway.  In our own case, it was set aside


                  because we were concerned with what levels were



                  present in the Canadian food supply, so that was


                  our first priority.  Now that that is completed, we


                  do have the two methods which we wish to compare,











                  not only against FDA's method, but also any


                  industry methods which are out there, and we will


                  be doing that through the round robin study.


                            DR. MILLER:  Dr. Russell.



                            DR. RUSSELL:  Yes.  Following up to Dr.


                  Chin, I also had wondered about the sweet potato,


                  but I was wondering if the soup, it says soups in


                  both the FDA data and your data, I was wondering if


                  there was any clues that could be gotten with the



                  types of soups.


                            There was a three- or four-fold variation.


                  What types of soups were looked at?


                            DR. FORSYTH:  I believe there was--I hope


                  there was a listing of the actual products that we



                  tested included in your information package.  In


                  any case, we have tested 30 products so far.  We


                  hope to be testing more in the near future but for


                  the time being, we will be participating in the


                  round robin study first.



                            DR. MILLER:  Dr. Chesney.


                            DR. CHESNEY:  Again, just for


                  clarification, and this may seem like a very











                  simplistic question, but is it correct that the


                  furan is created by oxidation of the ascorbic acid


                  products and the polyunsaturated fatty acids, it's


                  a product of oxidation of those entities, am I





                            DR. FORSYTH:  I think with the ascorbic


                  acid, I would view it more as a thermal degradation


                  as opposed to an oxidation per se, whereas, with


                  the lipids, it is a radical-mediated oxidation



                  mechanism, yes.


                            DR. MILLER:  Dr. Nelson.


                            DR. NELSON:  Following up on Dr. Dwyer's


                  comment about the equivalence of method or


                  recognition of each other's method, I guess, would



                  the food supplies be considered equivalent enough


                  for us to sort of accelerate the database by again


                  sharing the activity?  I don't know if we need the


                  same trading area, we have to have a NAFTA





                            DR. FORSYTH:  Is that related to me?


                  Presumably, in Canada, we find that we have


                  different branding as opposed to U.S. foods, but in











                  cases where you have the same manufacturer, I


                  personally can't see any reason why the data


                  couldn't be used.


                            DR. MILLER:  Dr. Downer.



                            DR. DOWNER:  I just wanted to respond to


                  Johanna's question about the chili.  I think in


                  Canada, they may be using Spam from looking at the


                  database here, so maybe that is where the


                  difference is.



                            Thanks for a good presentation.  I am just


                  wondering about Dr. Morehouse's presentation when


                  he looked at no furan detected in some of the


                  different groups of foods, particularly foods from


                  animal sources.  I was thinking that perhaps



                  because it was a bit lower in fat.


                            But on one of your slides, when you talked


                  about the effect of canned beef formation and you


                  added fats, it was really the opposite.  Could you


                  talk a little bit about perhaps the differences



                  that were seen there?


                            DR. FORSYTH:  Actually, in retrospect,


                  when you look at their study versus our own results











                  on canned luncheon meats, I have concerns.  It was


                  a 1974 survey, well, different analytical


                  capabilities than we have now, so I believe that


                  that work does bear out the results that we were



                  finding in terms of the presence of fat promoting


                  the increase in furans.


                            However, I can't reconcile the findings


                  that they reported in that publication with our own


                  and also with FDA's current findings on luncheon



                  meats, which I would have felt should be




                            DR. MILLER:  Dr. Dwyer.


                            DR. DWYER:  Just a question again about


                  methods.  Is there a standard method, or are you



                  driving toward a standard method instead of


                  everybody having their own method especially in


                  North America, it seems like this might be


                  something to agree about one way or another?


                            DR. FORSYTH:  There has been a few



                  factors, time being one of the largest.


                  Essentially, with the time constraints that all of


                  the organizations have had, you basically begin











                  with the people that you have who know how to do


                  these types of analyses, and you ask them to come


                  up with a working method, and I believe that is


                  essentially what has happened here.



                            The next phase will be these organizations


                  to have, and this is being done as we speak, a


                  round robin study in which case we all examine the


                  same food commodities and see if we get, hopefully,


                  roughly the same answers.  Depending upon the



                  results of that study, there would then be either


                  adjustments made or discussions amongst the various


                  organizations to determine why, if there are indeed


                  any, there are differences in our results on these


                  particular food commodities.



                            DR. MILLER:  Why don't we move on.  Thank


                  you very much.


                            The next speaker is Dr. Glenda Moser.  You


                  have got 25 minutes.


                             Scientific Overview of Furan in Foods



                            DR. MOSER:  Thank you.


                            Well, I would like to begin by thanking


                  the committee for inviting me today and say that my











                  talk is going to be a little different than those


                  that have been presented up until this point, and,


                  in particular, I am going to be talking about some


                  in-life studies that we did to try and determine as



                  best we could a mechanism for furan-induced liver


                  tumors in mice.


                            In a two-year NTP bioassay, there were


                  both non-neoplastic and neoplastic findings in rats


                  and mice of both sexes.  In particular, there were



                  neoplasms, cancer, in the liver in the rats, and


                  cholangiocarcinomas in the rats, as well as


                  mononuclear cell leukemia.


                            The important thing here is that at 2


                  mg/kg, there was approximately a 90 percent



                  incidence of cholangios.  In mice, there was an


                  increased incidence of benign tumors of the adrenal


                  gland, as well as hepatocellular tumors at both 8


                  at 15 mg/kg.


                            Here, what you see is more of what I am



                  going to be talking about today, is the incidence


                  of your hepatocellular adenomas or your benign


                  tumors, hepatocellular carcinomas, and then the











                  adenomas and the carcinomas together in female mice


                  and in male mice, and you see a dose-dependent


                  increase in both the males and the females.


                            Another important factor for the study



                  that I am going to be talking about is the


                  incidence of spontaneous liver tumors in both male


                  and female mice.  Historically, in your B6C3F1


                  mice, you will have somewhere between 20 and 60


                  percent incidence of liver tumors in control



                  animals.  Usually, it's much lower in your female


                  mice.  It is for that reason that we conducted our


                  two-year study in female mice.


                            Sometimes it is difficult when you have 40


                  or 50 percent incidence of spontaneous liver tumors



                  to find an increase in male mice.


                            Cancer, as we are well aware, is a highly


                  complex, multistage process that is operationally


                  divided into three stages, namely, initiation,


                  promotion, and progression.  Initiators or



                  genotoxic agents directly damage DNA.  They change


                  the primary sequence of the DNA.


                            Genotoxic agents can be carcinogenic after











                  a single exposure, and, in general, it is found


                  that genotoxic agents are better carcinogens if


                  they also induce cell proliferation, so they can


                  fix those mutations.



                            Metabolism, there are many carcinogens,


                  not only liver carcinogens, but carcinogens in


                  other systems, the parent compound is not


                  carcinogenic, but it is metabolized or intoxicated


                  to its toxic moiety.



                            In the case of furan, you have the


                  cytochrome 2E1 in the liver that metabolizes the


                  furan to its toxic moiety. There are a variety of


                  mechanisms of genotoxicity which we are not going


                  to talk about today.



                            What we have here is somewhat of a summary


                  of assays for genotoxicity after furan exposure.


                  We have those that are negative, those that are


                  positive, those that are highlighted in mammalian


                  systems, and those that aren't, are either in your



                  Salmonella or your Drosophila.


                            In spite of the fact that there are some


                  positive tests for genotoxicity, furan is generally











                  considered to be non-genotoxic.  Non-genotoxic or


                  epigenetic agents, they are generally believed to


                  clonally expand those initiated cells.


                            They provide an environment in which those



                  particular cells opportunistically grow and expand,


                  that, in general, your non-genotoxic agents require


                  multiple exposures, sometimes over the course of


                  the entire life span of the animal, and generally,


                  with your tumor-promoting agents or your



                  non-genotoxic agents, it requires high doses.


                            In the early stages, tumor promotion is


                  generally reversible, so that in a 13-week study


                  with furan, the animals were dosed at 8 and 15 mg,


                  then, they were held for either 6 months, 9 months,



                  or 15 months, and 18 months, and evaluated.


                            In these particular studies, furan was not


                  reversible, particularly the cholangiocarcinomas.


                            It is important for us that B6C3F1 mouse


                  is the mouse used by the National Toxicology



                  Program.  Part of the reason that it is used is


                  that it is sensitive to cancer, and that's both


                  spontaneous cancers, as well as chemically induced.











                            In particular, the liver, of the 500


                  compounds that the NTP has evaluated, approximately


                  50 percent of them are carcinogenic in the mouse


                  liver.  Really, that was the reason that we



                  conducted these studies was to try and make some


                  association of what relevance are these mouse liver


                  tumors to humans.


                            There are a whole variety of non-genotoxic


                  mechanisms, we are not going to talk about them



                  today.  The one I do want to talk about is the


                  cytotoxicity in cell proliferation, that furan, in


                  short-term studies, is necrotic to liver cells,


                  hepatocytes.  It kills them.


                            After this, in order for the liver to try



                  and maintain its homeostasis, we have regenerative


                  or compensatory cell proliferation.  There are


                  certain hypotheses that believe that the mutations


                  that you may find in the H-ras gene or some of the


                  other genes are secondary to this cytotoxicity and



                  cell proliferation, that the DNA is believed to be


                  inordinately sensitive to mutation when it is


                  dividing.  It is kind of spread out there, kind of











                  opening itself up, if you will.


                            Liver cytotoxicity, how do we determine if


                  a chemical is cytotoxic for the hepatocytes?


                  Commonly, that is done by clinical chemistry, by



                  evaluating serum ALT, alanine aminotransferase, or


                  sometimes SDH levels, sorbitol dehydrogenase, and


                  secondly, by histopathology, that liver sections


                  are stained with hematoxylin and eosin, your H & E


                  stained section, you will commonly find pycnotic



                  nuclei that generally the nuclei of the hepatocytes


                  are blue, and those cells that have been exposed to


                  a cytotoxic agent, their chromatin and the nucleus


                  is sometimes so blue that it is almost black.


                            You will find an inflammatory response.



                  You have the recruitment of both your mononuclear


                  and your polymorphonuclears to kind of clean up the


                  debris as a result of this cytotoxicity, and


                  thirdly are the degenerated hepatocytes or the


                  cytoplasmic vacuolization.



                            We conducted a 13-week study in male


                  B6C3F1 mice.  These animals were exposed by gavage


                  intergastrically 5 times a week.  The dose levels











                  were 0.5, 2, 4, 8, and 15 mg/kg.  We quantified


                  cell proliferation by BrdU.  In these particular


                  studies, we used an osmotic pump, a 7-day.  The


                  advantage of that is that the liver--the life span



                  of a hepatocyte in a mouse is generally about 200


                  days, so at any one point in time, you are only


                  going to have 0.5 percent of your hepatocytes




                            So, if we go over the course of 7 days,



                  then, we accumulate all the cells that are divided,


                  all the cell replication that occurred in those 7




                            So, this is an H & E stained liver section


                  of the mouse, and what you see here, you see the



                  inflammation that is common after exposures to


                  cytotoxic agents.  You have the influx of your


                  morphonuclear, of your mononuclear and your


                  polymorphonuclear neutrophils.


                            After you look at the incidence of liver



                  cytotoxicity, after 1, 3, 6, and 13 weeks, you will


                  see that the highest doses, you have a greatly


                  increased incidence of cytotoxicity, also, at 8











                  mg/kg you have a significant increase, and at 4,


                  you have an intermediate response.


                            Cell proliferation.  One of the markers,


                  the ways of quantifying cell proliferation is the



                  labeling index that measures the S phase of the


                  cell cycle.  There are a variety of methodologies.


                  You can look at mitotic figures, quantify those.


                  You can look at KI67 gene, you can look at PCNA, a


                  proliferating cellular nuclear antigen that is part



                  of a quaternary complex.


                            For us, we used BrdU, bromodeoxyuridine.


                  It's a thymidine analogue, so when the DNA is


                  replicating, in place of the thymidine, a certain


                  percentage of the BrdU will be incorporated.



                            Then, we have an antibody to the BrdU, so


                  we immunohistochemically stain for cells that have


                  incorporated this BrdU.  There are a variety of


                  routes of administration depending upon what the


                  endogenous cell proliferative rate is.  You can use



                  a pulse.  So, for instance, if you are looking at


                  cell proliferation in the skin, you may inject IP


                  an hour later euthanize the animals.











                            For us, there is the cumulative is the


                  advantage, as I said earlier, because you can find


                  out the cell proliferation that has occurred over a


                  period of time.



                            Quantifying, how do we quantify this?


                  Well, by light microscopy, we look at these


                  immunohistochemically stained sections.  [Off




                            I am sorry, excuse me.  At the 4 mg/kg,



                  these are the cells that have incorporated the


                  BrdU.  Over here, at 8, you will see many more of


                  them.  So, what we do is we evaluate 2,000 hepatic


                  nuclei and determine the percentage for those


                  nuclei that have incorporated this stain.



                            In our 13-week study, you will see that we


                  have a significant increase at the 15 mg/kg at all


                  three time points, 1, 3, 6, and 13 weeks.  At our 8


                  mg/kg, which I found to be interesting that your


                  calculations were 8 mcg/kg, you have a significant



                  increase at 1, 3, and 6 weeks.  At 3 weeks, you


                  also have an increase in 2 and 4.


                            We also conducted a study in female mice











                  in which they were exposed to 00.5, 1, 2, 4, and 8


                  mg/kg, and you will see that the highest dose of 8


                  mg/kg produced a significant increase in your ALT


                  levels with an intermediate response in the 4.



                            The SDH was elevated in both the 4 and the


                  8 mg/kg.  If you looked at the hepatic labeling


                  index, again, you saw a significant increase in


                  female mice at the highest dose of 8 mg/kg, and no


                  other increases.



                            In light of this data, we conducted a


                  carcinogenicity study, a two-year study.  It was in


                  female B6C3F1 mice.  Our dose levels were 0, 0.5,


                  1, 2, 4, and 8.  Eight, you will recall was the


                  dose that produced both cytotoxicity and an



                  increase in labeling index, that we had 50 to 100


                  animals per group, particularly in our lower groups


                  we wanted to be able to detect the significant


                  increase if there was one, so we increased the


                  number of animals.



                            They were exposed for two years.  They


                  were exposed by gavage, and this was 5 times per













                            We conducted necropsies on these animals


                  after two years, and what you will see here, on the


                  left, is a normal mouse liver.  This was an animal


                  that was exposed to 8 mg/kg, and at gross necropsy,



                  you will often find these masses.


                            We quantified the incidence of these


                  masses at necropsy.  There was a significant


                  increase, 100 percent of the animals had liver


                  masses at the final necropsy, and there was also a



                  significant increase at 4 mg/kg.


                            We evaluated H & E stained liver sections


                  for inflammation.  As before, you will see that at


                  your 8 mg/kg, that there was an increased incidence


                  of livers with both moderate and marked subcapsular



                  inflammation or cytotoxicity, and there was an


                  intermediate response at 4 mg/kg.


                            This is an H & E stained liver section,


                  and what we have here is a hepatocellular tumor.


                  It is a metastatic one as it ended up being.  You



                  can see how there is loss, there is disruption of


                  the normal liver architecture.  So, we evaluated H


                  & E stained sections for the presence of











                  hepatocellular adenomas, carcinomas, and foci.


                            You will see at 4 mg/kg, there was a


                  significant increase in foci.  Foci are believed to


                  be pre-neoplastic liver lesions that have the



                  ability to progress on to become benign or


                  malignant liver tumors.


                            At your 8 mg/kg, you had a significant


                  increase in the incidence of foci, adenomas, and





                            What we say here is that there is a very


                  good correlation between cytotoxicity as measured


                  by ALT or SDH, and labeling index, and the


                  incidence of liver lesions.  So, when those, at 8


                  mg/kg, where you had an increased incidence of



                  cytotoxicity, you had an increase in labeling


                  index, you also had an increase of masses at


                  necropsy, and adenomas and carcinomas




                            At 4 mg/kg, you had somewhat ambiguous



                  intermediate responses in the short-term assays,


                  and you had an increased incidence of lesions or


                  masses at necropsy, and an increased incidence of











                  pre-neoplastic lesions by light microscopy.


                            So, in conclusion, what we can say is that


                  this study demonstrated a dose-dependent increase


                  in furan-induced liver tumors in female B6C3F1



                  mice, and a relationship between the dose,


                  cytotoxicity, compensatory cell proliferation, and


                  tumor induction.


                            An overview.  In this particular study, we


                  have reproduced the results of the NTP bioassay.



                  In the NTP bioassay, they used 8 and 15 mg/kg.


                  They had an increased incidence of liver tumors as


                  did we.


                            What we noticed here is that there is a


                  threshold that doses below 4 mg/kg did not increase



                  the incidence of liver tumors or produce the


                  short-term effects that would suggest that they


                  would be hepatocarcinogenic.


                            At 13 weeks, I did not show the data, but


                  at 13 weeks, we saw that there was an increase in



                  cytotoxicity, there was an increase in labeling


                  index.  We have a stop group, so they were exposed


                  for 13 weeks, then, they were held for an











                  additional 4 weeks.  Both the labeling index and


                  the cytotoxicity returned to normal in that group.


                            There are other chemicals with the same


                  proposed mechanisms - chloroform, carbon



                  tetrachloride, theocitamide [ph], a whole variety,


                  that the mutations or the other events that we saw


                  in these genotoxicity assays may be secondary to


                  hepatocyte cytolethality or increased cell





                            This is a biologically plausible


                  mechanism.  It makes sense that cells are killed,


                  that new cells are produced, and that these


                  particular cells may be more susceptible to DNA or


                  genetic damage.



                            The furan-induced effects after short term


                  exposure are inhibited by p450 inhibitors.  We said


                  that furan is metabolized by cytochrome p450-2E1 to


                  its toxic metabolite, it's a dialdehyde.  If you do


                  studies in which you give the animals the furan and



                  the p450 inhibitor, you do not get an increase in


                  labeling index at these doses.  You do not get an


                  increase in cytotoxicity.











                            There are similar pharmacokinetics in the


                  mouse and in the human in vitro, and in general,


                  the rat metabolizes furan slower than does either


                  the mouse or the human.



                            Future areas of interest.  It would be


                  interesting to know that if you gave your animals


                  p450 inhibitors or maybe developed a transgenic


                  mouse in which that particular gene was knocked


                  out, would you get mouse liver tumors.



                            Are liver tumors due to the bolus dose?


                  So, unlike food where you are taking a little bit


                  in all the time, we gave them their furan in one


                  big dose the first thing in the morning.


                            Are the positive genotoxic results, are



                  they due to direct damage to the DNA, as some of


                  the genotoxicity assays indicated, are they due to


                  the high doses, or are they secondary to cell


                  proliferation or other phenomenon?


                            What are the molecular or the gene



                  expression changes in liver tumors?  That is


                  something that we have been looking at is to try


                  and find out are there growth factors, are there











                  other things, surrogate markers that we could find


                  in the blood that might help us identify those


                  chemicals that are possibly carcinogenic, in


                  particular, liver carcinogenesis, and, in



                  particular, those that do a biocidal toxic




                            Do these same mechanisms occur for


                  cholangiocarcinomas?  Is there a threshold for--and


                  I am sorry, these say cholangiosarcomas, they



                  should be cholangiocarcinomas--is there a threshold


                  for cholangiosarcomas, similar to what we found


                  with the mouse liver?


                            Is the mode of action of the


                  cholangiocarcinomas similar to that of the mouse?



                  Do biliary tract epithelial cells have


                  pharmacokinetic parameters similar to that of


                  hepatocytes?  It is the biliary epithelial cells


                  that are believed to be the precursors of the


                  cholangiocarcinomas. What is the relevance of the



                  mouse liver findings to cholangiocarcinomas and


                  leukemia in humans?


                            This particularly has to do with the











                  pharmacokinetic parameters.  What are the


                  concentrations in the organ systems of interest?


                            Are there populations of humans that are


                  susceptible to furan-induced effects and is age a



                  factor, whether it be the baby food or the elderly?


                  There is a lot of evidence that indicates that


                  infants don't have the same intoxification or


                  detoxification systems as adults, so does that make


                  them more susceptible or less susceptible?



                            Finally, I would like to thank my


                  colleagues, those who actually did the work,


                  particularly, the--well, anyway, my


                  colleagues--particularly the toxicology technicians


                  and the animal care and the laboratory assistants



                  who certainly did 99 percent of this work, the ILS


                  Histology Department for the H & E stained liver


                  sections, Dr. Robert Maronpot at NIEHS, who read


                  the liver sections, Julie Foley at NIEHS, who


                  helped with the cell proliferation studies, and Dr.



                  Tom Goldsworthy.


                            Questions, please?


                                   Questions of Clarification











                            DR. MILLER:  Dr. McBride.


                            DR. McBRIDE:  I have two questions.


                  Firstly, is there any data in the mice that is age


                  related?  Secondly, if I am remembering right, you



                  had the only slide that showed any changes at or


                  below 2 mg/kg dose was the one slide on


                  cytotoxicity, I forget how that was measured, and


                  if I am understanding you right, that was


                  reversible at least in time.



                            DR. MOSER:  That was in the 13-week study,


                  so let me see if we can find that data.  Do you


                  know, was that in the 13-week?


                            DR. McBRIDE:  Yes, at the 13-week.


                  Although it was reversible, it might still be of



                  import because, of course, as we are looking for


                  any change, we are looking for the lowest dose,


                  especially in humans where there may be multiple


                  factors that affect risk.


                            In other words, was this the only finding



                  that you found at 2 or lower mg/kg?


                            DR. MOSER:  I think that we had some cell


                  proliferation at 3 weeks in this 13-week study, so











                  there was a significant increase at 2 weeks at 2


                  mg/kg at 3 weeks, and that is the only significant


                  finding that we had.


                            DR. McBRIDE:  But on your other slide, it



                  was a 0.5 mg/kg, the one before that.


                            DR. MOSER:  Okay, and I think what it was


                  there, it's a statistical thing, we only had 10


                  animals per group, and we had 2 animals that did


                  show some evidence of cytotoxicity.  These slides



                  were read blind.  It is not a significant increase,


                  but there is something.


                            DR. McBRIDE:  And the question of age of




                            DR. MOSER:  Age of mice.  All the



                  short-term studies that we did, the mice were 6 to


                  8 weeks old when we started, and that is because


                  the liver continues to divide and is really not


                  mature until about 10 weeks of age, so we tried to


                  make sure that all of our studies were done the



                  same, be they the short term or the long term.


                            So, that our short-term studies and


                  long-term studies, they all started exposure at the











                  same age.  It is just that with your 2-year


                  studies, of course, we went out to the end.


                            DR. MILLER:  Dr. Gray.


                            DR. GRAY:  I think something that we want



                  to try and learn about here that is really


                  important for thinking about this food situation is


                  something you touched on in one of your last


                  slides, and that is this question of dose rate.


                            Is there any other data to help us



                  understand that, because as you mentioned, other


                  compounds that are thought to act in this way show


                  a strong dose rate effect. For example, chloroform


                  gives you very similar mouse liver carcinogenesis


                  by gavage.



                            Ninety percent, 100 percent response, you


                  give the same dose in drinking water over the


                  course of the day, no tumors at all.


                            DR. MOSER:  Right.


                            DR. GRAY:  And if dose rate is an



                  important factor here, that is something we need to


                  know because that is a big difference between our


                  animal studies here and the way in which people are











                  likely to be exposed.


                            So, I mean I don't know if there is


                  something in the literature you can help us


                  understand, or if it is something that we need to



                  think about as a study going forward is


                  understanding whether there is a strong dose rate


                  effect for liver tumors and the other tumors that


                  are out there.


                            DR. MOSER:  Let me say, and that is why I



                  put it up there, chloroform has a mechanism of


                  inducing liver tumors that appears to be very


                  similar to that of furan.  If you give chloroform


                  by gavage, the same way we gave the furan, you will


                  get liver tumors.  You give them the very same dose



                  in the water, you do not get liver or kidney




                            So, that indicates that maybe small


                  amounts over the course of time doesn't have the


                  same effect as just one huge dose, and particularly



                  maybe first thing in the morning.  They are


                  nocturnal, you know, they move, they do things at


                  night.  So, who knows?  But that is a very, very











                  important thing.


                            DR. GRAY:  And at this point, there really


                  isn't anything in the literature to help us on the


                  furan front on this?



                            DR. MOSER:  I think that Greg Kadaras [ph]


                  has done a little bit of work, and I think it has


                  been inhalation, and I will have to check on that,


                  but he has done work that I believe has been by a


                  mechanism other than gavage.



                            DR. MILLER:  Dr. Krinsky.


                            DR. KRINSKY:  Thank you for the nice


                  cancer cell biology review.  You used the term


                  "threshold" and "dose dependent."  Those are not


                  identical, and I think that is important in terms



                  of human consumption, because if, in fact, your


                  data indicates that there is a threshold level


                  prior to seeing toxicity, that may have very


                  important implications as far as human consumption


                  is concerned.



                            DR. MOSER:  I would agree.  The idea of


                  the threshold is that there is, from my definition,


                  okay, in this study, is there is a dose below which











                  you really don't see the cytotoxicity, you don't


                  see the compensatory cell proliferation, and you


                  don't see the liver tumors, as compared to dose


                  response, which means, you know, a low dose you get



                  a low response, medium dose, medium, high dose,


                  high response.


                            DR. KRINSKY:  And the mechanism for the




                            DR. MOSER:  What we would have to say is



                  that whether it's a matter of intoxication, you


                  know, that there is just so many mixed oxidase


                  function enzymes to produce the toxic metabolite,


                  or there is a detoxification mechanism, you know,


                  it is believed that glutathione is a way of



                  detoxifying the metabolite, and there may well be,


                  and we know that is the case, only so much


                  glutathione, so there is only so much to help us


                  cart that toxic metabolite out.  Beyond that level,


                  you may see toxicity.



                            But the truth of the matter is, when I


                  look at the literature, glutathione is the only


                  detoxification mechanism they have looked at.











                  There may be others.


                            DR. MILLER:  Dr. Russell.


                            DR. RUSSELL:  Again, thank you for that


                  presentation.  In thinking about populations of



                  humans that might be susceptible to furan-induced


                  effects, the one that comes to mind right away to


                  me is alcohol users, because it is such a potent


                  stimulator of p450-2E1.


                            DR. MOSER:  Exactly.



                            DR. RUSSELL:  I think that that ought to


                  be looked at in your model, but in epidemiologic


                  models if this gets carried on to humans, that this


                  really may be a population that is much more





                            DR. MOSER:  I think there was a study done


                  at CIIT, and it was a short-term study in which


                  they induced the cytochrome p450-2E1 by exposing


                  the animals to acetone, it was.  I don't think it


                  was the alcohol should have done the same thing.



                            DR. RUSSELL:  Yes.


                            DR. MOSER:  So, you have these higher


                  levels of the enzyme that is producing the toxic











                  metabolite, and we also know that certainly alcohol


                  consumption is a prerequisite to certain kinds of


                  liver damage and ever certain kinds of liver


                  cancer, so that is a very good point, and I



                  wholeheartedly agree.  Thank you.


                            DR. MILLER:  Dr. Callery.


                            DR. CALLERY:  Let me probe that 2E1 a


                  little further.  I want to know if you know that


                  the 2E1 in mice is the same as the human 2E1 is one



                  question, and then another is, I am probing my own


                  memory here, do you know Carlson's work at Purdue


                  on styrene?


                            DR. MOSER:  No, I am sorry, I don't.


                            DR. CALLERY:  I believe he has got a null



                  2E1 mouse.


                            DR. MOSER:  Oh, does he?


                            DR. CALLERY:  Where the styrene was still


                  converted to styrene oxide and had the same


                  viability for potential carcinogenicity.



                            DR. MOSER:  I think that's interesting


                  because it's like everything else, you know, we


                  have looked at the 2E1.  There may well be other











                  means of intoxication, we just haven't looked at




                            DR. CALLERY:  Or detoxification.


                            DR. MOSER:  Exactly.



                            DR. CALLERY:  The other is I am trying to


                  relate the mouse to the human, and especially in


                  the glutathione concentrations and such, and the


                  redox activity in the glutathione system.  I don't


                  know how that relates to the human.



                            DR. MOSER:  I don't know either.  Does


                  anybody know if the glutathione levels are


                  comparable in mice and in humans, or in the liver


                  anyway?  I am sorry, I don't know.


                            DR. CALLERY:  Then, I guess the last one



                  is you had mentioned that you were at 2 mg/kg and


                  we have an estimation that the human exposure might


                  be 1,000-fold less. Does that have any meaning to




                            DR. MOSER:  Well, there is species



                  extrapolation, as we all know, is extremely


                  difficult.  Not only that, we have got a tissue


                  extrapolation here, you know, that we are not











                  necessarily talking about, a high incidence of


                  liver cancer in this country, not to say that it is


                  not important, and most of the liver cancer that we


                  see in this country we believe to be more



                  associated with hepatitis and alcohol consumption.


                            So, does that mean that I think that those


                  levels are safer?  Not absolutely at all.  It does


                  appear that the mouse is more sensitive to liver


                  tumors than some of our other models.  Actually, a



                  better question is well, what about the


                  cholangiocarcinomas.  I mean they saw almost a 90


                  percent incidence at 2 mg/kg in your NTP bioassay.


                  Is that relevant?


                            Well, here, we don't know, at what dose do



                  you continue to get these cholangiocarcinomas.  As


                  I look at the literature, and I am not an expert on


                  that by any means, it does appear that the rat is


                  more sensitive to at least cholangiocarcinomas than


                  are mice or humans, that in all the reading I have



                  done in the last week or so trying to prepare for


                  this, there is only one study in which there was an


                  increased incidence of cholangiocarcinomas in mice,











                  and that was with PCBs, polychlorinated biphenyls,


                  and they were on some sort of a restrictive diet,


                  and the incidence was not as high as what you are


                  seeing in your rat.



                            So, again, we go back to the species is


                  extrapolation, is that data in rats relevant to


                  humans at that dose.  I wish I knew.


                            DR. MILLER:  Dr. Chesney.


                            DR. CHESNEY:  You just mentioned



                  hepatitis, and I was thinking that another human


                  population that might be at risk for furan are


                  patients with hepatitis B and hepatitis C, and


                  there is an animal model for hepatitis B, and I am


                  blocking on the species.  I think it might be the



                  prairie dog, but I am probably wrong about that.


                            DR. MOSER:  Is it the woodchuck?


                            DR. CHESNEY:  It's the woodchuck, that's


                  right.  Thank you.


                            DR. MOSER:  Don't ask me how I knew that.



                  That just came out of some deep recesses.


                            DR. MILLER:  I am going to call for a


                  break.  Can we be back here in 10 minutes.













                                        Public Comments


                            DR. MILLER:  The FDA received only one


                  request for public comment, that by the National



                  Food Processors Association.  It was to be


                  delivered by Dr. Richard Jarman.


                            Dr. Jarman has said that in the interest


                  of facilitating the discussion, he submitted a


                  statement which he intended to read and which you



                  have all received a copy of, and he is prepared to


                  allow the statement to stand in lieu of having to


                  make a presentation.


                            So, the statement will be incorporated in


                  the record.



                              Summary and Charge to the Committee


                            DR. MILLER:  Now we come to the nitty


                  gritty. Before we begin our discussions, Dr. Terry


                  Troxell of the FDA will summarize and re-present


                  the charge and reading of the questions, and then



                  we will proceed with our discussions.


                            DR. TROXELL:  Thank you.  My name is Terry


                  Troxell, Director, Office of Plant and Dairy Foods.











                            I don't want to take much of your time.  I


                  just want to recapitulate a little bit to


                  facilitate your discussion.  The main points here


                  are the actions we take and what are the data



                  needs, the charge, and the questions.


                            As we have said, we have developed the


                  method.  The method was posted on the web site.  We


                  are going to be doing a round robin, so we should


                  sort out any differences in levels being observed.



                            We did an exploratory survey, more than


                  160 foods were in our first round, and with 40 more


                  since we published a notice in the Federal


                  Register, and 30 from Canada.  We are now at 230


                  foods, and we will be testing a broader range of





                            The preliminary exposure assessment was


                  done.  We utilized the first 160 samples,


                  obviously, because things are moving so fast, we


                  were not able to incorporate all the data that we



                  pulled recently.


                            We also obviously did our call for data


                  and then we have established international











                  interactions.  We provided Canada with our method,


                  and they very quickly developed additional methods,


                  similar, so that we could generate a lot of data


                  together, and we also coordinated, so that we could



                  minimize duplication of effort, so that we can


                  maximize the results at this point.


                            The other thing you should be aware of is


                  that the EU has a heat tox project, which is to


                  look at investigating thermally processed induced



                  toxins in foods, and they are going to incorporate


                  furan in that process.


                            So, again, as with acrylamide, we are


                  trying to maximize our collaborations to try to


                  zero in on the problem as