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                              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


 

 

 

 

 

 

 

 

                                                                             2

 

 

                                          PARTICIPANTS

 

                  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.

 

                  FDA

 

                  Dr. Henry Kim


 

 

 

 

 

 

 

 

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                                        C O N T E N T S

 

                                                                          PAGE

 

                  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

 

                    Exposure

                    Jeremy Mihalov                                          36

 

                    Formation

                    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


 

 

 

 

 

 

 

 

                                                                             4

 

                                     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

 

                  deliberations.

 

                            When I call your name, will you please

 

 

                  just repeat your name and the institution with

 

                  which you are associated.

 

                            First, Dr. Acholonu.


 

 

 

 

 

 

 

 

                                                                             5

 

                            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

 

                  University.

 

                            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


 

 

 

 

 

 

 

 

                                                                             6

 

                  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

 

 

                  them.

 

                                 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.


 

 

 

 

 

 

 

 

                                                                             7

 

                  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

 

                  Committee.

 

                            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

 

                  Waslien.

 

                            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


 

 

 

 

 

 

 

 

                                                                             8

 

                  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.


 

 

 

 

 

 

 

 

                                                                             9

 

                  Miller.

 

                            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.


 

 

 

 

 

 

 

 

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                                        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


 

 

 

 

 

 

 

 

                                                                            11

 

                  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

 

                  study.

 

                            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

 

                  foods.

 

                            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,


 

 

 

 

 

 

 

 

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                  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.


 

 

 

 

 

 

 

 

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                            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


 

 

 

 

 

 

 

 

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                  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

 

                  foods.

 

                            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


 

 

 

 

 

 

 

 

                                                                            15

 

                  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


 

 

 

 

 

 

 

 

                                                                            16

 

                  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

 

                  response.

 

                            In the call for data we issued on May 7,


 

 

 

 

 

 

 

 

                                                                            17

 

                  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


 

 

 

 

 

 

 

 

                                                                            18

 

                  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

 

                  event.

 

                            3.  To determine whether the carcinogenic


 

 

 

 

 

 

 

 

                                                                            19

 

                  activity of furan is secondary to its hepatotoxic

 

                  effects.

 

                            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

 

                  safety.

 

                            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


 

 

 

 

 

 

 

 

                                                                            20

 

                  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

 

                  packages.

 

                            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.


 

 

 

 

 

 

 

 

                                                                            21

 

                            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

 

                  toxicology?

 

 

                            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


 

 

 

 

 

 

 

 

                                                                            22

 

                  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

 

 

                  Perfetti.

 

                            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


 

 

 

 

 

 

 

 

                                                                            23

 

                  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

 

                  laboratory.

 

                            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


 

 

 

 

 

 

 

 

                                                                            24

 

                  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

 

                  product.

 

                            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.


 

 

 

 

 

 

 

 

                                                                            25

 

                            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


 

 

 

 

 

 

 

 

                                                                            26

 

                  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


 

 

 

 

 

 

 

 

                                                                            27

 

                  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

 

                  vials.

 

                            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.


 

 

 

 

 

 

 

 

                                                                            28

 

                            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


 

 

 

 

 

 

 

 

                                                                            29

 

                  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


 

 

 

 

 

 

 

 

                                                                            30

 

                  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

 

                  jars.

 

                            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.


 

 

 

 

 

 

 

 

                                                                            31

 

                            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


 

 

 

 

 

 

 

 

                                                                            32

 

                  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.


 

 

 

 

 

 

 

 

                                                                            33

 

                            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

 

                  levels.

 

                            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

 

 

                  formulas.

 

                            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


 

 

 

 

 

 

 

 

                                                                            34

 

                  data in order to be able to come to that

 

                  conclusion.

 

                            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

 

                  differences.

 

                            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


 

 

 

 

 

 

 

 

                                                                            35

 

                  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

 

                  itself.

 

                            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

 

                  microwaved.

 

                            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


 

 

 

 

 

 

 

 

                                                                            36

 

                  also?

 

                            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.

 

 

                                            Exposure

 

                            MR. MIHALOV:  My name is Jeremy Mihalov,

 

                  Office of Food Additive Safety.  This also was done


 

 

 

 

 

 

 

 

                                                                            37

 

                  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

 

                  separately.

 

                            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


 

 

 

 

 

 

 

 

                                                                            38

 

                  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

 

                  corn.

 

 

                            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


 

 

 

 

 

 

 

 

                                                                            39

 

                  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

 

                  100.

 

 

                            Going back to discussing the model,

 

                  generally, when you do an exposure assessment,

 

                  there is a certain amount of uncertainty, and we


 

 

 

 

 

 

 

 

                                                                            40

 

                  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

 

                  exposure.


 

 

 

 

 

 

 

 

                                                                            41

 

                            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

 

                  weight/day.

 

                            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


 

 

 

 

 

 

 

 

                                                                            42

 

                  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

 

 

                  grow.

 

                            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.


 

 

 

 

 

 

 

 

                                                                            43

 

                            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

 

 

                  correction.

 

                            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


 

 

 

 

 

 

 

 

                                                                            44

 

                  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


 

 

 

 

 

 

 

 

                                                                            45

 

                  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

 

 

                  assumption?

 

                            MR. MIHALOV:  I couldn't say.

 

                            DR. MILLER:  Dr. Nelson.


 

 

 

 

 

 

 

 

                                                                            46

 

                            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

 

                  that?

 

                            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


 

 

 

 

 

 

 

 

                                                                            47

 

                  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


 

 

 

 

 

 

 

 

                                                                            48

 

                  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?


 

 

 

 

 

 

 

 

                                                                            49

 

                            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

 

                  furans.

 

                                           Formation

 

                            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

 

 

                  Ottawa.

 

                            I would like to take you through the

 

                  background as far as Health Canada is concerned on


 

 

 

 

 

 

 

 

                                                                            50

 

                  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


 

 

 

 

 

 

 

 

                                                                            51

 

                  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


 

 

 

 

 

 

 

 

                                                                            52

 

                  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,


 

 

 

 

 

 

 

 

                                                                            53

 

                  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,


 

 

 

 

 

 

 

 

                                                                            54

 

                  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

 

                  produced.

 

 

                            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

 

                  acid.

 

 

                            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


 

 

 

 

 

 

 

 

                                                                            55

 

                  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

 

                  trilinolenate.

 

                            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


 

 

 

 

 

 

 

 

                                                                            56

 

                  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

 

                  second.

 

                            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

 

                  method.

 

 

                            When you look at adult foods, we found

 

                  that the chili products had the highest levels

 

                  amongst those that we analyzed with levels ranging


 

 

 

 

 

 

 

 

                                                                            57

 

                  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


 

 

 

 

 

 

 

 

                                                                            58

 

                  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

 

                  available.

 

                            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


 

 

 

 

 

 

 

 

                                                                            59

 

                  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


 

 

 

 

 

 

 

 

                                                                            60

 

                  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

 

                  changing.

 

                            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

 

                  yet.


 

 

 

 

 

 

 

 

                                                                            61

 

                            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


 

 

 

 

 

 

 

 

                                                                            62

 

                  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

 

                  compound.

 

                            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


 

 

 

 

 

 

 

 

                                                                            63

 

                  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

 

                  also?

 

                            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


 

 

 

 

 

 

 

 

                                                                            64

 

                  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,


 

 

 

 

 

 

 

 

                                                                            65

 

                  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


 

 

 

 

 

 

 

 

                                                                            66

 

                  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

 

 

                  correct?

 

                            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

 

                  database.

 

 

                            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


 

 

 

 

 

 

 

 

                                                                            67

 

                  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


 

 

 

 

 

 

 

 

                                                                            68

 

                  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

 

                  comparable.

 

                            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


 

 

 

 

 

 

 

 

                                                                            69

 

                  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


 

 

 

 

 

 

 

 

                                                                            70

 

                  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


 

 

 

 

 

 

 

 

                                                                            71

 

                  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


 

 

 

 

 

 

 

 

                                                                            72

 

                  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


 

 

 

 

 

 

 

 

                                                                            73

 

                  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.


 

 

 

 

 

 

 

 

                                                                            74

 

                            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


 

 

 

 

 

 

 

 

                                                                            75

 

                  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


 

 

 

 

 

 

 

 

                                                                            76

 

                  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

 

                  dividing.

 

                            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

 

                  days.

 

                            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


 

 

 

 

 

 

 

 

                                                                            77

 

                  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.


 

 

 

 

 

 

 

 

                                                                            78

 

                            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

 

                  microphone.]

 

                            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


 

 

 

 

 

 

 

 

                                                                            79

 

                  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

 

                  week.


 

 

 

 

 

 

 

 

                                                                            80

 

                            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


 

 

 

 

 

 

 

 

                                                                            81

 

                  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

 

                  carcinomas.

 

 

                            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

 

                  microscopically.

 

                            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


 

 

 

 

 

 

 

 

                                                                            82

 

                  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


 

 

 

 

 

 

 

 

                                                                            83

 

                  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

 

                  proliferation.

 

 

                            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.


 

 

 

 

 

 

 

 

                                                                            84

 

                            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


 

 

 

 

 

 

 

 

                                                                            85

 

                  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

 

                  mechanism.

 

                            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


 

 

 

 

 

 

 

 

                                                                            86

 

                  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


 

 

 

 

 

 

 

 

                                                                            87

 

                            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


 

 

 

 

 

 

 

 

                                                                            88

 

                  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

 

                  mice?

 

                            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


 

 

 

 

 

 

 

 

                                                                            89

 

                  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


 

 

 

 

 

 

 

 

                                                                            90

 

                  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

 

                  tumors.

 

                            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


 

 

 

 

 

 

 

 

                                                                            91

 

                  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


 

 

 

 

 

 

 

 

                                                                            92

 

                  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

 

                  threshold?

 

                            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.


 

 

 

 

 

 

 

 

                                                                            93

 

                  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

 

                  susceptible.

 

 

                            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


 

 

 

 

 

 

 

 

                                                                            94

 

                  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


 

 

 

 

 

 

 

 

                                                                            95

 

                  means of intoxication, we just haven't looked at

 

                  them.

 

                            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

 

                  you?

 

                            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


 

 

 

 

 

 

 

 

                                                                            96

 

                  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,


 

 

 

 

 

 

 

 

                                                                            97

 

                  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.


 

 

 

 

 

 

 

 

                                                                            98

 

                            [Break.]

 

                                        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.


 

 

 

 

 

 

 

 

                                                                            99

 

                            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

 

 

                  foods.

 

                            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


 

 

 

 

 

 

 

 

                                                                           100

 

                  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