# MEETING SUMMARY AND RESEARCH NEEDS 

> **Federal Interagency Acrylamide Research Meeting**
>
> **September 24, 2002**
>
> **Center for Food Safety and Applied Nutrition**
>
> **Food and Drug Administration, College Park, MD**

## Welcome and Introduction, Desired Meeting Outcomes

> Dr. Bern Schwetz, Senior Science Adviser, Food and Drug Administration
> (FDA), opened the meeting with background on acrylamide (AC). He was
> followed by Dr. Terry Troxell, Director, Office of Plant and Dairy
> Foods and Beverages, Center for Food Safety and Applied Nutrition,
> FDA, who discussed desired meeting outcomes, especially the need to
> coordinate federal research on acrylamide to maximize results and
> effectively use scarce resources.
>
> **Environmental Protection Agency, Research Triangle Park, N.C.**
>
> Dr. Rob DeWoskin, National Center for Environmental Assessment,
> Environmental Protection Agency (EPA) spoke briefly about the IRIS
> review of AC in 1988. Development of IRIS files under the current EPA
> program includes preparation of comprehensive toxicological reviews.
> EPA has begun an IRIS review update in September 2002; internal review
> will be completed by August 2003, and release to the public is
> anticipated in June 2004.
>
> > **National Institute for Occupational Safety and Health, Cincinnati,
> > Ohio**
>
> > Mr. William J. Moorman summarized NIOSH research activities with
> > acrylamide. NIOSH has performed six Health Hazard Evaluations in the
> > past where AC was suspected as a worker problem. (These may be
> > accessed at <http://dshefs.niosh.cdc.gov/hetab/> ). Coal preparation
> > plant workers have reported neurotoxic symptoms and there is concern
> > regarding AC exposures associated with polyacrylamide flocculents
> > used to precipitate coal particles. NIOSH's study in coal
> > preparation plants will describe and evaluate worker exposure to AC,
> > solvents and manganese, and develop a database of neurotoxic
> > chemicals based on the National Occupational Health Survey of
> > Mining. Information will be obtained by focus group discussions with
> > chemical suppliers, mine operators, union and non-union workers.
>
> > NIOSH is also studying potential reproductive and neurological
> > effects of exposure to AC. Worker exposure to AC and congeners will
> > be evaluated using ambient area and personal sampling, dermal
> > sampling, reported exposure data and exposure biomarkers (urinary
> > metabolites, hemoglobin (Hb) adduct levels). In addition, male
> > reproductive health will be assessed (semen quality and sperm DNA
> > integrity, hormone levels, PSA levels and reported reproductive
> > health history). Neurobehavioral parameters will be assessed.
> > Protocol is available from Mr. Moorman of NIOSH.
>
> > Mr. Moorman pointed out several aspects of NIOSH research
> > potentially relevant to FDA. NIOSH will evaluate exposure to
> > hemoglobin (Hb) adducts in non-occupationally exposed people,
> > attempting to distinguish between smokers and those with regular
> > dietary uptake of foods containing high amounts of AC. NIOSH will
> > also assess the relative sensitivity of reproductive and
> > neurological effects. The study will analyze levels of a B6
> > metabolite in urine, as B6 supplementation has been shown to
> > antagonize AC neurotoxicity in rats. Genetic differences (*i.e.*,
> > polymorphisms for enzymes in the pathway) affecting AC metabolism
> > will be evaluated.
>
> **Center for Disease Control and Prevention, National Center for
> Environmental Health**
>
> Dr. Hubert Vesper described CDC's AC research. Currently CDC is
> developing a method to analyze Hb adducts (N-Val) of AC and its
> metabolite, glycidamide (GC). Peptide-based standards and calibrators
> will be developed and characterized. AC and GC adducts in people will
> be assessed in special studies and in NHANES. CDC's method will be
> based on procedures described by Springer *et al* (J. Tox. Environmen.
> Health 1993; 40:161-176) and Jeppsson *et al.* (Clin. Chem. Lab. Med.
> 2002; 40:78-89). The method currently in development is based on a
> well-established procedure which uses a well defined, specific and
> stable analyte, shows a good correlation between exposure and health
> risk, and reflects exposure over the last 3 months. The method also
> shows good precision and accuracy and is independent of fasting status
> and diurnal variation. In the future, CDC hopes to create reference
> materials, perform method comparisons, and establish relationships
> between other analytes (*i.e.*, DNA adducts, free serum AC) and Hb
> adducts.
>
> **National Center for Toxicological Research, FDA, Little Rock,
> Arkansas**
>
> Dr. Daniel Doerge presented a brief review of AC metabolism and
> disposition, and carcinogenicity. NCTR proposes to study AC DNA
> adducts using stable labeled analogs, a validated LC-/MS/MS method,
> and DNA from a short-term rodent exposure (leukocytes and target
> tissues). In an *in vivo* mutagenicity study in transgenic rats and
> mice (Big Blue), administered AC and GC (short-term exposure in
> drinking water), target tissues will be identified and correlated with
> GC-DNA adducts.
>
> NCTR also proposes to develop and validate a LC/MS/MS method for serum
> AC/GC and perform a toxicokinetic analysis for AC and GC, including
> looking at AC bioavailability (*i.v.* vs. oral gavage studies) in an
> AC-fortified diet. AC/GC Hb adducts (N-Val) will be determined in
> rodents after short-term exposure and correlated with rodent GC-DNA
> adducts.
>
> In human volunteers, "background" GC-DNA and AC/GC Hb adducts will be
> measured and compared to those in cigarette smokers. These data will
> be compared with rodent dose-responses for exposure estimation.
>
> > In addition, Dr. Fred Beland will be leading a two-year rodent
> > carcinogenicity bioassay using drinking water exposures to AC and GC
> > in male and female F344 rats and B6C3F1 mice. The benefits and need
> > for using feed-incorporation as the delivery mechanism will also be
> > evaluated. The study will be designed especially to yield a
> > dose-response relationship. GC-DNA adducts levels in target tissues
> > will be correlated with tumor incidences.
>
> > **National Institute for Environmental Health and Safety, Research
> > Triangle Park, N.C.**
>
> > Dr. Jack Bishop presented NIEHS/NTP GeneTox data on AC. A
> > reproductive assessment using continuous breeding has been conducted
> > on AC as well as several AC congeners (N-hydroxymethylacrylamide,
> > methacrylamide, and methylene bisacrylamide). Significant adverse
> > reproductive effects were seen in the absence of overt
> > neurotoxicity. Germ cell assays including the dominant lethal test,
> > the heritable translocation test, PAINT/DAPI 1^st^-cleavage embryo
> > chromosome damage (developed by Dr. Francesco Marchetti, Lawrence
> > Livermore National Laboratory, Livermore, CA), the specific locus
> > test and adduct binding, have been conducted on AC. The NIEHS/NTP
> > study showed that paternal exposure to AC significantly increased
> > the frequencies of zygotes with chromosomal abnormalities especially
> > during the last two weeks of spermatogenesis. There was no selection
> > against unstable aberrations between the first and second metaphase
> > stage. PAINT/DAPI analysis of zygotes and 2-cell embryos showed that
> > unstable aberrations are associated with embryo loss during
> > pregnancy and that stable aberrations are associated with heritable
> > translocations at birth.
>
> > Dr. Bishop also noted that, in a 13-week, multidose, drinking water
> > dominant lethal study on N-hydroxymethylacrylamide conducted by the
> > NTP, the induction of germ cell mutations appeared to be associated
> > with attainment of a total accumulated exposure dose of greater than
> > 1000 mg/kg. This could have important biological relevance for
> > chronic low dose AC exposures in food
>
> Dr. Bishop noted that the review by Dearfield (Mutation Res. 1995
> 330:71-99) has summarized information showing that AC is negative in
> Salmonella, causes chromosome aberrations *in vitro* and *in vivo*, is
> positive in the rat and mouse dominant lethal test, is positive in the
> mouse heritable translocation test, and generally causes reproductive
> and developmental toxicity. However, most of the *in vivo* tests of AC
> have been conducted in mice using the *i.p.* route of exposure and at
> relatively high doses of 50-150 mg/kg.
>
> Dr. Bishop recommended that human epidemiology studies (for example,
> those under development by NIOSH) should include collection of sperm
> for sperm FISH analysis and measure of adducts, protamine and DNA . A
> low-dose PAINT/DAPI study should also be conducted.
>
> **Center for Food Safety and Applied Nutrition, Food and Drug
> Administration, College Park, MD**
>
> Dr. Richard Canady summarized CFSAN's data on AC. The agency's initial
> response was to perform a hazard assessment using the Swedish data on
> AC levels in foods, U.S. consumption rates and FDA dose-response
> evaluations developed for AC in food packaging contact issues. This
> assessment has indicated that the Swedish data is probably correct,
> further action is needed, and that the hazard is not clearly
> insignificant.
>
> FDA's occurrence data shows that the range of AC levels is similar to
> that reported previously at the WHO Consultation, and that cooking
> time and temperature make a difference in AC levels.
>
> In 2003, the Total Diet Study will include AC monitoring. FDA will
> encourage collaboration between government, trade groups, consumer
> groups, and academia to achieve public health improvements.
>
> FDA will hold a public meeting on September 30, 2002 and a Joint
> Institute for Food Safety and Applied Nutrition (U of MD/CFSAN
> consortium) meeting October 28-30 in Chicago in which members of the
> food industry will participate.
>
> Since AC levels seem to increase with frying or baking, the need
> exists to clarify nutritional needs vs. risk aversion choices. For
> example, if we reduce exposure, what negative impact will this action
> cause on nutrition, microbial risk and other added risk factors? It is
> important to FDA that these risk management alternatives be discussed
> in public meetings, such as the meeting planned September 30, 2002.
>
> Dr. Canady also summarized the conclusions of the World Health
> Organization Consultation on Acrylamide. Analytical methods are judged
> adequate to confirm occurrence. The formation mechanism of AC in foods
> in unknown. Exposure is in the sub to low mcg/kg/day range, with
> children possible receiving several fold higher levels. Neurotoxicity
> lowest observed adverse effect levels (LOAELS) are well above current
> observed consumer exposures. AC is an animal carcinogen and may also
> induce heritable damage. The consumer message to date is to reinforce
> dietary guidelines (*i.e.*, consumption of a balanced diet) with
> limited advice on cooking.
>
> The WHO Consultation listed the following research needs: 1) define
> GC-DNA binding as a marker of toxicity/risk; 2) describe the
> relationship of Hb adduct to DNA adducts in different organs; 3)
> describe the susceptibility to AC, as influenced by metabolism
> variations, age, gender, other, etc.; 4) evaluate human exposure using
> biomarkers which are correlated and calibrated with intake; 5) look at
> other sources of exposure; 6) acquire toxicity/carcinogenicity data
> for GC; 7) identify mechanisms for germ cell damage and
> linearity/non-linearity of genotoxicity; and 8) conduct an
> epidemiology study of cancer and testicular effects in workers that
> had neurotoxic signs and high AC-Hb adducts measures.