# SUMMMARY OF DISCUSSIONS OF BREAKOUT GROUPS

**BREAKOUT GROUP I. Toxicity, Toxicokinetics and Bioavailability of
Acrylamide (AC)**

Question 1: What research is needed to improve the risk characterization
of AC relevant to food exposures?

The group stated the need to better define germ cell mutations generated
by AC and/or glycidamide (GC). Low and multiple dose exposure effects
need to be defined; a dose response needs to be established. Results
from acute, subchronic and chronic exposures should be assessed; out
comes should be compared when AC is administered in a food matrix or in
the drinking water. Past routes of exposure to AC have largely been i.p.
and/or skin painting.

It was pointed out that FDA/CFSAN has never regulated a substance based
on positive germ cell mutations in animals. The group recommended that
other types of mutations resulting from AC and particularly GC, be
re-assessed. Some members believed that the Big Blue Mouse, Big Blue Rat
and Tk+/- animal models would be appropriate to use in such assessments
along with standard assays, such as micronuclei, etc.

The group agreed that neurotoxicity had been well documented for AC.
However, when conducting the chronic studies with AC and GC, notice
should be taken of neurotoxic endpoints, such as distal peripheral
axonopathy or synaptopathy, energy pathways, e.g., mitochondrial energy
pathways.

All members thought that hemoglobin (Hb) adduct determination was an
excellent way to assess exposure, in both animal models and in humans.
The need to measure protamine adducts in germ cells was also discussed.
Every effort should be made to involve the NHANES database and to make
certain that measurements for AC be included in future NHANES as soon as
possible. Though NHANES is a cross-sectional study that provides only
limited information on dietary behavior, it allows the assessment of the
general AC exposure of the U.S. population and provides data on smoking
and occupation, two other possible sources of AC exposure. Furthermore,
it provides information on diseases and health conditions such as anemia
(low Hb concentrations in blood) or diabetes, (formation of Hb adducts
of glucose that may compete with AC adduct formation) which could
confound results obtained with Hb adducts. However, the importance of
these possible confounders needs to be assessed. Further specialized
studies investigating AC exposure from food need to be performed.
Perhaps susceptible populations or populations known to consume large
amounts of fried foods should be over-sampled. Many group members
believed that an epidemiology study would be extremely difficult given
the wide variety of "rodent carcinogens" and other "tumor promoter"
substances known to be in most American diets.

The determination and identification of DNA adducts by NCTR in upcoming
rodent studies was strongly supported, and some believed it important to
attempt to determine if such adducts are present in humans, perhaps
using human lymphocytes.

The importance of comparing and contrasting metabolic and
pharmacokinetic studies of AC and GC in rodents was discussed. The issue
of polymorphism was discussed. It was noted that only induction of
particular isozymes, *e.g.*, 2E1, was likely in rodents, but that
polymorphism would certainly be an issued for humans exposed to AC. Some
members of the group believed that human metabolism and pharmacokinetic
studies should be considered.

The group supported the need to conduct a well-controlled chronic
toxicity/carcinogenicity study that would establish a dose response,
thus providing CFSAN with data that would be the basis for conducting a
scientifically sound risk assessment. Many believed that it was
particularly important to conduct cancer studies in the mouse because of
metabolic similarities to humans. Parallel studies with AC and GC were
deemed important, both in the rat and in the mouse model. All agreed
that it was important to determine differences in bioavailability
between AC/GC administration in drinking water vs. a food matrix. The
consensus of the group was that FDA's nomination of AC and GC to the
National Toxicology Program as FDA's Fiscal Year '03 priority chemical
was appropriate. It was agreed that assessment of germ cell damage
(adduct and structural/numerical chromosome damage) should be included
in the NTP study.

It was also agreed that the FDA-sponsored studies should include hormone
level assessment, particularly those hormones that modulate organ and
cell growth in the sex organs. Several discussants mentioned work being
done by researchers outside government to assess the effect of AC on
endocrine modulation, focusing on LH/FSH levels and on prolactin,
estrogen and progesterone levels. Hormone studies should also be done in
humans; NIOSH intends to study hormones in the male reproductive studies
in workers exposed to AC.

**Research needs may be summarized as follows:**

**Germ cell mutations:**

-   There is a need to define more completely germ cell mutations
    generated by AC and/or GC. The current mouse germ cell data
    (dominant lethal, heritable translocation, morphological specific
    locus and Paint/Dapi) define well the type of damage induced by AC
    and GC at high doses. The issue is only relevant to low dose
    exposures.

    -   Assess low dose and multiple dose exposure effects;

    -   Establish a dose response;

    -   Assess the results from acute, subchronic and chronic exposures,
        and

    -   Compare the outcomes when administered in a food matrix or in
        drinking water.

-   Oral route of exposure judged to be most relevant, as opposed to
    past studies which used i.p. and or skin painting.

-   Incorporation of germ cell toxicity into NIOSH epidemiology study.

-   Accumulation of defects over time from repeated exposures.

Regulatory importance of this endpoint: Germ cells may be the most
sensitive target. Exposure assessments may demonstrate significant germ
cell accumulations that extrapolate to significant germ cell damage
and/or significant germ cell damage in chronic low dose exposure studies
may be demonstrable. If these prove to be true, is CFSAN willing to
regulate a compound based on positive germ cell mutations in animals?

Other Types of Mutations:

-   Re-assess the types of mutations that result from AC and
    particularly GC.

    -   Use Big Blue Mouse, Big Blue Rat and Tk+/- animal models.

    -   Use in addition standard Salmonella tests, micronuclei,
        chromosomal aberrations, etc. models.

## Neurotoxicity

Include neurotoxic endpoints, such as distal peripheral axonopathy,
synaptopathy, energy pathways, (*e.g.*, mitochondrial) when conducting
the chronic studies with AC and particularly with GC.

**DNA and Hemoglobin Adducts:**

-   Use Hb adduct determination as best way to assess exposure, in both
    animal models and in humans.

-   Include measurements for AC in future NHANES assessments as soon as
    possible.

-   Determine if an epidemiology study is feasible, given the difficulty
    of distinguishing between effects caused by AC/GC and other "rodent
    carcinogens" and "tumor promoters" in the American diet.

-   Determine if such adducts are present in humans, perhaps using human
    lymphocytes.

-   Measure protamine adducts in sperm.

**Metabolism and Pharmacokinetic Studies:**

-   Compare and contrast general metabolism and pharmacokinetic studies
    of AC and GC in rodents.

-   Evaluate AC induction of particular isozymes (*e.g.*, CYP2E1) in
    rodents to help interpretation of relevance of induction (by AC and
    other inducers) in humans.

-   Evaluate likely effect of CYP2E1 polymorphism for humans exposed to
    AC. Consider performing human metabolism and pharmacokinetic
    studies.

**Subchronic and Chronic Studies:**

-   There is a need for a well-controlled chronic
    toxicity/carcinogenicity study, establishing a dose response.

-   Recognizing the past two industry-sponsored studies in rats, cancer
    studies in the mouse are particularly important to conduct because
    of metabolic similarities to humans.

-   Parallel studies with AC and GC are important, both in the rat and
    the mouse model.

-   Differences between AC/GC administered in drinking water and in a
    food matrix should be determined.

-   Both AC and GC are being nominated by FDA to the National Toxicology
    Program as FDA's Fiscal Year 03 priority chemicals. Such studies
    will be conducted at NCTR under an interagency agreement between the
    FDA/NCTR and the NIEHS/NTP.

Alternative Modes of Action for Carcinogenicity:

There is ongoing research outside government to assess the effect of AC
on endocrine modulation, focusing on LH/FSH levels and on prolactin,
estrogen and progesterone levels. Studies conducted by the government
should include hormone level assessments, particularly those hormones
that modulate organ and cell growth in the sex organs. Interface with
non-governmental researchers should be maintained.

**Reproductive and Developmental Studies:**

It was generally agreed that measurement of protamine and DNA adducts in
sperm, and Paint/Dapi and sperm FISH assessment of numerical and
structural chromosome damage in sperm would be included in any NTP
bioassay of AC or GC.

## *Question 2. What are the priority needs; what sequencing of research is needed?*

The first four research projects may be viewed as basic to the other
suggested research areas and are therefore ranked as top priority.

Cooperation and data sharing:

Communication and sharing of data between government, academia and
industry, as well as European research groups, are top priorities.

Method of analysis for Hb adducts:

Hb adducts appear to be the current most appropriate biomarker of AC
exposure. A method for analysis of adducts in human and rodent tissues
must be developed and tested collaboratively. In addition, the effect of
cumulative exposure on adduct formation should be tested.

Bioavailability studies:

These studies should include effects of storage, various cooking
temperatures and conditions, etc. on levels and bioavailability of AC
and GC in rodent and human diets and drinking water.

Germ cell mutations:

-   Incorporate germ cell toxicity observations into NIOSH epidemiology
    study. Do defects accumulate over time from repeated exposures to
    AC?

-   Current mouse germ cell data (dominant lethal, heritable
    translocation, morphological specific locus and PaintDapi) define
    the type of damage induced by AC and GC at high doses. However, low
    dose and multiple dose exposure effects should be assessed, and a
    dose response should be established. Outcomes should be compared
    when the doses are administered in food or in drinking water as
    opposed to i.p. administration and/or skin painting.

The remaining research projects will require significantly more time and
effort and, to some extent, depend on successful completion of the above
three areas.

Metabolism and disposition of AC:

-   Examine AC/GC Hb adduct formation/elimination kinetics when doses
    have been administered from the diet.

-   Determine rodent GC-DNA and AC/GC Hb adducts.

-   Determine polymorphism of P450 2E1 and its significance in response
    to AC.

-   Synthesize and characterize GC-DNA nucleoside adducts, develop and
    validate an analytical method (LC-ES/MS/MS) for these adducts. Use
    leukocytes and target tissues in rodents to determine DNA adduct
    levels from short-term exposure.

*In vivo* mutagenicity:

Use transgenic mice (Big Blue, Tk+/- mice) with short-term drinking
water exposures to AC and GC. Identify target tissues and correlate with
GC-DNA adducts.

Biomarkers in humans:

-   Measure "background" GC-DNA and AC/GC Hb adducts in "normal" and
    smoking volunteers. Compare these levels with rodent dose-responses
    to estimate exposure. Investigate effect of diet, such as high AC
    foods, on adduct levels. There are urinary markers of AC exposure in
    humans. Recent human exposure has been evaluated on levels of
    mercapturic acids (AC metabolites with an estimated 8 hour
    half-life) in mid- to late-workweek, and post-shift urine samples in
    AC-exposed workers.

Carcinogenicity bioassay:

Drinking water exposure to AC and GC in male and female mice and rats.
Correlate GC-DNA adduct levels in target tissues with tumor incidences.

*Question 3. Identify any areas of overlap and potential coordination
between the agencies or with outside parties (where such research
efforts are known to exist) for planned research.*

Results of the recent Joint Institute for Food Safety and Applied
Nutrition (JIFSAN -- consortium between University of Maryland and
FDA/CFSAN) conference in Chicago should help in elucidating what areas
industry is pursuing in AC research. Results of the meeting will be
available in the near future. An acrylamide working group including
members of the food industry, academia, and the federal government has
been formed. Data are to be shared through the CFSAN Risk Assessment
Consortium. It is important that industry research results be made
available to federal researchers so that duplication of effort may be
avoided and industry results may be evaluated in the light of federal
efforts. It would be helpful if industry and federal research groups
would use a similar method for analyzing foods for AC so that results
would be comparable. Similarly, if industry is analyzing samples of
blood or other tissue from humans for either the Hb adduct or the DNA
adduct, the methods used by industry researchers should be compatible
with those used by federal agency laboratories.

Research on DNA adducts should continue. Dr. Beland of NCTR indicated
that an analytical method should be available in about one year.

Ongoing communication should be maintained between federal agencies (and
with industry) to prevent overlap and to efficiently utilize resources.
One agency may be expected to benefit in the conduct of its AC/GC
research by knowing the progress and findings of other federal agencies.

NIOSH is conducting an assessment of both neurotoxicity and male
reproductive health. The neurobehavioral assessment will include
measurements of tactile sensitivity, postural stability, manual
dexterity, and simple reaction time. The male reproductive assessment
will include semen, hormone, and prostate specific antigen analysis, as
well as reported reproductive health history, of both exposed and
unexposed workers. Exposure will focus on biomarkers of exposure and
effects. Project officer is William Moorman, NIOSH/CDC.

There is a need to establish a federal interagency workgroup as a means
of facilitating communication and, where possible, of enabling
coordination (through an IAG or other means) of research across federal
agencies.

BREAKOUT GROUP II -- Measuring and evaluating exposures to acrylamide
through biomarkers (exposure or effect)

Question 1. What research is needed to improve the risk characterization
of acrylamide relevant to food exposures?

The group identified the following research needs:

-   Measuring chronic exposure to AC and its association to health
    outcomes and germ mutations

-   Establishing correlation between animal studies and human health

-   Epidemiological studies (cross-sectional and longitudinal).

The first area of research for developing biomonitoring markers should
focus on protein adducts for AC and GC, since this methodology is fairly
well developed to date. A key problem is variability; that is, multiple
analyses of the same sample may produce widely varying levels of AC.
Detection limits need to be established and adducts should be
characterized across species, laboratory animals vs. humans. Analytical
standards need to be acquired and Round Robin analysis trials will be
necessary.

After the methods have been validated, samples from NHANES and other
appropriate populations should be sampled for AC and GC. The NHANES
sampling will not only permit the establishment of a reference range for
humans, but also provide opportunities to look at exposure pathways,
such as smoking and possible dietary intake. It may also be possible to
segment out more susceptible populations of exposure for further study.

Research on DNA adducts should also continue as work on protein adducts
for AC proceeds. Leukocytes and hair were mentioned as possible matrices
for DNA adduct research.

FDA/CFSAN may need to address the issue of cumulative exposure of the
consumer to AC, *e.g.*, from smoking, from consuming fried foods, from
food contact packaging materials, etc.

## *Question 2. What are the priority needs; what sequencing of research is needed?*

Priority should be given to development of a reliable method for
measuring AC Hb adducts. CDC should put together a meeting as described
above including federal, European, academic and industry researchers by
either December 2002 or January 2003. Coordinator is Dr. Gary Myers of
CDC.

At the same time, development of the DNA adduct analysis method should
proceed, as well as the nomination of AC and GC for the NTP bioassay
program.

Communication between federal agencies, industry and academia involved
in AC research is a priority.

*Question 2. Identify any areas of overlap and potential coordination
between the agencies or with outside parties (where such research
efforts are known to exist) for planned research.*

Possibilities exist for collaboration between CDC and NIOSH on a cohort
study and with NCTR on methodological issues. It was suggested that a
more permanent interagency working group on AC exposure research be
established to increase efficiency in governmental research efforts.

It was agreed that CDC should convene a select working group of
laboratory experts to look at measurement issues needed to improve and
standardize AC measurement results in humans. This meeting should
include representatives of industry, European countries, academia and
federal agencies to get the maximum benefit from available laboratory
resources. Due to the urgency, CDC will attempt to convene such a
meeting in Dec. 2002. Coordinator is Dr. Gary Myers of CDC.