1. National Toxicology Program’s Acrylamide Bioassay:
Risk Assessment Considerations
James R. Coughlin, Ph.D.
President, Coughlin & Associates
Aliso Viejo, California
jrcoughlin@cox.net
www.linkedin.com/in/jamescoughlin
SYMPOSIUM
“The Long-awaited NTP Acrylamide Bioassay:
Where Do We Go from Here?”
Institute of Food Technologists
Annual Meeting, New Orleans
June 14, 2011
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4. Acrylamide Snapshot: Chemistry and Toxicology
 Occupational neurotoxin in humans; genotoxic & mutagenic in
cell cultures
 Foods: mainly formed in the Maillard Browning Reaction from
glucose or fructose and the common amino acid asparagine
 Known rat carcinogen, “probable human carcinogen”
 Metabolized to glycidamide, an epoxide animal carcinogen
 Protective enzymes can detoxify acrylamide and glycidamide
 Can chemically react with DNA to increase carcinogenic
potential.
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5. U.S. National Toxicology Program (NTP) Bioassay
of Acrylamide [April 2011]
 FDA nominated acrylamide and glycidamide for complete
toxicology testing in Nov. 13, 2002 letter:
“…FDA requires a properly designed (dose response considerations
and accounting for the food matrix through which humans are
exposed), well-conducted, GLP-compliant bioassay. Results from
such studies will provide the agency with sound scientific data by
which more accurate risk assessments can be conducted.”
 2-year cancer bioassay of acrylamide in rats and mice fed in
drinking water (untreated control + 4 treatment doses), with
many ancillary studies on metabolism, genotoxicity and
toxicokinetics
 Draft Technical Report No. 575 released mid-Feb 2011 was
peer-reviewed by the NTP Peer Review Panel on April 5.
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6. NTP “Levels of Evidence” of Carcinogenic Activity
 CLEAR Evidence: “dose-related increase of neoplasms”
 SOME Evidence: “a chemical-related increased incidence of
neoplasms” where the “strength of response is less than that
required for clear evidence.” Also designated as “were also
related”
 EQUIVOCAL Evidence: “a marginal increase of neoplasms
that may be chemical related.” Also designated as “may have
been related.”
 NO Evidence: “no chemical-related increases in malignant or
benign neoplasms.”
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7. Acrylamide Bioassay Conclusions: “Clear Evidence of
Carcinogenicity” in Male & Female Rats and Mice
 Male rats: “Clear” - malignant mesothelioma of epididymis & testes;
malignant schwannoma of heart; benign adenoma / malignant carcinoma
of thyroid
“Some”- benign adenoma of pancreatic islets
 Female rats: “Clear” - benign fibroadenoma of mammary gland; benign
papilloma of oral cavity; skin neoplasms; adenoma / carcinoma of thyroid
“Some” - benign liver adenoma; clitoral gland malignant carcinoma
“Equivocal” - malignant schwannoma of heart (“may have been related”)
 Male mice: “Clear” - benign adenoma of Harderian gland and lung; benign
papilloma of forestomach; no increases in malignant tumors
 Female mice: “Clear” - benign adenoma of Harderian gland and lung;
malignant carcinoma of mammary gland; benign neoplasms of ovary;
malignant neoplasms of skin
 “Some” - benign papilloma of forestomach.
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8. Formal Comments by Food and Chemical Industries
 Food Industry (GMA Coalition) – Coughlin, written and oral comments
 North American Polyelectrolyte Producers Association (NAPPA) –
written comments only
 Dr. Joseph Haseman (retired NTP Dir. of Statistics) provided
his critique of many key tumor findings and factual errors;
 Chairman twice referred to Haseman’s comments as “very
detailed” but that “we can’t address them all”; in fact, they
addressed none of the criticisms
 SNF / France (major acrylamide manufacturer) – written comments
 Dr. Marvin Friedman (toxicology consultant) also presented
oral comments; co-author of two previous acrylamide rat
bioassays (1986, 1995)
 NTP Peer Review Panel – I believe a very cursory review; did not
address or modify conclusions based on industry comments.
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9. Key Issues in Industry Comments
 Glycidamide: Draft Acrylamide TR should not be finalized until the
Glycidamide TR is peer reviewed; Panel rejected this advice, even
though Glycidamide’s results can help interpret acrylamide
 Draft Acrylamide TR, page 33: “To test these hypotheses and
to provide data for meaningful risk assessments, studies were
conducted to compare the extent and types of tumors in
B6C3F1 mice and F344/N rats treated chronically with either
acrylamide or glycidamide.“
 We learned that the Glycidamide Draft TR is delayed by 2 years, so at
this time we are not going to get the benefit of this significant,
expensive NTP bioassay to assist in acrylamide’s risk evaluation
 Maximum Tolerated Dose (MTD) was exceeded based on very poor
animal survival over 2 years; some tumor findings may have been
caused by excess acrylamide CNS toxicity rather than by acrylamide
inherently acting as a carcinogen – but no discussion of this.
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10. Key Issues in Industry Comments (cont’d)
 Historical Controls for tumors were poorly chosen, even though they are
meant to be used to help interpret the current study; they give the %
incidence of spontaneous tumors in untreated control animals over
several years of testing in all NTP contract labs
 Most of the NCTR historical control data were 20-23 years old, while
other NTP contract labs have done so many more recent studies; NCTR
has no rat drinking water studies and only one mouse drinking water
study in its database
 We argued that the more recent and extensive NTP historical control
database (March 2010 at NTP’s website) is more appropriate to use
 NCTR’s Dr. Beland defended by simply claiming their controls have been
“stable” since the beginning of their rat / mouse colony 30 years ago; but
no such rat / mouse spontaneous tumor stability is known in decades of
NTP historical controls (significant drifting upward has occurred).
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11. Female Rat Mammary Fibroadenomas (Benign)
 Our Historical Controls argument is best illustrated here
 These benign tumors in acrylamide-dosed rats were unlikely to
be caused by acrylamide, since even the top acrylamide dose
produced less tumors (65%) than seen in the more recent NTP
drinking water, untreated historical controls (74% + 12%); only
the top acrylamide dose (65%) was statistically significantly
increased over concurrent control (33%):
% Tumor Incidence
Acrylamide study concurrent controls: 33%
NCTR historical controls: 35% (27.1 - 42.6%)
Recent NTP “Oral/Dr. Water” controls: 74% + 12%
Acrylamide (lowest to highest dose) 38% 52% 47% 65%*
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12. Risk Assessment Considerations (Rat)
 JECFA used preliminary, non-peer reviewed NTP rat and mouse
tumor data in their February 2010 evaluation and risk assessment of
acrylamide (published March 2010)
 These NTP female rat benign mammary fibroadenoma tumors were
used as the pivotal tumor endpoint in calculating the highest risk
value based on rats
 We believe we demonstrated to NTP that all four acrylamide test
doses produced mammary tumor incidences below the highest
spontaneous background incidence of untreated NTP historical
controls; in addition, this type of tumor occurs in rats but not in
humans, and benign human tumors do not progress to malignancy
 JECFA should reevaluate acrylamide’s risk based on the lower
incidences of malignant rat tumors relevant to humans.
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13. Risk Assessment Considerations (Mouse)
 The NTP male mouse benign Harderian gland adenoma tumors
were used by JECFA as the pivotal tumor endpoint in calculating
the highest risk value based on mice or rats
 The Harderian gland is a tear-producing gland in the third eyelid
of some mammals and rodents but is not found in humans; many
toxicologists & pathologists believe that these tumors are not a
scientifically justified endpoint for human risk assessment
 JECFA acknowledged this: “As humans have no equivalent
organ, the significance of these benign mouse tumors in the
Harderian gland is difficult to interpret with respect to humans.
However, in view of acrylamide being a multisite carcinogen in
rodents, the Committee was unable to discount the effect in the
Harderian gland.”
 JECFA should reevaluate acrylamide’s risk based on the lower
incidences of malignant mouse tumors relevant to humans.
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14. Future Risk Assessment Considerations
 JECFA and other public health authorities should consider
reevaluating the current acrylamide risk assessment after
dismissing consideration of the NTP’s benign tumors in the rat
mammary gland and mouse Harderian gland as not scientifically
relevant to human risk assessment
 These two most sensitive tumor endpoints are not malignant
tumors, and these tumor types are not relevant to humans
 JECFA and others should reevaluate acrylamide’s potential for
human risk based on the lower incidences of relevant NTP
malignant rat and mouse tumor endpoints
 I firmly believe that acrylamide is too important and widespread a
contaminant in the human diet to have its risk determined by
scientifically irrelevant rodent tumor endpoints.
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17. Proposition 65: Risk Assessment Reevaluation is Needed
 The current acrylamide No Significant Risk Level (NSRL) (set 1990):
 NSRL = 0.2 µg/day
 Based on the oral cancer potency estimate of 4.5 per mg/kg-d,
derived by U.S. EPA using only the tumor results from the
Johnson et al. (1986) rat chronic drinking water bioassay
 Linearized multistage analysis of combined incidence data for all
tumors in the CNS, mammary and thyroid glands, uterus and oral
cavity in female F344 rats (no CNS or uterus tumors were
increased in NTP rats)
 The NSRL should be reevaluated using state-of-the-art physiologically-
based toxicokinetic (PB-TK) modeling:
 NTP Bioassay results for rats and mice supersede the two old rat
bioassays
 Use rat and mouse tumors that are more relevant to humans
 Use updated comparative metabolic and toxicokinetic data.
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20. For a Full Risk Evaluation, We Need More Focus on:
 Going beyond just counting tumors and modeling the results
 Acrylamide can also react with amino acids and proteins in the
human body, which can keep it from subsequent reactions with
critical DNA targets inside cells (see USDA Mendel Friedman’s
key reviews):
 Blood hemoglobin adducts are well-known biomarkers of
exposure, but hemoglobin and other blood / organ proteins
can be considered “sinks” for acrylamide, destroying the
molecule
 Dietary proteins may reduce acrylamide uptake during
digestion in humans.
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21. Food for Thought…
Risk-Benefit Assessment of
Acrylamide-containing Foods
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22. “Risk-Benefit” Assessment Considerations
 Interpretation of rodent cancer bioassays of extreme chemical doses
has often been shown to be overly conservative, especially if the
Maximum Tolerated Dose may have been exceeded
 Assessing individual food chemicals has been our focus in the past,
but we now need to consider the risks and benefits of whole foods
using a “Holistic Approach”
 Failure to give proper weight to epidemiology studies showing little or
no increased risk of foods containing the chemical, such as acrylamide
 Past failure to consider the POSITIVE health benefits of foods
containing only trace levels of carcinogens
 Cancer-protective substances:
 nutrients like fiber and vitamins
 antioxidants
 inducers of detoxification enzymes.
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23. General Scheme of Maillard Browning Reaction
Ammonia Melanoidins
Alkyl amines (pigments)
Amine Amino acids
Proteins HEAT
Phospholipids
Amino-Carbonyl
Volatile Compounds
Interaction
(aroma chemicals)
(Amadori Products)
Aldehydes
Ketones Carbonyls
Carbonyl Sugars Esters
Furans Oxazoles Amides (Acrylamide)
Carbohydrates Pyrroles Imidazoles
Lipids Thiophenes Pyridines
Heterocyclic Compounds
Thiazoles Pyrazines
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24. “Maillard Browning Reaction” –
Possible Beneficial Health Effects
 Flavors, aromas, colors and texture of browned foods depend on the
Maillard Browning Reaction, but carcinogens are also formed
 However, Antioxidants (AOX) produced by Maillard Reaction may
protect against diseases linked to oxidative damage (cancer, diabetes,
atherosclerosis, arthritis, inflammation, etc.)
 Specific Maillard Reaction Products (MRPs), including the brown
melanoidin polymers (they are polyphenolic AOX) and heterocyclic
flavor compounds, are known to have antioxidant, anti-carcinogenic
and anti-mutagenic effects
 Some MRPs also induce protective detoxification enzymes, including
the acrylamide detoxification enzyme, glutathione-S-transferase (GST).
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25. Does Acrylamide in Food Pose a Real Risk
to Human Health?
 Risk characterization traditionally includes:
 Rodent cancer bioassay results (like the NTP bioassay)
 Biomarker and metabolic studies in animals and humans
 Reliable data on human intake estimates
 But for acrylamide in heated foods, we must consider…
 Bioavailability may be less in human diets than in water
 Consideration of thresholds for some of the NTP tumors and
non-linear dose modeling; hormonal mechanisms?
 Dietary epidemiology studies generally support lack of
significant risk globally
 Consideration of health-protective, beneficial components of
acrylamide-containing foods.
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26. Thank You!
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