1. National Toxicology Program Acrylamide Bioassay
and Risk Assessment Considerations
James R. Coughlin, Ph.D.
President, Coughlin & Associates
Aliso Viejo, California
jrcoughlin@cox.net
www.linkedin.com/in/jamescoughlin
Snack Food Association Acrylamide Conference
June 7, 2011
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4. Acrylamide Snapshot: Chemistry and Toxicology
 Occupational neurotoxin in humans; genotoxic & mutagenic in cell cultures
 Known rat carcinogen, classified as “probable human carcinogen”
 Metabolized to glycidamide, an epoxide animal carcinogen
 Mainly formed in the Maillard Browning Reaction from glucose or fructose
and the common amino acid asparagine
 Protective enzymes shown to detoxify acrylamide and glycidamide
 Acrylamide & glycidamide can chemically react with DNA to increase
carcinogenic potential
But for a Full Risk Evaluation, We Need More Focus on:
 Acrylamide & glycidamide can also react with amino acids and proteins in
the human body, which keeps them from later reacting with critical DNA
targets inside cells (see USDA’s Mendel Friedman key reviews):
 Blood hemoglobin adducts are well-known biomarkers of exposure, but
they and other blood / organ proteins are “sinks” for acrylamide
 Dietary proteins may reduce acrylamide uptake during digestion in
humans.
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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 (tox consultant) also presented oral
comments; co-author of two previous acrylamide rat
bioassays (1986, 1995)
 NTP Peer Review Panel – very cursory review; did not address or
modify one conclusion based on industry comments.
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9. Key Issues in Industry Comments
 Glycidamide: The 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 the 2 years; some tumor findings may have
been caused by excess acrylamide CNS toxicity rather than by
acrylamide acting as a carcinogen – but no discussion of this.
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10. Key Issues in Industry Comments (cont’d)
 All 3 commenters argued that Historical Controls for tumors were
poorly chosen, even though they are 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 labs
 Most of the NCTR historical controls 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 tumor stability is known in decades of
NTP historical controls (significant drifting up has occurred).
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11. Female Rat Mammary Fibroadenomas (Benign)
 Where our Historical Controls argument is best illustrated:
 These benign tumors in the 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)
 The 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
 JECFA should reevaluate acrylamide’s risk based on the much
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 believe that tumors of this gland 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 much 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 reevaluate 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 tumors do not occur in humans
 JECFA and others should reevaluate acrylamide’s potential for
human risk based on the much lower incidences of relevant
malignant rat and mouse tumor endpoints (there are several
relevant types of malignant rat & mouse tumors)
 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 rat tumor results from the
Johnson et al. (1986) 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
 Rat and mouse tumors that are relevant to humans
 Updated comparative metabolic and toxicokinetic data.
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18. Food for Thought…
Risk-Benefit Assessment of
Acrylamide-containing Foods
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19. “Risk-Benefit” Assessment Considerations
 Interpretation of rodent cancer bioassays of extreme chemical
doses has 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,
as well as antioxidants and 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. “Risk-Benefit Considerations of Mitigation Measures on Acrylamide
Content of Foods – A Case Study on Potatoes, Cereals and Coffee.”
Seal et al., Br. J. Nutr. 99 [Suppl 2]: S1-S46 (2008).
Expert Report commissioned by the International Life Sciences
Institute/Europe Process Related Compounds Task Force (12
collaborating institutes, universities and companies)
1. To summarize and evaluate the impact of pre-harvest, post-
harvest and processing conditions on acrylamide formation in
potatoes, cereals and coffee.
2. To consider the nutritional value and beneficial health impact of
consuming these commodities.
3. To calculate the impact of mitigation using probabilistic risk-
benefit modeling to demonstrate the principle of this approach.
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26. Dietary Epidemiology Studies of Acrylamide
 Pelucchi et al. 2011. “Exposure to Acrylamide and Human Cancer - A
Review and Meta-analysis of Epidemiologic Studies.” Annals of
Oncology, January.
 The summary Relative Risks for an increase of 10 µg/day of
acrylamide intake were close to 1.0 for all the cancers considered,
ranging from 0.98 for esophageal cancer to 1.01 for colon,
endometrial, ovarian and kidney cancer. None of the associations
was statistically significantly increased.
 “Conclusions: Available studies consistently suggest a lack of an
increased risk of most types of cancer from exposure to acrylamide.”
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27. 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 and non-linear dose modeling
 Dietary epidemiology studies support lack of risk globally
 Consideration of health-protective, beneficial components of
acrylamide-containing foods.
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28. IFT Symposium, New Orleans AM, June 14, 2011
“The long-awaited NTP acrylamide bioassay: Where do we go
from here?”
 “Risk Assessment Considerations” – James R. Coughlin
 “Risk Management Considerations: FDA Update on
Acrylamide in Food” – Nega Beru, FDA
 “Risk Communication Considerations” – Ron P. Guirguis,
Fleishman-Hillard, NY
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