National Toxicology Program Acrylamide Bioassay and Risk Assessment Considerations

James R. Coughlin, Ph.D.President, Coughlin & Associates

Aliso Viejo, California

jrcoughlin@cox.netwww.linkedin.com/in/jamescoughlin

Snack Food Association Acrylamide ConferenceJune 7, 2011

1

2

3

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.4

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.

5

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.”

6

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.7

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 commentsDr. 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.

8

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.

9

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).

10

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%

11

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.

12

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.

13

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.

14

15

16

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.

17

Food for Thought…

Risk-Benefit Assessment of Acrylamide-containing Foods

18

“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.

19

20

21

22

23

AmmoniaAlkyl aminesAmino acidsProteinsPhospholipids

AldehydesKetonesSugarsCarbohydratesLipids

CarbonylsEstersAmides (Acrylamide)Heterocyclic Compounds

Amine

Carbonyl

Amino-CarbonylInteraction

(Amadori Products)

HEATHEAT

Furans OxazolesPyrroles ImidazolesThiophenes PyridinesThiazoles Pyrazines

Melanoidins(pigments)

Volatile Compounds(aroma chemicals)

General Scheme of Maillard Browning Reaction

“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).

24

“Risk-Benefit Considerations of Mitigation Measures on AcrylamideContent 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.

25

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.”

26

Does Acrylamide in Food Pose a Real Riskto 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 considerBioavailability may be less in human diets than in water

Consideration of thresholds and non-linear dose modeling

Dietary epidemiology studies support lack of risk globallyConsideration of health-protective, beneficial components of

acrylamide-containing foods.27

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

28