Translational Toxicology and Exposomics for Food Safety

Risk Management

Yongning WU China National Center for Food Safety Risk Assessment，

Beijing，100050，China

2

Introduction

• Risk Analysis Paradigm

• Translational Toxicology & TT21C

• Exposome & Exposomics

• Perspectives

Food Safety Law 2009

- Protection of people’s health is the ultimate priority

- Take measures on scientific basis and in consideration of

international trends and public sentiment

Basic principle

- National and local authorities

- Food-related business operators

- Consumers

Responsibilities and roles of persons concerned

- Policy formulation based on the risk assessment

- Obligation for risk communication

Basic direction for policy formulation:

Implementation of risk analysis method

Establishment of Food Safety Commission

Risk Analysis Paradigm

Scientific advice and

information analysis Regulation

and control

Dialog with

all stakeholders

Risk Assessment

(Science)

Risk Management

(Policy)

Risk Communication

Risk Assessment

What is the nature and magnitude of

the health risk associated with a

particular chemical?

How should the risk be managed and

communicated to those affected?

Risk Management

Food Safety Risk Analysis

2. Management option assessment

3. Implementation of management decision

4. Monitoring and review

i) Hazard identification

ii) Hazard characterization

iv) Ｒｉｓｋ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ

iii) Exposure assessment

Interactive exchange of information and opinions

throughout the risk analysis process about risk and

related issues

Risk Management Risk Assessment

1. Preliminary Risk Management activities

Risk Communication

interaction

Risk Assessment Process

2.Hazard characterisation (dose response relationship)

1. Hazard identification (adverse effects)

4. Risk characterisation (comparison of

exposure to health-based reference values)

3. Exposure Assessment

(population exposure)

The Nature of Advice to Risk Managers

• Quantitative advice

- level of risk at given exposure (difficult to obtain)

- ADI/ TWI (most common)

- margin of exposure (useful in some cases)

• Qualitative advice

- conditional approval (eg, only particular production

method allowed; only certain uses allowed)

- minimize exposure (as low as reasonable achievable)

- avoid intake by certain groups

Quantitative risk assessment

• Known quantitative relationship between hazard and effect in studied population (human, animal)

• Extrapolation to low levels: how many cases of [effect] will occur at an estimated given range exposure

• Linked to the concept of „virtual safe dose“ – A additonal case of [effect] caused

if 106 humans ingest for their whole life the „virtual safe dose“

– Risk management, societal, political decision

9

Hazard Assessment (Toxicity Assessment)

= Hazard Identification

+ Dose-response Assessment

Hazard identification • Epidemiology

• Biochemistry

• Toxicology

• Analytical chemistry

• Chemistry, biochemistry

• Biological background

• Metabolism

• Food technology

• Food science

• Forensic science

• Agronomy

High doses in animals

Low doses in

humans

Linear

extrapolation

often

used

Threshold

What is Toxicology

• Toxicology is NOT Philately (stamp collecting) – Any undergraduate can make a list of effects at escalating

doses and pick a NO(A)EL – Simply collecting different types of studies in predetermined

animal species is not evaluation – Competent toxicologists must be able to EVALUATE studies

and endpoints/findings observed within them.

• Toxicology is COMPARATIVE everything – Physiology – Anatomy – Biochemistry – Immunology – Reproductive physiology – General and histopathology – etc

The stairway toward a new regulatory toxicology

“Evidence-based

medicine goes

toxicology!”

• Can we determine small effects in the experiment?

• Margin of exposure (MoE)

• Quantitative risk assessment

• No observed (adverse) effect level

• Benchmark dose

Hazard Characterisation

Understanding the dose-response relationship

Dose

Severity

of response

X

X

X

X

NOEL

LOEL

NOAEL

LOAEL Health-Based Reference

Value (HBRV)

Acceptable daily intake (ADI)

- food additives,

pesticides,

veternary drugs

Tolerable intake (TI)

- contaminants,

natural toxins

Upper safe level (UL)

- nutrients

Hazard characterization

• Dose-response assessment

• Limited database (usually no standard long term toxicological study in animal

• Toxic mechanism without a threshold mechanism (mutagenic carcinogen)

• Assessment of low level comtamination (analytical sensitivity)

Wächter (1986)

Risk characterization – the MoE

• Margin of exposure, BMD, QRA – their role?

• What‘s the implication of a MoE?

• Examples from JECFA

– Aflatoxin

– Acrylamide

– Methyleugenol

Margin of exposure High doses in animals

Low doses in

humans

BMD

Benchmark dose (BMD)

• Modelling based on sufficient experimental data

• mathematical model is selected, based on the data that are being analysed and the characteristics of the response

• Generally the more limited the database, the more simple the model

• Databases with larger numbers of dose groups and a greater experimental complexity will be better suited for more complex models.

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Example for QRA : Aflatoxin

JECFA 1997 49th

Examples for MoE

Acrylamide 1 mg/kg bw vs 0.4 µg/kg bw 2‘500

Aflatoxin

(Europe) 20 µg/kg bw vs 0.25 ng/kg bw 80‘000

(Far East) 20 µg/kg bw vs 2 ng/kg bw 10‘000

Methyleugenol

Flavouring 700‘000

Food 100‘000

Eating „pesto“ (basil based Italian sauce) 10‘000

MoE = dose causing tumour / human exposure

The distribution curve of inorganic arsenic (iAs) in overall rice samples

800.0700.0600.0500.0400.0300.0200.0100.00.0

iAs C o n c . n g /g

400

300

200

100

0

Fre

quen

cy

99th

95th

90th

Me a n=112

S td.De v=69N=1289

Wu et al. (2012) , : CX/CF 12/6/8 Proposed draft Maximum Levels for Arsenic in Rice (at Step 4),

Joint FAO/WHO Food Standards Programme- Codex Committee on Contaminants in Food

Diet Exposure for Inorganic Arsenic (iAS) in Rice for various Cluster Diet (mg/Kg.bw per day)*

Cluster Diet A B C D E F G H I J K L M

Rice Consumption （g）

91.0 31.6 94.6 33.2 12.7 12.7 376.9 64.3 38.0 74.3 238.4 381.3 34.6

Average iAs Intake

0.17 0.06 0.17 0.06 0.02 0.02 0.69 0.12 0.07 0.14 0.44 0.70 0.06

P90 iAs Intake

0.30 0.11 0.32 0.11 0.04 0.04 1.26 0.21 0.13 0.25 0.79 1.27 0.12

P99 iAs Intake

0.46 0.16 0.47 0.17 0.06 0.06 1.88 0.32 0.19 0.37 1.19 1.91 0.17

BMDL0.5 : 3.0 μg/kg bw per day with the range of 2–7 μg/kg bw per day

from lung cancer epidemiological studies (JECFA 2010)

MOE: Only 1?

Wu et al. (2012) , : CX/CF 12/6/8 Proposed draft Maximum Levels for Arsenic in Rice (at Step 4),

Joint FAO/WHO Food Standards Programme- Codex Committee on Contaminants in Food

Dietary iodine intake of women and men aged 18 – 50y (excluding outliers) in four coastal provinces in China in 2009, by

percentile and compared to EAR, RDA & UL

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6

n=310

mean±SD=278±200

Wo

me

n, n

Dietary iodine, mg/d

RDA

50th

75th

90th percentile

UL

120

EAR

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

Me

n, n

Dietary iodine, mg/d

RDA

75th

90th percentile

UL

EAR n=289

mean±SD=322±231

1.4 1.5 1.6

50th

Wu et al., J Nutrition, 2012

Translational Toxicology & TT21C

TT21C: NAS Report and Project Paper

TT21C

Translation Framework

Hubal, et. al. JTEH 2010

Translational Toxicology: Biomarker Development

• Translational toxicology: a review of the status of both qualification and validation of translational biomarkers.

• Biomarker use in translational medicine is predicated upon preclinical qualification and validation – 2 distinct steps in the biomarker development process.

• Prior to issue in 2009 (EMA) and 2010 (FDA) of the renal-specific DRAFT qualification guidelines, there was no clear direction FDA/EMA of how companies should qualify new biomarkers for disease progression or clinical trial endpoints.

• The trend in biomarker use is multivariant analysis, the tracking of subtle changes in multiple biomarkers simultaneously, often utilizing various tissue types. While the new guidance addresses biomarker qualification, analytical validation of new biomarkers remains undefined. U.S. Food and Drug Administration (FDA)

European Medicines Agency (EMA)

Translational Toxicology Dealing with all aspects of toxicology including

(but not limited to);

• In vitro, in vivo and mechanistic toxicology

• Human biomonitoring and risk assessment

• Biochemical and molecular effects of toxicants

• Development of biomarkers for monitoring the toxicant induced injury

• In silico approaches for predictive toxicity testing

• Innovative methods and approaches in risk assessment

• Sensors, devices and chips for detection of biomolecules, toxicants and contaminants

• Nanomedicine, nanotoxicology, and much more.

Organizations participating on Translational Toxicology and the Work of the

Predictive Safety Testing Consortium

Aflatoxin Example: A 50-Year Odyssey of Mechanistic and Translational Toxicology

Time line for key events in the discovery, toxicological evaluation, molecular epidemiology, and regulation of aflatoxins (AFT)

Biotransformation pathways for aflatoxin B1

Products measured in biofluids for use as biomarkers in epidemiological and intervention studies

are highlighted in green (urine) and red (serum).

Key steps in the development of HCC by viral (HBV and HCV) and chemical (AFT) factors

Blood- and urine-based biomarkers used in etiological studies are indicated.

Aflatoxin Risk Assessment

• Aflatoxins are genotoxic carcinogens

• Occur on mainly on maize and peanuts

• Major health and trade issue

• 0.1 cancers/year per 1,000,000 population per ng aflatoxin/kg body weight

• 30 times higher for HBsAg pos individuals

JECFA Potency Estimates

Corn and Peanut Consumption by GEMS/Food Regions (g/person/day)

Exposure Assessment Senarios for Aflatoxin B1 in Peanut

• Samples > 10 ųg/kg excluded

• Samples > 15 ųg/kg excluded

• Samples > 20 ųg/kg excluded

• No samples excluded

Estimated Population Risk of Cancer for Aflatoxin B1(cases/year/109 people)

0

20

40

60

80

100

120

10 ug 15 ug 20 ug No Limit

the GEMS/Food

European-type Diet

38

39

40

41

10 ug 15 ug 20 ug

0

1000

2000

3000

4000

5000

6000

10 ug 15 ug 20 ug No Limit

1350

1400

1450

1500

1550

1600

1650

1700

10 ug 15 ug 20 ug

the GEMS/Food

Far East-type Diet

the GEMS/Food

Far East-type Diet

the GEMS/Food

European-type Diet

Mortality from HCC in Qidong, China (A) Survival curve following diagnosis

of HCC in Qidong. Median survival is < 3months (Chen et al. , 2003).

(B) Age-specific incidence of HCC in Qidong and Beijing, China, where both HBV infection is the same, while AFT exposure (more prevalent in Qidong), could underlie the elevated risk of HCC. This regional dichotomy highlights opportunity to reduce incidence of HCC simply by reducing the AFT effects (Kensler et al. , 2003).

(C) Mortality rates of HCC by year ( Qidong Liver Cancer Inst).

(D) Projected mortality rates from HCC over the next 50 years on the basis of existing vaccination programs against HBV infection (red line) and coupled with possible reductions in AFT exposures (blue line).

• The impact of vaccination is modeled on the basis of the current experience in Taiwan, where near universal vaccination was implemented in 1984 (Chang et al., 2009). • Effect of attenuated AFT exposure is modeled from results of animal intervention studies, where reduction and delay of incidence observed (Kensler et al., 1997)

Multi-Media Exposure: Pulling it together • Viewing exposure as the

summation of ALL routes – Both Direct & Indirect are combined

– A real possibility “double counting”

– A form of aggregate exposure

• Determining how each route will affect the final receptor – Not all routes are equal

– Justification for route elimination

Air

Soil

Water

Beef

Fish

Vegetables

Dermal

EXPOSURE

Exposome & Exposomics

Food Chemical Exposure

• With the birth of U.S. regulatory agencies in the 1970s, interest in the environmental origins of human diseases waned, and exposure scientists focused instead upon levels of selected contaminants in food and water.

• Toxic chemicals enter the body not only from exogenous sources (air, water, diet, drugs, and radiation) but also from endogenous processes, including inflammation, lipid peroxidation, oxidative stress, existing diseases, infections, and gut flora.

• Thus, even though current evidence suggests that non-genetic factors contribute about 90% of the risks of chronic diseases, we have not explored the vast majority of human exposures that might initiate disease processes.

Exposure assessment Variability!

• Diet: composition and amount

• Level of contamination

• Specific population groups

• Few rules for exposure assessment

• Mathematical models

• Rather case-by-case

Risk Assessment

• Risk pathway

– Source (hazard)

– Pathway (Exposure)

– Person (target/receptor)

Multi-Media Exposure: Potential Pathways

Emissions from

Source

Downwind

Modeled Air

Concentration

Direct Inhalation

Exposure

Dry & Wet

Deposition of

Compounds

Surface Water

Contamination

Soil

Contamination

Plant Uptake

via Roots

Groundwater

Contamination

Plant Surface

Contamination Exposure via Plant

Ingestion

Fish

Contamination Exposure via Fish

Consumption

Exposure via

Drinking Water

Animal

Consumption

of Plants etc.

Exposure via Dairy

Products

Exposure via Meat

Consumption

Total Exposure

Soil Ingestion

Exposure via

Drinking Water

Dermal Exposure

and Absorption

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Multi-Media Exposure

• Biota Transport

– From dry & wet deposition on plants

– Uptake from soil contamination

– From groundwater/

surface water Uptake

– Consumption by animals

– Consumption by humans

The concept of the exposome

• Representing the totality of exposures received by a person during life,

• Encompasses all sources of toxicants

• Therefore, offers scientists an agnostic approach for investigating the environmental causes of chronic diseases.

• In this context, it is appropriate to regard the “environment” as the body's internal chemical environment and to define “exposures” as levels of biologically active chemicals in internal environment.

What is the exposome? • Success in mapping the human genome has fostered the

complementary concept of the "exposome".

• The exposome can be defined as the measure of all the exposures of an individual in a lifetime and how those exposures relate to health.

• An individual’s exposure begins before birth and includes insults from environmental and occupational sources.

• Understanding how exposures from our environment, diet, lifestyle, etc. interact with our own unique characteristics such as genetics, physiology, and epigenetics impact our health is how the exposome will be articulated.

Exposomics • is the study of the exposome and relies on the application of internal and

external exposure assessment methods.

• The extent to which internal and external exposure assessment will contribute to our understanding of the exposome is being debated as each approach has certain merits.

• Internal exposure relies on fields of study such as genomics, metabonomics, lipidomics , transcriptomics and proteomics. The fields include

1) use of biomarkers to determine exposure, effect of exposure, disease progression, and susceptibility factors,

2) use of technologies that result in large amounts of data and

3) use of data mining techniques to find statistical associations between exposures, effect of exposures, and other factors such as genetics with disease.

• External exposure assessment relies on measuring environmental stressors. Common approaches include using direct reading instruments, laboratory-based analysis, and survey instruments.

A key factor in describing the exposome

• is the ability to accurately measure exposures and effect of exposures.

• Many of the "omics" technologies have the potential to further our understanding of disease causation and progression.

• Metabonomics and adductomics (DNA and protein adduct measurement) have been used in the past to establish exposure-disease relationships.

• Research is needed to determine the utility of the "omics" technologies in defining the exposome.

Thought leaders in the exposome

• To narrow the focus to include only the study of metabonomics.

• Many of these small molecular weight compounds act as signals to regulate biological systems.

• To show promise in deciphering disease mechanisms.

• While metabonomics has great potential to contribute to the study of the exposome, it has not been established that it is the only approach needed to clearly articulate the important aspects of the exposome.

Why should we study the exposome? • One of the promises of the human genome project

was that it could revolutionize our understanding of the underlying causes of disease and aid in the development of preventions and cures for more diseases.

• Unfortunately, genetics has been found to account for only about 10% of diseases, and the remaining causes appear to be from environmental causes.

• So to understand the causes and eventually the prevention of disease, environmental causes need to be studied.

challenges & limit in this field

• An individual’s exposome is highly variable and dynamic throughout their lifetime.

• The impact of exposures can also vary with the individual’s stage of life. E.g.,

• exposure to the drug thalidomide during specific

developmental periods in utero causes malformation of limbs;

• exposure to lead in infants and early childhood can lead to cognitive deficiencies.

• Exposures during early years may also predispose an individual to certain chronic diseases later in life.

Mapping an entire exposome for an individual will be difficult, if not impossible because of the complexity of a life-time of

exposure.

• The impact of exposures can be different for each individual because of differences in genetic and other personal factors.

Some people will develop a disease

another person with the same or greater exposure will not.

The exposome may help to determine the underlying causes for this difference.

• Specific exposures can be difficult to measure due to lack of sensitive methods or not knowing that an exposure has even occurred.

• Even when the exposure is known, measuring that exposure can be difficult since the indicators of exposure may be transient, such as for most chemicals, which are rapidly excreted and only a short time frame exists to directly measure them.

• In other cases, past exposure can be defined using legacy biomarkers.

• A common example of a legacy biomarker is antibodies produced by exposures to environmental or occupational insults.

• To explore the exposome, it makes sense to employ a top-down approach based upon biomonitoring (e.g. blood sampling) rather than a bottom-up approach that samples air, water, food, and so on.

• Because sources and levels of exposure change over time, exposomes can be constructed by analyzing toxicants in blood specimens obtained during critical stages of life.

Perspectives

Aflatoxin: A 50-Year Odyssey of Mechanistic and Translational Toxicology

• Since their discovery 50 years ago, the aflatoxins have become recognized as ubiquitous contaminants of the human food supply throughout the economically developing world.

• The adverse toxicological consequences of these compounds in populations are quite varied because of a wide range of exposures leading to acute effects, including rapid death, and chronic outcomes such as hepatocellular carcinoma.

• Furthermore, emerging studies describe a variety of general adverse health effects associated with aflatoxin, such as impaired growth in children.

• Aflatoxin exposures have also been demonstrated to multiplicatively increase the risk of liver cancer in people chronically infected with hepatitis B virus (HBV) illustrating the deleterious impact that even low toxin levels in the diet can pose for human health. The public health impact of aflatoxin exposure is pervasive.

• Aflatoxin biomarkers of internal and biologically effective doses have been integral to the establishment of the etiologic role of this toxin in human disease through better estimates of exposure, expanded knowledge of the mechanisms of disease pathogenesis, and as tools for implementing and evaluating preventive interventions.

• Key Words: aflatoxin; biomarkers; molecular epidemiology

Using a Tiered Approach SCREENING EXPOSURE ASSESSMENT (worst-case exposure)

below

unacceptable

DONE REFINE EXPOSURE ASSUMPTIONS

( more realism )

below standard

unacceptable

• REFINE ASSUMPTIONS • MAKE CRITICAL DECISIONS

below

DONE

unacceptable

DONE !!

(best estimate)

Study sites of total diet study in China

South 2

North 1 North 2

South 1

Specific objectives

To estimate the dietary intakes of chemical contaminants by Chinese population；

To compare the intakes of chemical contaminants with HBRV；

To monitor the trends of chemical contaminants in Chinese diet；

To provide scientific basis for developing food safety regulations and standards.

Extended Risk Assessment software

• Probabilistic Exposure Assessment

• Acute and Chronic risks

• Uncertainty analysis by means of bootstrap

• Web-based

• Can combine data from different provinces (E-platform)

• To be extended with algorithms for effect modeling

Monte-Carlo sampling

Sample 1

Sample i

Sample m

Monte-Carlo

Dietary Exposure

Distribution

In dietary exposure assessment ,

Monte Carlo Model is used to qualify

the variability of data.

For Variability Analysis

Consumption Data Concentration Data PF and VF

parametric model nonparametric model

Intake amount 1

Intake amount i

Intake amount m

In dietary exposure assessment ,

Bootstrap is needed to evaluate the

uncertainties of the parameters and

distribution interested.

Bootstrap For Uncertainty Analysis

Consumption Data Concentration Data

Bootstrap Sampling Monte-Carlo Sampling

Monte-Carlo Sampling

Monte-Carlo Sampling

The Distribution

of Uncertainty

bootstrap iteration 1

bootstrap iteration i

bootstrap iteration m

PF and VF

exposure science goes exposome

• Initial investigations could use archived blood from prospective cohort studies to measure important classes of toxic chemicals, notably, reactive electrophiles, metals, metabolic products, hormone-like substances, and persistent organic compounds.

• The exposome offers health scientists an avenue for integrating research that is currently fractured along lines related to particular diseases and risk factors, and can thereby promote discovery of the key exposures responsible for chronic diseases.

• By embracing the exposome as its operational paradigm, exposure science can play a major role in discovering and mitigating these exposures.

Risk characterization & Food Safety Risk Managment

• Depends on the contaminant, its properties, toxicity, can it be removed, reduced

• Advice, how to reduce exposure of vulnerable groups, how to manage occurrence

• Good practice guidance may be part of it