Dr Cliff Elcombe Biomedical Research Centre Ninewells Hospital

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Dr Cliff ElcombeBiomedical Research
CentreNinewells Hospital Medical
SchoolUniversity of Dundee

• Risk Perception, Risk Assessment and the Role of
  Mechanisms

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Father of Modern Toxicology PARACELSUS 1564
Dose THE KEY CONCEPT in Toxicology

• All things are poisonous, only the dose makes it
  non-poisonous.
• (Dose alone determines toxicity)

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ALL Interactions between Chemicals and
Biological Systems follow a Dose-Response
Relationship
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Dose-Response Relationship

• The quantitative relationship between the
  concentration of a xenobiotic in the body and the
  magnitude of the biological effect it produces.
• The magnitude of the effect of a xenobiotic is a
  function of the amount of xenobiotic a person is
  exposed to (i.e., The Dose Makes the Poison).

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Dose-Response RelationshipThe Dose Makes the
Poison
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A Selection of Natural Carcinogens
anise apples bananas brocolli brussel
sprouts cabbage carrots cauliflower celery cinnamo
n cloves cocoa comfrey tea fennel grapefruit
juice
honey dew melon horseradish kale mushrooms mustard
orange juice parsely parsnips peaches black
pepper pineapples radishes raspberries tarragon tu
rnips
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The science of Toxicology helps people make
informed decisions and balance RISKS vs.
BENEFITS
The study found the highest levels of
pesticide residues in peaches, apples, pears.
AND Spinach.
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Hazard and Risk

• Hazard
• the potential for harm
• Intrinsic property of the chemical
• Risk
• Risk is the chance (probability) that harm will
  actually occur
• Hazard x exposure (dose and time)

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• Dose Determines Whether a Chemical Will Be
  Beneficial or Poisonous

Beneficial Dose Toxic Dose Aspirin 300 1,000
mg 1,000 30,000 mg Vitamin A 5000
units/day 50,000 units/day Oxygen 20 (Air) 50
80 (Air)
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Concentration Analogies
One Part Per Million (ppm) is

• one automobile in bumper-to-bumper traffic from
  Cleveland to San Francisco
• one minute in two years

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Concentration Analogies
One Part Per Billion (ppb) is

• one 4 inch hamburger in a chain of hamburgers
  circling the earth at the equator two-and-a-half
  times (4x10 9 inches)
• one second of time in 32 years

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Which Results in the Largest Exposure?

• 1000 ng/mL
• 1 mg/mL
• 1 ppm
• 1000 ppb

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Perceived Risk Ratings
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Carcinogenic Risk of "Alar- Contaminated" Apple
Juice
15 pints of apple juice per day has a
carcinogenic risk equivalent to
1 mushroom (15g) per day
1/3 of a peanut butter sandwich
100g celery
100g cabbage
1/100 pint (6.5ml) of beer
1/2 teaspoon (2.5ml) of wine
9.6 litres

15 minutes in a swimming pool
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The Stages of Risk Assessment
Hazard Identification
Risk Estimation
Risk Evaluation
Risk Management
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Hazard Identification

• Human Studies
• epidemiology
• retrospective
• low statistical power
• uncertain exposure estimates
• volunteer studies
• prospective
• usually not ethical
• Short Term Tests and Structure Activity
• surrogate measurements and qualitative
• Animal Studies
• high to low dose extrapolation route of exposure

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Animal Studies in Toxicological Evaluation

• Acute Toxicity ("LD50")
• rats/mice
• Irritancy/corrosivity/sensitization
• guinea pigs/rabbits
• Mutagenicity, clastogenicity
• in vivo and in vitro
• Subacute/subchronic toxicity
• rats/mice/dogs

• Carcinogenicity
• rats and mice
• Reproductive toxicology
• rats/mice/rabbits
• teratology
• foetal toxicity
• multigeneration

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Risk Estimation
Safety Factor Approach
Mathematical Models
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Risk Estimation - Safety Factor Approach

• ADI NOEL / SF
• ADI acceptable (allowable) daily intake
• NOEL no observable effect level
• SF safety factor

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Hypothetical Dose-response
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30
25
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Tumour Incidence ()
15
10
5
0
0
200
400
600
800
1,000
1,200
Dose (mg/kg)
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Application of a 100-fold safety factor to
convert a NOAEL in animals into an ADI for humans
10 fold
10 fold
Human variability
Species differences
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Risk Estimation- Mathematical Models

• Virtually Safe Dose
• Linear Extrapolation
• One-hit Models
• Multi-hit Models
• Multistage Models
• Weibull Model
• Physiologically-based Pharmacokinetic Models

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Linearised Model
1,000,000
100,000
10,000
1,000
Risk per 1,000,000
100
10
1
1
100
1,000
10,000
100,000
10
Dose (ppm in diet)
VSD ("virtually safe dose")
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Metabolic Saturation!
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Interspecies Comparisons

• Scaling Factors
• need to account for
• absorbtion, distribution, metabolism and
  excretion
• size
• lifespan
• Fraction of Diet Scaling
• Body Weight Scaling
• Surface Area Scaling
• PB PK
• Mechanisms of Toxicity

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The Exposure-Dose-Response Paradigm for
Carcinogens and Toxicants
oral inhalation dermal
Exposure
Blood concentration
Tissue dose of toxic moiety
metabolic activation/deactivation accumulation/exc
retion
Toxic moiety-target interaction
eg. transcriptional activation, cofactor
depletion, mutation, enzyme inhibition, etc.
acute, eg. cell death subacute, eg. organ
growth chronic, eg. cancer
Toxic response
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Biologically-based Model Linking Mechanisms of
the Exposure-dose-response Continuum
Exposure
PB-PK
Mechanisms
Models
Tissue Dose
Toxicant-target
Interaction
Mechanisms
Models
Toxicant-tissue Interaction
Tissue-response
Mechanisms
Models
Toxic Response
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PB-PK Models

• Animal experiment to determine actual kinetics of
  uptake and elimination
• Derive mathematical model using actual data and
  literature values
• Use model to predict human kinetics using human
  in vitro data and literature data

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Physiologically-based Pharmacokinetic Model for
Volatile Organic Chemicals
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Risk Evaluation and Management

• What level of risk is acceptable?
• how safe is safe enough?
• risk-benefit analysis
• perception of relative risk
• technical considerations
• socioeconomics
• politics

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Mechanisms in Hazard and Risk Assessment
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Classification of Carcinogens
Carcinogen
Non-genotoxic
Genotoxic
Cytotoxic
Non-cytotoxic
Endocrine
Direct mitogen
disruptor
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Liver Growth Carcinogens

• Hyperplasia
• Stimulate cell proliferation (acute and/or
  chronic)
• Inhibit apoptosis
• Hypertrophy
• Organelle proliferation
• SER
• Peroxiosmes

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Peroxisome Proliferators

• Clofibrate
• Ciprofibrate
• Fenofibrate
• Bezafibrate
• Halofenate
• WY-14,643
• BR-931
• Tiadenol
• Gemfibrozil
• Tibric acid
• Clobuzarit
• Aspirin
• LY-171883
• Methyclofenapate
• Dehydroepiandrosterone acetate
• Halothane

• 2,4-D
• 2,4,5,-T
• MCPA
• Dimethrin
• Lactofen
• Formesafen
• Haloxyfop
• Trichloroethylene
• Perfluorinated fatty acids
• 2-ethylhexanoic acid
• Trimethylpentane
• DEHA
• DEHP
• Phytol
• Citral
• Chlorinated paraffins
• HCFC 225

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Characteristics of the Peroxisome Proliferation
Phenomenon in Rodents

• Hepatomegaly
• Proliferation of peroxisomes and smooth
  endoplasmic reticulum
• Induction of peroxisomal fatty acid oxidising
  enzymes
• Induction of CYP 4A1
• Stimulation of replicative DNA synthesis
• Inhibition of apoptosis
• Hepatocellular tumours in long term studies

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Key Events in the Mode of Action for PPARa
Agonist-Induced Rodent Liver Tumours
PPARa Agonist
Activation of PPARa
Oxidative stress
Cell proliferation Decreased apoptosis
Preneoplastic foci
Clonal expansion
Liver tumours
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Model of Peroxisome Proliferator Action
Peroxisome Proliferation, Growth Regulation and
Hepatocarcinogenesis
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PPARa Knockout Mouse

• No hepatomegaly
• No peroxisome proliferation
• No peroxiosomal enzyme induction
• No CYP4A induction
• No stimulation of DNA synthesis
• No inhibition of apoptosis
• No hepatocellular tumours

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Diethylhexyladipate Toxicology

• Very low acute toxicity
• Not irritant/corrosive/sensitizer
• Non-mutagenic
• No reproductive effects
• Hepatocarcinogen in mice not rats
• Potential Human Carcinogen?

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Safety/Risk Assessment
Parallelogram Paradigm
Animal data
Animal in vitro
Human in vitro
Human Hazard
Exposure
Human risk
Animal in vivo
Human in vivo
Mechanism
?
Actual risk?
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Metabolism of Diethylhexyladipate (DEHA)
HOOC(CH2)4COOH

HOOCCHCH2CH2CHCH3
HOOCCHCH2CH2CH2CH3
CH2CH3
CH2CH3
OH
HO0CCHCH2CH2CCH3
O
CH2CH3
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Peroxisome Proliferation in Mouse Hepatocyte
Cultures - DEHA and Metabolites
3,000
EHA
2,500
2,000
EH
MEHA
1,500
Peroxisome Proliferation ( control)
1,000
OH-EHA
Keto-EHA
500
EHdiA
AA
0
0
1,200
200
400
600
800
1,000
Concentration (microMolar)
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Species Differences in Response - EHA
3,000
Mouse
2,500
2,000
1,500
Peroxisome Proliferation ( control)
1,000
Rat
Human
500
Guinea pig
Marmoset
0
0
200
400
600
800
1,000
1,200
Concentration (microMolar)
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Dose-response to Peroxisome Proliferators
Liver growth
Tumours
Response
1
3
2
Dose
Threshold for early events
Tumour threshold
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Summary

• Hazard and risk are not the same
• Perception of risk does not necessarily relate to
  actual risk
• Risk assessment is an imprecise procedure
• Mathematical models must not be used to provide a
  spurious precision or to create an impression of
  scientific sophistication
• Mechanisms of toxicity and biologically-based
  models should play a major role in risk assessment

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Resources

• A Journalist's Handbook on Environmental Risk
  Assessment
• http//ruby.fgcu.edu/Courses/Twimberley/IDS3920/
• main.html
• http//www.agius.com/hew/resource/hazard.htm
• http//www.sis.nlm.nih.gov/ToxTutor.html