(How) Can In silico and/or - PowerPoint Presentation

Slide1

(How) Can In silico and/or In vitro Testing be used for Toxicity Assessment instead of in vivo Approaches?

Timothy J. Shafer2012 SOT In vitro Lunch

Disclaimer: The views expressed

in this presentation

are solely those of the Author. They do not represent Agency

Policy.Slide2

What is the Current Toxicity Testing Paradigm?Batteries of in vivo tests in various mammalian species-

when agencies have regulatory authority to require testing

870.1100

Acute oral toxicity

–

rat870.1200Acute dermal toxicity870.1300Acute inhalation toxicity – rat870.2400Primary eye irritation – rabbit870.2500Primary dermal irritation870.2600Dermal sensitization870.6100Delayed neurotoxicity (acute) – hen870.6200Acute neurotoxicity – ratSubchronic Testing870.310090–day Oral – rodent870.315090–day Oral - non-rodent870.320021/28–day Dermal870.325090–day Dermal870.346590–day Inhalation – rat870.610028–day Delayed neurotoxicity-hen870.620090–day Neurotoxicity – ratChronic Testing870.4100Chronic oral – rodent870.4200Carcinogenicity - two rodent species - rat and mouse preferred

Developmental Toxicity and Reproduction870.3700Prenatal Developmental toxicity - rat and rabbit, preferred870.3800Reproduction and fertility effects870.6300Developmental neurotoxicityMutagenicity Testing870.5100Bacterial reverse mutation assay870.5300870.5375In vitro mammalian cell assay870.5385870.5395In vivo cytogeneticsSpecial Testing870.7485Metabolism and pharmacokinetics870.7200Companion animal safety870.7600Dermal penetration870.7800Immunotoxicity

27 Different Studies-Does not include Eco

http://www.epa.gov/pesticides/regulating/data_requirements.htmSlide3

Draize eye test (Ocular Toxicity)Use validated alternativesConsideration of chemical propertiesAlternative tests for corrosive compounds (acids/bases)Alternative in vitro tests (hens egg chorioallantoic

membrane (HET-CAM) assay; human corneal cells in cultureIn vitro tests prior to in vivoGenetic Tox assays: Ames Test (Mutagenicity/Carcinogenicity)Tests mutagenicity using bacteria

Is 70% predictive of carcinogens for general classes

Nearly 100% prediction for PAH, Aromatic and heterocyclic amines, nitro aromatics

Poorly predictive for chlorinated organic (chlorophenols chlorinated hydrocarbons)

Rat and mouse are 70% predictive of humans; and 70% predictive of each otherAre there any success stories?Slide4

Large number of chemicals for which we do not have complete toxicity informationNeed to assess mixturesNeed to assess all endpoints and life stagesEfficiencyCosts associated with in vivo testing

$15-20 M to register 1 food use pesticide$1M and 1.5yr for a Developmental Neurotoxicity (DNT) guideline studyHumane reasons- use fewer animals and minimize distress

Why do we need a new testing paradigm?

Developmental Neurotoxicity < 1%Slide5

REACH legislationRegistration, Evaluation, Authorisation and Restriction of

Chemical substances“Under REACH, animal testing is to be avoided in favour of alternative methods and registrants can only carry out tests involving the use of animals as a last resort. “ (http://ec.europa.eu/environment/chemicals/reach/reach_intro.htm

)

.

NAS Toxicity in the 21

st Century reportLimitations of current paradigm are high to low dose extrapolations and animal to human extrapolations = limited ability to predict what happens in humans exposed to low doses.StakeholdersPublic wants safe use of chemicalsIndustry wants reduced costsAnimal welfare groups want reduced use of animalsWho says we need a new testing paradigm?Slide6

Allow a new compound to be produced.Risk/Benefit (e.g. new drug) vs safety (food additive)Replace or choose between compounds (Risk/Risk).Regulate levels released into the environment or use conditions for a compound (e.g. pesticide labels).

Extent of clean up at a hazardous waste site.Setting exposure levels (RfD, RfC, etc), ambient air quality standards, allowable levels of contamination (e.g. in drinking water).

What kinds of decisions do regulators have to make?Slide7

Who is exposed and to how much?What is the hazard?Organ systemLifestageEcologicalWhat are the properties of the chemical?Will it move in the environment?

Will it bioaccumulate?Is it reactive?What is the chemical used for and are there alternatives?

What kind of information do regulators need to make these decisions?Slide8

How do we improve the current paradigm?

Source: Figure 2-3, NAS, 2007Slide9

What is a Toxicity Pathway anyhow?

Exposure

ADME

Altered

Na

ChannelFunctionAlteredNeuronalExcitabilityCell LevelAdverseClinicalOutcomeAltered NeuronalPathwaysAlteredNetworkPatternsTissue LevelOrgan LevelKey EventAcute Neurotoxicity of Pyrethroid Insecticides

??AdverseClinicalOutcome ?????Developmental Neurotoxicity of Pyrethroid InsecticidesNa ChannelFunction

Neuronal

Excitability

Normal

Function

Neuronal

Pathways

Network

Firing

PatternsSlide10

Developing Predictive Models for Toxicity

QSAR

Hepatotoxicity

Neurotoxicity

Developmental toxicity

Renal toxicityCarcinogenicImmunotoxicityCardiotoxicityChemical CharacteristicsHTS Assays for Toxicity PathwaysExisting Chemicals

Quantitative Structure-Activity Relationships (QSAR)New ChemicalSlide11

1. Is it a realistic goal to replace all animal testing? (In what timeframe?)2. What criteria must be fulfilled for an in vitro approach to replace an in vivo approach?

3. Does the context of the decision to be made, or the level of information required matter?4. What are the challenges to human risk

? How can these be addressed?

5

. (How) Can

an in vitro approach be useful if the toxicity pathway is not completely understood?6. If we replace, what are the scientific questions (uncertainties) that we need to be concerned about?7 Would the public accept and be comfortable with decisions made using in vitro data? What if the decision was made entirely on the basis of in vitro data?Questions