Transcript Can In silico and/or In vitro Testing be used for Toxicity Assessment

Timothy J. Shafer
2012 SOT In vitro Lunch
Disclaimer: The views expressed in this presentation are solely those of the
Author. They do not represent Agency Policy.
Batteries of in vivo tests in various mammalian species-
when agencies have regulatory authority to require testing
870.1100 Acute oral toxicity – rat
870.1200 Acute dermal toxicity
870.1300 Acute inhalation toxicity – rat
870.2400 Primary eye irritation – rabbit
870.2500 Primary dermal irritation
870.2600 Dermal sensitization
870.6100 Delayed neurotoxicity (acute) – hen
870.6200 Acute neurotoxicity – rat
Subchronic Testing
870.3100 90–day Oral – rodent
870.3150 90–day Oral - non-rodent
870.3200 21/28–day Dermal
870.3250 90–day Dermal
870.3465 90–day Inhalation – rat
870.6100 28–day Delayed neurotoxicity-hen
870.6200 90–day Neurotoxicity – rat
Chronic Testing
870.4100 Chronic oral – rodent
870.4200
Carcinogenicity - two rodent species rat and mouse preferred
Developmental Toxicity and Reproduction
Prenatal Developmental toxicity - rat
870.3700
and rabbit, preferred
870.3800 Reproduction and fertility effects
870.6300 Developmental neurotoxicity
Mutagenicity Testing
870.5100 Bacterial reverse mutation assay
870.5300
In vitro mammalian cell assay
870.5375
870.5385
In vivo cytogenetics
870.5395
Special Testing
870.7485 Metabolism and pharmacokinetics
870.7200 Companion animal safety
870.7600 Dermal penetration
870.7800 Immunotoxicity
27 Different StudiesDoes not include Eco
http://www.epa.gov/pesticides/regulating/data_requirements.htm

Draize eye test (Ocular Toxicity)
Use validated alternatives
Consideration of chemical properties
Alternative tests for corrosive compounds (acids/bases)
Alternative in vitro tests (hens egg chorioallantoic membrane (HETCAM) assay; human corneal cells in culture
◦ In vitro tests prior to in vivo
◦
◦
◦
◦

Genetic 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 other






Large number of chemicals for which we do
not have complete toxicity information
Need to assess mixtures
Need to assess all endpoints and life stages
Efficiency
Developmental
Costs associated with in vivo testing
Neurotoxicity <
◦ $15-20 M to register 1 food use1%pesticide
◦ $1M and 1.5yr for a Developmental
Neurotoxicity (DNT) guideline study
Humane reasons- use fewer animals and
minimize distress

REACH legislation

NAS Toxicity in the 21st Century report

Stakeholders
◦ Registration, 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).
◦ Limitations 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.
◦ Public wants safe use of chemicals
◦ Industry wants reduced costs
◦ Animal welfare groups want reduced use of animals






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).


Who is exposed and to how much?
What is the hazard?
◦ Organ system
◦ Lifestage
◦ Ecological

What 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?
Source: Figure 2-3, NAS, 2007
Acute Neurotoxicity of Pyrethroid
Insecticides
Exposure
ADME
Key Event
Cell Level
Tissue Level
Organ Level
Altered
Na Channel
Na Channel
Function
Function
Altered
Neuronal
Neuronal
Excitability
Excitability
Altered
Network
Network
Firing
Patterns
Altered
Neuronal
Neuronal
Pathways
Pathways
Adverse
Normal
Clinical
Function
Outcome
??
??
??
Adverse
Clinical
Outcome ?
Developmental Neurotoxicity of Pyrethroid
Insecticides
Existing Chemicals
Chemical
Characteristics
HTS Assays for
Toxicity
Pathways
Hepatotoxicity
Cardiotoxicity
Neurotoxicity
Quantitative StructureActivity Relationships (QSAR)
Developmental toxicity
Renal toxicity
Carcinogenic
New Chemical
Immunotoxicity
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?