2n McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Highlighting the Need for AOPs in Streamlining Hazard Assessment Methods

(for cancer assessment)

Catherine Willett, PhD

Director, Regulatory Toxicology

Humane Society of the United States

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

1. Rodent Cancer Bioassay: cost/benefit?

2. Advantages of mechanism based approaches

3. AOP approaches and activities at OECD

Outline

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

• Minimally 400 animals, 2 – 4 million USD and 3 years*

• Often performed in two species (drugs, food additives)

• Or rat bioassay plus shorter-term transgenic mouse

assays1

– ras H2 + p 53 +/- : use half the number of animals and for 6 months

– Reduced animal use mitigated by need for creation and

maintenance of multiple lines

– Only improves identification if both used (geno and non-genotoxic)

no animal savings, minimal cost savings

*Thomas RS, Pluta L, Yang L, Halsey TA. Toxicol Sci 2007. 97(1):55-64.

1Alden et al. Veterinary Pathology. 2011. 48(3):772-784.

1. Rodent Cancer Bioassay: Cost

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

• Drugs (review of 533)

– False positives: 80%

– False negatives: 27%

– Sensitivity: 73%

• 12 out of 44 chemicals with human concern not seen in either rodent

species

“When tested repeatedly, most molecules….had conflicting test results.”

Alden et al. Veterinary Pathology. 2011. 48(3):772-784

1. Rodent Cancer Bioassay: Benefit?

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Other chemicals (e.g. NTP studies)

– 82% of 500 studies resulted in noncommittal classifications*

– Only 57% of chemicals tested multiple time give consistent results

– 22% of all chemicals tested test positive1

• Largely due to findings at MTD and irrespective of biological plausibility

• Nevertheless results in classification as “possible human carcinogen”

– Positive correlation?

• 9/10 known human carcinogens have tested positive either rats or mice in NTP studies

• All known human carcinogens have tested positive in some rodent assay, with the possible exception

of arsenic

“This result says more about the persistence of toxicologists than about the

ability of a standard (rodent) protocol to predict human carcinogenicity.”1

* Wasted Money Wasted Lives. People for the Ethical treatment of Animals. 2006. http://www.mediapeta.com/peta/pdf/Wasted-money-PDF.pdf1 Ennerver and Lave. Reg. Toxicol. Pharma. 2003. 38: 52-57

1. Rodent Cancer Bioassay: Benefit?

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

400 animals2 – 4 million USD3 years

1. Rodent Cancer Bioassay: Cost / benefit

2False Positives: 80%False Negatives: 27%

or

82% noncommittal classifications

Too high

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

2. Advantages of mechanistic approaches

1. Refine design and interpretation of animal studies

2. Demonstrate human relevance (or not)

3. Improve predictivity for both human health and other

target species

4. Design assessment strategies that are not

dependent on animal testing

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

2. Early adoption of mechanistic approaches

DNA reactive vs non-DNA reactive

– Mouse transgenic lines

– 3Rs value highly debatable

– Chemical alerts for DNA reactivity

– Used to flag chemicals

– Genotoxicity battery

– Currently used to exclude chemicals

– In vitro tests have high false positive rates

– in vivo tests are insensitive

– Battery misses non-DNA reactive chemicals

– Cell transformation assays

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

2. Early adoption of mechanistic approaches

Human Relevance Frameworks

- Characterize MoA of each class of carcinogens

- Determine which rodent MoA is possible relevant to humans

- Built using case studies

Timeline stolen from V. Dellarco:• EPA & IPCS 1999-2001. Conceptual Framework for Evaluating a Mode of Action for

Chemical Carcinogenesis.• ILSI 2003. Framework for human relevance analysis of information on carcinogenic modes

of action.• ILSI 2005. Extends Framework to non-cancer outcomes & life stage information.• IPCS 2006 & 2008 Adopts Human Relevance Framework: Boobis, et al. IPCS framework

for analyzing the relevance of a non-cancer mode of action for humans. Crit Rev Toxicol. 2008;38(2):87-96.

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Short-term screening tests*

– Step 1: 90-day “screen”

• E.g. hepatocarcinogenicity: hepatocellular necrosis, hypertrophy, cytomegaly, increased liver weight

– Step 2: mechanistic screens (most 90-days)

• Histopathology• Serum enzymes• Acyl Co-A oxidase • CYP induction• CAR, PSR, AHR binding• ER binding (or histologic evaluation of endocrine-sensitive organs)• Iron staining• Reversibility• Metabolic activation

*Cohen, S.M. Toxicol. Pathol. 2010. 38(3):487-501.

Proposals to improve efficiency of rodent-based assessment

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Testing strategy to rule out carcinogens*

– Negative genotoxicity in standard battery

– Negative hormonal perturbation in chronic studies

• effects on endocrine-related tissues, hormone levels

– Lack of histopathologic risk factors in 6 month rat study

• neoplasia in any tissue

– Negative in 6 month transgenic mouse

• Both genotox and non-genotox models required?

– Positives go into a standard rat bioassay

*Sistare et al. Toxicol. Path. 2011.39: 716-744.

Proposals to improve efficiency of rodent-based assessment

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

2. Molecular approaches

Gene expression profiling following 13 week exposure*

– Predict lung tumors in B6C3F1 female mice

– Overall accuracy 77.5% in predicting mouse lung tumors

– 25 chemicals sufficient to develop predictive model • (could create predictive models for 8 organs using 200 chemicals)

– Gene profiles placed into biologic process categories1

• Used to calculate BMD values for liver or lung

• Some transcriptional and tumor incident BMD values correlate well

transcriptional BMD values could potentially be used as points of departure for cancer as well as non-cancer risk assessment

* Thomas, R.S. et al. Toxicol. Sci. 2009. 112(2): 311-321.1 Thomas, R.S. et al. Toxicol. Sci. 2011. 120 (1): 194-205.

Could the predictive capacity of ‘omics be improved by AOPs?

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

3. Adverse Outcome Pathway Approaches

Adverse Outcome Pathway: a chemical and biological description of what

occurs when a substance interacts with a living organism and results in

an adverse reaction – a biological map from the initiating event through

the resulting adverse outcome that describes both mechanism and

mode of action.

From: Ankley et al. Environ.Toxicol.Chem. 2010. 29 (3): 730–741.

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

3. Adverse Outcome Pathway Approaches

AOPs can be useful for:

Near-term:

– Developing chemical categories and structure activity relationships

– Increasing certainty of interpretation of both existing and new information

– Developing integrated testing strategies that maximize useful information

gained from minimal testing

Longer-term:

– Identifying key events for which non-animal tests can be developed, thereby

facilitating mechanism-based, non-animal chemical assessment

– Creating predictive toxicological assessments with low uncertainty and high

human relevance

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Adverse Outcome Pathway

QSAR focus area

Chemicals

ReceptorBinding

ER Binding

Liver CellsAlteredProtein

Expression

Vitellogenin

LiverAltered proteins

GonadOva-testis;

Sex-reversed;Fecundity

Sex reversal;

Altered behavior;

Repro.

ER-mediated Reproductive Impairment

In vivo

MOLECULAR Target

CELLULARResponse

TISSUE/ORGAN INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

In vitro Assayfocus area

Toxicity Pathway

3. AOP activities at OECD: expert consultation on the ER decision framework in Feb 2009

P. Schmieder, McKim conference 2008.

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

3. AOP activities at OECD

2010 Workshop on using mechanistic information in forming

chemical categories near-term recommendations:

1) Develop AOPs for well-established effects (e.g., skin sensitization) as well as

several longer term health and ecotoxicological endpoints.

3) Establish, populate and maintain an accessible, electronic repository e.g.

Effectopedia.

4) Develop a strategic plan including:

–an information template for developing and assessing AOPs

–guiding principles for assessing completeness and acceptance of an AOP

–a format for attaining mutual acceptance of an AOP.

5) Harmonize terminology associated with AOPs.

6) Integrate AOPs in the OECD QSAR Toolbox.

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

3. AOP activities at OECD: Sensitization draft AOP

OECD 2011. (Draft) The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

3. AOP activities at OECD

Agreed at last Joint Meeting in June 2011 that AOPs were to

become the cornerstone for all future projects in the test

guidelines programme.

Under development:

• A process for development and maintenance of AOPs

• A Guidance Document for Developing and Assessing Completeness of

AOPs

2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment | 8 – 10 May 2012 | Baltimore, MD

Thank you.

Catherine Willett, PhDDirector, Regulatory Toxicology,

Risk Assessment and Alternativeskwillett@humanesociety.org

t +01.240.599.6785 

The Humane Society of the United States2100 L Street NW    Washington, DC 20037

humanesociety.org/animalsinlaboratories