The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.
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TestSmart Endocrine Disruptors
February 25-26
Hyatt Fair Lakes Hotel
12777 Fair Lakes Circle
Fairfax, VA
Program Committee:
Richard A. Becker, American Chemistry Council
Alan M. Goldberg, Johns Hopkins University
Pamela J. Lein, Johns Hopkins University
Ellen K. Silbergeld, Johns Hopkins University
Gary Timm, US Environmental Protection Agency
James D. Yager, Johns Hopkins University
Proceedings for TestSmart -- Endocrine Disruptors
Screening and Testing Chemicals for Endocrine Activity
Richard A. Becker, Ph.D., DABT
American Chemistry Council
Alternatives to Animal Testing
- Industry is supportive of development efforts
- However, validated alternatives are not presently available for every type of toxicity test
- Research to reduce, refine or replace the need for laboratory animals should continue
- When animal testing is necessary:
- Necessary to follow guidelines for humane treatment
- the number of animals should be minimized to the greatest extent practicable without compromising the scientific integrity of the method
Reliance on laboratory animals can be minimized by:
- Integrating various chemical evaluation initiatives and, when scientifically valid, grouping chemicals with similar characteristics, reducing duplication of testing
- Standardizing and validating - all new and refined screens and tests - both animal and non-animal
- International harmonization and mutual acceptance of data
- Tiered testing - results-based prioritization to focus testing on chemicals of greatest concern, thus reducing the total amount of testing
- Using hazard and exposure data to prioritize substances for further testing thereby making more efficient use of resources, including laboratory animals
Tiered Hierarchical Framework for Hormonally Active Substances
- Prioritizing chemicals for screening and testing
- Validated screening assays to identify substances with hormonal activity & prioritize for more definitive testing
- Validated tests to characterize dose response of adverse effects mediated by the endocrine system
Hierarchical Approach for Hormonally Active Agents
EDSTAC Tiered Approach
- Tier 1 - Screening assays to identify agents (estrogen, androgen or thyroid) that may have hormone-related activity
- Tier 2 - Definitive testing to characterize adverse effects and dose response relationships for agents positive in Tier 1
- Validation consistent with ICCVAM principles
Tier 1 Screening Assays:
- maximal sensitivity (minimize false negatives)
- range of vertebrate organisms
- detect relevant modes of action
- diverse and complementary endpoints for specific modes of action
- fast and cost-effective
Tier 2 Tests
- Protocols designed to evaluate adverse health effects
- Data to characterize nature, incidence, severity and dose-response relationship of any adverse effects
- Include critical life stages, appropriate doses & administer by relevant route
- Results supersede screening assay results
- "Gold standard" — multigeneration reproduction toxicity test
ICCVAM Validation Criteria:
- Rationale for the method
- Clear statement of proposed use
- Demonstration - within assay variability & assay reproducibility within and across laboratories
- Performance - demonstrated with representative reference chemicals, including known positives and known negatives
- Description of method limitations
- Ideally, validation program for an assay conducted in accordance with Good Laboratory Practices (GLP's).
Tier 1 In Vitro ER and AR Binding or Transcriptional Activation Assays
- Needs systematic evaluation for inter-lab variability, sensitivity, reproducibility
- Recommend standardization & validation of a single methodology
- "Gold Standard" by which to compare alternative protocols & validation of QSAR
- Validation — multiple labs, identical protocols, reference chemicals, GLP, Data interpretation (pass/fail criteria)
Tier 1 In Vitro Steroidogenesis Assay
- Confounding by cell toxicity is major limitation
- (Powlin et al. 1998) — correctly detected only 4 of 9 substances
- Results indicate that the assay would not be useful as a routine screen
Tier 1 Uterotrophic & Hershberger Assays
- Validation activities in progress via OECD (industry & research institute labs)
- European & Japanese industry supporting chemical repository
- ACC is actively contributing
- Uterotrophic assay - Wil Laboratories, Cincinnati, OH
- Hershberger assay — RTI, RTP North Carolina
- OECD program expected to result in guidelines that are internationally harmonized and accepted
Tier 1 Fish Gonadal Recrudescence Assay
- Industry studies
- Indicate numerous practical difficulties
- e.g., tubercle quantitation, hormone measurements, gonad weights
- Indicate numerous practical difficulties
- Not suitable as Tier 1 screen (endpoints affected by multiple factors that will be difficult to interpret)
- Pre-validation studies on alternative approaches in progress in Europe
Tier 1 Frog Metamorphosis Assay
- EPA development studies to assess assay in progress
- Results reveal several issues
- Redundant (thyroid hormone assessments included in mammalian Tier 1 assays)
- Practical problems in quantitation of endpoint (tadpole tail resorption)
- Lack of specificity (corticosterone and prolactin also involved in addition to thyroid hormone)
- Method remains unproven - low priority for further development
Tier 1 Intact Adult Male Assay
- Systematic evaluation > 15 reference substances representing diverse endocrine mechanisms
- Good sensitivity and specificity
- Preferred approach that can be incorporated in a Tier 1 battery, utilizes fewer animals and is more cost effective than the EDSTAC-recommended battery
Tier I Male Battery
- Model:
- 10-Week old intact male rats
- n = 15/group
- 15-Day test (oral)
- Endpoints:
- Organ weights - liver, testes, thyroid, epididymides, prostate, seminal vesicles, ASG unit
- Histopathology - testis, epididymides, thyroid, liver?
- Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH
- Biochemical - preparation of hepatic microsomes
Tier I Male Battery: Fingerprint of Unknowns
Advantages of Tier I Using Intact Male Assay
- Comprehensive mode-of-action screen
- Run in parallel with uterotrophic assay and in vitro receptor binding assays
- Redundancy of endpoints
- Tier I focuses direction of any further testing
- Design allows integration of new endpoints
- Intact endocrine system
- Cost effective because it integrates
- Intact male assay replaces three assays in EDSTAC battery
- Uses less animals than EDSTAC battery
Tier 1 - Pubertal Assays
- Endpoints can be influenced by non-endocrine factors
- Dose selection is critical -- many physiologic and toxicologic mechanisms can affect pubertal onset. Compounded if MTD used
- Inherent variability -- influences interpretation of "small" (< 2 days) changes in age at VP or PPS
Concerns: Tier 2 non-mammalian tests
- Fish - intermediate tier needed
- Fish - lack of specificity of endpoints (FFLC)
- Avian - inadequate triggers
- Mysid - inappropriate species - need to show plausible biolog. mechanism (such as estrogen specific receptor) otherwise inappropriate triggering criteria for the test
Tier 2 Mammalian Test
- Mammalian multiple generation reproduction study -- definitive Tier 2 test for human health risk assessment
- 1998 revision of OPPTS 870.3800 — added many hormonally sensitive end points
- testis and epididymal sperm count, sperm morphology & sperm motility
- estrous cyclicity evaluation
- weights and histology of primary and accessory reproductive organs in parents and offspring
- puberty onset in F1 offspring, and anogenital distance
Tier 2 Mammalian Test
EDSTAC suggested consideration of additional endpoints
However
- Any significant modifications must take into account international harmonization -- any changes to the guideline will affect it globally
- Already a complex study — need to consider logistics and value of new endpoints
Tier 2 Mammalian Test
- Some modifications could be demonstrated and validated in a relatively short period of time, examples:
- enhanced clinical observations
- thyroid weight & histopath
- whole brain weight and brain histology
- Major modifications would be problematic
Hazard Identification & Characterization .Weight of Evidence Approach
Integration- relevant studies- assessment of overall adequacy and concordance of the data set:
- higher quality studies are given more weight than lower quality studies
- substances with inadequate data sets - additional screening or testing data??
- lack of concordance — base decisions on preponderance of available data
- longer-term definitive tests outweigh or supercede screening assays
- adequate & concordant data sets - no additional testing, proceed to risk assessment and risk management processes as appropriate
Hazard = Risk
- Screening data — insufficient for risk characterization
- Risk characterization requires testing data — integration of:
- Hazard
- Understanding of the hazard data endpoints and inferences
- Dose-response
- Exposure