Non-Animal Methods

PETA, in partnership with scientists at the Fund for the Replacement of Animals in Medical Experiments and the Dr. Hadwen Trust for Humane Research in England, has proposed the following completely animal-free framework for screening and testing chemicals for endocrine-mediated adverse effects:

Step 1: Sorting and Prioritization Based on Existing Information

• Physical and chemical properties (molecular weight, pH, reactivity, volatility, potency)
• Environmental fate and transport (use and release pattern, persistence, exposure, bioaccumulation, etc.)
• Hazard information (robustness of database, existing evidence of adverse effects, etc.)
• Chemical grouping (based on physical/chemical properties, mode of action, etc.)

Step 2: In Vitro Barrier and Metabolism Studies and Biokinetic Modeling

• Measure absorption through the gut (Caco-2 cells) and skin (OECD test guideline 428 for dermal penetration in vitro)
• Evaluate metabolic activation/deactivation (phase I and II conjunctive metabolism)
• Identify active/toxic metabolites for further study in vitro
• Perform computerized biokinetic modeling of probable distribution of a chemical and/or metabolites throughout the body

Step 3: Receptor-Mediated Mechanistic Assays

• Sub-cellular estrogen/androgen receptor-binding studies (recombinant human receptors)
• Transcriptional activation/reporter gene assays (T47D-KBluc human breast cancer cell line or LUMI-CELL™ assay to measure anti/estrogenic effects and MDA-kb2 cell line or AR-Calux bone cell line to measure anti/androgenic effects)

Step 4: Nonreceptor-Mediated Mechanistic Assays

• Thyroid biochemistry (FRTL-5 rat thyroid cell line to measure the production of thyroid hormone, thyroglobulin and the enzymes thyroid peroxidase and deiodinase)
• Steroidogenesis and aromatase inhibition (H295R or KGN human cell lines)
• Vitellogenin induction (using liver cell lines from birds, fish and amphibian species)

Step 5: Assays to Detect Adverse Effects

• In vitro fertilization (sperm penetration and cell division, sperm motility, morphology, etc.)
• Embryonic stem cell test (validated method using mouse derived stem cells to evaluate the potential for chemically-induced embryotoxicity)
• In vitro genetic toxicity (various internationally accepted OECD test guidelines to evaluate the potential for chemically induced genetic mutations to occur and be passed on to future generations)

The strategy outlined above, if adopted, could vastly reduce the exorbitant costs associated with the EPA’s Endocrine Disruptor Screening Program––both financial and in terms of animal suffering and death. In vitro (cell-based) assays using human-derived tissue are generally more sensitive, reliable, and relevant to people than animal-based methods and produce results more quickly and at a much lower cost. In vitro assays are excellent models for investigating the mechanism of action of potential hormone-disrupting chemicals and can be performed either individually or in great numbers through automated “high throughput” screening.