Proceedings for TestSmart -- Endocrine Disruptors

The Endocrine Disruptor Screening Program - Animal Protection Concerns

Martin L. Stephens, Ph.D.
The Humane Society of the United States

Outline of Concerns

Heavy Reliance on Animal Testing
Under-Emphasis on Alternatives
Shortcuts on Validation

Heavy Reliance on Animal Testing

Scale of animal use
- 80,000+ chemicals to be evaluated
- 600,000 to 1.2 million animals per 1,000 chemicals
- Several thousand chemicals likely to be tested
- Final toll: millions of animals
- For a "screening" program???
Scope for animal wastage
- Wastage in single-sex tests?
- Males when Uterotrophic is being conducted
- Females when Hershberger is being conducted
- Too few endpoints in some tests?
- Pubertals: R&D focusing on E & A, but not T effects - the class of hormone it was intended to assess
Scope for pain and distress
- Some tests involve lengthy, complicated, invasive, or repetitive procedures (e.g. ovariectomy, castration, testosterone implants, repeated sc/ip injections)
- Uterotrophic: a proposed OECD TG allows use of either intact or ovariectomized animals
  - Recent evidence that there is no real difference in performance b/w castrated/ovariectomized vs. intact animals
  - If true: then there is no justification for the flexibility seen in the proposed TG re: the need to use surgically altered animals
Human population studies needed to assess nature, severity & scope of problem:
- From the NAS's ED report:
  "Recommendation: Prospective and cross-sectional studies, using common protocols and strict quality control, be conducted in human populations suspected of being affected by [hormonally active agents]".
- "Determining the risk of humans from contact [with potential ED] chemicals in the environment is difficult because ordinary exposure to these agents has not been routinely monitored. Determining what these exposures are is, therefore, of primary importance." — Knobil, chair, NRC Committee on HAAs in the Environment

Twenty years from now we will have killed millions of animals, spent millions of dollars, and we still won't know how [EDs] affect humans. The entire process to date...has been focused on testing — not on determining the extent of the problem. We need to take a step back, get the right minds together, and focus on what the problem is.
- B. Schwetz, Acting Principal Deputy Commissioner, FDA

EDMVS Mtg, Oct. '01: EPA plans to fund research that will help in determining the extent to which humans & wildlife are exposed to chemicals that may act as EDs.
Will the outcome of the this work affect the course of animal testing in EDSP?
Diff. b/w human & animal endocrine systems:
- Endocrine system is extremely complex & there are many important differences b/w humans and rodents.
- E.g., in contrast to humans, rodents do not produce sex hormone-binding globulin following parturition, resulting in reduced hormonal clearance.
Diff. b/w adults & developing organisms:
- "There are important differences among species and between adult and developing organisms in their responses [to EDs]. These differences could have important implications when assessing toxicity studies or extrapolating data from one species to another." - NAS
Difference among strains
- One study found Fischer 344 rats were up to 10,000x more sensitive to the effects of the known ED chemical, bisphenol A than the closely related Sprague-Dawley strain.
Difference from housing & social rank.
- E.g. submissive males in mixed-sex cages develop smaller prostates than dominant males, confounding any ED effects.
Difference from womb position
- A male positioned b/w 2 males will have differing hormone levels than a male positioned b/w females
Difference from lack of test standardization
- E.g.: In UT assay, uterus must be cut out & weighed. Scientists have warned that different labs will cut the uterus in slightly diff. ways, thereby compromising test results.
- Dr. Matanoski, former chair of EPA's SAB: "You can't go across labs because each lab tends to be unique. I find that a big problem."
Low Dose Theory
- Scientists have failed repeatedly to repeat von Salle's low dose observations
- Low dose phenomenon seemingly does not exist or it is so subtle as to be toxicologically insignificant
- EPA & OECD should not be pressured into including extra low doses in all animal-based EDSP protocols

Under-Emphasis on Alternatives

The only non-animal assays apparently under consideration are ER/AR binding, steroid hormone synthesis, and possibly transcriptional activation (vs. the large number of animal-based assays).
High-Throughput Pre-Screen (HTPS):
- Prematurely cast aside (after only $70K of R&D), despite need for a priority-setting test
- After one feasibility study, EPA concluded in its Report to Congress: "the approach needed more development before routine use"
- Japan is virtually alone in its efforts to develop the HTPS
- Can QSARs for ER/AR binding fill the breach?
Nov. '01: $4M Congressional appropriation to EPA for dev. of non-animal methods in its testing programs:
"The Committee is concerned that the Agency has paid little attention and provided fewer resources to the development of alternative test methods ...The Committee, therefore, has provided $4 million ...for the [R&D], and validation of non-animal, alternative, chemical screening and prioritization methods, such as rapid, non-animal screens and ... QSAR ..., for potential inclusion in EPA's ...chemical evaluation programs."

Shortcuts on Validation

Misuse of flexibility?
- Hershberger and Uterotrophic assays have been around for many years ...
- But they require more than a rudimentary validation since they have never before been intended to be used for regulatory purposes for ED
- "It has ... been said that the validation phase of the new test development and acceptance sequence should be applied flexibly in this situation. ...Validation is concerned with the reliability and relevance of methods for a particular purpose. How can there possibly be flexibility about whether or not methods are reliable and relevant and about what they should be used for? What would be the value of the data such tests would provide, and with what confidence could they be applied in making decisions?" — M. Balls, ECVAM
Assessing in vitro tests via ICCVAM, in vivo tests via EPA: A potential double standard
- NTP Adv. Committee on Alt. Tox. Methods twice issued unanimous statements advising against this approach: "The Committee's primary concern is that both in vitro and in vivo methods be subjected to the same rigorous peer review and validation process to ensure the highest likelihood of acceptance by the regulatory agencies, the scientific community, and the public."
Lower validation standards at EPA?
- EPA indicated their intention to not produce standard detailed review papers for male & female pubertals, mammalian 2-gen reproductive toxicity study, or the 14-day intact male assay, and had considered providing EDMVS with only executive summaries of validation studies for peer review, rather than the complete studies themselves.
EDMVS was established to provide advice and recommendations to EPA regarding:
- The development of initial protocols
- Prevalidation and validation study designs
- The integration of prevalidation and validation study results into Tier 1 & 2 methods documents suitable for external peer review
Yet many EDMVS members admittedly have little validation experience and expertise

Concluding Remarks

Animal protection concerns re: EDSP remain largely the same as discussed at last year's TestSmart meeting:
- Heavy reliance on animal-based testing
- Under-Emphasis on alternatives methods
- Potential double standards in validating in vivo vs. in vitro procedures

Proposed EDSP Tier 1 & 2 Screens and Tests

Tier 1 Screening Battery includes:

Estrogen receptor binding/transcriptional activation assays (non-animal)
Androgen receptor binding/transcriptional activation assays (non-animal)
Steroidogenesis assay
Hershberger assay
Pubertal female assay
Frog metamorphosis assay
Fish reproductive screen
Uterotrophic assay

Tier 2 Testing Battery:

2-generational mammalian reproduction toxicity study
Avian reproductive toxicity study
Fish life cycle toxicity study
Invertebrate life cycle toxicity study
Amphibian developmental and reproductive toxicity study

TestSmart Endocrine Disruptors