Grantee Research Project Results
Final Report: Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption
EPA Contract Number: EPD04054Title: Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption
Investigators: Fort, Douglas J.
Small Business: Fort Environmental Laboratories Inc.
EPA Contact: Richards, April
Phase: II
Project Period: April 1, 2004 through June 30, 2005
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2004) Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , Ecological Indicators/Assessment/Restoration , SBIR - Monitoring
Description:
Concerns regarding both the presence of endocrine disruptors in food, water or other environmental media, and the potential risk they pose to humans and wildlife have been growing in recent years. Passage in 1996 of the Food Quality Protection Act and Amendments to the Safe Drinking Water Act reflected these concerns and required the U.S. Environmental Protection Agency (EPA) to develop a screening program, using appropriately validated test systems and other scientifically relevant information, to determine whether certain substances may have an endocrine effect in wildlife and humans.
Overall, although many pesticides and some industrial chemicals have undergone extensive toxicological testing, this testing may have been inadequate to determine whether they interact with the endocrine system and whether additional testing is needed for the EPA to assess and characterize both human health and ecological risk. Notwithstanding recognition that the scientific knowledge related to endocrine disrupting chemicals (EDCs) is still evolving, there is appropriate widespread agreement that the development of a screening and testing program is needed. By standardizing and validating the Xenopus tropicalis life cycle assay (XLCA) system for EDC testing, Fort Environmental Laboratories Inc. (FEL) is providing the scientific community, chemical and pharmaceutical producing industries, appropriate regulatory agencies and, ultimately, the public with a versatile amphibian reproduction, development and growth assay. Further, this model will enhance understanding of the significance of the effects of EDCs on the reproductive systems of amphibians. The technical feasibility of the XLCA model to evaluate the effects of EDCs is high. Although more work will be required to validate the model and prepare the guidance document, the XLCA model appears to fit the criteria established for emerging Tier 2 test method that are currently being sought by EPA’s Endocrine Disruptor Screening Program (EDSP). The model is relatively straightforward and could be commercialized following method standardization and validation. Results from the Phase I studies have shed light on attributes of the model in that it is capable of rapidly and cost-effectively testing materials with widespread endocrine activity for effects on development, growth and reproduction, each of which may be affected by EDCs. The ability to rapidly and cost-effectively screen for and evaluate the mechanisms of EDCs is an attractive alternative to the laborious and expensive testing systems used today. Increasing concerns over the widespread finding of EDCs in the environment have dramatically increased the need for standardized assays, such as the XLCA, since no other assay of this type is available today. Female reproductive function could be affected by EDCs at a number of target sites, including the brain, pituitary, gonad, liver and oviduct (nonmammalian species) (1 to18). Gonadal effects of EDCs have considerable potential to impair the reproduction of female amphibians and have been reported in other lower vertebrate wildlife groups (19 to 24).
An ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was established in 1994 by the National Institute of Environmental Health Sciences to develop a report recommending criteria and processes for validation and regulatory acceptance of toxicological testing methods that would be useful to federal agencies and the scientific community. In late 1998, the ICCVAM initiated an evaluation of the Frog Embryo Teratogenesis Assay - Xenopus (FETAX). FETAX represents a partial life cycle test method capable of evaluating early developmental effects. From the ICCVAM, two overwhelming recommendations were brought forth. The first suggestion was to expand the scope of testing with Xenopus to include both partial and full life cycle test methods to effectively exploit the true versatility of this widely used model species. The second recommendation was to strongly consider replacing the currently used X. laevis with a species with greater potential, X. tropicalis. Unfortunately, only X. laevis was available for use during the development of the FETAX model. The ICCVAM expert panel cited several lines of reasoning for the change, including some distinct advantages of using X. tropicalis. Being diploid, X. tropicalis also represents a genetic advantage over X. laevis, which is oligotetraploid. The smaller size of X. tropicalis compared to X. laevis represented practical advantages for the assay. For instance, less test material was required for the small organism and more organisms were accommodated in each replicated treatment. X. laevis produced a greater quantity of waste products during the assay than X. tropicalis (approximately 1.5 mg/L NH3-N versus <0.5 mg/L NH3-N; FEL, unpublished data). Increased clutch size also created a significant production advantage for the FETAX model. One of the most exciting advantages was the increased capacity to create transgenic lines of X. tropicalis and the use of cDNA microarray technology.
In addition to the aforementioned anticipated advantages of X. tropicalis, the greatest advantage was the shorter life cycle, which allowed more expedient and practicable means of using this model to evaluate the entire life cycle. It was also possible to create inbred strains of X. tropicalis, which will likely decrease the genetic variability observed in X. laevis. Developing inbred lines of X. laevis has been difficult at best. The end result of the proposed research was the development of a rapid, relatively inexpensive, and predictive amphibian life cycle model system. Further, this model filled the need for a whole animal, but nonmammalian (rodent) system for developmental toxicity testing. The market for this technology is similar to that of the originally developed FETAX model, but the new chronic exposure model will be larger due to the high demands of the EDSP process.
The primary objectives of Phase II studies were to: (1) validate the standard protocol developed for the XLCA using known EDCs, suspected EDCs, compounds with unknown EDC activity, and materials with no apparent EDC activity; (2) prepare a guidance document regarding the performance of this potential EDSP Tier 2 test method; and (3) continue efforts to commercialize the proposed assay system. These objectives flowed directly as an extension of the Phase I objectives, which were to develop and prepare the assay for validation and commercialization.
Summary/Accomplishments (Outputs/Outcomes):
Results from Phase I studies strongly indicated that chronic exposure to estradiol (E2) slightly delayed development, had a marginal effect on the normalcy of early larval development, dramatically skewed sex ratios toward the female gender, induced gonadal malformations, which included some intersexual development at low test concentrations, and induced liver abnormalities. Further, chronic E2 exposure impaired both female and male reproductive fitness. In females, a concentration-dependent decrease in the total number of oocytes present in the ovaries and increase in oocyte necrosis was observed. In males, a concentration-dependent reduction in testis weight and sperm cell count, as well as an increase in sperm cell dysmorphology, was noted. Chronic exposure to E2 decreased breeding success in both sexes, decreased fertilization in exposed females, and decreased embryo-larval viability in the F1 progeny. Results from these Phase I studies also suggested that chronic exposure to androstenedione marginally inhibited development similar to E2, had a marginal effect on the normalcy of early larval development, had only a slight effect in skewing sex ratios toward the male gender, induced gonadal malformations, which included some intersexual development at low test concentrations, and caused liver abnormalities. Androstenedione appeared to stimulate maturation of the oocytes in vivo. In males, a concentration-dependent increase in testis weight and sperm cell count were noted. Chronic exposure to androstenedione did not markedly alter breeding success in both sexes or decrease embryo-larval viability in the F1 progeny, in contrast to the effects of E2.
Of the eight compounds tested, each altered sexual development either in terms of sex ratios or in the induction of abnormal gonadal development. Evaluation of the effects of the tested materials indicated that Tier 1 test material ethinyl estradiol skewed phenotypic sexual maturation toward the female gender, whereas the aromatase inhibitor, 4-hydroxyandrostenedione (formestane), and the 5α-reductase inhibitor, dihydrofinasteride, skewed phenotypic sexual differentiation toward the male and female gender, respectively. None of these test materials significantly increase abnormal development of the gonads. Of the Tier 3 test materials, bisphenol A skewed phenotypic sex ratios somewhat toward the female gender. Octylphenol increased the frequency of undifferentiated testes, but did not alter the sex ratios appreciably. Atrazine, bromodichloromethane (BDCM) and diethylhexyl phthalate (DEHP) had no marked effect on phenotypic sex ratios, however, each increased the frequency of abnormal gonadal development or differentiation. Atrazine induced an increased number of intersex individuals, but did not induce gonadal abnormalities at frequencies appreciably above that of the controls. Like octylphenol, DEHP appeared to more sensitively target the developing male gonad, resulting in abnormal testicular development. Bisphenol A slightly increased abnormal female gonad development. BDCM primarily increased the incidence of abnormal testicular development, but to a lesser extent repressed ovary development. By standardizing and validating the X. tropicalis life cycle assay system for EDC testing, FEL is providing the scientific community, chemical and pharmaceutical producing industries, appropriate regulatory agencies and, ultimately, the public with a versatile amphibian reproduction, development and growth assay.
Conclusions:
Results from the present studies suggested that each of these test materials were capable of altering gonadal developmental and sexual differentiation in a concentration-dependent manner with varying degrees of potency, but with different signature responses. Intra- and inter-laboratory variability was relatively low in testing performed at the two study sites. These results indicated that the technology developed in the present project was transferable to outside laboratories. Overall, results suggested that the proposed XLCA model could be useful for evaluating impacts of test substances on gonad development, sexual differentiation and, ultimately, reproductive fitness.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 2 publications | 2 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Fort DJ, Guiney PD, Weeks JA, Thomas JH, Rogers RL, Noll AM, Spaulding, CD. Effect of methoxychlor on various life stages of Xenopus laevis. Toxicological Sciences 2004; 81(2):454-466. |
EPD04054 (Final) 68D03024 (Final) 68D03044 (Final) |
not available |
Supplemental Keywords:
endocrine disrupting chemicals, Endocrine Disruptor Screening Program, Xenopus tropicalis, X. laevis, amphibian, reproduction, development, gonadal development, growth, life cycle, estradiol, androstenedione, ethinyl estradiol, formestane, dihydrofinasteride, octylphenol, atrazine, bromodichloromethane, diethylhexyl phthalate, bisphenol A, EPA, small business, SBIR,, RFA, Health, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, Environmental Chemistry, Endocrine Disruptors - Environmental Exposure & Risk, Chemicals, endocrine disruptors, Monitoring/Modeling, Biochemistry, Environmental Monitoring, Endocrine Disruptors - Human Health, Biology, amphibian lifecycle testing, assays, altered sexual development, endocrine disrupting chemicals, sexual development, animal model, developmental biology, modeling, amphibian lifecycle test, androstene dione, ecological modelsSBIR Phase I:
Development and Preliminary Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption | Final ReportThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.