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Fish Screening Assay Detailed Review Paper NACEPT Endocrine Disruptor Methods Validation Subcommittee March 2002 Les Touart

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Fish Screening Assay Detailed Review Paper NACEPT Endocrine Disruptor Methods Validation Subcommittee March 2002 Les Touart. Detailed Review Paper: FISH SCREENING ASSAYS FOR ENDOCRINE DISRUPTION. WORK PERFORMED BY On behalf of the United States Environmental Protection Agency

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slide1

Fish Screening Assay

Detailed Review Paper

NACEPT

Endocrine Disruptor Methods Validation Subcommittee

March 2002

Les Touart

detailed review paper fish screening assays for endocrine disruption
Detailed Review Paper:FISH SCREENING ASSAYS FOR ENDOCRINE DISRUPTION

WORK PERFORMED BY

On behalf of the United States Environmental Protection Agency

EPA CONTRACT NUMBER 68-W-01-023

methods used in this analysis
METHODS USED IN THIS ANALYSIS
  • On-line Literature Search (August 9th 2001)
    • “Dialog” On-Line search with database Biosis Previews Aquatic Science and Fisheries Abstracts
    • Endocrine disruptor screening methods for fathead minnows, zebrafish, and Japanese medaka
    • Key Words “estrogen* or testosteron* or endocrin* or antiandrogen* or androgen* or hormon* or thyroxin* or * thyroid * method, protocol etc…
    • Approximately 10000 records were refined down to 500 papers that were reviewed
slide4
OVERVIEW AND SCIENTIFIC BASIS OF FISH REPRODUCTIVE SCREENING ASSAY (ENDOCRINE CONTROL OF REPRODUCTION)
  • Fish differ in steroid profiles from mammals
  • Estrogen receptor in fish differ structurally and functionally from mammals
  • Steroid receptors in eggs and hepatic vitellogenin (VTG) have no known analogous receptors in mammals
  • Fish Reproductive Screen Assay is essential to address these known endocrine differences.
test species
Test Species
  • Fathead minnow (Pimephales promelas)
  • Japanese medaka (Oryzias latipes)
  • Zebrafish (Danio rerio)
    • small size at maturity
    • ease of culture
    • maintenance costs
    • asynchronous spawners
fathead minnow family cyprinidae
Fathead MinnowFamily Cyprinidae
  • 35 to 75 mm length
  • Extensive aquatic toxicity in USA
  • generation time about 4 months
  • sexually dimorphic
  • can be kept in breeding condition all year
  • females produce 50 to 250 embryos per spawn
fathead minnow
Strengths

Large enough to collect individual blood plasma samples

Distinct secondary sex characteristics in both sexes

Large historical regulatory database

Many laboratories are familiar with culture and testing

Spawn on a substrate

High fertilization rate

Indigenous to North America

Weaknesses

Relatively long life cycle

Relatively high variability in fecundity

Relative size of the fish requires more space for culture and testing

Intersex condition is less frequently observed compared to other fishes.

Genome poorly characterized

Fathead Minnow
japanese medaka family cyprinodontidae
Japanese medakaFamily Cyprinodontidae
  • indigenous to Japan, Taiwan, and southeastern Asia
  • Generation interval of 2 to 3 months
  • sexually dimorphic
  • 25 mm to 50 mm length
  • females produce 10 to 30 eggs per spawn
  • estimated to be over 500 cultivated strains
    • Genetically Engineered / Inbred Strains in Toxicity Testing
japanese medaka
Strengths

Relatively short life cycle

Relatively small fish, making culture and testing possible in smaller space

Female sex determined during embryo stage vs. male sex determined after hatch

Sex-linked color strain

Weaknesses

Smaller size reduces individual blood sample volumes compared to fathead minnow

Less distinctive secondary sex characteristics

Regulatory data base less extensive compared to fathead minnow.

Limited use in short-term tests in the U.S.A.

Japanese medaka
zebrafish family cyprinidae
ZebrafishFamily Cyprinidae
  • Native to East India and Burma
  • 4 cm to 5 cm in length
  • Extensive aquatic toxicity in Europe
  • Difficult to sex zebrafish
  • Sexual maturity in 10 to 12 weeks
  • 150 to 400 eggs per female
  • Development of transgenic zebrafish
zebrafish
Strengths

Short life cycle

Small fish, making culture and testing possible in smaller spaces

Male fish go through a hermaphroditic phase as juveniles

Widely used in other medical and genetic research

Frequently used in Europe for regulatory purposes

Transgenic fish increasingly available

Anticipated that entire genome will be sequenced soon.

Weaknesses

Small size makes individual blood plasma samples not likely

Minimal secondary sex characteristics

Limited US regulatory data base

Limited testing experience in the US

Zebrafish
routes of administration of chemical exposure
Routes of Administration of Chemical Exposure
  • Aqueous
  • Dietary exposures
  • Direct injection techniques

-intraperitoneal

-intramuscular

-dorsal sinus

measurement endpoints
Measurement Endpoints
  • Growth and Morphological Alterations
    • Gonadosomatic Index
    • Histology Techniques
    • Sexual Differentiation
    • Secondary Sex Characteristics
  • Biochemical Measures
    • Vitellogenin Induction
    • Tissue Steroid Concentrations
    • Thyroid hormones
  • Measures of Reproductive Performance
    • Fecundity
    • Gamete Viability
    • Fertilization Success
measurement of biochemical endpoints
MEASUREMENT OF BIOCHEMICAL ENDPOINTS
  • Sex Steroids in Tissues Estrogens/Androgens/Progestins
    • Radioimmunoassay (RIA)
    • Enzyme-linked Immunosorbent Assay (ELISA)
    • Liquid/Gas Chromatography with Mass Selective Detection (LC/GC-MS)
measurement of vitellogenin
Measurement of Vitellogenin
  • Indirect Quantification of Vitellogenin Protein
    • Alkaline-labile Phosphate Assay
  • Direct Quantification of Vitellogenin Protein
    • RIA
    • Enzyme-linked Immunosorbent Assay
      • Antibody-capture
      • Antigen-capture
    • Direct Enzyme-linked Immunosorbent Assay
    • Sandwich Enzyme-linked Immunosorbent Assay
  • Quantifying Vitellogenin mRNA
    • Ribonuclease Protection Assay (RPA)
    • Quantitative Reverse Transcription-Polymerase Chain Reaction (QRT-PCR)
  • Mass spectrometry (MS)
candidate protocols
CANDIDATE PROTOCOLS

1) 21-day reproductive assay (as described in Ankley et al., 2001)

2) 14-day fish reproductive assay (modified from the version described in Ankley et al., 2001)

3) 14-day fish non-reproductive screen (OECD Draft-31 December 2001)

21 day fish reproductive assay see hand out
21-Day Fish Reproductive Assay (See Hand Out)
  • Chemical Exposure – 21 Days
  • 14 day pre-exposure spawning quantitative measures
  • Exposure Measurement Endpoints
    • Adult survival, reproductive behavior, secondary sexual characteristics, GIS and gonadal history, plasma VTG and sex steroids (b-estradiol, testosterone, 11-KT) concentrations, fecundity, fertility, embryo hatch, and larval survival
  • Acceptance Criteria: D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls and successful egg production in controls
14 day fish reproductive assay see hand out
14-Day Fish Reproductive Assay(See Hand Out)
  • Chemical Exposure – 14 Days
  • 14 day pre-exposure spawning observations
  • Exposure Measurement Endpoints
    • Adult survival, reproductive behavior, secondary sexual characteristics, GIS and gonadal history, plasma VTG and sex steroids (b-estradiol, testosterone, 11-KT) concentrations, fecundity, fertility, embryo hatch, and larval survival
  • Acceptance Criteria:
    • D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls and successful egg production in controls
  • Similar to the 21 day Fish Reproductive Assay with out quantitative measures of fecundity or embryo viability (e.g., hatchability) are made during the 14 day pre-exposure
14 day fish non reproductive assay see hand out
14-Day Fish Non-Reproductive Assay (See Hand Out)
  • Chemical Exposure - 14 Days
  • Exposure Measurement Endpoints
    • Non-specific:
      • Survival,
      • Behavior
    • Endocrine specific:
      • Gross morphology (GSI)
      • VTG
      • Gonadal history
  • Acceptance Criteria: D.O. ≥ 60% saturation; Mean temp. 25°C ± 2°C; 90% survival in the controls
  • (No reproductive Endpoints)
significant data gaps
Significant Data Gaps

• Male-specific effects of estrogen agonists other than VTG induction.

• The effects of anti-estrogens, especially pure or Type II anti-estrogens in sexually mature test species.

• The effects of androgenic and anti-androgens in sexually mature test species, specifically, endpoints other than secondary sex characteristics that may be more sensitive to (anti-) androgens.

• Baseline data for thyroid hormone levels during reproduction in test species.

• The effects of thyroid hormone agonists (or thyroid stimulation) on reproduction.

implementation considerations
IMPLEMENTATION CONSIDERATIONS
  • Pre-validation studies following the ICCVAM validation process
    • Recommend Comparison of three candidate protocols with like chemicals
    • Recommend Evaluation of Vtg methods
    • Recommend Consideration be given to the lack of data on Thyroid – none of candidate protocols adequately measure thyroid function/performance. Consider smoltification assay
  • Validation of the study design through interlaboratory comparisons
questions
Questions
  • Does the DRP provide adequate background and rationale to support further validation of a fish reproduction assay for screening potential endocrine disrupting chemicals?
  • Does the EDMVS have suggestions to improve the DRP?
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