# Presentation: 117296

## A Fish Two Generation 

**Toxicity Test **
**Detailed Review Paper**
**Endocrine Disruptor Methods **
**Validation Subcommittee**
**December 2002**
**Leslie Touart**

## Detailed Review Paper:A Fish Two Generation Toxicity Test Detailed Review Paper

**WORK PERFORMED BY:**
**and**
***SpringbornSmithers Laboratories LLC ***
**On behalf of the United States Environmental Protection**
**Agency EPA CONTRACT NUMBER 68-W-01-023**

## METHODS USED IN THIS ANALYSIS

- On-line Literature Search
- “Dialog” On-Line search with database Biosis Previews Aquatic Science and Fisheries Abstracts
- Endocrine disruptor screening methods for fathead minnow, zebrafish, medaka, sheepshead minnow
- Key Words “estrogen* or testosteron* or endocrin* or antiandrogen* or androgen* or hormon* or thyroxin* or * thyroid * method, protocol etc...
- 7453 records were refined down to 601 records

## METHODS USED IN THIS ANALYSIS Cont.

- Interviews With The Following Experts
- Tom Hutchinson
- Reynaldo Patino
- Taisen Iguchi
- Alf Lundgren
- Dan G. Cyr
- Nancy Denslow

## METHODS USED IN THIS ANALYSIS Cont.

- External/Internal Peer Review
- Dr. Dave Hinton – Duke Unv. USA
- Dr. John Sumpter – Brunell Unv. UK
- Dr. Gary Thorgaard  - Univ. of Wash. USA
- EPA Technical Experts

## OVERVIEW AND SCIENTIFIC BASIS

- Evidence exists that EDCs affect sexual differentiation, development, and reproduction in fish
- Fish life cycle test methods have been standardized for decades
- Existing methods do not assess transgenerational effects and lack relevant biochemical, morphological, and behavioral endpoints
- The proposed Fish Two Generation Test addresses these EDC relevant omissions

## Test Species

- Fathead minnow (*Pimephales promelas*)
- Medaka (*Oryzias latipes*)
- Zebrafish (*Danio rerio*)
- Sheepshead minnow (*Cyprinodon variegatus)*
- small size at maturity
- ease of culture
- maintenance costs
- asynchronous spawners

## Fathead MinnowFamily Cyprinidae

- 35 to 75 mm length
- Extensive aquatic toxicity in USA
- generation time about 4 months
- sexually dimorphic
- 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

## MedakaFamily Adrianichthyidae

- 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

## 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.

## ZebrafishFamily 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

## Sheepshead MinnowFamily Cyprinodonitidae

- Native to Atlantic and Gulf of Mexico estuaries
- 35 mm to 50 mm length
- Tolerates wide ranges in temperature (0 to 40oC) and salinity (0.1 ppt to 149 ppt)
- Sexually dimorphic
- Sexual maturity in 60 days
- Low variability in fecundity
- Large historical regulatory database

## Sheepshead Minnow

- _Strengths_
- Very short life cycle (<60days to sexual maturity), seawater costs may be offset by shorter exposure times for testing
- Males large enough for individual blood plasma samples
- Distinct sexual dimorphism
- Relatively low variability in fecundity
- Relatively large historical regulatory database
- Many laboratories are familiar with culture and testing
- Relatively small fish making culture and testing possible in smaller space

- _Weaknesses_
- Estuarine/marine species, salinity of 15 to 30 ppt recommended, however, lower salinity may be possible (5 ppt)
- Culture requires a large number of females to produce enough eggs in a 24-hr period to initiate a life-cycle test
- Limited information on reproductive endocrinology
- Small size makes individual blood plasma samples not likely

## Routes of Administration of Chemical Exposure 

- Aqueous
- Dietary exposures
- Direct injection techniques
- Intravascular
- intraperitoneal

## Measurement Endpoints

- Growth and Morphological Alterations
- Gonadosomatic Index
- Histology Techniques
- Sexual Differentiation
- Secondary Sex Characteristics
- Measures of Reproductive Performance
- Fecundity
- Gamete Viability
- Changes in Spawning Behavior
- Biochemical Measures
- Vitellogenin Induction
- Tissue Steroid Concentrations
- Thyroid hormones

## 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

- 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

**Partial Life-Cycle Test   **
**	****{Adult (P) to Juvenile (F1)}**
- 2) **Full Life-Cycle Test **
**    ****{Egg (P) to Juvenile (F1)}**
- 3) **Multi-Generation Test**
**    ****{Egg (P) to Juvenile (F2)}**** **
**4) Two Generation Test **
**    ****{Adult (P) to Juvenile (F2)}**

## Partial Life-Cycle Test {Adult (P) to Juvenile (F1)}

- A partial life-cycle toxicity test, which exposes P adult, sexually mature fish and the early life cycle of F1 fish, ( can be used to estimate the NOEC)
- A pre-exposure reproductive evaluation is conducted on the P fish.
- Biological endpoints evaluated:
- P pre-exposure, secondary sexual characteristics and fecundity/reproduction (e.g., eggs/female)
- P post-exposure, survival, secondary sexual characteristics, fecundity/reproduction (e.g., eggs/female), GSI, histopathology, and protein and sex steroid biomarkers (e.g., VTG)
- F1 hatching success, survival, growth (length and weight)

## Full Life-Cycle Test {Egg (P) to Juvenile (F1)}

- A full life cycle test developed (Benoit 1981- fathead minnows ) and (Hansen et al. 1978 - sheepshead minnow).
- Initiated with fertilized eggs (P) and the fish are continuously exposed through reproductive maturity, followed assessment of the early development of the F1 generation.
- Biological endpoints evaluated:
- P  embryo time-to-hatch, hatching success, larval survival and length, weight of thinned fish, survival, secondary sexual characteristics, fecundity/reproduction (e.g., eggs/female), growth.
- F1  hatching success, survival, growth (length and weight)

## Multi-Generation Test  {Egg (P) to Juvenile (F2)}

- Multi-generation toxicity test, exposes all life-stages of two generations of fish
- Test is initiated with eggs and two full generations of fish are exposed during the test (can estimate the NOEC)
- Biological endpoints evaluated:
- C	P and F1 hatching success, survival, growth (length and weight), time-to-maturity, sex ratio, secondary sexual characteristics, fecundity/reproduction (e.g., eggs/female), and protein and sex steroid biomarkers (e.g., VTG).
- F2 hatching success, survival and growth.

## Two Generation Test  {Adult (P) to Juvenile (F2)}

- Two generation life-cycle toxicity test, exposes the adult P, full F1generation,and measures F2 viability
- Variables including the time-line of the test, the number of fish required in the test, and obtaining endpoints such as Vtg plasma levels will be associated with the different species.
- Biological endpoints evaluated:
- P  Survival, secondary sex characteristics, reproductive behavior, spawning activity, fecundity, fertilization success
- F1 hatching success, survival, growth (length and weight), time-to-maturity, sex ratio, secondary sexual characteristics, fecundity/reproduction (e.g., eggs/female), and protein and sex steroid biomarkers (e.g., VTG).
- F2 hatching success, survival and growth.

## Timeline forTwo-Generation Protocol  with the Fathead Minnow

| Parental | → | → |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|     F1 |  |  | →  // | → |  |  |
|     F2 |  |  |  |  | → |  | |
|  |  |  |  |  |  |  |

- Time (weeks)

**-3           0           3            18         21        25**

## Significant Data Gaps

- •	Male-specific effects of estrogen agonists other than VTG induction.
- •	The effects of anti-estrogens
- •	The effects of androgen agonists and antagonists
- •	Baseline data for thyroid hormone levels in test species.
- •	The effects of thyroid hormone agonists (or thyroid stimulation) on reproduction.
- Published methods of sexual differentiation for fathead and sheepshead minnows

## IMPLEMENTATION CONSIDERATIONS

- Pre-validation studies following the ICCVAM validation process
- Recommend evaluating the increased sensitivity of a two-generation design over the existing fish full life-cycle standard practice
- Recommend determining and optimizing specific two generation protocol variables for the candidate species
- Recommend demonstration of sensitivity, reliability and reproducibility for each species in the recommended protocol
- Validation of the study design through interlaboratory comparisons