COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE DISRUPTING CHEMICALS COMPARATIVE EVALUATION OF FATHEAD COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING MINNOW ASSAYS FOR DETECTING ENDOCRINE ENDOCRINE DISRUPTING CHEMICALS DISRUPTING CHEMICALS Endocrine Disruptor Methods Validation Subcommittee August 2003 Presented by:

Dr. Irv Schultz and Michael L. Blanton 2 COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING ENDOCRINE DISRUPTING CHEMICALS COMPARATIVE EVALUATION OF FATHEAD COMPARATIVE EVALUATION OF FATHEAD MINNOW ASSAYS FOR DETECTING MINNOW ASSAYS FOR DETECTING ENDOCRINE ENDOCRINE DISRUPTING CHEMICALS DISRUPTING CHEMICALS WORK PERFORMED BY On behalf of the United States Environmental Protection Agency EPA CONTRACT NUMBER 68 W 01 023 3 Background Background Background EDSTAC ( 1998) recommends inclusion of fish ( fathead minnow) recrudescence/ reproduction assay as one of five Tier 1 screening tests for EDCs that could affect the HPG axis Evaluations conducted by EPA scientists at Duluth Mid

Continent Ecology Division ( MED; 1998 1999) suggest that recrudescence assay would be problematic for routine testing due to logistic constraints Alternative short term reproduction assay with fathead minnow described by Ankley et al. ( 2001; Environ. Toxicol.

Chem. 20: 1276)

Detailed standard guaidance published by USEPA ( 2002; A Short Term Test Method for Assessing the Reproductive Toxicity of Endocrine Disrupting Chemicals using the Fathead Minnow ( Pimephales promelas), EPA/ 600/ R

01/ 067) 4 Background cont.

Background cont.

Background cont.

Assay system evaluated/ validated using materials reflective of MOA of concern:

Estrogens ( methoxychlor, Ankley et al. 2001; E2, Harries et al. 20001)

Androgens ( methyltestosterone, Ankley et al. 2001;

20032)

Anti androgens ( vinclozolin, Makyenen et al. 20003;

flutamide, Jensen et al. 4)

Aromatase inhibitor ( fadrozole, Ankley et al. 20025)

1Environ. Sci. Technol. 34: 3003 ( conducted with pairedspawners

2Environ. Toxicol. Chem. 22: 1350

3 Aquat. Toxicol. 48: 461

4Aquat. Toxicol. Submitted

5Toxicol. Sci. 67: 121 5 Background cont.

Background cont.

Background cont.

Organization for Economic Cooperation and Development ( OECD) forms EDTA ( Endocrine Disruptor Testing Advisory) oversight group with goal of international harmonization of EDC testing ( 1998) Validation Management Ecology ( VMG eco)

assembled as working group under EDTA ( 2001)

to focus on EDC testing with non mammalian vertebrates ( and invertebrates)

Fish Drafting Group ( FDG) one of several formed to provide technical input to VMG eco ( 2001)

International ring testing of draft fish protocols initiated ( 2003) 6 Background cont.

Background cont.

Background cont.

Draft protocols provided for consideration to FDG for fish EDC testing:

 Short term ( 14 d) juvenile vitellogenin induction assay ( fathead minnow)*

 Short term ( 21 d) reproduction assays with fathead minnow and medaka as described by Ankley et al.

( 2001)

 Zebrafish ( 40 60 d) early developmental assay

 Non spawning ( 21 d**) assay with fathead minnow and medaka conducted under conditions similar to spawning assay

* Emphasis decreased due to limitations in MOA detected ( estrogens)

* * Initially proposed for 14 d as opposed to 21 d 7 Purpose of this Study Purpose of this Study Purpose of this Study Evaluate short term screening assays designed to detect substances that interfere with the estrogen and androgen systems of fish

evaluate the transferability and sensitivity of short term reproduction assays with the fathead minnow to identify specific modes of action of endocrine disruptors using four model compounds

conduct a side by side comparison of the 21 day fathead minnow short term reproduction assay ( EPA 2001) with two separate 14 day assays: one a shortened version of the 21

day assay with less intensive monitoring of reproductive performance and the other an assay using non spawning fathead minnows ( OECD Draft 31 December 2001) 8 Model Chemicals used and Modes of Action Model Chemicals used and Model Chemicals used and Modes of Action Modes of Action Methoxychlor is considered a weak estrogen that is biotransformed into an estrogen like metabolite.

Trenbolone is an anabolic steroid that mimics 11 KT and testosterone. This is expected to cause masculinization of females and perhaps enhance growth.

Flutamide is an established mammalian anti androgen. If this effect occurs in fish, then the normal effectiveness of testosterone and 11 KT would be reduced.

Fadrozole inhibits aromatase, which is the key step in E2 synthesis. Methods Methods Methods 10 Experimental Test Concentrations and Chemical Analyses Experimental Test Concentrations and Experimental Test Concentrations and Chemical Analyses Chemical Analyses Methoxychlor: analyzed by gas chromatograph with an electron capture detector ( GC ECD)

Trenbolone : control and low samples analyzed by GC with mass selective detection ( MSD); mid and high samples analyzed by highperformance liquid chromatograph ( HPLC) analysis with a fluorescence detector Flutamide : analyzed by high performance liquid chromatography ( HPLC) with a UV/ VIS detector at the 220 nm wavelength Fadrozole : analyzed by HPLC with a UV/ VIS detector Methoxychlor & flutamide prepared using a saturator column ( Kahl et al.

1999) 5.0 2.5 1.0 Methoxychlor 50 25 5 Fadrozole 650 350 6.0 Flutamide 1.0 0.5 0.1 Trenbolone High Mid*

Low Chemical ( ug/ L) 11 Proportional Diluter Proportional Diluter Proportional Diluter Continuous flow proportional diluter used for chemical delivery Adjusted to deliver three concentrations ( including control) with four replicates per concentration for the EPA assays.

Second diluter modified for the nonspawning assay to deliver four concentrations ( including control) with two replicates per concentration.

Chemical stock solution metered into the mixing cell of the diluter using a fluid metering pump. Diluter set to add chemical laden water to the test chamber every 12 min, equal to six volume exchanges of water per day. 12 Animal Husbandry Animal Husbandry Animal Husbandry 4 month old P. promelas were obtained from EC& T for use in the methoxychlor experiments Based on methoxychlor results it was decided that younger P.

promelas should be purchased and a in house culture established at Battelle 30 to 60 day old P. promelas were obtained from ABC Laboratories for use in the trenbolone, flutamide,

and fadrozole experiments   Water conditions were maintained at 24 ° C to 26 ° C. A flow through system provided adequate volume replacement while maintaining required constant temperature. Gentle aeration was provided to the tanks. P. promelas housed in 55   gal tanks 13 Summary of the 14 day Nonspawning Assay Summary of the 14 Summary of the 14 day Nonspawning Assay day Nonspawning Assay Protocol : OECD draft proposal   31 December 2001 ( OECD 2001)

Healthy, sexually dimorphic nonspawning adults ( males and females contained in separate chambers

Two replicate tanks ( one for each gender)

per treatment with four treatments: a dilution water control, and low, medium,

and high concentrations  Gross morphological conditions, GSI determinations and histological analyses were performed  Plasma samples analyzed for sex steroids and VTG.

Excising gonads 14 Summary of the 14 day EPA Assay Summary of the 14 Summary of the 14 day EPA Assay day EPA Assay Pre exposure ( 7days) four females/ two males No quantitative measures of fecundity Larval hatching conducted once during preexposure and once during chemical exposure Successful breeding pairs transferred to the chemical exposure Four replicate containers, a dilution water control, and a low and high concentration Behavior, fecundity and routine water chemistry measurements assessed Gross morphological conditions, GSI and histological analyses were performed Plasma samples analyzed for sex steroids and VTG. Terracotta tiles and screen used for egg collection Eggs on tile 15 Summary of 21 day EPA Assay Summary of 21 Summary of 21 day EPA Assay day EPA Assay 21 day assay paralleled 14 day assay with some exceptions:

Larval hatching once during preexposure phase and three times during chemical exposure ( Days 7, 14 and 21)

Pre exposure conducted for 14 days;

quantitative counts of fecundity Successful breeding pairs used for chemical exposure Test conducted using same stock solution, proportional diluter, and water table as the 14 day EPA assay a) View of pre exposure tanks b) fecundity counts   Appearance, behavior, and fecundity assessed daily   Gross morphological conditions, GSI and histological analyses performed   Plasma analyzed for sex steroids and VTG. Histology Histology Histology 17 Female Histology Methods Female Histology Methods Female Histology Methods General Ovarian Staging

General development scored from 1 to 5 ( 18 sections)

Quantitative Staging

Oogonia and oocytes were typed; 100 cells from each of three sections were rated according to developmental stage Atretic Follicles

Proportion of atretic follicles/ 100 cells was determined Corpora Lutea

Proportion of corpora lutea/ 100 cells was determined 18 Male Histology Methods Male Histology Methods Male Histology Methods General Testes Staging  General development scored from 1 to 5 ( 12 sections)

Quantitative Testes Staging  spermatic cells were typed; 100 cells were from each of the three slides were rated according to developmental stage.

Diameter of the seminiferous tubule was measured Other changes were noted, including changes to the interstitial tissues  proliferation of Sertoli or Leydig cells  premature shedding of spermatocytes  presence of any ovatestes or patterns of testicular atrophy  foci of necrotic spermatocytes Methoxychor Methoxychor 20 Methoxychlor Cumulative Fecundity Methoxychlor Cumulative Fecundity Methoxychlor Cumulative Fecundity EPA 21 Day Assay 0 5,000 10,000 15,000 20,000 25,000  15  10  5 0 5 10 15 20 Day Cumulative Number of Eggs Control Low High 21 Methoxychlor Cumulative Fecundity Methoxychlor Cumulative Fecundity Methoxychlor Cumulative Fecundity EPA14 Day Assay 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Day Cumulative Number of Eggs Control Low High 22 Methoxychlor Male Vitellogenin ( mg/ mL)

Methoxychlor Male Vitellogenin Methoxychlor Male Vitellogenin ( mg/ mL)

( mg/ mL) NS NS NS Significant ? 0.22 0.16 9   10.89 1 0.65 0.56 4 High 0.001 0.001 10             Medium 0.003 0.002 10 0.001 0.001 7 < 0.001 < 0.001 7 Low 0.001 < 0.001 10 < 0.001 < 0.001 7 < 0.001 < 0.001 7 Control SD Mean n SD Mean n SD Mean n Non Spawning Adult 21 Day 14 Day 23 Methoxychlor Steroid Results Methoxychlor Steroid Results Methoxychlor Steroid Results MED Data: 5 µ g/ L   E2 in Females; T/ 11KT in males.

EPA 21 Day assay: no dose related effects.

EPA 14 Day assay: no dose related effects; low dose reduced E2 in females and T in males.

Non spawning Adult assay: no dose related effects;

mid ( 2.0 µ g/ L) dose increased T in females.

High variability within treatments. 24 Methoxychlor Histology Results Methoxychlor Histology Results Methoxychlor Histology Results EPA 21 Day assay: no noticeable effects on gonad histology.

EPA 14 Day assay: no noticeable effects on gonad histology.

Non spawning Adult: Low ( 0.8 µ g/ L) and mid ( 2.0 µ g/ L) concentrations had noticeable effects on male gonad histology. 25 Effects of Methoxychlor on Fathead Effects of Methoxychlor on Fathead Minnow Reproduction EPA Minnow Reproduction EPA   MED MED  15  10  5 0 5 10 15 20 25 Time ( d)

0 1000 2000 3000 4000 5000 6000 7000 8000 Eggs Spawned Cumulative Number of Methoxychlor ( µ g/ L)

Control 0.5 5 * 26 Methoxychlor Effects on Male VTG EPA MED Methoxychlor ( µ g/ L)

Control 0.5 5 .0000 .0025 .0050 4 5 6 Vit ellogenin (

mg/

ml) * Trenbolone Trenbolone 28 Trenbolone Cumulative Fecundity Trenbolone Cumulative Fecundity Trenbolone Cumulative Fecundity EPA21 Day Assay 0 5,000 10,000 15,000 20,000 25,000 30,000  14  12  10  8  6  4  2 1 3 5 7 9 11 13 15 17 19 21 Day Cumulative Number of Eggs Control Low High 29 Trenbolone Cumulative Fecundity Trenbolone Cumulative Fecundity Trenbolone Cumulative Fecundity EPA 14 Day Assay 0 2,000 4,000 6,000 8,000 10,000 12,000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Day Cumulative Number of Eggs Control Low High 30 Trenbolone Female Vitellogenin ( mg/ mL)

Trenbolone Female Vitellogenin Trenbolone Female Vitellogenin ( mg/ mL)

( mg/ mL) Control > High; Medium Low > Medium *

Control > High Low > High Control > High Low > High Significant ? 0.15 0.11 9 0.27 0.11 15 0.08 0.04 16 High 0.24 0.13 9             Medium 1.12 0.91 10 1.09 1.22 14 1.80 2.24 14 Low 1.02 2.81 8 1.16 1.29 13 0.59 2.61 16 Control SD Mean n SD Mean n SD Mean n Non Spawning Adult 21 Day 14 Day 31 Trenbolone Steroid Results Trenbolone Steroid Results Trenbolone Steroid Results MED Data: 0.5 µ g/ L   E2, T in females EPA 21 Day assay: low dose ( 0.041 µ g/ L) reduced T in females; high dose ( 0.60 µ g/ L) reduced E2 in females versus low dose.

EPA 14 Day assay: high dose ( 0.78 µ g/ L) reduced E2 and T in females.

Non spawning Adult assay: low dose ( 0.071 µ g/ L)

reduced E2, T, and KT in males, perhaps reduced T in females; mid dose ( 0.45 µ g/ L) reduced E2 in females and T in males and perhaps females; high dose ( 0.86 µ g/ L) reduced E2 and T in females.

High variability within treatments. 32 Trenbolone Histology Results Trenbolone Histology Results Trenbolone Histology Results EPA 21 Day assay: High dose ( 0.60 µ g/ L) reduced the proportion of corpora lutea and increased the proportion of atretic follicles in the ovaries.

EPA 14 Day assay: High dose ( 0.78 µ g/ L) reduced the proportion of atretic follicles in the ovaries compared to the control and low dose; progression to late vitellogenic stage inhibited. Testes in males from the high dose showed a later general developmental stage than those from the control and low dose.

Non spawning Adult: Low ( 0.07 µ g/ L), mid ( 0.45 µ g/ L), high ( 0.86 µ g/ L) doses less advanced ovarian staging; low reduced atretic follicles. 33 Effects of Trenbolone on Fathead Minnow Effects of Trenbolone on Fathead Minnow Reproduction EPA Reproduction EPA MED MED 0 2 4 6 8 10 12 14 16 18 20 Exposure ( d)

0 800 1600 2400 3200 4000 Cumulative Number of Eggs Spawned Control 0.005 0.05 0.5 5.0 50 Trenbolone ( µ g/ L) *

*** 34 Control 0.005 0.05 0.5 5.0 50 0 5 10 15 20 25 Tubercle Score * * * *

Trenbolone ( µ g/ l)

Effects of Trenbolone on Female Tubercles EPA MED 35 Control 0.005 0.05 0.5 5.0 50 0 6 12 18 24 30 Vitellogenin (

mg/

ml) a a b c c b, c Trenbolone ( µ g/ l)

* * * *

Trenbolone Effects on Females EPA MED 36 Masculinization by Trenbolone EPA MED Control male and female Female 0.05 µ g/ L Flutamide Flutamide Flutamide 38 Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity EPA 21 Day Assay 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000  14  12  10  8  6  4  2 1 3 5 7 9 11 13 15 17 19 21 Day Cumulative Number of Eggs Control Low High 39 Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity EPA 14 Day Assay 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 20,000 0 2 4 6 8 10 12 14 Day Cumulative Number of Eggs Control Low High 40 Flutamide Steroid Results Flutamide Steroid Results Flutamide Steroid Results MED Data: T   in Females.

EPA 21 Day assay: high dose ( 510 µ g/ L) reduced E2 and increased T and KT in males; increased T in females.

EPA 14 Day assay: high dose ( 519 µ g/ L) reduced E2 in males versus low dose ( 46 µ g/ L) .

Non spawning Adult assay: no dose related effects.

High variability within treatments. 41 Flutamide Histology Results Flutamide Histology Results Flutamide Histology Results EPA 21 Day assay: high dose ( 510 µ g/ L) increased the proportion of atretic follicles in the ovaries compared to the control and low dose.

EPA 14 Day assay: high dose ( 519 µ g/ L) increased the proportion of atretic follicles and decreased the proportion of corpora lutea in the ovaries. Males from high dose showed more advanced testicular staging.

Non spawning Adult: no noticeable dose related effects on histology; several males in all three doses had abnormal testes histology. 42 Summary of EPA Flutamide Results Summary of EPA Flutamide Results Summary of EPA Flutamide Results Dose of 650 µ g/ L slightly increased VTG in males and females, and slightly increased plasma T in females.

Dose of 650 µ g/ L increased early stage follicles and atretic oocytes relative to controls.

Dose of 650 µ g/ L caused abnormal gonadal histology in males, with increased incidence of pychnotic and degenerating spermatocytes among healthy cysts. 43 Summary of Battelle Flutamide Results Summary of Battelle Flutamide Results Summary of Battelle Flutamide Results There was no apparent effect on plasma VTG in males or females from any of the three designs.

The high dose ( 510 519 µ g/ L) decreased plasma E2 in males, increased T in females ( 21 Day assay), and KT in males ( 21 Day assay).

The high dose ( 510 519 µ g/ L) increased the proportion of atretic follicles in the ovaries compared to the control and low dose.

Non spawning assay showed abnormal male histology that was not related to flutamide dose. 44 0 2 4 6 8 10 12 14 16 18 20 22 Exposure ( d)

0 1000 2000 3000 4000 5000 6000 Cumulative Number of Eggs Spawned Control 62.8 649 Flutamide ( µ g/ L)

Effects of Flutamide on Fathead Minnow Reproduction EPA MED * 45 Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity Flutamide Cumulative Fecundity EPA 14 Day Assay 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 20,000 0 2 4 6 8 10 12 14 Day Cumulative Number of Eggs Control Low High Fadrozole Fadrozole Fadrozole 47 Fadrozole Cumulative Fecundity Fadrozole Cumulative Fecundity Fadrozole Cumulative Fecundity EPA 21 Day Assay 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000  14  12  10  8  6  4  2 0 2 4 6 8 10 12 14 16 18 20 Day Cumulative Number of Eggs Control Low High 48 Fadrozole Cumulative Fecundity Fadrozole Fadrozole Cumulative Fecundity Cumulative Fecundity EPA 14 Day Assay 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 20,000 0 2 4 6 8 10 12 14 Day Cumulative Number of Eggs Control Low High 49 Fadrozole Female Estradiol ( pg/ mL)

Fadrozole Female Estradiol Fadrozole Female Estradiol ( pg/ mL)

( pg/ mL) Control > High *

Control > Low Control > High Low > High Significant ? 129 142 10     0 86 39 16 High 457 320 9             Medium 524 727 10 756 1,343 7 468 1,088 16 Low 1,132 1,519 10 1,492 2,861 16 1,176 2,064 15 Control SD Mean n SD Mean n SD Mean n Non Spawning Adult 21 Day 14 Day 50 Fadrozole Female Vitellogenin ( mg/ mL)

Fadrozole Female Vitellogenin Fadrozole Female Vitellogenin ( mg/ mL)

( mg/ mL) Control > High; Medium Low > High; Medium Control > High; Low Low > High Control > High; Low Low > High Significant ? 0.02 0.01 9 0.011 0.005 16 0.002 0.002 16 High 0.03 0.02 10             Medium 0.45 0.79 10 0.75 1.20 16 0.70 1.15 16 Low 2.62 4.93 10 2.25 5.95 16 1.06 5.36 16 Control SD Mean n SD Mean n SD Mean n Non Spawning Adult 21 Day 14 Day 51 Fadrozole Steroid Results Fadrozole Steroid Results Fadrozole Steroid Results MED Data:   E2 in Females; T, 11KT   in Males at 50 µ g/ L.

EPA 21 Day assay: low ( 5.2 µ g/ L) and high ( 55.7 µ g/ L)

doses increased T in females and males and increased KT in males. High dose increased KT in females. Low dose decreased E2 in females.

EPA 14 Day assay: low ( 5.5 µ g/ L) and high ( 59.9 µ g/ L)

doses increased T in females and perhaps males and increased KT in males and perhaps females. High dose decreased E2 in females.

Non spawning Adult assay: High dose ( 57.0 µ g/ L) perhaps increased KT and T in males, increased KT and reduced E2 in females.

High variability within treatments. 52 Fadrozole Histology Results Fadrozole Histology Results Fadrozole Histology Results EPA 21 Day assay: high ( 55.7 µ g/ L) dose females showed less advanced staging; low ( 5.2 µ g/ L) and high doses had increased proportions of atretic follicles in the ovaries; high decreased proportion of corpora lutea. Males from high dose had increased tubule diameter. Some males had abnormal testes histology.

EPA 14 Day assay: low ( 5.5 µ g/ L) and high ( 59.9 µ g/ L)

doses showed less advanced staging and decreased corpora lutea; high increased proportion of atretic follicles in the ovaries.

Non spawning Adult: mid ( 31.7 µ g/ L) and high ( 57.0 µ g/ L)

doses showed less advanced quantitative staging; high increased proportion of atretic follicles. Several males in all three doses had abnormal testes histology. 53 Fadrozole Effects on Fathead Minnow Reproduction EPA MED  20  18  16  14  12  10  8  6  4  2 0 2 4 6 8 10 12 14 16 18 20 Exposure ( d)

0 2 4 6 8 10 (

Thousands)

Cumulative Number of Eggs Spawned Control 2 10 50 Fadrozole ( ug/ L) *

** 54 8 0 2 4 6 E2 (

ng/

ml) * *

0 10 20 Vtg (

mg/

ml) * * *

Control 2 10 50 Fadrozole effects on female Fathead Minnow EPA MED Fadrozole ( ug/ l) Spawning Tile and Egg Dish Comparison Spawning Tile and Spawning Tile and Egg Dish Comparison Egg Dish Comparison 56 Proportional Difference of Eggs on Tiles Versus Eggs on Dishes Proportional Difference of Eggs on Tiles Versus Proportional Difference of Eggs on Tiles Versus Eggs on Dishes Eggs on Dishes High < Control High < Low High < Control NS NS Significant ? 9%   41%

14%   60%

21%   83%

6%   342%

CVs 0.72 0.69 0.61 0.72 High 0.87 0.91 0.88 0.87 Low 0.87 0.94 0.92 0.90 Control EPA 21 Day NS NS NS NS Significant ? 20%   95%

9%   54%

15%   68%

23%   115%

CVs 0.65 0.86 0.86 0.75 High 0.86 0.91 0.93 0.92 Low 0.89 0.90 0.91 0.86 Control EPA 14 Day Fadrozole Flutamide Trenbolone Methoxychlor 57 Major Conclusions Major Conclusions Major Conclusions Good agreement between EPA and Battelle for key apical and diagnostic data generated for four different chemicals using 21 d reproduction protocol for fathead minnow

Methoxychlor

Fecundity reductions comparable

Effects on male VTG induction qualitatively similar

Trenbolone

Fecundity reductions comparable

Changes in secondary sex characteristics comparable

Decreases in female VTG similar

Flutamide

Fecundity reductions comparable

Subtle VTG effects and histological responses not similar

Fadrozole

Fecundity reductions comparable

VTG and E2 reductions in females similar 58 Suggested Protocol Suggested Protocol Suggested Protocol 14 day spawning protocol shows promise for use as a fish endocrine disruptor screening protocol based on:

Consistency in overall response to the 21 day protocol

Insufficient need for quantitative pre exposure fecundity measurements

Reduced cost compared with the 21 day protocol

Lack of response at the low exposure level and frequent negative responses at the high exposure level in the non spawning protocol. This observation suggests a higher risk of false negative results, particularly with weak acting endocrine disruptors. 59 It is recommended that additional development of the abbreviated 14 day spawning protocol be performed to evaluate how well the assay performs in comparison to the full 21 day protocol with weaker acting and/ or high log P compounds ( agonists/ antagonists spanning range of MOA ( including mixed MOA), as well as " negative" chemicals)

A Mutli Chemical Evaluation is currently underway with the following chemicals to help provide additional information.

° Atrazine ° DDE ° Bisphenol A ° Perchlorate ° Dibutyl Phthalate ° Cadmium Additional Research, Testing and Questions Additional Research, Testing and Additional Research, Testing and Questions Questions 60 Work on endpoint measurement techniques to standardize/ reduce variability

Fecundity ( capture of detached eggs?)

Histopathology ( quantification?)

Steroids ( RIA vs. ELISA?)

Others? Additional Research, Testing and Questions cont.

Additional Research, Testing and Additional Research, Testing and Questions cont.

Questions cont.


[Federal Register: September 2, 2010 (Volume 75, Number 170)]
[Notices]
[Page 53969]
[DOCID:fr02se10-60]

=======================================================================
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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

[Docket No. FDA-2010-E-0061]

Determination of Regulatory Review Period for Purposes of Patent
Extension; ONGLYZA

Correction

    In notice document 2010-21583 beginning on page 53315 in the issue
of Tuesday, August 31, 2010, make the following correction:
    On page 53316, in the second column, in the fifth line from the
top, ``[insert date 180 days after date of publication in the Federal
Register]'' should read ``February 28, 2011''.

[FR Doc. C1-2010-21583 Filed 9-1-10; 8:45 am]
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[Federal Register Volume 81, Number 130 (Thursday, July 7, 2016)]
[Notices]
[Pages 44356-44357]
[FR Doc No: 2016-16039]

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SECURITIES AND EXCHANGE COMMISSION

Submission for OMB Review; Comment Request

Upon Written Request, Copies Available From: Securities and Exchange
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Extension:
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Robert W. Errett,
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[FR Doc. 2016-16039 Filed 7-6-16; 8:45 am]
 BILLING CODE 8011-01-P