- lnteragency Coordinating Committee on the Validation of Alternative Methods

Department of the Interior

Alternative Toxicological Test Method Activities

Barnett Rattner, Jennifer Brennan, Jessica Leet, Joel Putnam, Rachel Riegerix

- lnteragency Coordinating Committee on the Validation of Alternative Methods

Mission Protect and manage the Nation’s natural resources and cultural heritage

Provides scientific and other information about those resources

Honors trust responsibilities & commitments to American Indians, Alaska Natives and affiliated island communities

- lnteragency Coordinating Committee on the Validation of Alternative Methods

Some Applied Toxicological Research Limited regulatory authority on “chemicals”

1. Research with direct application to natural resource management

2. Environmental contaminant biomonitoring

3. Natural Resource Damage Assessment

4. Registration of chemicals used in aquaculture

5. Alternatives to “lead shot” used in hunting

Embrace 3R’s

- lnteragency Coordinating Committee on the Validation of Alternative Methods

Diphacinone SSD Red-toothed Triggertish Clotting Times 200

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largemouth ba$SA N

fathead minnow 4 ~ + 0.8

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pig !i EE moooe 0' 0.6 ... 60 American Kellrel ! ~:

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Log LD50 Diphacinone (log/g ) Concentrations (µg ARI g of fish body weight) µg Chlorophaclnone/kg BWt-day ww

Anticoagulant Rodenticide Bait Application and Risk to Non-target Species

Global use - pest species eradication for ecosystem restoration on DOI properties

Toxicity and ADME studies to assess risk to non-target raptorial birds and fish

Up Down Test Procedure LC50 in fish Sequential dosing procedure LD50 in American kestrel Sublethal exposure thresholds (TRVs) for coagulopathy Data for ecological risk assessments and EIS

BMD50~40 µg CPN/kg BWt-day

- lnteragency Coordinating Committee on the Validation of Alternative Methods

• Sediment, mg equivalents

10"' 10-3 10-2 10-1 100 101 102

0 120-~ , e 100 :!::=+-• T T

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E2, pg

Testing Environmental Samples for Endocrine or Dioxin-like Activity in vitro

• Luciferase transactivation cell bioassays – 4 stably transfected human cell lines

• Estrogen (ERα, ERα/ERβ) pathways

• Androgen/Glucocorticoid/Progesterone pathways

• Androgen/glucocorticoid pathways

– 1 stably transfected rodent cell line • Aryl hydrocarbon pathways

• 384-well plate format

• Activity in cells normalized to protein content

- lnteragency Coordinating Committee on the Validation of Alternative Methods

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Date of Experiment

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Anastrozole, (M)

Behind the goal…. • Assay Optimization

– Medium/serum selection

– Treatment conditions

• Performance – Tracking assay parameters over time

• Characterization – Unexplored capabilities of existing assays

– Selective agonists/antagonists in multiple-receptor cell lines

• Streamlining – Increasing automation on a modest budget

– Increasing assay while maintaining integrity

BioTekTM Synergy 4 with dual injector, BioTekTM MultiFlo FX with washer unit.

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- lnteragency Coordinating Committee on the Validation of Alternative Methods

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High-Content Tier I Screening

fli1 egfp zebrafish 72 hours post fertilization

FITC image

fli1-egfp zebrafish 72 hours post fertilization

Transmitted light image

384 well format

Molecular Devices ImageXpress High Content Imaging system

• Developmental cardiovasculartoxicity assay at 72 hpf – Body length

– Pericardial area

– Intersegmental vessel area

– Heart Rate

– Circulation

• Targeted assessment oftoxicity – LC50 and mode of action information

• Rapid image acquisition, data extraction, and analysis

- lnteragency Coordinating Committee on the Validation of Alternative Methods

High-Content Tier I Screening • HCS to formulate hypotheses & prioritize compounds for further testing

• Utilizes pre-feeding fish embryos in microtiter plate format to reduce:

– Animal use

– Test compound needed

– Waste

– Labor

• Highly adjustable platform to incorporate a variety of endpoints

• HCS assays can provide evidence to justify larger-scale studies to determine actual risk versus perceived risk of contaminants.

lnteragency Coordinating Committee on the Validation of Alternative Methods

Fish Toxicant Database “modeling/screening compounds”

Species

Tetramethyl thiuram disulfide Ziram

24 hour LC10 (ppb)

Bluegill 610 850

Fathead minnow 170 380

Rainbow trout 310 470

24 hour LC99 (ppb)

Bighead carp 510 614

Silver Carp 414 640

Grass Carp >2000 390

• Database:

>25 chemical parametersMany biological endpoints

– Tetramethy thiuram disulfide would be selective for carp

• Modeling indicated in vivo trials of Ziram would have similar effects

• Two compounds used to identifynew invasive species toxicant

• No additional animals needed to initiate species selective cellular assays

- lnteragency Coordinating Committee on the Validation of Alternative Methods

I

- - -- - -- - --

Cell viability using CellTiter-Glo® 150% 140% 130% 120%

Reduced activity -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0

110% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Log [TC-29], M

BHC fry BHC gill BLG fin/skin BLG fry FHM skin LST fry PAH gill RBT fry SVC fry SVC gill

Rapid selectivity screening • Fish cell lines

– 7 fish species

– Multiple organs from some species

– Paddlefish and Sturgeon toxicresponse without using liveanimals

• TC-29 (new biocide)

– Selected using toxicity models

• Results

– Bighead carp and rainbow troutare similarly sensitive to TC-29

– TC-29 stimulates the productionof ATP in lake sturgeon

– Traditional in vivo trials for both studies (TTS, Ziram, TC-29)would require more than 8,400fish. We used 1,005 fish to validate the entire selection process

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- lnteragency Coordinating Committee on the Validation of Alternative Methods

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~bn.11u 1• 2, 2010

Pan JO

Environmental Protection Agency Slxl)"•Flfth Rrpctl'1 of lhc TSCA lm~ney TQlln,c Cummllltt lo the Admlnlstn.lOr ol the Cmiro~W Prol'-"'ll)n ~acy. Rcc:clpc of Rcpon :a.nd lk:q~t for r.A".m1.m c n1-,; ~oflcc

Registration of Non-toxic Shot Lead shot replacements: iron (steel) iron-tungsten bismuth-tin copper-clad iron corrosion-inhibited copper tungsten-bronze tungsten-iron tungsten-matrix tungsten-nickel-iron tungsten-polymer tungsten-tin-bismuth tungsten-tin-iron tungsten-tin-iron-nickel

Bottom Line – many shot types registered using existing information, risk assessment and no toxicity test (harmonized with Canada)

11

- lnteragency Coordinating Committee on the Validation of Alternative Methods

Challenge… extrapolating toxic/therapeutic effects among diverse groups of species

• Diverse exposure pathways

• Differences in life history and behavior

• Differences in ADME among species that can affect toxicity

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