characterization of murine hepatocarcinogenesis

Characterization of Murine Hepatocarcinogenesis Induced by theHepatocarcinogenesis Induced by the

Nitrification Inhibitor Nitrapyrin: Mode of Action, Human RelevanceMode of Action, Human Relevance Framework, and Risk Assessment

Implications

RASS WebinarFebruary 10, 2016February 10, 2016

Kerry Hastings D Phil Risk Assessor Dow AgroSciencesKerry Hastings, D.Phil Risk Assessor, Dow AgroSciencesJessica LaRocca, PhD Toxicologist, Dow AgroSciencesMatthew LeBaron, PhD Toxicologist, Dow Chemical

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Agenda• Background/overview• Liver MoA evaluationLiver MoA evaluation• Application of the HRF• Risk assessment overview• Risk assessment overview

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Nitrapyrin• (2-chloro-6-trichloromethyl pyridine)• Registered in the US since 1974Registered in the US since 1974• Nitrification inhibitor• Nitrogen stabilizer• Nitrogen stabilizer

ClN Cl

ClCl

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Cl

Nitrate Loss

Ammonium Nitrite NitrateNit Nit b t

NH4(+) positive

h

NO2(-) soil negative

h

NO3(-) negative

Nitrosomonasbacteria

Nitrobacterbacteria

Nitrogen loss

charge charge( ) g

charge

Nitrogen lossthrough

denitrification

Nitrogen lossthrough nitrate leaching

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through nitrate leaching

Nitrapyrin Stabilizes Nitrogen

Ammonium

NH4

Nitrite

NO2

Nitrate

NO3

Nitrosomonasbacteria

NitrobacterbacteriaX X XX(+) positive charge (-) soil negative charge

3(-) negative charge

X X XX

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STABILIZED NITROGENReadily Available

Liver Tumors250

150

200 No adverse effects

Systemic toxicity

g/da

y)• 250 mkd: ↑ adenomas and adenomas/carcinomas in ♂and♀↑ absolute and relative liver weights in ♂and♀

100 Tumors

ose

(mg/

kg

↑ absolute and relative liver weights in ♂and♀• 125 mkd: ↑ adenomas and adenomas/carcinomas in ♀

↑ absolute and relative liver weights in ♂and♀

0

50Do

• 125/250 mkd: ↓ bw at 12 and 24 months in ♂and♀• 250 mkd: ↓ bwg at 12 mo (29.6%), 24 mo (26.9%) in ♂

Rat(1989)

Mouse (1990)

Mouse (1997)

0

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What is the MoA for nitrapyrin-mediated mouse liver tumors and is it relevant to

humans?

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Approach:• Question 1: Can we assimilate/generate

data to define an MoA for nitrapyrin-data to define an MoA for nitrapyrinmediated mouse liver tumors?

• Question 2: Can we exclude other MoAs?

• Question 3: Is the MoA relevant to humans?• Question 3: Is the MoA relevant to humans?

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Question 1: MoA

Assessed potential MoAs by evaluating• Assessed potential MoAs by evaluating previous toxicity data

• Generated additional MoA data to rule in or rule out nuclear receptor activationpKey events (NR activation, proliferation) Recovery after removal of treatmentRecovery after removal of treatment

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Nitrapyrin Liver MoA Study0, 75, 250, 400 mg/kg/day

nitrapyrin0 mg/kg/day nitrapyrin (recovery)

14Days of exposure 7 14 plus 21 day recovery4

Endpoints:• Gene expression of biomarkers of NR activation (AhR, CAR, PXR, PPAR-α)

• Protein and enzyme activity• Liver weight and histopathology• Hepatocellular proliferation (via BrdU osmotic pumps)• Assess recovery after treatment cessation

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Assess recovery after treatment cessation

Key Event #1: NR Activation

0 75 250

400 0 75 250

400 0 75 250

400 0 75 250

400

Cyp1a1AhR

Cyp2b10CAR

Cyp3a11PXR

Cyp4a10 PPAR-

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Key Event #1: CAR Activation (Cyp2b10)tr

olov

er c

ond

chan

ge

p2b1

0fo

ldC

yp

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mg/kg/day nitrapyrin

N=6-9 mice/timepoint/dose

Key Event #1: Liver Weight Increasesro

lov

er c

ontr

ncre

ase

oPe

rcen

t In

P

N=6 9 mice/timepoint/dose

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mg/kg/day nitrapyrin N=6-9 mice/timepoint/dose

Similar responses for liver hypertrophy

Cyp2b10 and Expected PROD Activity7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent)

Activation downstream genes/pathways

Cyp2b10C b2b10

PB

CARCyb2b10

Cyp2b10

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Key Event #1: CYP Enzyme Induction on

trol

7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent)e

Ove

r Co

d C

hang

e

Cyp2b10Western Blot

Fold

mg/kg/day nitrapyrin

←Cyp2b10

Cyp2b10 Western Blot

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N=6 mice/timepoint/dose

Suicide Inhibition• Inhibition of cytochrome activity (irreversible)• Phenobarbital (PB)-induced liver microsomes used to

investigate the role for suicide inhibition

Inhibitor

Enzyme

Binds irreversibly, removes active enzyme from system

Enzyme

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Suicide Inhibition• Inhibition of cytochrome activity (irreversible)

Activation downstream genes/pathways

Cyp2b10C b2b10

N ClCl

ClCl

I hibi

CAR NRCyb2b10

Inhibitor

N ClCl

ClClCl

Cyp2b10

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CYP Enzyme Induction/Suicide InhibitionPB

Cyp2b10

C

Cyp2b10

N ClCl

ClCl

Cyp2b10

| 18Cyp2b10 enzymatic inhibition similar to what was seen in vivo

Key Event #2: Increased Hepatocellular Proliferation C

ells 4.3

Per 1

,000

4 9Fold increases

ive

Cel

ls P

2

4.9

3.5

rdU

Posi

ti

2.4

Br

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mg/kg/day nitrapyrin N=6-9 mice/timepoint/dose

Summary Key Events #1 and 2• Nitrapyrin exposure in mice causes:

Key Event #1 – CAR activation─Cyp2b10 gene and protein expression─Liver weight increases─Liver hypertrophy─Suicide inhibition of PROD

Key Event #2 – Hepatocellular proliferation─BrdU Labeling Index

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Question 1: Conclusion

Can we assimilate/generate data to define an gMoA for nitrapyrin-mediated mouse liver

tumors?tumors?

YESKey events #1 (CAR) and #2 (Proliferation)y ( ) ( )

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Question 2• Can we exclude other MoAs?

• Is CAR necessary for nitrapyrin mediated• Is CAR necessary for nitrapyrin-mediated liver effects (proliferation)?

• Addressed this question with a CAR-KO qmouse study

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CAR-KO Mouse Study Design

0, 250 mkd nitrapyrinEndpoints:

C 2b10• Cyp2b10

• Liver weight increases, CAR-KOWT

ghistopathology

• Hepatocellular

4Days of Exposure

Hepatocellular proliferation

4

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Gene expression in WT and CAR-KO Liversro

lov

er c

ontr

chan

ge o

Fold

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mg/kg/day nitrapyrin (mouse strain)N=6 mice/strain/dose

Relative liver weight increasesea

serc

ent I

ncr

Per

mg/kg/day nitrapyrin (mouse strain)

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g g y py ( )

Histopathologic changes in WT and CAR-KO Mice H t h V li ht li ht V li ti V li ht

6/6 6/6

ecte

d

Hypertrophy; Very slight or slight

5/66/6

ecte

d

Vacuolization; Very slight

Per

cent

Affe

Per

cent

Affe

Increased number of mitotic figures

0/6 0/6P

mg/kg/day nitrapyrin (mouse strain)

0/6 0/6P

mg/kg/day nitrapyrin (mouse strain)Increased number of mitotic figures

Affe

cted

5/6

Per

cent

A

1/60/6 0/6

| 26mg/kg/day nitrapyrin (mouse strain)

0/6

Hepatocellular Proliferation in WT and CAR-KO Miceol

ver c

ontr

och

ange

ov

Fold

c

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mg/kg/day nitrapyrin (mouse strain)N=6 mice/strain/doseNitrapyrin-induced proliferation requires CAR activation

Alternative MoAsAlternative MoAs were evaluated for plausibility and coherence by Bradford Hill Criteria:

•DNA Reactivity•Not plausible•No coherence

•AhR, PXR, PPARα Activation•Not plausible•No coherence

•Cytotoxicity (1 Wk – 12 Mo)•Plausible•No coherence: based on magnitude of effect, entirety of data

•Increased Apoptosis•Not plausible•No coherence

•Estrogens, Statins, Metals, Infectious•Not plausible•No coherence

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Key Event 1 Key Event 2Apical Endpoints:

Temporality

Dose (mkd)

Key Events After Recovery

Apical Endpoints:Increased

Hepatocellular Tumors and Altered Foci

Causal: CAR Activation(Cyp2b10

Transcript &

Hepatocellular Proliferation( ) p

Protein)

4-14 Days 4-14 Days 14 Days Plus 21 Days Recovery 2 Yrs

5 -25 -

Dose

75 - - - -125 +,+# -250 + + - +

250 CAR KO - -

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+ Indicates effect present, - indicates effect absent at indicated duration of treatment. Blank cell = No data.

#Data only from 1-year interim sacrifice

Question 2: Conclusion

Can we exclude other MoAs?

YESCAR is necessary for nitrapyrin-induced

hepatocellular proliferationhepatocellular proliferation

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Question 3: Relevance to Humans?• CAR activation has been shown to be not relevant

to humans:Key Events Evidence in Rodents Evidence in Humans

Activation of CAR Yes Yes

#1CYP Enzyme

InductionAssociated Liver

Hypertrophy

Yes; unclear if critical step or indicator of

activity secondary to CAR activation

Yes; different enzymes induced compared to

rodents

#2 Hepatocellular Proliferation Yes

No evidence of increased cell proliferation in the human liver (limited in vitro and in vivo data)vitro and in vivo data).

Apical Endpoints

Selective Clonal Expansion (Foci) Yes No; none reported

O f

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Apical Endpoints Occurrence of Hepatocellular

TumorsYes No; based on

epidemiological data

Relevance to Humans for Nitrapyrin?• Wanted to generate nitrapyrin-specific data

• How?Mouse vs. human hepatocyte proliferation study

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Study Design

• Mouse hepatocytes treatedMouse hepatocytes treated with 0, 1, 3, 10 µM nitrapyrin

• Human hepatocytes treated p ywith 0, 3, 10, 30, 100 µM nitrapyrin

• Positive control EGF• DNA synthesis analyzed via

EdU t i i (fl tEdU staining (fluorescent alternative to BrdU)

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Hepatocyte Proliferation in Mice and HumansMouse Hepatocytes Human Hepatocytes

* p<0.05*

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N=2 human donors, 2-3 technical replicates/donor/dose

N=5-7 technical replicates

Hepatocyte Proliferation in Mice and HumansMouse Hepatocytes Human Hepatocytes

* p<0.05

*

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N=2 human donors, 5-6 technical replicates/donor/dose

N=5-7 technical replicatesConclusion: Nitrapyrin does not increase hepatocellular proliferation in human hepatocytes

Conclusions: Nitrapyrin MOA and Relevance to HumansPOD =75 mg/kg/day

spon

se Hepatocellulartumors in mice

Res

N t di t d i

Nitrapyrin Dose

No tumors predicted in CAR-KO mice

No relevance to humansNitrapyrin Dose

Key event #1 Mice

Key event #1

Key event #2 Mice

Key event #2 Mouse Hepatocytes

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Key event #1 CAR-KO mice

Key event #2 CAR-KO mice

Key event #2Human Hepatocytes

ILSI/IPCS Mode-of-Action/Human Relevance Framework

Q1: Is the weight of evidence sufficient to establish the MoA in animals?

Yes No

Assume MoA

Assume

Q2: Fundamental qualitativedifferences in key events?

Relevant to Humans

Assume MoA NOT Relevant

NoYes

to HumansNo

Q3: Fundamental quantitative differences in

key events?

| 37As described by Meek et al. (2003) and revised by Seed et al.(2005).

key events?

Nitrapyin MoA/HRF• Data support CAR activation as MoAKey Event #1 CAR ActivationKey Event #1 – CAR ActivationKey Event #2 – Hepatocellular Proliferation

• Alternative MoAs can be excluded

D t lit ti diff M A f• Due to qualitative differences, MoA for nitrapyrin is not relevant to humans

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Conclusion – Part #1

Questions on the MoA/HRFQuestions on the MoA/HRF evaluation?

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Regulatory Reviews • EPACPRC (1992) - Not classifiableCPRC (1992) - Not classifiable CARC (2000) - LikelyCARC (2005) Lik lCARC (2005) - LikelyCARC (2012) - Suggestive EvidenceCARC (2017) – Under Review

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Dietary Risk Assessment• Dietary exposure is estimated using

Food consumption data (from NHANES surveys)

P t ti l l f ti id id i diff t f d (t l ) Potential values for pesticide residues in different foods (tolerances)

NHANES = National Health and Nutrition Examination Survey

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NHANES National Health and Nutrition Examination Survey

nitrapyrin tolerances

Dietary Risk Assessment

• Diet Residues of concern in food:

nitrapyrin 6-chloropicolinic acid

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Dietary Risk Assessment

• Diet Dietary cancer risk assessment:

nitrapyrin 6-chloropicolinic acid

No residues ever detected in food commodities

[Former] Cancer endpoint only relevant to nitrapyrin; not 6-CPA

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Dietary Risk Assessment Chronic RfD = 0.03mg/kg bw/day (based on NOAEL of 3mg/kg/day

(chronic feeding – dog) and uncertainty factor = 100; FQPA = 1)cPAD RfD÷FQPA 0 03mg/kg bw/day cPAD = RfD÷FQPA = 0.03mg/kg bw/day Exposure ≤1% cPAD for US population and all subgroups p p g p

Table 3 extracted from Nitrapyrin Chronic Dietary

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Exposure Assessment for the Registration Eligibility

Decision. D. Soderburg, EPA 2004. D299299

Dietary Risk Assessment

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Occupational Risk Assessment

• Occupational Potential exposure through mixing, loading and application:

Exposure is calculated using unit exposure values from specific studies (such as those conducted by the Agricultural Handlers Exposure Task Force).p )

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Occupational Risk Assessment

Exposure = AR (lb ai/acre) x AT(acre/day) x unit exposure (mg/lb ai) body weight (kg)

AR = application rateAR = application rateAT = area treated

Exposure (in mg/kg/day) is used to assess risk in two ways:

Cancer and non-cancer

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Occupational Risk Assessment

Cancer: Lifetime Average Daily Dose (LADD) calculated from daily exposure LADD used to calculate risk:exposure. LADD used to calculate risk:

LADD = total exposure x no. of days exposed per year* x 35 working years

365 days per year 70 year lifetime

*3 day per year for private applicators; 30 day per year for commercial applicators

Risk = Q1* [4.25 x 10-2 (mg/kg/day)-1 human equivalents] x LADD.

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Occupational Risk Assessment Cancer risks marginally higher than LOC (LOC = 1 x 10-6) but risk vs. benefit

recommends no additional mitigation beyond long pants, long sleeves and gloves for mixing/loading

(extracted from occupational t 2005)

C b d i k t t i d i 2012 ( l ifi ti )

exposure assessment, 2005)

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Cancer-based risk assessment not required since 2012 (re-classification)

Occupational Risk Assessment

Non-cancer: the MOE is calculated:

MOE = NOAEL (mg/kg/day) Need MOE ≥ 100 for pass Exposure (mg/kg/day)

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(extracted from occupational exposure assessment, 2005)

Occupational Risk Assessment

All MOEs ≥ 100 when long pants, long sleeves and gloves worn for mixing/loading

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AcknowledgementsDow AgroSciences Human Health Assessment

Dow Toxicology & Environmental Research & Consulting (TERC) Laboratories

Reza RasoulpourB Bhaskar Gollapudi

Johnson ThomasLynea Murphy B. Bhaskar Gollapudi

Dave Eisenbrandt H. Lynn Kan

Lynea Murphy Kamin Johnson Nico Viscontiy

Melissa Schisler Val Marshall

Lindsay Sosinski Dave DeLine

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