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Characterization of Murine Hepatocarcinogenesis Induced by the Hepatocarcinogenesis Induced by the Nitrification Inhibitor Nitrapyrin: Mode of Action, Human Relevance Mode of Action, Human Relevance Framework, and Risk Assessment Implications

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Characterization of Murine Hepatocarcinogenesis Induced by the Hepatocarcinogenesis Induced by the Nitrification Inhibitor Nitrapyrin: Mode of Action, Human Relevance Mode of Action, Human Relevance Framework, and Risk Assessment Implications

RASS Webinar February 10, 2016 February 10, 2016 Kerry Hastings D Phil Risk Assessor Dow AgroSciences Kerry Hastings, D.Phil Risk Assessor, Dow AgroSciences Jessica LaRocca, PhD Toxicologist, Dow AgroSciences Matthew LeBaron, PhD Toxicologist, Dow Chemical

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SLIDE 2

Agenda

  • Background/overview
  • Liver MoA evaluation

Liver MoA evaluation

  • Application of the HRF
  • Risk assessment overview
  • Risk assessment overview
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SLIDE 3

Nitrapyrin

  • (2-chloro-6-trichloromethyl pyridine)
  • Registered in the US since 1974

Registered in the US since 1974

  • Nitrification inhibitor
  • Nitrogen stabilizer
  • Nitrogen stabilizer

Cl N Cl Cl Cl

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Cl

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SLIDE 4

Nitrate Loss

Ammonium Nitrite Nitrate

Nit Nit b t

NH4

(+) positive h

NO2

(-) soil negative h

NO3

(-) negative

Nitrosomonas bacteria Nitrobacter bacteria

Nitrogen loss

charge charge ( ) g charge

Nitrogen loss through denitrification Nitrogen loss through nitrate leaching

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through nitrate leaching

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SLIDE 5

Nitrapyrin Stabilizes Nitrogen

Ammonium

NH4

Nitrite

NO2

Nitrate

NO3

Nitrosomonas bacteria Nitrobacter bacteria

X X X X

(+) positive charge (-) soil negative charge

3

(-) negative charge

X X X X

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STABILIZED NITROGEN

Readily Available

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SLIDE 6

Liver Tumors

250 150 200

No adverse effects Systemic toxicity

g/day)

  • 250 mkd: ↑ adenomas and adenomas/carcinomas in ♂and♀

↑ absolute and relative liver weights in ♂and♀ 100

Tumors

  • se (mg/kg

↑ absolute and relative liver weights in ♂and♀

  • 125 mkd: ↑ adenomas and adenomas/carcinomas in ♀

↑ absolute and relative liver weights in ♂and♀ 50

Do

  • 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)

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SLIDE 7

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

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SLIDE 8

Approach:

  • Question 1: Can we assimilate/generate

data to define an MoA for nitrapyrin- data to define an MoA for nitrapyrin mediated 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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SLIDE 9

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 activation p

  • Key events (NR activation, proliferation)

Recovery after removal of treatment

  • Recovery after removal of treatment
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SLIDE 10

Nitrapyrin Liver MoA Study

0, 75, 250, 400 mg/kg/day nitrapyrin 0 mg/kg/day nitrapyrin (recovery) 14 Days of exposure 7 14 plus 21 day recovery 4

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

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SLIDE 11

Key Event #1: NR Activation

7 5 2 5 4 7 5 2 5 4 7 5 2 5 4 7 5 2 5 4

Cyp1a1 AhR Cyp2b10 CAR Cyp3a11 PXR Cyp4a10 PPAR-

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SLIDE 12

Key Event #1: CAR Activation (Cyp2b10)

trol

  • ver con

d change p2b10 fold Cyp

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mg/kg/day nitrapyrin

N=6-9 mice/timepoint/dose

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SLIDE 13

Key Event #1: Liver Weight Increases

rol

  • ver contr

ncrease o Percent 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

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SLIDE 14

Cyp2b10 and Expected PROD Activity

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

Activation downstream genes/pathways

Cyp2b10 C b2b10

PB

CAR Cyb2b10 Cyp2b10

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SLIDE 15

Key Event #1: CYP Enzyme Induction

  • ntrol

7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent) e Over Co d Change

Cyp2b10 Western Blot

Fold mg/kg/day nitrapyrin

←Cyp2b10

Cyp2b10 Western Blot

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N=6 mice/timepoint/dose

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SLIDE 16

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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SLIDE 17

Suicide Inhibition

  • Inhibition of cytochrome activity (irreversible)

Activation downstream genes/pathways

Cyp2b10 C b2b10

N Cl Cl Cl Cl

I hibi CAR NR Cyb2b10 Inhibitor

N Cl Cl Cl Cl Cl

Cyp2b10

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SLIDE 18

CYP Enzyme Induction/Suicide Inhibition

PB Cyp2b10

C

Cyp2b10

N Cl Cl Cl Cl

Cyp2b10

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Cyp2b10 enzymatic inhibition similar to what was seen in vivo

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SLIDE 19

Key Event #2: Increased Hepatocellular Proliferation

Cells

4.3

Per 1,000

4 9 Fold increases

ive Cells P

2

4.9 3.5

rdU Positi

2.4

Br

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mg/kg/day nitrapyrin

N=6-9 mice/timepoint/dose

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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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SLIDE 21

Question 1: Conclusion

Can we assimilate/generate data to define an g MoA for nitrapyrin-mediated mouse liver tumors? tumors? YES Key events #1 (CAR) and #2 (Proliferation) y ( ) ( )

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SLIDE 22

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

q mouse study

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SLIDE 23

CAR-KO Mouse Study Design

0, 250 mkd nitrapyrin

Endpoints: C 2b10

  • Cyp2b10
  • Liver weight increases,

CAR-KO WT

g histopathology

  • Hepatocellular

4 Days of Exposure

Hepatocellular proliferation

4

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SLIDE 24

Gene expression in WT and CAR-KO Livers

rol

  • ver contr

change o Fold

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

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SLIDE 25

Relative liver weight increases

ease rcent Incr Per mg/kg/day nitrapyrin (mouse strain)

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g g y py ( )

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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

ected

Hypertrophy; Very slight or slight 5/6 6/6

ected

Vacuolization; Very slight

Percent Affe Percent Affe

Increased number of mitotic figures 0/6 0/6

P mg/kg/day nitrapyrin (mouse strain)

0/6 0/6

P mg/kg/day nitrapyrin (mouse strain)

Increased number of mitotic figures

Affected

5/6

Percent A

1/6 0/6 0/6

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mg/kg/day nitrapyrin (mouse strain)

0/6

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SLIDE 27

Hepatocellular Proliferation in WT and CAR-KO Mice

  • l

ver contro change ov Fold c

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

Nitrapyrin-induced proliferation requires CAR activation

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SLIDE 28

Alternative MoAs

Alternative 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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SLIDE 29

Key Event 1 Key Event 2 Apical 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

  • |

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

#Data only from 1-year interim sacrifice
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Question 2: Conclusion

Can we exclude other MoAs?

YES CAR is necessary for nitrapyrin-induced hepatocellular proliferation hepatocellular 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 #1 CYP Enzyme Induction Associated 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 Tumors Yes No; based on epidemiological data

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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 treated

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

  • Human hepatocytes treated

p y with 0, 3, 10, 30, 100 µM nitrapyrin

  • Positive control EGF
  • DNA synthesis analyzed via

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

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Hepatocyte Proliferation in Mice and Humans

Mouse 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

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SLIDE 35

Hepatocyte Proliferation in Mice and Humans

Mouse Hepatocytes Human Hepatocytes

* p<0.05

*

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N=2 human donors, 5-6 technical replicates/donor/dose N=5-7 technical replicates

Conclusion: Nitrapyrin does not increase hepatocellular proliferation in human hepatocytes

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SLIDE 36

Conclusions: Nitrapyrin MOA and Relevance to Humans

POD =75 mg/kg/day

sponse Hepatocellular tumors in mice Res N t di t d i Nitrapyrin Dose No tumors predicted in CAR-KO mice No relevance to humans Nitrapyrin 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 #2 Human Hepatocytes

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SLIDE 37

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 qualitative differences in key events?

Relevant to Humans Assume MoA NOT Relevant No Yes to Humans No

Q3: Fundamental quantitative differences in key events?

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

key events?

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SLIDE 38

Nitrapyin MoA/HRF

  • Data support CAR activation as MoA
  • Key Event #1

CAR Activation

  • Key Event #1 – CAR Activation
  • Key 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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SLIDE 39

Conclusion – Part #1

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

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SLIDE 40

Regulatory Reviews

  • EPA
  • CPRC (1992) - Not classifiable
  • CPRC (1992) - Not classifiable
  • CARC (2000) - Likely

CARC (2005) Lik l

  • CARC (2005) - Likely
  • CARC (2012) - Suggestive Evidence
  • CARC (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

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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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  • Decision. D. Soderburg, EPA 2004. D299299
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SLIDE 45

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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SLIDE 47

Occupational Risk Assessment

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

  • Exposure (in mg/kg/day) is used to assess risk in two ways:
  • Cancer and non-cancer
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SLIDE 48

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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SLIDE 49

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)
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SLIDE 50

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)

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SLIDE 51

Occupational Risk Assessment

  • All MOEs ≥ 100 when long pants, long sleeves and gloves worn for

mixing/loading

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SLIDE 52

Acknowledgements

Dow AgroSciences Human Health Assessment Dow Toxicology & Environmental Research & Consulting (TERC) Laboratories

  • Reza Rasoulpour

B Bhaskar Gollapudi

  • Johnson Thomas

Lynea Murphy

  • B. Bhaskar Gollapudi
  • Dave Eisenbrandt
  • H. Lynn Kan
  • Lynea Murphy
  • Kamin Johnson
  • Nico Visconti

y

  • Melissa Schisler
  • Val Marshall
  • Lindsay Sosinski
  • Dave DeLine
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