1 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Predicting pyrethroids target tissue exposure across ages A pplication of in vitro -in vivo extrapolation and physiologically based pharmacokinetic modeling Miyoung Yoon and Harvey J. Clewell The Hamner Institutes for Health Sciences Research Triangle Park, NC USA

1 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 

# Hypothesis #2: PBPK models can be used to predict target tissue levels in human adults and children

23 The Hamner Institutes for Health Sciences | August 5, 2010 

### 1yr 5 yr 10 yr Adult AgeBrainCmax(ng/ml)



# Provide a tool to predict target tissue exposure in children and adults Deltamethrin , 0.005 mg/kg 

# The objective

3 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)4 The Hamner Institutes for Health Sciences | August 5, 2010 4 

# Our approach In vitro to in vivo extrapolation ( IVIVE )

Predicting in vivo kinetics based on in vitro/in silico metabolism and other kinetic data

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)5 The Hamner Institutes for Health Sciences | August 5, 2010 

# The framework

Human Early Age PBPK model ( Hamner ) Human metabolism data in vitro ( LFR ) Rat metabolism data in vitro ( LFR ) Rat Early Age PBPK model ( Refinement of Tornero -Velez et al. model) 

### ( Hamner ) IVIVE

In vivo PK data in growing rats ( UGa ) IVIVE approach validation Human enzyme ontogeny ( MCoW/ Hamner /Published data) IVIVE

Prediction of target tissue dose in the age of concern at environmentally relevant exposure Application of the approach

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# Target tissue dosimetry in AOP- based risk assessment
• Internal exposure at the target site of action is critical in defining accurate concentration - effect relationship for early cellular events in Adverse Outcome Pathway (AOP) framework
• Physiologically based pharmacokinetic (PBPK) modeling provides a tool to predict target tissue exposure and link it to estimated or observed effect concentrations from toxicity studies to inform human safe exposure

6 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# Potential impacts of age-specific pharmacokinetics on tissue dosimetry and sensitivity

• Rapidly changing physiology and biochemical processes are expected to affect chemical kinetics in the growing body
• Resulting differences in target tissue exposure may lead to age - associated sensitivity to chemical effects during rapid growth period

7 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)8 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea



# PBPK modeling to predict age-specific tissue dosimetry PBPK models serve as a platform to coherently integrate diverse, independent data sets and provide predictive/extrapolation power • Parameters (inputs) for PBPK models:

‒ Exposure (age - dependent; chemical - dependent)

‒ Physiology such as body weight, tissue volumes, blood flows etc. (age - dependent; chemical - independent)

‒ Biochemical processes such as metabolism, partitioning, protein binding etc . (age - dependent; chemical - dependent)

8 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# IVIVE - PBPK modeling: Taking into account key determinants of early life dosimetry Age-specific internal dose 

# Life stage PBPK MODEL

Age - dependent physiological changes Age - dependent biochemical changes Age - appropriate exposure (CARES or SHEDS) 

# Requires in vitro data - based parameterization

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## • The bottom - up PBPK modeling concept has been used in environmental chemical risk assessment started with volatiles and later on for other compounds including pesticides (Clewell, 1993; Clewell and Andersen, 1996; Kedderis and Lipscomb, 2001; Hinderliter et al., 2011; Yoon et al., 2014) • The approach is widely accepted for drugs because of general PBPK modeling tools for drugs ( Simcyp ) and rapid advances in in vitro metabolism technologies • Pediatric applications, initially used for pharmaceuticals, have successfully been adopted for environmental chemicals in in vitro - based risk assessment initiatives ( Nong et al., 2005; Price et al., 2011; Wetmore et al., 2014)

10 

# The bottom - up ( IVIVE ) PBPK modeling approach is an already widely accepted concept

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# IVIVE - PBPK application to pediatrics: A source to outcome modeling for chlolopyrifos

Copyright 2013 by The Hamner Institutes for Health Sciences. May not be reproduced without permission -2 -1 0 1 2 10 - 4 10 - 3 10 - 2 10 - 1 10 0 10 1 Chlorpyrifos

Chlorpyrifos oxon } 3 Year Olds } 30 Year Olds } 3 Year Olds } 30 Year Olds Z-Score (3-97th Percentiles)Blood Concentration (ng/L)

-2 -1 0 1 2 10 - 5 10 - 4 10 - 3 10 - 2 } 3 Year Olds } 30 Year Olds

Z-Score (3-97th Percentiles)RBC AChE Inhibition (% basal)

-2 -1 0 1 2 10 - 6 10 - 5 10 - 4 10 - 3 } 3 Year Olds } 30 Year Olds

Z-Score (3-97th Percentiles)Brain AChE Inhibition (% basal)

CARES LifeLine Daily LifeLine Seasonal Price et al., 2011, Regulatory Toxicology and Pharmacology Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)12 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea Chemical Median C ss for Healthy Population Median Css for Most Sensitive Most Sensitive Chemical -Specific Pharmacokinetic Adjustment Factors ( PK-AF) % Contribution of Metabolism to Average PK-AF Acetochlor 0.026 0.063 Pediatric 2.5 80 Azoxystrobin 0.099 0.22 Pediatric 2.2 86 Bensulide 0.241 0.35 Pediatric 1.5 66 Carbaryl 0.043 0.17 Pediatric 4.0 88 Difenoconazole 0.201 0.30 Renal Insufficiency 1.5 58 Fludioxonil 0.38 1.45 Pediatric 3.0 86 Haloperidol 0.029 0.04 Pediatric 1.5 83 Lovastatin 0.001 0.003 Pediatric 2.1 92 Tebupirimfos 0.107 0.16 Renal Insufficiency 1.5 20

12 

# IVIVE - PBPK application to pediatrics: High -throughput IVIVE to address susceptible subpopulations

Copyright 2013 by The Hamner Institutes for Health Sciences. May not be reproduced without permission Adapted from Table 4 in Wetmore et al., 2014, Toxicological Sciences Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# Color coding used for this presentation IVIVE - PBPK modeling – background Human IVIVE - PBPK – general platform Rat IVIVE - PBPK – case study Human IVIVE - PBPK – case study Concepts Application Proof of concept case study Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# In vitro to in vivo extrapolation - based PBPK modeling for early life dosimetry

Human in vitro metabolism

data: age-specific Rat in vitro metabolism

data: age-specific

Proof of concept case studies Application of the approach 14 

## Rat Adult PBPK Model Human Adult PBPK Model

Human Early Life PBPK Model Rat Early Life PBPK Model

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

## Human Age -dependent PBPK Model Human age - specific parameters in vitro Human Growth Physiology 

### Human Age - relevant Exposure (CARES or SHEDS) 

## Rat Age - dependent

PBPK Model Rat Growth Physiology

Rat age - specific parameters in vitro IVIVE Step 1: Rat modeling for case compounds Validation of the concept IVIVE Step 2: Human modeling for case compounds Application of the approach to humans 

### General PBPK model for pyrethroids in humans across ages

Step 4: Exercising the general modeling platform for pyrethroid risk assessment in different ages of humans 

# Building a general model platform for pyrethroids for risk assessment

15 Step 3 : Read across strategies for model parameterization built upon the experience with case studies Targeted in vitro studies for compound specific key model parameters

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# Targeted in vitro experiments for pyrethroid IVIVE

16 Types of studies Rat – case study Human - case study Human - general model Purpose * Metabolism In vitro Microsomes and cytosol; age - specific donors ( LFR ) Microsomes and cytosol; age sepecific donors - DLM only; Recombinant CYP /CES enzymes ( LFR ) Recombinant CYP /CES enzymes ( LFR ) P Enzyme ontogeny of expression In vitro; Published database n/a Published database #; CAPHRA data - CESs, CYP2C8, 1A2 ( MCoW ; Hamner ) Published database #; CAPHRA data - CESs, CYP2C8, 1A2 ( MCoW ; Hamner ) P Partition coefficient In vivo Steady- state PK in you ng and adult rats ( UGa ) Adopt rat parameters Adopt rat parameters Read - across based on

underlying biology P Plasma protein binding In vitro Plasma from age specific donors ( UGa ) Plasma from age - specific donors ( UGa ) Plasma from adults; Read - across by QSAR P PK time course In vivo PK studies in young and adult rats ( UGa ) n/a n/a P;V * P indicates that the purpose of the study is for model parameterization and V for model validation # The published database include those are used in Simcyp and those are available from a series of publications from Dr. Ron Hines lab. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# M ajor challenges in IVIVE for human early ages for environmental chemicals
• Human in vivo data is necessarily limited (in contrast to pharmaceuticals), leading to a reliance on in vitro data. However:

– Pediatric tissues samples are very difficult to obtain and sample quality is generally uncertain

– Addressing human variability is challenging • IVIVE with recombinant enzymes in conjunction with enzyme ontogeny of expression is selected as an alternative approach in CAPHRA

17 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# In vitro metabolism studies for pyrethroid IVIVE

18 

# Microsomes

Cytosol Subcellular fractions Age-specific liver donors Human recombinant enzymes 

# Individual CYP or CES enzymes CESs CYPs + CESs Recombinant enzymes 

# Human Rat

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)19 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Biological scaling is necessary in using in vitro data

• Extrapolation of in vitro metabolic parameters to in vivo systems requires:

– Relating the total amount of enzyme present in the system (capacity of metabolism, Vmax )

– Relating the available substrate concentration for enzyme reaction (affinity of metabolism, Km)

19 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# Biological scaling for different in vitro systems System Vmax or Clint in the System Scaling factors to whole liver Microsomes nmol /min /mg protein MPPGL x Liver weight Cytosol nmol /min /mg protein CPPGL x Liver weight Expressed

Enzyme μl/min / pmol enzyme ( ISEF xEnz abundance 

### ) x MPPGL x Liver weight Liver nmol/min/g liver Liver weight Whole Body nmol /min/whole liver
20 Yoon et al., 2012 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)21 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Human Recombinant CYPs and CESs with enzyme ontogeny data Application and validation of the alternative IVIVE approach Human Subcellular fractions from liver tissue bank with different ages Application of the IVIVE approach to humans Rat Subcellular fractions from young and adult rats Validation of IVIVE approach for model parameterization 

# Deltamethrin modeling as a proof of concept

21 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)22 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Post- proof of concept IVIVE work flow (recombinant enzyme IVIVE )

22



# Screening & Measuring Metabolic rates for each active CYP or CES ( CAPHRA) 

# Biological scaling Average adult Clints for each active enzyme ( CAPHRA + Published database) 

# Extrapolation across ages Incorporating ontogeny of enzyme abundance Distribution of age specific Clints for each enzyme ( CAPHRA + Published database) 

# Integration with PBPK modeling Distribution of age specific total hepatic clearance for a given pyrethroid ( CAPHRA)

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# IVIVE with recombinant enzymes A bottom - up modeling approach used by SimCyp for conducting population based PBPK modeling for diverse human populations including pediatrics

23 (Rostami - Hodjegan and Tucker, 2007) Hepatic CYP Isozyme Abundance in Healthy Adults (% of Total) 

### Clint in vivo 

### = Clint rEnz 

### x EnzAbundance x Scaling factor

Clint rEnz determined in recombinant CYPs and CESs CYP and CES enzyme abundance in population 

## Screening and Measuring Biological Scaling

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# Recombinant enzyme IVIVE for pyrethroids

24 (Hines et al., submitted to Tox Sci ) 

# • Collecting data for ontogeny and biological scaling factors for CESs and CYPs that are important for pyrethroids, but are currently not available in Simcyp

• Metabolism assays to determine pyrethroid clearance rates for each enzyme that is active for the given pyrethroid (Song et al., in preparation) 

### CES ontogeny

CYP2C8 ontogeny

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)25 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Characterizing human variability in metabolism based on in vitro

25 Inter-individual variations in expression levels (abundance) of each enzyme result in population variability in the given pyrethroid clearance Individual 1 Individual 2 Individual 3 Individual 4

Individual i Clint for each involved enzyme Abundance of each involved enzyme in each individual 

### Clint_in vivo 20 year old Clint_in vivo

1 year old 

## Extrapolation across Ages

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)26 

# Subpopulation - based in vitro-to- in vivo extrapolation

CYP1A2
CYP2D6

CYP2C8 CYP2E1

UGT1A4
CYP3A5 CYP3A4
CYP2C19
UGT1A1 CYP2C9

CYP2B6 UGT2B7 Primary Hepatocytes - 5 -4 - 3 - 2 - 1 0 1 2 3

0 50 100 150Ln Conc (uM)

Time (min) Hepatic Clearance Cl in vitro Plasma Css General Population CYP1A2

CYP... CYP3A4

UGT... Cl rCYP1A2

Cl rCYP... Cl rCYP3A4

Cl rUGT... Plasma Css for: Neonates

Northern Europeans

Asians

Children And so on... Hepatic Intrinsic Clearance rCYP1A2
rCYP3A4

rCYP...

rUGT ... Wetmore et al., 2014, slide courtesy of Dr. Barbara Wetmore An example for early life IVIVE for environmental chemicals

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 2627 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 0 20 40 60 80 -4 -3 -2 -1 0 1 CYP3A4 CYP3A5 CYP2C19 CYP2C8 CYP2C9 CYP1A2 CYP2C18 Time (min)Ln Concentration (uM)

0 20 40 60 80 -0.4 -0.2 0.0 0.2 0.4 0.6 UGT1A4

Time (min)Ln Concentration (μM) 0 20 40 60 80 -10

-8 -6 -4 -2 0 2 CYP2E1 CYP2C9 CYP1A2 CYP2C19 CYP2B6
Time (min)Ln Concentration (μM)

No UGT metabolism detected Carbaryl Difenoconazole 

# Recombinant enzyme clearance rates

(Wetmore et al., 2014) An example for early life IVIVE for environmental chemicals

27 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)28 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Comparison of c ss 

# values derived across multiple subpopulations

Upper 95 th percentile C ss Carbaryl
Gen Jap Ntrn Eur Diabetic

Renal Insuff. 6-20 2-60.5-20-0.5 0.01

0.1

1Css at1μM (μM)(5th-95th %ile)

Subpopulation (Age (yr) or Ethnic) Subpopulation (Age (yr) or Ethnic) (Wetmore et al., 2014) An example for early life IVIVE for environmental chemicals

28 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)29 

# Evaluation of IVIVE approach for developing age - specific PBPK models 

# Deltamethrin PBPK

model for growing rats Rat Subcellular fractions from young and adult rats Validation of IVIVE approach for model parameterization

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)30 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Deltamethrin in vivo kinetic studies for model evaluation

• Plasma time course in adult rats (PND 90)

– single oral bolus dose of 0.05 - 5.0 mg DLM/kg in 5 ml corn oil (CO)/kg

– serial micro - blood samples taken arterial cannula for up to 96 hours post dose

– 4- 5 animals per timepoint • Tissue-distribution study

– s ingle oral bolus dose of 0.1, 0.25 or 0.5 mg DLM/kg in 5 ml corn oil (CO)/kg

– plasma and tissues were collected at terminal sacrifice

– plasma, brain and liver analyzed • Plasma and tissue samples were analyzed by GC- MS • Time-course data analyzed to estimate standard PK parameters
30 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)31 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Plasma kinetics in adult rats single oral dose of DLM (in 5 ml/kg corn oil)

31 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)32 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Pharmacokinetics in adult rats

Dose mg/kg 5 1 0.5 0.1 0.05 Tmax min 420 360 420 420 300 C max ng/ml 337 ± 126 94.7 ± 51.8 49.4± 18.4 10.4± 10.1 4.7 ± 2.6 C max Dose 67 95 99 104 94 Effective Half- life hr 3.9 4.5 3.7 4.1 3.0

AUC ∞ ng* hr/ml 3069 937 521 148 61 AUC ∞ Dose 614 937 1042 1476 1225 

### • Single oral dose of DLM in 5 ml/kg corn oil to PND90 Rats with serial blood sampling • Linear plasma kinetics over the 100-fold range of doses (0.05 to 5 mg DLM/kg ). • Parallel plasma absorption, distribution and elimination profiles • Peak concentrations (Cmax) and area under the concentration –time curve (AUC) increased proportionately with dose • Absorption rate - limited kinetics • Broad peaks with Tmax values of 300-420 minutes • Bioavailability orally in corn oil is low (5.5 to 9.9 %)

32 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)33 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Comparison of Cmax in plasma and brain a cross a ge

Adult PND15 Adult PND15PlasmaCmax(ng/g)BrainCmax(ng/g)

33 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)34 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Pharmacokinetics in adult and juvenile Rats

Plasma Brain Dose 0.5 mg/kg 0.25 mg/kg 0.1 mg/kg 0.5 mg/kg 0.25 mg/kg 0.1 mg/kg Cmax ( ng/g) Adult 50.4± 24.3 36.4 + 12 14.6 ± 11.9 7.63 + 1.59 2.52 + 0.31 <LOQ PND15 284 + 52 128 + 36 21 + 6.6 16.3 + 5.7 10.7 + 1.4 3.1 + 0.8 AUC ( ng- hr/mL) Adult 258 327 96 113 29 <LOQ PND15 1172 858 148 144 95 42 

### • Single oral dose of DLM in 5 ml/kg corn oil to PND90 Rats with terminal blood and brain sampling
• Plasma and brain levels higher at PND15 that PND 90 for both plasma and brain for all doe levels. • Cmax 2 to 5 -fold • Note : At lowest dose of 0.1 mg/kg, no DLM was detected in PND90 rats, indicating rapid and complete elimination.

34 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)35 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Deltamethrin in vitro metabolism studies for model parameterization
• Microsomal and cytosolic fractions were prepared from PND15, 21 and 90 SD rat liver samples • Kinetic studies were performed to determine Vmax and Km in

– Microsomes • CYP enzymes • CES enzymes

– Cytosol • CES enzymes • These in vitro-derived metabolic constants were extrapolated to in vivo according to biological scaling steps and used in the PBPK model 35 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)36 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# In vitro measured age-specific metabolic capacity used for IVIVE

Age-specific metabolic capacity (Vmax) values in vitro The data are collected by LFR in CAPHRA research efforts. Km values range from 0.8 to 1 μM before adjusting for non - specific binding Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)37 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Rat model parameterization supported by CAPHRA data

Parameters Values Adult (PND15) Liver cytochrome P450 (CYP ) V max_LCYP ( umol /h/kg tissue) 786 ( 142) Km _LCYP ( umol /l) 0.739 (0.952) Liver carboxylesterase (CaE ) V max_Lest_m ( umol /h/kg tissue) 168 ( 56) Km _Lest_m ( umol /l) 1.007 (2.016) V max_Lest_c ( umol /h/kg tissue) 170 ( 33) Km _Lest_c ( umol /l) 0.51 (0.562) Plasma carboxylesterase (CaEP ) V max_PLest ( umol /h/kg tissue) 1264 ( 195) Km _PLest ( umol /l) 1 ( 1) Parameters Values Adult (PND15) Partition coefficients Blood to plasma ratio ( BPR ) 1.15 Liver/plasma ( DRL ) 0.8047 Fat/plasma ( DRF ) 68.7 GI/plasma ( DRGI ) Same as Liver Brain/plasma ( DRBR ) 0.22 ( 0.25) Slowly perfused/plasma ( DRS ) 3.94 (1.02) Rapidly perfused/plasma ( DRR ) Same as Liver Plasma protein binding Total unbound fraction ( FFPlas ) 0.2 Parameters Values Adult (PND15) Oral absorption related parameters KA , uptake rate constant (h - 1 ) 0.35 K 12 , rate constant, from corn oil to lumen (h - 1 ) 0.2 K 21 , rate constant, from lumen to corn oil ( h - 1 ) 0.000026 K FEC, Fecal excretion (h - 1 ) 0.025 Tissue permeability area-cross product (L/ hr

/tissue weight^0.75) Fat ( PAFC ) 1.8 Brain ( PABRC ) 0.05 Slowly perfused ( PASC ) 0.08
Note: The full parameter table can be found in the overview document Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# IVIVE Ageappropriate metabolism parameters

- in vitro 

# Rat deltamethrin model

38 Refined from Tornero -Velez et al, 2010 based on the new data collected so far in CHPHRA Rapidly Liver QBR QF QS QR QH
GI tissue QGI Metabolism (CES)

Metabolism (CES and CYP) Plasma

Vehicle Oral dose Fecal excretion Lumen Brain Fat Slowly

QGI QL QBR QF QS QR

Lymphatic absorption 

## Rat Growth Physiology
Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)39 The Hamner Institutes for Health Sciences | August 5, 2010 

# Describing in vivo free concentration in the model
DLM chylomicron 

# DLM chylomicron


# DLM DLM
VLDL or HDL 

# DLM VLDL or HDL 

# Plasma Tissue

Plasma proteins /lipids Partition to tissue Lipolysis by lipoprotein lipases

39 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)40 The Hamner Institutes for Health Sciences | August 5, 2010 

# Recapitulating dose-dependent kinetic profiles of deltamethrin

PND90 (new CAPHRA data) The data are collected by UGa in CAPHRA research efforts. Serial sampling from plasma was performed ; 5 mlg/kg corn oil used as vehicle 40 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 

## PND90 (new CAPHRA data) The data are collected by UGa in CAPHRA research efforts. 5 mlg/kg corn oil used as vehicle



# Recapitulating deltamethrin kinetics in adult rats

41Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 

# Recapitulating deltamethrin kinetics in adult rats

PND90 (published data) The data are from Kim et al., 2008 1 ml/kg glycerol formal used as vehicle

42Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 

# Recapitulating deltamethrin kinetics in juvenile rats

PND15 (new CAPHRA data) The data are collected by UGa in CAPHRA research efforts. 5 mlg/kg corn oil used as vehicle 43Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 

# Recapitulating deltamethrin kinetics in juvenile rats

PND10 (published data) The data are from Kim et al., 2008 1 ml/kg glycerol formal used as vehicle Note that PND15 metabolic constants were used here. 4445 

# Application of IVIVE approach to develop age - specific PBPK models in humans 

# Deltamethrin PBPK

model for humans at different ages Human Subcellular fractions from adult liver with CES ontogeny data Application of the IVIVE approach to humans

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# Developing human early age models for deltamethrin

• Applies the in vitro - based parameterization approach validated in the rat modeling • Human in vitro metabolism data –for demonstration of the approach with deltamethrin , subcellular fractions from adults and CES enzyme ontogeny were used • Integration of information – human growth physiology (Clewell et al., 2004), in vitro - derived metabolic constants ( LFR data in CAPHRA ), and exposure levels are coherently integrated in PBPK models

46 Copyright 2014 by The Hamner Institutes for Health Sciences. May not be reproduced without permission Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# Human PBPK model for deltamethrin

47 Human relevant Exposure 

## IVIVE Age-appropriate metabolism parameters from **in vitro** data Adult liver metabolism by CES ( LFR )
CES ontogeny ( MCoW ) 

# IVIVE Rapidly Liver QBR QF QS QR QH
GI tissue QGI Metabolism (CES and CYP) Plasma

Oral dose Fecal excretion Lumen Brain Fat Slowly

QGI QL QBR QF QS QR

Lymphatic absorption Human Growth Physiology

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)

# IVIVE steps for human deltamethrin modeling
• The ultimate plan is to use recombinant enzyme - based data, but p reliminary simulations were conducted using the average adult Clint from adult subcellular fractions data (CES- 1 is the major contributor)

48 

### Measuring metabolic rates Clint_microsomes = 24 ml/min/mg Clint_cytosol = 6 ml/min/mg 

### ( LFR, preliminary)

Average adult in vivo Clint = 86896 L/ hr/kg liver in adults (mainly CES- 1 mediated) 

### IVIVE CES- 1 ontogeny Mature at birth 

### (Hines et al.,

submitted) Ontogeny on Liver weight and blood flow (Clewell et al., 2004) Ontogeny on the scaling factor ( MPPGL) ( Barter et al., 2007;2008 )

Copyright 2013 by The Hamner Institutes for Health Sciences. May not be reproduced without permission 

### Clint_in vivo All ages
The estimated average Clints for each age are shown 

### PBPK modeling

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)49 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Human model parameterization based on IVIVE supported by CAPHRA data
Parameters Value Metabolism CLCAEC (L/h/kg liver )* 86896 CLCYPC (L/h/kg liver) 0 * Sum of microsomal and cytosolic CESs Parameters Value Partition coefficients Blood to plasma ratio ( BPR ) 1.15 Liver/plasma ( DRL) 0.8047 Fat/plasma ( DRF ) 68.7 GI/plasma ( DRGI ) Same as Liver/plasma Brain/plasma ( DRBR ) 0.25 Slowly perfused/plasma ( DRS ) 3.94 Rapidly perfused/plasma ( DRR ) Same as Liver/plasma Erythrocyte/plasma ( DRRBC ) 1.34 Parameters Value Oral absorption KA , Uptake rate constants (h - 1 ) 0.35 K FEC, Fecal excretion (h - 1 ) 0 Protein binding Total unbound fration ( FFPlas ) 0.1 Tissue permeability area-cross product (L/ hr

/tissue weight^0.75) Fat ( PAFC ) 1.8 Brain ( PABRC ) 0.05 Slowly perfused ( PASC ) 0.08 Note: The full parameter table can be found in the overview document Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)50 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Predicting deltamethrin internal exposure in humans across age

50 Simulation is performed using a single dose of deltamethrin at 0.005 mg/kg Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)51 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Which parameters have the most influence over the deltamethrin brain concentration?

• The relative influence of each of the model parameters on brain deltamethrin concentrations was evaluated by a sensitivity analysis • This information can determine key data requirements and identify studies needed for read - across for additional pyrethroids

51 Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)52 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Sensitivity analysis of the human deltamethrin model

• The analysis was performed for

– 8 hr post- exposure (around Cmax for all the ages simulated) to a single dose at 0.005 mg/kg

– two ages, adult (20 year old) and young (1 year old) • The sensitivity coefficient (SC) is defined as the percent change in the dose metric ( brain DLM concentration) resulting from a 1% change in the particular parameter. • Thus, an SC of 1 represents 1:1 relationship between the change in the parameter and the internal dose metric. A negative SC indicates and inverse relationship. • Sensitivity coeffieicnts are grouped as high (absolute value greater than or equal to 0.5), medium (absolute value greater than or equal to 0.2 but less than 0.5) or low (absolute value greater than or equal to 0.1 but less than 0.2); parameters with sensitivities less than 0.2 are not listed (IPCS guideline). • To determine the overall importance of a parameter, both sensitivity and uncertainty have to be considered together.
Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)53 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# Model parameter sensitivity Sensitivity coefficient 

### Adult (20 year old) 

### Young (1 year old) Age- related data available? 8 hr post exposure (How uncertain we are in the parameter?) parameter SC designation SC value SC designation SC value Chemical

specific parameters Maximum rate of metabolism CLCAECM * H 0 H 0 Age - specific in vitro data (Low uncertainty) Brain permeability PABRC H 0.86 H 0.86 Estimated based on in vivo PK data/ Lipoprotein lipase distribution (Medium uncertainty) Physiological

parameters Cardiac output QCC H -0.85 H -0.94 Published database available ( Low uncertainty) Liver blood flow QLIVC H -0.8 H -0.89 Published database available (Low uncertainty) Brain volume VBRNC M -0.22 M -0.22 Published database available (Low uncertainty) Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)54 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

# IPCS Sensitivity/Uncertainty matrix UNCERTAINTY High Medium LowSENSITIVITYHigh



### — Brain permeability Metabolic Clearance Cardiac Output Liver Blood FlowMedium



### — Brain volumeLow


### — Body weight, Other t issue weights and blood flows * From the International Programme on Chemical Safety ( IPCS ) Harmonization Project Document on ‘Characterization and Application of Physiologically based Pharmacokinetic Models in Risk Assessment’ Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)55 The XIII International Congress of Toxicology | July 2, 2013, Seoul, Korea 

## • Predict brain and plasma deltamethrin concentrations

• in different ages of humans • under different exposure scenarios – capability to connect to and use outputs from exposure models (CARES or SHEDS) • Use model - predicted brain dosimetry • to develop CSAF for early age subpopulation • to compare with effective concentrations observed in neurotoxicity studies to inform margin of safety
55 Copyright 2014 by The Hamner Institutes for Health Sciences. May not be reproduced without permission 

# Examples of model application

Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)56 The Hamner Institutes for Health Sciences | August 5, 2010 

# Simulation of dose-dependent brain Cmax profiles in vivo neurotoxicity studies

Note that the x- axis is in a log scale. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 5657 The Hamner Institutes for Health Sciences | August 5, 2010 

# Reverse dosimetry to estimate human equivalent doses RAT HUMAN Age External Dose Brain Cmax (from model) External Dose based on Brain Cmax Target Juvenile PND15 1 mg/kg 

# * 48 ng/mL 2.0 mg/kg Child Adult PND90 1 mg/kg 

# * 11 ng/mL 0.2 mg/kg Adult * Rat NOAEL for both juvenile and adult rats Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 5758 The Hamner Institutes for Health Sciences | August 5, 2010 

# Distribution of simulated brain Cmax in early ages vs adults

Single dose of deltamethrin 0.005 mg/kg was used for Monte Carlo simulation

Horizontal line bisecting large rectangle, median; large rectangle, lower and upper quartiles; whiskers, 5 th and 95 th percentiles 

### 1yr 5 yr 10 yr Adult AgeBrainCmax(ng/ml)

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# Acknowledgement Modeling • The Hamner Institutes • Huali Wu • Gina Song • Miyoung Yoon • Harvey Clewell • David Billings • Alina Efremenko 

## Data collection • LFR Molecular Sciences • Brian G. Lake Lab 

## • University of Georgia • James Bruckner Lab 

## • Medical College of Wisconsin • Gail McCarver/Ron Hines Lab 59 

## CAPHRA discussion

• Thomas Osimitz (Science Strategies, LLC) • Paul Hinderliter (Syngenta) • Other members in the Consortium


# Funding The Council for Advancement of Pyrethroid Human Risk Assessment, LLC ( CAPHRA ) Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA)