Pyrethroid/Pyrethrins Technical Working Group 

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Study Title 

Response to US EPA’s Request Soliciting Proposals for Study Designs and Protocols to Evaluate 

Potential Differential Sensitivity of Pyrethrin and Pyrethroid Insecticides between Juveniles and 

Adults (Letter from Tina Levine dated February 16, 2010) - REVISED 

Data Requirement

FIFRA Guideline: None 

Authors

 Tom Osimitz, David Kim, Larry Sheets, Walter Schmitt, Dan Minnema, Gary Mihlan, 

Satheesh Anand, Harvey Clewell, Ron Hines, Tom Lewandowski, Lorenz Rhomberg, Charles 

Breckenridge, Bruce Young, Richard Allen, Nasser Assaf, David Carlson, Matthew Brooks, Kazuki 

Mikata, Ryozo Tsuji 

Completed on

June 18, 2010 

Submitted by

Pyrethroid/Pyrethrin Technical Working Group 

Chair, Thomas G. Osimitz 

Science Strategies LLC 

600 E. Water Street, Suite G 

Charlottesville, VA 22902 
Pyrethroid/Pyrethrins Technical Working Group 

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GOOD LABORATORY PRACTICE CERTIFICATION 

Information in this document summarizes technical data submitted in support of registration or 

provides risk assessments using these data and is not subject to Good Laboratory Practice Standards. 

Company: Pyrethroid/Permethrin Technical Working Group 

Company Agent: Date: June 18, 2010 

 Thomas Osimitz 

 Chair PPTWG 
Pyrethroid/Pyrethrins Technical Working Group 

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TABLE OF CONTENTS

1 INTRODUCTION.......................................................................................................................... 5

2 REVIEW OF AGE-RELATED SENSITIVITY TO PYRETHROIDS AND 

PYRETHRINS ............................................................................................................................... 7

2.1 High-dose phenomenon........................................................................................................ 7

2.2 Knowledge Gaps and Proposed Studies - Assessment of Age-Dependent Sensitivity 

(Study Block #1)................................................................................................................... 8

3 EXAMINATION OF FACTORS THAT COULD LEAD TO AGE-RELATED 

SENSITIVITY ............................................................................................................................. 12

3.1 Metabolic Differences ........................................................................................................ 12

3.2 Physiological Differences................................................................................................... 13

3.3 Knowledge Gaps and Planned Studies (Study Block #2)................................................... 16

4 ESTIMATION OF INTERNAL DOSE TO TARGET TISSUES ............................................... 17

4.1 PBPK Modeling of Pharmacokinetics in Humans (adult and non-adult)........................... 18

4.1.1 Rationale ............................................................................................................ 18

4.1.2 Proposed approach for characterizing internal dose in humans ......................... 18

4.1.3 Evaluation of the Human PBPK Model ............................................................. 19

4.1.4 Implementation Considerations.......................................................................... 19

4.2 Evaluation of the in vitro/silico Human PBPK Modeling Strategy.................................... 20

4.2.1 Current model structure and physiological parameters...................................... 20

4.2.2 Chemical-specific parameters for the rat PBPK model...................................... 21

4.3 Experimental determination of parameters for PBPK models (Study Block #3)............... 23

5 SUMMARY OF KNOWLEDGE GAPS AND STUDY PLANS................................................ 25

6 SUMMARY ................................................................................................................................. 27

7 REFERENCES............................................................................................................................. 28

Appendices 

Appendix 1. Suggested Refinements to the Rat PBPK Model for Deltamethrin (Tonero, et al., 

2010)........................................................................................................................... 33

Appendix 2. Company Membership of PPTWG Task Forces ............................................................. 34
Pyrethroid/Pyrethrins Technical Working Group 

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TABLE OF CONTENTS (continued) 

Tables

Table 1. Active ingredients represented by Consortia Companies......................................................... 6

Table 2. Lethality (LD50) results for pyrethroid insecticides in male Long-Evans hooded rats............ 8

Table 3. Milestones in the development of Sprague-Dawley and Wistar rats...................................... 10

Table 4. Study Design Elements for Testing Acoustic Startle ............................................................. 10

Table 5. Study Design Elements for Testing Toxicokinetics and Dynamics* ..................................... 11

Table 6. Ontogeny of enzyme activities in humans (relevant to pyrethrin/pyrethroid 

metabolism)................................................................................................................ 13

Table 7. Chemical Specific Parameters for PBPK Models of Deltamethrin in Rats (from 

Tornero-Velez et al. 2010) ......................................................................................... 22

Table 8. Proposed Pyrethrin and Pyrethroid Insecticides for Studies .................................................. 26

Figures

Figure 1 PBPK model for the maturing rat (Tornero-Velez et al., 2010)................................. 21
Pyrethroid/Pyrethrins Technical Working Group 

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1 INTRODUCTION

The Pyrethroid Working Group (“PWG”), Pyrethrin Joint Venture (“PJV”) and Permethrin Data 

Generation II (“PDGII”), representing the interests of pyrethroid and pyrethrin insecticide registrants 

have established the Pyrethroid/Pyrethrin Technical Working Group (PPTWG) to respond to the 

request of US EPA soliciting proposals for study designs and protocols to evaluate potential 

differential sensitivity of pyrethrin and pyrethroid insecticides between juveniles and adults (letter 

from Tina Levine dated February 16, 2010). 

These three consortia comprising the Technical Working Group, together with Valent USA 

Corporation representing the Sumitomo Chemical Corporation, represent registrants holding 

registrations for almost all pyrethrin and pyrethroid insecticides registered in the USA (Table 1). 

This document is a response to the above-mentioned letter from EPA. The Agency reports their 

evaluation of data from developmental neurotoxicity studies and consideration of juvenile 

susceptibility (Scollon, 2010) and cites studies from the published literature that indicate juveniles 

may be more susceptible than adults to acute pyrethroid toxicity (lethality). The Agency also 

summarizes the literature that supports age-dependent sensitivity in rats at high doses and 

appropriately states that: 

“It is unknown whether such sensitivity would occur at lower doses more relevant for human 

health risk assessment”. 

The EPA letter informed registrants that the FIFRA Science Advisory Panel (SAP) would be meeting 

to: 

“...discuss experimental studies designed to evaluate potential for comparative sensitivity 

between juvenile and adult rats exposed to synthetic pyrethroids and naturally-occurring 

pyrethrins. 

Further: 

“The Agency is soliciting proposals for study designs/protocols to evaluate potential 

differential sensitivity between juvenile and adult rats.” 

We appreciate that the Agency is reaching out to registrants for input on this topic and are pleased to 

respond. However, the Agency’s request only addresses part of the information needed for a robust 

assessment of potential human risk. Thus, our response necessarily goes beyond the relatively narrow 

focus of this request and addresses the broad issue of assessing possible age-related sensitivity in 

humans. Embedded in this exercise is the development of data that will determine whether an FQPA 

safety factor is needed to account for potential increased susceptibility of children. 

Our response consists of four sections: 

 Basis for concern about age-related sensitivity to pyrethroids and pyrethrins; 

 Factors that could lead to age-related sensitivity; 

 Estimation of internal dose; 

 Identification of knowledge gaps and a plan to fill them. 

An important outcome from the proposed series of activities will be the determination of the need to 

maintain the FQPA safety factor for age-related sensitivity based upon the age groupings typically 
Pyrethroid/Pyrethrins Technical Working Group 

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used in aggregate or cumulative exposure assessments. If age-related differences in toxicity were 

found to occur, both differences in toxicity and exposure need to be integrated for each relevant age 

interval. 

Table 1. Active ingredients represented by Consortia Companies

Consortia Active Ingredients 

Pyrethroid Working Group Beta-Cyfluthrin 

Bifenthrin 

Bioresmethrin 

Cyfluthrin 

Cyhalothrin 

Cypermethrin 

Deltamethrin 

Esfenvalerate 

Fenpropathrin 

Fenvalerate 

Gamma-Cyhalothrin 

Lambda-Cyhalothrin 

Permethrin 

Resmethrin 

Tefluthrin 

Tralomethrin 

Zeta-Cypermethrin 

Permethrin Data Generation II Permethrin 

Pyrethrin Joint Venture Pyrethrins (pyrethrin 1, pyrethrin 2, cinerin 1, 

cinerin 2, jasmolin 1, jasmolin 2) 

Valent USA Corporation on behalf of Sumitomo 

Chemical Corporation 

Allethrin stereoisomers 

Bioallethrin 

Cyphenothrin 

Imiprothrin 

Metofluthrin 

Phenothrin 

Prallethrin 

S-Bioallethrin 

Sumithrin (D-phenothrin) 

Tetramethrin 
Pyrethroid/Pyrethrins Technical Working Group 

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2 REVIEW OF AGE-RELATED SENSITIVITY TO PYRETHROIDS AND PYRETHRINS

The issue of age-related sensitivity to pyrethrins and pyrethroids has been extensively discussed. 

However, a careful consideration of the data indicate that juvenile or neonatal sensitivity as evidenced 

by acute lethality is limited only to high dose levels and only following gavage dosing (at a high 

dose) in rats. Such a dosing regimen likely exceeds the limited capacity of the non-adult animals to 

metabolize a large bolus dose. More relevant to humans are the studies conducted at lower dose 

levels that illustrate no difference in sensitivity to these pyrethroids. These data are consistent with 

the limited reported findings in the offspring from developmental neurotoxicity studies with 

pyrethroids following dietary administration. Indeed, neurotoxic signs are generally not seen in 

dietary studies with pyrethroids in adult animals, except at very high doses, as the dose is delivered at 

a relatively lower rate, compared with a bolus gavage dose. Below, we cite examples of pyrethroids 

for which this age-dependent difference is limited, or does not occur, even at lethal dose levels, and 

discuss the literature that explains the biological basis for such differences. 

2.1 High-dose phenomenon

The Agency cites examples of age-dependent differences in the sensitivity of rats to pyrethroids at 

high dose levels. For example, the acute oral LD50 of deltamethrin in 11-, 21- and 72-day-old male 

rats is 5, 11 and 81 mg/kg, respectively, with a similarly large age-dependent difference in sensitivity 

to an acute lethal dose of cypermethrin at these ages (Sheets et al., 1994 and Sheets 2000). By 

comparison, an age-related difference in sensitivity to lethality was not evident with permethrin or 

cismethrin (Table 2, based on Sheets, 2000). 

Cantalamessa (1993) reported a similar age-related sensitivity difference in acute lethality in rats 

treated with cypermethrin. The oral LD50 in 8- and 21-day-old rats that was 17- and 9-fold lower 

than in the adult, respectively. In addition, a relatively modest difference in LD50 with permethrin 

(4-fold lower than in the adult) was observed at both ages. Collectively, these results provide limited 

evidence of age-dependent sensitivity of rats to pyrethroids that have an α-cyano phenoxybenzoyl 

moiety (Type II, deltamethrin and cypermethrin) and a limited (e.g., 0- to maximum 4-fold) 

difference for pyrethroids that lack this moiety (Type I, cismethrin and permethrin). 

In contrast to the non-specific coarse measure of high-dose toxicity that is represented by the lethality 

endpoint, the acoustic startle response has been used to measure the acute neurotoxic potency of 

pyrethroids in juvenile rats (Sheets et al., 1994; Sheets 2000) at non-lethal dose levels, for 

comparison with the adult (Crofton and Reiter, 1988). The results from these studies demonstrate that 

21-day-old rats are not more sensitive to neurotoxic dose levels of representative Type I or Type II 

pyrethroids compared with adult rats. These investigations established that doses of deltamethrin, 

cypermethrin and permethrin that produced a 50% change (ED50) in the startle response were 

comparable in juvenile and adult rats, while the juvenile was actually less sensitive than the adult to 

cismethrin. 

Most important, because the acoustic startle response is representative of a specific neurological 

response it should be considered as the endpoint of interest for use in human risk assessments rather 

than lethality. 
Pyrethroid/Pyrethrins Technical Working Group 

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Table 2. Lethality (LD50) results for pyrethroid insecticides in male Long-Evans hooded rats

PND 11 PND 21 Adult 

Cismethrin 119 (87 - ?) 32 (26 - 38) 55 (44 - 63) 

Permethrin 254 (181 - 322) 201 (131 - 299) 406 (100 - 524) 

Deltamethrin 5 (3 - 8) 11 (8 - 14) 81 (59 - 95) 

Cypermethrin 18 (15 - 23) 73 (60 - 88) 439 (342 - 583) 

Values represent LD50 estimates (mg/kg), based on 6 or 7 males per dose group at each age. The 

95% confidence limits, where calculable, are shown in parentheses (from Sheets, 2000). 

2.2 Knowledge Gaps and Proposed Studies - Assessment of Age-Dependent Sensitivity (Study 

Block #1)

Given the relatively limited dataset, the PPTWG proposes to investigate this phenomenon using a 

broad range of commercial pyrethroid and pyrethrin insecticides (see Table 8 ), under circumstances 

that are suited for human risk assessments. The approach described below involves measuring the 

concentrations of representative pyrethroids in brain tissue that are associated with a specific acute 

neurotoxic effect; namely a change in the auditory startle response. 

The auditory startle response (ASR) is well suited to assess age-dependent sensitivity to pyrethroids, 

as it has been shown with pyrethroids to provide a robust and sensitive measure of neurotoxicity in 

adult and juvenile rats. Brain concentrations of deltamethrin have been measured at doses that 

reduced the ASR by 50%, to provide an internal dose metric to compare the sensitivity of young (21-

day-old) and adult male rats (Sheets et al., 1994). 

Test Parameters and Metrics: Animals will be tested at the time-of-peak effect following dose 

administration, based on the time-course of clinical signs at higher dose levels and pyrethroid brain 

concentrations. Test conditions will be selected to minimize within-session habituation, in order to 

provide a representative measure of the startle response across a number of trials. The peak 

(maximum) startle response amplitude will be measured for comparison, with the time-to-peak 

amplitude (msec) used to verify the measured value represents the primary startle response. Within a 

few minutes after the startle test, whole-brain tissue will be collected, snap-frozen and stored at -70°C 

(maximum). 

Route of Administration: Oral administration will be used, due to direct relevance for risk 

assessment and providing the means to deliver a specific dose under controlled conditions. While 

dietary exposure is generally preferred for relevance to human exposure, relative to a bolus dose, it 

does not provide for the management of dose delivery (dose level and testing at the time-of-peak 

effect) that is required for precise comparison. Moreover, the effects of pyrethroids on the nervous 

system are transient and not cumulative. Therefore, for practical reasons we propose to use a bolus 

dose, with animals tested at the time-of-peak effect following treatment. 
Pyrethroid/Pyrethrins Technical Working Group 

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Dose Selection: Dose levels will be selected to avoid overt toxicity and to provide data suited for 

benchmark dose analysis (e.g., BMD10). Dose levels that produce overt toxic signs (e.g., postural 

changes, motor incoordination, profuse salivation & urination, and decreased body temperature) 

interfere with a more specific measure of effects on the startle response. Moreover, testing at lower 

doses avoids age-dependent differences that are specific to high doses, due to their limited capacity to 

metabolize a high, bolus dose. 

Dose Volume: We propose to use a dose volume of 5 ml/kg, rather than 1 ml/kg, for practical reasons 

and to avoid further exaggerating the toxicity due to delivery in a low dose volume. With respect to 

practicality, we propose to use a constant dose volume, to facilitate comparisons across all 

pyrethroids and pyrethrins that will be tested. While some of these compounds are sufficiently potent 

to allow delivery in a low-dose volume of 1 ml/kg, a higher dose volume of 5 ml/kg is required for 

others. Regarding toxic potency, Wolansky and co-workers (2007) determined that the toxic potency 

of bifenthrin, as shown by effects on motor activity and pyrethroid-specific clinical signs at a given 

dose, was 2-fold greater at a dose volume of 1 mL/kg, compared to 5 mL/kg. We consider that 

administration as a bolus dose in corn oil already exaggerates the acute toxicity of these compounds, 

relative to environmentally-relevant exposures (e.g., dermal, inhalation and dietary). While delivery 

in a bolus dose is not preferred, due to limited relevance to human exposure circumstances, the 

proposed study design requires precise control over the delivery of a measured dose, at a specific time 

before animals are tested and tissue samples are collected. Thus, we plan to administer the 

compounds in corn oil, at a sufficient, but moderate dose volume of 5 ml/kg. 

Gender and Age: To conserve animals, we plan to test males only, since there is no evidence of 

gender differences and the relevant literature is almost exclusively with male rats. With respect to 

age, while rats have the minimum coordination and strength for evaluation of the startle response as 

early as PND 17, we consider Day 21 as a more appropriate age for quantitative assessment and 

comparison with the adult. However, if it is considered necessary or important to evaluate this 

phenomenon in younger animals, we will determine whether a robust acoustic startle response in 

animals as young as Day 14 can be measured. Table 3 summarizes some of the relevant 

developmental milestones in the rat. 

Toxicokinetics and Dynamics: We propose to measure the concentration of representative (model) 

pyrethroids in plasma and whole-brain tissue in 21-day-old and adult rats in order to fully characterize 

the time-course of pyrethroids in these compartments. Characterization of the concentration-time 

profiles in plasma and brain will aid in the assessment and selection of the dose metric appropriate for 

evaluation of the toxicodynamics. Plasma and brain concentration measures will include the time of 

peak effect on the ASR to provide a measure of the relative sensitivity of the nervous system (the 

target tissue). This approach follows that reported by Sheets et al. (1994), who used deltamethrin as a 

model pyrethroid. Brain concentration will be measured at equally-toxic or effective dose levels 

(e.g., ED20 or ED30) in adult and 21-day-old male rats. 
Pyrethroid/Pyrethrins Technical Working Group 

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Table 3. Milestones in the development of Sprague-Dawley and Wistar rats

PND Milestones Comments 

10 Thermoregulation and motor skills – 

poorly developed 

Sensory function: no audition or vision 

Motor skills – Very limited; “paddling gait”; weight 

generally not supported by limbs (rest on abdomen) 

Sensory – Eyelids and pinnae closed 

12 Thermoregulation – developing 

Audition – pinnae detachment 

Motor skills – Limited improvement from PND 10 

Sensory – Respond to acoustic stimuli 

13 Acoustic Startle Response Sensory and motor development sufficient to elicit a 

startle response 

15 Eyelids open Motor skills – Mobile (walking and climbing) 

Sensory – Respond to acoustic and visual stimuli 

17-18 Motor and Sensory: Substantive improvement in 

motor skills and coordination 

21 Brain development equivalent to 1-

year-old child 

Proven capability to express a robust acoustic startle 

response by many laboratories. 

Table 4. Study Design Elements for Testing Acoustic Startle 

Animals1 Male SD rats (21-days and 6-8 weeks old) 

Number of pyrethroids Limited (Model) ; a.i. based on FOB study, if possible 

Group Size 8-10 / dose level at each age 

Number of dose levels 5 or 6 each, including vehicle control 

Ideal effective dose range ED: 0, 10, 20, 30 & 40%, or 

ED: 0, 5, 10, 15, 20 & 30% 

Vehicle Corn oil (5 ml/kg); based on FOB study & solubility 

Time-to-Peak effect Based on FOB results (supplement to verify, as needed) 

Test Conditions (ASR) Repeated measures (e.g., 40-50 trials) to provide a reliable measure 

of response amplitude, without habituation) 

Measures Response amplitude (latency to peak to verify); 

Animals1 Male SD rats (21-days and 6-8 weeks old) 

1

Selection of rat strain depends on the specific purpose / circumstances 
Pyrethroid/Pyrethrins Technical Working Group 

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Table 5. Study Design Elements for Testing Toxicokinetics and Dynamics* 

Animals1 Male SD rats (21-days and 6-8 weeks old) 

Number of pyrethroids Limited (Model) ; a.i. based on FOB study, if possible 

Group Size 6-8 / dose level 

Number of dose levels one or two each 

Ideal effective dose range ED30; or 

ED10 & ED30 

Vehicle Corn oil (5 ml/kg); as per ASR study 

Time-to-Peak effect Based on ASR results 

Euthanasia Anesthesia: Light (CO2/O2) with decapitation 

Whole-brain collected and snap frozen (liquid CO2) 

Storage: -70C (minimum) until assayed 

Homogenization, methods for analysis, etc. 

Measures Pyrethroid concentration (parent); based on tissue weight (g) and protein 

Animals1 Male SD rats (21-days and 6-8 weeks old) 

*Design details (e.g., age, time and dose levels) based on results from the ASR study. 

1

Selection of rat strain depends on the specific purpose / circumstances 
Pyrethroid/Pyrethrins Technical Working Group 

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3 EXAMINATION OF FACTORS THAT COULD LEAD TO AGE-RELATED 

SENSITIVITY

As stated above, the Agency reports their evaluation of data from developmental neurotoxicity studies 

and consideration of juvenile susceptibility (Scollon, 2010) and cites studies from the published 

literature that indicate juveniles may be more susceptible than adults to acute pyrethroid toxicity 

(lethality). The authors of these papers suggest that this age-dependent difference is mainly due to 

pharmacokinetic mechanisms. 

A clear understanding of the development and, most important, the differences between humans and 

rats in this regard are essential to answering the most important question: is age-related sensitivity to 

pyrethrins and pyrethroids of concern for humans? We have commissioned an extensive review of the 

scientific literature with respect to critical developmental changes in physiology and metabolism that 

rats and humans undergo from the fetal period through adolescence. We have learned a great deal 

from this exercise, some of which is summarized below. 

3.1 Metabolic Differences

Given that the metabolic detoxification and elimination of pyrethroids and pyrethrins is a critical key 

event in their toxic action, species differences in both the enzymes involved and the developmental 

trajectory or ontogeny of those enzyme systems must be taken into account when evaluating 

approaches to better understanding age-dependent sensitivity. Substantial species differences 

between the rat and human exist in the enzymes responsible for the disposition of the pyrethroids and 

pyrethrins. Thus, serum esterases exhibit significant activity in the rat, but not the human (Crow et 

al., 2007) while in the human, intestinal carboxylesterase 2 (hCES2) is important for the hydrolysis of 

some, but not all pyrethroids, whereas rat intestinal carboxylesterases exhibit little activity (Crow et 

al., 2007). In the liver, both oxidative and hydrolytic pathways can be important, depending on the 

pyrethroid, and species differences are observed in the preferred route of metabolism in this organ. 

Studies to evaluate the ontogeny of several of the rat cytochromes P450 were reviewed by Rich and 

Boobis (1997). These studies would suggest that though inducible, the constitutive expression of 

CYP1A2 is delayed and is absent or expressed at low levels through 15 days after birth. Somewhat 

similarly, CYP2C6 increases slowly after birth, reaching adult levels by 3 to 6 weeks of age. In 

contrast, rat CYP3A1 is apparently expressed at adult levels in as little as 2 weeks after birth. If the 

CYP1A2 and 2C6 enzymes have a dominant role in the clearance of pyrethroids in rat, at all ages, 

these data would suggest exposure during very early life stages of the rat and would represent an 

increased risk for toxicity relative to the adult. 

Considerable information is available regarding the ontogeny of human CYP2C19 (Koukouritaki et 

al., 2004) and CYP3A4 (Stevens et al., 2003), whereas limited information is available on the 

ontogeny of hCE1 and hCE2 (Yang et al., 2009; Zhu et al., 2009). Mean hepatic CYP2C19 levels 

increase during the first 5 months of life to levels that are approximately 80% of adult values from >5 

months to 10 years and thereafter are present at adult levels. Although more delayed, CYP3A4 also is 

expressed at or near adult levels by 1 to 2 years of age. Based on hCE1 and hCE2 mRNA levels that 

showed good correlation with protein levels, both of these gene products are being expressed at adult 

levels by 3 months of age. 
Pyrethroid/Pyrethrins Technical Working Group 

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Table 6 summarizes the relative activity of the various CYP and hCE isoforms at different stages of 

postnatal development. From the data, it is clear that multiple CYP and hCE isoforms that are at full 

activity in adults are also available to varying degrees at stages of development. 

Moreover, it is apparent from other studies that multiple CYP and hCE isoforms are involved in 

pyrethrins and pyrethroid metabolism; the relative role varies by compound (Scollon et al., 2009). 

Given the ontogeny data summarized in Table 6, the role of a specific CYP or hCE isoforms will thus 

vary according to age and compound. 

It is important to note that the internal dose of the pyrethrins or pyrethroids that is achieved is 

dependent not on the level of a single CYP or hCE isoform: rather, clearance depends upon the action 

of the entire suite of CYP and hCE enzymes available. Thus, even developing children (especially at 

>3 months of age) have a significant compliment of enzymes available to metabolize pyrethrins and 

pyrethroids. Moreover, given the relatively low exposure levels to be expected, it is likely that the 

enzyme activity that is present, even if not fully at adult levels, is not a limiting factor in metabolism 

and elimination and that the resulting internal dose in the nervous system will be very small. 

Table 6. Ontogeny of enzyme activities in humans (relevant to pyrethrin/pyrethroid 

metabolism) 

Activity in children, as % of adult 

Adult 12-18 

years 

6-9 

years 

3-5 

years 

1-2 years 3-12 

months 

<3 months 

Cytochrome P450 

CYP1A1 100% 

CYP1A2 40% 10 - 20% low to 

10% 

CYP2B6 Low 

CYP2C8 Unknown 

CYP3A4 >adult 110-

120% 

35 - 100% 10 - 30% 

CYP2C9 100% 30% low to 

50% 

CYP2C19 100% 

Carboxylesterase 

hCE-1 100% 110% 65% 65% 45% 

hCE-2 100% 80% 90% 90% 70% 

Note: the ranges indicated for 3-12 months represent increasing activity during that age span, not 

uncertainty or variability 

Empty cells represent no known data 

3.2 Physiological Differences

During the course of development after birth through adolescence and into adulthood, various 

physiological and biochemical parameters and functions undergo major changes. Most importantly 

the time courses of development vary significantly across species. Understanding these differences is 

essential to the estimation of the internal dose, a critical factor in risk assessment. Comparisons of 

several of the changes that occur as rats and humans develop are given below. Some of these 
Pyrethroid/Pyrethrins Technical Working Group 

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parameters can be directly accounted for in PBPK models without additional experimentation. Others, 

such as plasma protein binding are compound-specific and will be determined for individual 

compounds of interest (see section 4.3). 

 Blood-Brain Barrier: Of particular concern for chemicals that exhibit neurotoxicity, the 

blood-brain barrier in human infants is relatively undeveloped and displays greater 

permeability to drugs and other exogenous compounds until about three to four months of age 

(Statz and Felgenhauer, 1983). Little data exist to quantify the function of the blood brain 

barrier at earlier ages (Webster, 2008). It is well recognized that development of the bloodbrain barrier takes comparatively longer in the neonatal rat than in humans, with development 

between one and four weeks of age, and maturity at about four weeks (Calabrese, 1983; 

NRC, 1993), well after weaning at approximately 3 weeks of age. For this reason, the young 

rat is generally regarded as a poor model for evaluating potential human health effects of 

chemicals that are normally impeded by the blood brain barrier (Webster, 2008). 

 Body Water/Body Fat: Body water and body fat percentages may play an important role in 

determining how a chemical is distributed in the body and may therefore affect its half-life. 

In humans, tissue hydration decreases with age, from about 70-74% in the full-term neonate, 

to 60% at one year, 65% at eight years, and approximately 55-63% in the adult (Alcorn and 

McNamara, 2003; NRC, 1993). In rats, tissue hydration also decreases with age, but starts at 

higher levels: 87% at birth, 80% at one week, 70% at one to two months, and 65-66% in the 

adult (Altman and Dittmer, 1974). Thus, while the volume of distribution for a water-soluble 

chemical would be roughly equivalent in adult rats and humans, it would be somewhat higher 

in neonatal rats than in neonatal infants. 

Body fat generally follows a more complex pattern in humans, being relatively low at birth 

(14%), rising during the first 12 months to 23%, declining around puberty to 15%, then 

increasing during adulthood in females (31%), but not in males (13%) (NRC, 1993). Rats 

have lower percentages of body fat overall, with 3-4% from birth to three weeks, 10% at 

eight weeks, and 7-18% in adults, depending on strain and age (Clewell et al., 2003; Brown et 

al., 1997). Thus, the volume of distribution for a fat-soluble chemical such as a pyrethroid 

would be higher in humans than in rats at all ages, resulting in lower plasma levels than 

would be predicted using parameters derived from the rat. 

 Brain Myelination: The lipid-rich myelin sheath that surrounds nerve cell axons prevents 

the entrance of xenobiotics in the nerve cell. The process of brain myelination begins 

comparatively earlier in humans than in rats. In humans, the process begins during gestation 

at week 12, with peak activity during the last trimester and the first year after birth. 

Thereafter, the pace of myelination slows, but continues through adolescence (Rice and 

Barone, 2000; de Graaf-Peters and Hadders-Algra, 2006). In rats, the myelination process 

begins at birth, peaks during the second week post-natal, and continues through adolescence 

(Rice and Barone, 2000; Butt and Berry, 2000). Thus, human infants are relatively more 

protected from chemical insults to the brain compared to developing rats 

 Liver Volume and Perfusion: Because the liver is the key organ for metabolism of 

pyrethroids, developmental changes in both liver volume and liver perfusion will be critical 

for quantifying pyrethroid metabolism. It is important to note that, although we often 

compare metabolic rates across species using specific activities (on a per mg microsomal 
Pyrethroid/Pyrethrins Technical Working Group 

Page 15 of 34 

protein basis), the key factor for determining the impact of a chemical on an organism is total 

metabolic capability. This is a function of specific activity, amount of metabolic tissue 

available (e.g., liver size) and blood perfusion. Data from a number of studies suggest that 

relative liver volume (liver weight as a fraction of body weight) is similar in both rats and 

humans during development (approximately 3 to 4% of body weight) although relative liver 

volume declines to about 2% of body weight in human adults but does not decline in the rat 

(Schoeffner et al., 1999; Björkman, 2005; Downie et al, 2005). Liver perfusion appears to be 

somewhat less in rats compared to humans at birth, although data are lacking to determine 

whether this finding persists during rat development. 

 Plasma Protein Binding: Binding of chemicals to plasma proteins can strongly influence 

their distribution and elimination. Age-related differences in plasma protein binding affinity, 

plasma protein concentrations, and the availability of competing endogenous ligands may all 

be important. In blood, the principal binding proteins that are not compound-specific are 

albumin and alpha-acid glycoprotein (Ginsberg et al., 2004). At birth, levels of plasma 

binding proteins in both rats and humans are lower than in adults, leading to less serum 

binding capacity. In humans, alpha-acid glycoprotein levels in serum are approximately 25% 

of adult levels at birth but rise quickly, reaching about 75% of adult levels by six months of 

age (Alcorn and McNamara, 2003). Serum concentrations of albumin are about 75% of adult 

human levels at birth, rise gradually, and approach adult levels after the first year of life 

(Alcorn and McNamara, 2003). The binding capacity of plasma proteins may also be lower 

at birth. For certain drugs, binding capacity is as little as 40-50% of adult human capacity on 

a per gram of protein basis (Ehrnebo et al., 1971). Compared to humans, neonatal rats have 

lower concentrations of plasma binding protein relative to adults. At birth, rats have 

approximately half of adult total plasma protein levels, half of adult albumin levels (de Zwart 

et al., 2008), and half of adult levels of alpha-1 globulins (of which alpha-acid glycoprotein is 

a subset) (Grabowski, 1966). These differences in the relative amounts of rat and human 

plasma binding proteins during development may complicate extrapolations from the rat 

model to humans. 

 Sodium and Calcium Channels: Voltage-gated sodium (Na+) channels (VGSC) are a 

primary target of pyrethroids (Soderlund et al., 2002; Shafer et al., 2005) and thus are a 

potential cite of toxicodynamic differences in age-dependent sensitivity. While some work 

has been done in the rat (Shafer et al., 2005; Meacham et al., 2008; Erecinska et al., 2004), 

little is known about the timing of Na+ channel development in humans and no studies 

investigating development of various VGSC isoforms in humans could be located. 

Voltage-gated calcium channels (VGCCs) are structurally related to the voltage-gated sodium 

channels (VGSCs) and are widely distributed in the central nervous system. VGCCs play 

crucial roles in several different processes in neurons, including neurotransmitter release, 

gene transcription, and propagation of Ca-dependent action potentials in some regions of the 

CNS. Although work has been done addressing Calcium channels in rat brains and human 

embryonic kidney (HEK) cells, their activity in human brain vs. rat brain cells, at multiple 

age groups, has not been addressed. 
Pyrethroid/Pyrethrins Technical Working Group 

Page 16 of 34 

3.3 Knowledge Gaps and Planned Studies (Study Block #2)

Metabolic differences are the most likely causes of the age-related sensitivity observed in the rat. 

Given the central role that metabolism plays in the detoxification of pyrethroids and pyrethrins, 

species differences in the enzymes involved and the ontogeny of those enzyme systems must be taken 

into account when evaluating approaches to better understanding age-dependent sensitivity. 

We propose to evaluate age-dependent in vitro metabolism of pyrethrins and pyrethroids in human 

liver tissue. We will use both hepatic microsomes and hepatocytes depending upon their availability 

and the specific studies being performed. 

Microsomal metabolism assays with the pyrethroids have previously been conducted using the parent 

compound depletion approach (Scollon et al., 2009). Briefly, a mixture of Trizma buffer, microsomal 

protein, and NADPH is pre-incubated at 37°C for 10 min. Assays are initiated by the addition of 

pyrethroid in different concentrations. Aliquots are removed from the mixture over the time-course of 

the experiment and the parent compound remaining is quantified by LC/MS. The substrate remaining 

is used to estimate the amount of metabolites formed and plotted versus time to produce a reaction 

velocity. Plotting reaction velocities against initial concentrations yield Vmax and KM estimates for the 

test chemical. The intrinsic clearance is calculated by dividing the Vmax by KM. 

Hepatocytes are generally regarded to be better for in vitro quantitative/qualitative predictions of in 

vivo metabolism than subcellular fractions such as microsomes (Hewitt et al., 2007). The advantages 

of using primary hepatocytes over microsomes have been well documented in the context of 

quantitative predictions of in vivo metabolism. Experiments using hepatocytes can be performed in 

commercial laboratories such as at Life Technologies (Durham NC). These experiments may be 

valuable for confirming results from Scollon et al. (2009). Additionally, donated hepatocytes and/or 

microsomal fractions from adult and pediatric patients may be used to estimate metabolic clearance in 

vitro. The sample size from these donor pools will allow for analysis of population variability. For 

microsomal analysis, sufficient samples sizes in the postnatal, 1-12 months, and 1-10 year age groups 

will allow for investigation of population variability in metabolic clearance (personal communication 

with Dr. Ronald Hines, Medical College of Wisconsin). For hepatocyte analysis, there are a 

sufficient number of samples in the 11+ years age range to measure clearance for sub-sets of the 

population (personal communication with Life Technologies, Durham NC).
Pyrethroid/Pyrethrins Technical Working Group 

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4 ESTIMATION OF INTERNAL DOSE TO TARGET TISSUES

The assessment of target-tissue concentrations of the compound of interest is essential to reduce the 

uncertainties in the evaluation of exposure-response relationships and to produce the most robust 

estimate of human exposure and risk (US EPA, 2006). Pharmacokinetic data and PhysiologicallyBased Pharmacokinetic (PBPK) models allow for quantitative estimation of target tissue 

concentrations. The structure of a PBPK model is made up of a series of mass-balance differential 

equations describing the time-course of a chemical in various tissues, including the target tissue. 

Below is an example of a mass-balance differential equation that describes the time-course of 

chemical in a perfusion limited tissue compartment: 















  

blood:tissue

tissue

tissue ARTERIAL

tissue

tissue P

C CQ

dt

dC V

where Vtissue is the volume of the tissue compartment, Ctissue is the concentration of chemical in the 

tissue, Qtissue is blood flow to the tissue, Carterial is the concentration of chemical in arterial blood, and 

Ptissue:blood is the tissue:blood partition coefficient. PBPK models have been used for extrapolation 

between test animals and humans (e.g., rat to human), within human populations (e.g., gender and 

age), routes of exposure (e.g., oral to dermal), duration adjustment (e.g., acute to chronic), and from 

high dose to low-dose levels. 

In recent years, several PBPK models for select pyrethroids have been published (Mirfazaelian et al., 

2006, Tornero-Velez et al., 2010, Godin et al., 2010), which make it possible for assessment of 

internal dose levels in various organs and tissues. The aim of these previously published PBPK 

models has been to simulate the pharmacokinetic profile of deltamethrin in rats (Godin and coworkers 

scaled their rat model to humans, but the model itself was not evaluated against human 

pharmacokinetic data). Of these models, the most relevant for simulating the age-dependent 

pharmacokinetic behavior of pyrethroids is the model by Tornero-Velez and coworkers. The 

Tornero-Velez model is a modification of the Mirfazaelian model, and includes descriptions for agespecific weights and age-dependent changes in the metabolic clearance of deltamethrin. Comparisons 

of brain concentrations using the Tornero-Velez PBPK model revealed that pups (PND 10) have a 3.8 

fold higher Area Under the Curve (AUC) value than mature rats. Whether this difference is observed 

in human populations was not investigated. 

The current research proposal builds upon EPA's work in the area of pyrethroid PBPK modeling by 

focusing on the simulation of pharmacokinetic profiles of pyrethroids in human populations. Godin 

et al. (2010) proposed a human PBPK model; this model will be modified to include human-relevant 

physiological and chemical-specific parameters for both adults and juveniles. The new human PBPK 

model will be formulated using physiological and chemical-specific parameters obtained from 

existing databases and in vitro/silico inputs. Parameters will be optimized against previously 

published pharmacokinetic data (e.g., Woollen et al., 1992). However, we recognize that there is a 

paucity of such pharmacokinetic data that could be used for PBPK model evaluation. Therefore, 

additionally, we will evaluate the proposed in vitro/silico PBPK modeling strategy for humans in the 

rat by replacing chemical-specific parameters in the Tornero-Velez and Godin deltamethrin PBPK 

models with parameter values determined in vitro/silico. This strategy is consistent with the NAS 

report on Toxicity Testing in the 21st Century (NRC, 2007). 
Pyrethroid/Pyrethrins Technical Working Group 

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4.1 PBPK Modeling of Pharmacokinetics in Humans (adult and non-adult)

4.1.1 Rationale

Several factors point to the need to focus our efforts on the development of a PBPK model for 

humans. As discussed earlier in this document, meaningful differences exist in the compoundspecificity and ontogeny of enzymes in rats and humans. Thus, it is unlikely that clearance of a 

compound in a neonatal rat would be predictive of clearance in a human infant. There are also other 

age-dependent physiological factors that can affect the clearance of compounds during early life, such 

as the blood flow to the liver, intestinal absorption, renal clearance, etc. (Clewell et al., 2002). 

Reliance on animal studies to infer human infant and child sensitivity cannot account for these 

factors. However, in silico/in vitro/in vivo studies can be used to identify the specific ADME 

parameters that influence the pharmacokinetic behavior of pyrethroids in humans and can be used to 

estimate the internal dose of the compound of interest at the target tissue. 

4.1.2 Proposed approach for characterizing internal dose in humans

An approach that has the potential to overcome the limitations in the animal-based studies is to 

develop a PBPK model for predicting the relationship in the human between internal dose in the adult 

and internal dose in non-adults using chemical-specific properties. For pharmaceutical substances, 

pediatric pharmacokinetics have been successfully predicted using PBPK models based on adults and 

extrapolated to children using age-appropriate parameters (Björkman, 2005; Edginton et al., 2006a).

A similar approach has been demonstrated for other environmental compounds (Clewell et al., 2004; 

Gentry et al., 2003; Edginton & Ritter, 2009). Given the central role that metabolism plays in the 

detoxification of pyrethroids and pyrethrins, species differences in the enzymes involved and the 

ontogeny of those enzyme systems must be taken into account. Thus the development of appropriate 

PBPK models based on direct measurement of metabolic clearance in vitro using hepatocytes from 

adults and infants/children is critical (Johnson, 2003; Hines 2008; Björkman, 2006; Edginton et al. 

2006a; Alcorn and McNamara, 2008). 

The literature data on physiological values for the rat and human during early life have recently been 

organized into a database by ILSI, on behalf of the EPA (Gentry et al., 2004). This database can be 

used to refine parameter estimates for use in PBPK modeling of early life. In the approach previously 

demonstrated in the human (Clewell et al., 2004), chemical-specific parameters (partition 

coefficients, metabolism rate constants) were taken from existing PBPK models for adults; partition 

coefficients were assumed to be independent of age, while neonatal metabolic parameters were scaled 

from the adult values based on the age-dependent activity of the enzymes responsible for the 

metabolism of the particular compound. This same approach could be used in the case of pyrethroids. 

As described in section 3.3, the PPTWG is proposing a comprehensive block of studies on 

representative pyrethrins and pyrethroids to be conducted in microsomes and/or hepatocytes derived 

from human liver tissue (both adult and non-adult). 

A first task toward the development of such a capability is to develop the age-dependent 

physiological framework for the rat and test it using a variety of pyrethroids, following an approach 

similar to that used with deltamethrin (Mirfazaelian et al. 2006; Anand et al., 2006). These case 

studies would be used to validate the predictive reliability of the modeling approach. The human 

version of the model can then be applied to evaluate early life sensitivity to untested pyrethroids by 
Pyrethroid/Pyrethrins Technical Working Group 

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estimating the chemical-specific parameters using QSAR or in vitro studies, and then running the 

model with the appropriate age-dependent pharmacokinetic and exposure parameters. 

4.1.3 Evaluation of the Human PBPK Model

The development and use of the human PBPK model will follow good modeling practices as for 

example described in Loizou et al. (2008). In the proposed approach of extrapolating from adult 

humans to children, an important step is the verification of the output of the human model by 

comparison to respective experimental data. As shown by Edginton et al. (2006a) a good model for 

adult humans assures an extrapolation to younger humans with high accuracy. Previously published 

pharmacokinetic data for adult humans (e.g., Woollen et al, 1992) will be used for model evaluation. 

However, given the paucity of human in vivo data, the following procedures are proposed to 

complement existing datasets. 

As suggested in Loizou et al. (2008) the aim is to use a generic model structure for all compounds. 

The model structure and the compound specific parameterization may be verified by applying the 

generic model to simulate the dosimetry of compounds that have properties similar to those of 

pyrethroids and pyrethrins for which human PK data can be found in the literature. This compound 

specific parameterization will follow common practices in order to minimize uncertainty. A 

sensitivity analysis will be performed for characterizing the remaining uncertainty and identifying the 

most sensitive parameters for determining the time-course profiles of pyrethroids and pyrethrins. 

The influence of human inter-individual variability will be investigated by applying Monte Carlo 

simulations together with the human model (for adults and children) following approaches described 

in the literature (Price et al., 2003; Willmann et al., 2007; Beaudouin et al., 2010). 

4.1.4 Implementation Considerations

The parameters in a PBPK model can be categorized into four types: exposure, physiological, 

partitioning, and metabolism. The exposure parameters are determined solely by the characteristics 

of the exposures and the physiological parameters are available from the literature (Arms and Travis, 

1988; Brown et al., 1997). Additionally, there are public databases containing physiological 

parameters in humans. An example is POPGen (http://xnet.hsl.gov.uk/popgen/), a web application 

that generates physiological parameters of human populations for use in PBPK-modeling. Parameter 

sets can be obtained for user-defined sub-populations (definable population parameters: Number of 

individuals, gender, ethnicity and ranges for age, body height, body weight and BMI). POPGen 

contains data for children of all age groups (i.e., body composition information and ontogeny of 

various enzymes, Hines, 2007). 

Physiological parameters are not chemical-specific, and the values used in the evaluation of an 

untested compound would be the same as those used for well-characterized compounds. Partitioning 

and kinetic parameters, however, are chemical-specific and need to be estimated for untested 

compounds. Methods using QSAR techniques and physical properties can be used to estimate 

partitioning parameters (Hansch, 1979; Poulin and Krishnan, 1996a,b; Poulin and Theil, 2000; 

Beliveau et al., 2003; Rodgers et al., 2005; Rodgers and Rowland, 2006; Schmitt, 2008), and a 
Pyrethroid/Pyrethrins Technical Working Group 

Page 20 of 34 

variety of in vitro methods are available for estimating parameters for absorption and metabolism 

(reviewed in Clewell et al., 2007). 

4.2 Evaluation of the in vitro/silico Human PBPK Modeling Strategy

4.2.1 Current model structure and physiological parameters

A PBPK model for deltamethrin in the maturing rat has recently been published by Tornero-Velez et 

al. (2010). The structure of this PBPK model is shown in Figure 1. This model describes the 

pharmacokinetics of deltamethrin in rats of different ages between 10 d and 90 d reasonably well. 

The model includes compartments representing the brain, fat and liver, and combines the remaining 

tissues into rapidly-perfused and a slowly-perfused compartment. Brain, fat and slowly-perfused 

compartment are described as diffusion-limited compartments while the other compartments are 

perfusion limited. Some model parameters, particularly metabolic rates and tissue volumes, are 

considered age dependent by using continuous functions of age to calculate their values at a certain 

age. This age dependence is dynamically taken into consideration over the simulation time. The 

PBPK model as described above generates results that show overall a reasonable agreement with 

observed PK data from rats of different ages. 

As with many models, improvements can be made to the model structure to more accurately describe 

the pharmacokinetics of pyrethroids and pyrethrins in mammalian systems. The focus of the 

proposed research program is not to improve the published rat PBPK models for deltamethrin, but to 

give more confidence to extrapolation between and within species using a PBPK model. The 

proposed research program focuses on independent collection of in vitro experimental data that can 

be used within the PBPK modeling strategy (i.e., section 4.1) of predicting the pharmacokinetic 

behavior of pyrethroids and pyrethrins in human adults and children. At present, there is a lack of 

clarity on the reasons for significant changes in metabolism with age. The age-dependence of 

metabolic clearance needs to be considered in addition to age-dependent changes in blood flow and 

permeability cross-product to reliably extrapolate from rats to humans and adults to children. 

Particularly for the pyrethroids and pyrethrins, where it is known that there are significant metabolic 

differences between rats and humans, these differences need to be addressed appropriately in order to 

conduct an informative and accurate risk assessment. Therefore, it is proposed that the focus of the 

PBPK research program be on additional chemical-specific parameters that could inform the agedependent PBPK model with careful consideration of known physiological parameters for children. 
Pyrethroid/Pyrethrins Technical Working Group 

Page 21 of 34 

Figure 1 PBPK model for the maturing rat (Tornero-Velez et al., 2010)

4.2.2 Chemical-specific parameters for the rat PBPK model

Additional chemical-specific parameters will be incorporated into the Tornero-Velez PBPK model to: 

1) identify the specific parameters that are most sensitive and important for predicting the 

pharmacokinetics of pyrethroids and pyrethrins, and 

2) inform the strategy for building the human PBPK model for both adults and juveniles. 

These chemical-specific data will be independently measured using in vitro experimental systems. 

Relevant parameters include partition coefficients, absorption rates (gastrointestinal and dermal), 

metabolic clearance, and protein binding. A list of parameters specific to deltamethrin are shown in 

(Table 7) below (taken from Tornero-Velez et al., 2010). The ADME parameters obtained from 

these experiments, after appropriate scaling to in vivo conditions, can be incorporated into the 

recently published rat PBPK model for deltamethrin. 

The results of the in vitro experiments (see section 4.3 ) with pyrethrins and a broad set of pyrethroids 

should be compared against in silico (i.e., QSAR) estimates. In cases where in vitro experimental 

values cannot be obtained, or values are inconsistent with how pyrethroids are expected to behave, 

then in silico parameter estimates will be used to formulate the PBPK model. This strategy allows for 

application of the deltamethrin PBPK model to other pyrethroids by replacing deltamethrin-specific 

parameters with parameters specific to other pyrethrins/pyrethroids. 
Pyrethroid/Pyrethrins Technical Working Group 

Page 22 of 34 

The PBPK modeling strategy of using physiological parameters from the literature and biochemical 

parameters determined from in vitro/silico experiments will be evaluated against in vivo

pharmacokinetic data generated from rat studies. By using known physiological constants and 

experimentally-derived ADME parameters in the model, alternative descriptions of the 

pharmacokinetic behavior (e.g., perfusion-limited vs. diffusion-limited tissue compartments) may be 

investigated with confidence. Pharmacokinetic in vivo data may be obtained from published and 

unpublished sources. Alternative model structures (i.e., changes to the representation of organ/tissue 

compartments) can be used to compare simulated tissue concentration time-course profiles under each 

of the diffusion or perfusion limiting assumptions with different experimental data. Model structures 

different from previously published work will be evaluated for consistency with experimental 

pharmacokinetic measurements of pyrethroids. This process of evaluating the in vitro/silico PBPK 

modeling strategy will inform the development of the human PBPK model that will be used to 

extrapolate between adults and juveniles. In particular, the rat PBPK model will be used to identify 

the key parameters that most influence the dosimetry of pyrethrins and pyrethroids in plasma and 

target tissues/organs. In the formulation of the human PBPK model, these sensitive parameters will 

be replaced with ADME parameters measured experimentally using human cells and tissues. 

Table 7. Chemical Specific Parameters for PBPK Models of Deltamethrin in Rats (from 

Tornero-Velez et al. 2010) 
Pyrethroid/Pyrethrins Technical Working Group 

Page 23 of 34 

4.3 Experimental determination of parameters for PBPK models (Study Block #3) 

As discussed above, physiological and chemical-specific parameters in the rat PBPK model can be 

replaced with human relevant physiological parameters (e.g., blood flow rates and organ/tissue 

volume) as well as chemical-specific parameters determined experimentally using in silico

approaches and/or in vitro systems of human cells and/or tissues. Some examples of human in vitro

systems for measurement of ADME parameters include Caco-2 cells for quantifying intestinal 

permeability (Dokoumetzidis et al., 2007), protein binding using human plasma (Banker and Clark, 

2008), and investigation of metabolism using human hepatocyptes and/or microsomes as mentioned 

earlier in this document (Hines, 2008; Hewitt et al., 2007). The advantage of these in vitro systems is 

that cells/tissues from pediatric donors may be used to generate ADME data specific to juveniles. 

The parameters discussed below are important for the development of a robust PBPK model for rats 

and humans. The appropriate studies will be conducted with pyrethrins and a comprehensive set of 

pyrethroids (see Table 8). Note: because of the varying physical properties of the pyrethrins and 

pyrethroids being studied, it is possible that not all techniques described will work with all 

compounds. 

Dermal absorption - Dermal absorption can be quantified using in vitro systems by placing rat or 

human skin on static Franz diffusion cells (Franz, 1975). In these experiments, the donor 

compartment is placed on top of the dermatomed skin and secured with screw clamps. The test 

chemical is placed in the donor compartment. The receptor solution is filled with a buffer (pH  7.4) 

and temperature kept at 37 °C. Samples are collected from the receptor compartment to measure flux 

across the skin. At the end of the exposure, the amounts remaining in the donor cell and the skin are 

quantified. The diffusivity coefficient is determined from plots of cumulative mass per surface area 

vs. time; the diffusivity coefficient is calculated by dividing flux by the concentration of test chemical 

applied to the donor cell. Existing data on pyrethrins and the pyrethroids will be used when possible. 

The data from these studies should allow for re-analysis to calculate the diffusivity coefficient. 

Gastrointestinal absorption – This can be estimated for the rat by using experimentally measured 

values of intestinal permeability using in vitro systems similar to the Ussing chamber technique 

(Ussing and Zerahn, 1951). This technique utilizes small intestinal sheets that are mounted between 

two compartments: (1) donor where the chemical is applied and (2) receptor from which the absorbed 

chemical is sampled. The mucosal (i.e., luminal) and serosal (i.e., basolateral) compartments are 

usually supplied with Krebs-Ringer bicarbonate buffer that is continuously gassed with a O2:CO2

(95:5) mixture to provide sufficient oxygen tension for maintaining tissue viability. The pyrethroid is 

applied to either the luminal or basolateral side to quantify absorption in either the L-B or B-L 

direction. The apparent permeability coefficient is calculated from the appearance of the pyrethroid 

in the receptor compartment. The absorption rate constant (ka) in each GI segment is proportional to 

Peff (Dokoumetzidis et al., 2007). For human GI absorption, Caco-2 cells may be used (Sun et al, 

2008). Permeability across differentiated monolayers of Caco-2 is measured on fully differentiated 

cells grown for 3 weeks on permeable filter supports. The integrity of the monolayer is determined 

by measurement of TEER or by Lucifer Yellow permeability. Compounds are applied to the apical or 

basolateral side of the monolayer and incubated for 2 h. The amount of compound on each side is 

measured by LC/MS. The parameter Peff is calculated in the apical to basolateral and basolateral to 

apical directions. The ka is derived, for different regions of the GI tract, from Peff as described above 

(Dokoumetzidis et al., 2007) 
Pyrethroid/Pyrethrins Technical Working Group 

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Partition coefficients - (Ptissue:blood) for the rat can be measured in vitro using the ultra-filtration 

method, described by Jepson et al. (1994). Partition coefficients measured in rat tissue are comparable 

in value to human partition coefficients. Tissue:saline partition coefficients are determined for each 

test compound. Tissues may include (but are not limited to) brain, kidney, liver, muscle, fat, skin, 

and blood. Tissue:blood partition coefficients are determined by dividing the tissue:saline value by 

the blood-to-saline value. The modifications to the Jepson method include implementation of the 

negligible depletion solid phase microextraction (nd-SPME) technique, which does not require 

filtration (Tremblay et al., 2010). This method is required due to the low water solubility of 

pyrethroids. In this revised methods, only the free-phase compounds and not those that are tissuebound can partition into the SPME fiber coating. Tissue and blood are added to solutions spiked with 

the chemicals (phosphate buffered saline) and allowed to equilibrate for a minimum of 4 hrs at 37 °C. 

Free concentrations are measured by inserting a polydimethylsiloxane fiber (PDMS, 100 μm thick, 

variable length) directly into the solutions. The extracted compounds are then desorbed into a GC/MS 

for quantification. 

Plasma Protein Binding - Upon entering the systemic circulation, a chemical is eliminated by 

excretion and/or metabolism to one or more metabolites. It is only the unbound chemical, or free 

concentration of chemical, that is susceptible to these processes in vivo. Due to this, protein binding 

affinity plays an important role in a compound’s pharmacokinetic behavior. Equilibrium dialysis can 

be used to measure either the binding constant for a chemical entity, or to assess the extent of binding 

at a given incubation time point; this technique generally yields the most accurate estimates of protein 

binding. The pyrethroid is dissolved in a small volume of a suitable solvent and added at defined 

concentrations to the plasma; both rat and human plasma can be used to determine protein binding. 

After equilibration, the concentration in the plasma (C) and the dialysis buffer (Cu) are determined 

and used for calculation of the fu according to the relation fu = Cu/C×100. These calculations should 

be similar to the inverse value of the blood:saline partition coefficient measured in the row above. 
Pyrethroid/Pyrethrins Technical Working Group 

Page 25 of 34 

5 SUMMARY OF KNOWLEDGE GAPS AND STUDY PLANS 

Based on our analysis of the knowledge relating to the pharmacodynamics and pharmacokinetics of 

pyrethrins and pyrethroids we have identified a number of data gaps that need to be filled so that a 

robust estimate of internal dose and associated risk can be made: 

1) Study Block #1 - Further studies of possible age-related sensitivity in the rat; 

2) Study Block #2 – Age-dependent in vitro metabolism of pyrethrins and pyrethroids in human 

liver tissue 

3) Study Block #3 – Experimental determination of parameters for PBPK models; 

We propose to investigate a set of model compounds that were selected taking into account the 

following criteria: 

 Pyrethroids that are important for concern, due to the potential for children to be exposed; 

 Complexity of the material: In some cases the isomer profile of certain pyrethroids is very 

complex, having many different isomers. It will be necessary to ensure that a complex 

isomer profile does not overly complicate the assessment of metabolic clearance and 

interpretation of the results. 

 Class: Pyrethrins, α-cyano and non-α-cyano pyrethroids should be studied. 

 Type: The type of pyrethroid is based on clinical signs. Both type I and type II pyrethroids 

should be studied. It may also be relevant to investigate a pyrethroid exhibiting mixed 

properties (type I/II). 

 Time to Peak Effect: Some pyrethroids have a relatively short time to peak effect (2 hours or 

less) whereas others have a longer time to peak effect (8 hours). This time to peak effect 

(based primarily on motor activity and/or FOB data) could obviously reflect differences in 

toxicokinetics. Pyrethroids at both extremes of the range of time to peak effect should be 

selected. 

 Metabolism: The relative importance of hydrolytic and oxidative metabolism in the 

detoxification of pyrethroids and existing knowledge on the involvement of the different 

enzyme isoforms should be considered. 

 Dose: Some pyrethroids are considerably more potent than others. As a practical 

consideration this should be considered taking into account the analytical methodologies 

employed. 

The proposed model compounds are summarized in Table 8. In the first instance the PPTWG 

proposes to study effects with six chemicals. The program may be increased depending upon the 

need to address specific regulatory requirements for other pyrethroid insecticides and the results of 

the initial group of studies.
Pyrethroid/Pyrethrins Technical Working Group 

Page 26 of 34 

Table 8. Proposed Pyrethrin and Pyrethroid Insecticides for Studies 

Characteristic Pyrethrins Bifenthrin s-Bioallethrin Permethrin Deltamethrin α-cyano or non-α-cyano 

non-α-cyano non-α-cyano non-α-cyano non-α-cyano α-cyano 

Type I or II or mixed I I I I II Isomers 

primarily a mixture of 

pyrethrin I and 

pyrethrin II 

100% 

(Z, 1R cis) 

92% d-trans-d 

5.6% d-trans-l 

1.7% l-trans-d,l 

40% cis 

60% trans 

1:1 of 1R:1S 

100% 

1R, 3R, αS 

MW (grams/mole) 316 – 374 422.9 302.4 391.3 505.2 LD50: Adult 710 mg/kg 55 mg/kg 700 mg/kg 1200 mg/kg 67 mg/kg LMA ED30 (rats)* 3.2 mg/kg 90 mg/kg 43 mg/kg 2.5 mg/kg LMA (time of peak effect)* 4 hr 1 hr 1.5 hr 2 hr FOB Dose levels (rats)* 400-800 mg/kg 40-55 mg/kg 150-300 mg/kg 200-250 mg/kg 12.5-35 mg/kg FOB (time of peak effect) * 4 hr 8 hr 2 hr 4 hr 4 hr Use in Agriculture (0 → +++) 

+ ++ 0 +++ ++ 

Use in Residential (0 → +++) 

+++ +++ + +++ ++ 

Rationale of Use 

PYR are natural products that can be used for comparison to the pyrethroids. Extensive residential use and human pediculicide use. 

BIF is a non-αcyano pyrethroid having a relatively long time to peak effect based on both LMA and FOB data. 

s-BIO is a non-α-cyano 

pyrethroid having a relatively short time to peak effect based on both LMA and FOB data. Represents several residential use compounds. 

PRM is a non-α-cyano 

pyrethroid having a relatively moderate time to peak effect based on both LMA and FOB data. Widely used in agriculture and residential settings. Human pediculicide use as well. 

DLM is an α-cyano 

pyrethroid exhibiting Type II activity that has relatively moderate time to peak effect based on both LMA and FOB data. Data from DLM was the driver for this research effort. 

* Data from Wolanksy et al., (2006) and Weiner et al., (2009)
Pyrethroid/Pyrethrins Technical Working Group 

6 SUMMARY 

The PPTWG has taken a comprehensive and thoughtful look at the questions posed by the EPA in 

their recent communication. We acknowledge that uncertainty remains about the potential for agedependent sensitivity to pyrethrins and pyrethroids in humans particularly at lower doses more 

relevant for human health risk assessment. Based on this analysis, the PPTWG has outlined a series of 

studies that will address the key knowledge gaps that must be filled. Moreover, the PPTWG is 

prepared to take the resulting data and apply them to the development of a state-of-the-art PBPK 

model that will allow a robust assessment of internal dose at the target neurological tissue in adult and 

non-adult rats and humans. 

We have presented a PBPK modeling approach for predicting the pharmacokinetic behavior of 

pyrethrins and pyrethroids in humans, and to evaluate potential differential sensitivity between 

juveniles and adults. The PBPK strategy builds upon previously published models first by identifying 

the key ADME process that drives the observed kinetic behavior in rodents. Second, with this 

improved understanding of pyrethrin/pyrethroid pharmacokinetics in the rat, we propose formulating a 

human PBPK model with human relevant physiological and chemical-specific parameters. These 

parameters may be determined in silico or experimentally using in vitro systems of human cells and/or 

tissues. The human PBPK model will allow for the evaluation of potential pharmacokinetic-based 

aged-related sensitivity to pyrethrins and pyrethroids in humans. 

We expect that this significant amount of effort will take approximately two years to complete from 

initiation, especially as the need for additional data may become apparent once the results from the 

first studies are evaluated. We propose to set milestones through the duration of the proposed 

program to share results with the agency at regular intervals. 

Ultimately, this work will lead to the conduct of a robust risk assessment for pyrethrins and the 

pyrethroids that uses the best science available (consistent with the goals of the National Academy of 

Sciences – Toxicity Testing in the 21st Century) and is protective of public health. 

Page 27 of 34 
Pyrethroid/Pyrethrins Technical Working Group 

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Appendix 1. Suggested Refinements to the Rat PBPK Model for Deltamethrin (Tonero, et al., 

2010)

The following considerations are offered to reduce uncertainties in the rat PBPK model: 

 Blood flow rates: In the rat PBPK model, the blood flow rates in different tissues (Qtissue) are 

calculated relative to the age dependent cardiac output; the fractional flow to different tissues 

is considered independent of age. However, it is known that Qtissue scales with tissue volume 

rather than with cardiac output. This difference has probably the strongest impact on the 

simulated concentrations in the case of the target organ brain. In early post natal ages, the 

brain has a significantly larger fraction of the total body weight than at later ages. From the 

parameters reported for the age dependence of tissue weights (Edginton et al., 2006b), the 

brain weight as a percentage of body weight decreases from 2.4 % to 0.4 % from birth to adult 

age. Further, the percentage of cardiac output to the brain, Qbrain, will decrease from birth to 

adult age. For humans, cerebral perfusion rates measured in children of different ages support 

this assumption. It was found that Qbrain increases almost to adult level within the first year 

after birth while for other tissues this increase is much slower (see Edginton et al. (2006b) and 

references therein). 

 Tissue permeability × surface area products: Tornero-Velez et al. (2010) do not provide a 

description of their method for scaling tissue uptake rates (i.e., permeability × surface area 

(P×A) products) in diffusion limited tissues for different age groups. Scaling tissue uptake in 

diffusion-limited compartments is difficult; however, a simple guideline to following when 

scaling tissue uptake within and between species is to assume that permeability is independent 

of age, and surface area scales with tissue volume. Unfortunately the scaling law for surface 

area is not exactly known since the tissue surface area cannot be measured. An allometric 

scaling law (i.e., surface area = a × tissue volume0.75) may be appropriate for describing the 

relationship between surface area and tissue volume. 

 Perfusion vs. diffusion limited distribution: It may be reasonable to consider distribution 

into all tissues to be diffusion limited, as was the case for the recent deltamethrin PBPK model 

for adult rats (Godin et al., 2010). The ratio P×A/Q can be used to determine whether 

distribution into a tissue is diffusion or perfusion limited (i.e., if P×A/Q < 1, then diffusion 

limited; if P×A/Q > 1, then perfusion limited). This ratio can also be used to determine if 

distribution changes with age and/or body size since the scaling laws for the two factors are 

different. It may be the case that certain tissues are perfusion limited in newborns and 

diffusion limited in adults; this may happen since Q increases faster with size than P×A. This 

transition is also compound specific as permeability depends on lipophilicity and molecular 

volume of a substance. This has two implications on the use of PBPK models for 

extrapolations across ages and species that have significantly different body sizes. For 

extrapolation from smaller organisms to larger ones, the question whether perfusion limitation 

is conserved requires evaluation. This potential difference in distribution is not a limitation 

for extrapolations from adults to younger animals or humans (i.e., within species). Those 

cases where tissues are combined require careful treatment as well. The tissue specificity of 

the age/size dependence of distribution rates has to be considered when extrapolating the 

parameters of lumped tissues. If possible, the use of separate compartments for tissues that 

influence the kinetics (i.e. tissues with significant capacity to take up the substance) is 

preferred.

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Pyrethroid/Pyrethrins Technical Working Group 

Page 34 of 34 

Appendix 2. Company Membership of PPTWG Task Forces

PWG Member Companies 

AMVAC Chemical Corporation 

Bayer CropScience 

DuPont Crop Protection 

FMC Corporation, Ag Products Group 

Pytech Chemicals GmbH 

Syngenta Crop Protection, Inc. 

Valent USA Corporation 

PJV Member Companies 

BASF, The Chemical Company 

Botanical Resources Australia 

S.C. Johnson & Son, Inc. 

McLaughlin Gormley King Co. 

Prentiss Incorporated 

Pyrethrin Board of Kenya 

Societe du Pyrethre au Rwanda (SOPYRWA) 

Pyrethrum Company of Tanzania 

Valent BioSciences Corporation 

PDGII Member Companies 

AMVAC Chemical Corporation 

Bayer Environmental Science 

FMC Corporation 

Gharda Chemicals Limited 

LG Life Science, LTD. 

Meghmani Organics Limited 

United Phosphorus, Inc. 

Valent Biosciences Corp.