Pyrethroid/Pyrethrins Technical Working Group

Page 1 of 33 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)

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 May 21, 2010

Submitted by Pyrethroid/Pyrethrin Technical Working Group Chair, Thomas Osimitz Science Strategies LLC 600 E. Water Street, Suite G

Charlottesville, VA 22902Pyrethroid/Pyrethrins Technical Working Group

Page 2 of 33 GOOD LABORATORY PRACTICE CERTIFICATION

Inform ation 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

Co mpany Agent: Date: May 21, 2010

Thomas Osimitz

Chair PPTWGPyrethroid/Pyrethrins Technical Working Group

Page 3 of 33 TABLE OF CONTENTS

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

2 BASIS FOR CONCERN ABOUT AGE-RELATED SENSITIVITY TO

PYRETHROIDS AND PYRETHRINS ......................................................................................... 7

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

3 FACTORS THAT COULD LEAD TO AGE-RELATED SENSITIVITY.................................... 9

3.1 Metabolic Differences .......................................................................................................... 9

3.2 Physiological Differences................................................................................................... 10

4 ESTIMATION OF INTERNAL DOSE ....................................................................................... 13

4.1 Uncertainties and Refinements to the Rat PBPK Model.................................................... 13

4.1.1 Model structure and phys iological parameters................................................... 13

4.1.2 Improving the rat PBPK model.......................................................................... 16

4.2 PBPK Modeling of Early Life Pharmacokinetics in Humans ............................................ 17

4.2.1 Rationale ............................................................................................................ 17

4.2.2 Proposed approach for characterizi ng early-life sensitivity in the

human................................................................................................................. 18

4.2.3 Implementation Considerations.......................................................................... 19

4.2.4 Human in vitro Experiments .............................................................................. 19

5 IDENTIFICATION OF KNOWLEDGE GAPS AND PLAN TO FILL THEM ......................... 21

5.1 Study Block #1 - Assessment of Rela tive (Age-Dependent) Sensitivity............................ 23

5.2 Study Block #2 - Experimental determina tion of parameters for PBPK models ............... 25

5.3 Study Block #3 - Age-dependent in vitro metabolism of pyrethrins and pyrethroids in

human liver tissue............................................................................................................. .. 26

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

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

Appendices

Appendix 1. Company Membership of PPTWG Task Forces ............................................................. 33Pyrethroid/Pyrethrins Technical Working Group

Page 4 of 33 TABLE OF CONTENTS (continued)

Tables

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

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

Table 3. Ontogeny of enzyme activities in humans (relevant to pyrethrin/pyrethroid metabolism) ................................................................................................................ 10

Table 4. Chemical Specific Parameters for PBPK Models of Deltamethrin in Rats (from

Tornero-Velez et al. 2010) ......................................................................................... 17

Table 5. Proposed Pyrethrin and Pyrethroid Insecticides for Studies .................................................. 22

Table 6. Study Design Elements for Testing Acoustic Startle ............................................................. 24

Table 7. Study Design Elements for Testing Toxicokinetics and Dynamics* ..................................... 24

Figures

Figure 1 PBPK model for the maturing rat (Tornero-Velez et al., 2010) ................................. 14Pyrethroid/Pyrethrins Technical Working Group

Page 5 of 33 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 Tec hnical 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 Corportation representing the Sumitomo Chemical Corporation, represent registrants holding registrations for almost all pyrethrin and pyre throid insecticides registered in the USA ( Table 1 ).

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

evaluation of data from devel opmental neurotoxicity studies a nd consideration of juvenile susceptibility (Scollon, 2010) and cites studies from the published literature that indicate juveniles may be more susceptible than adults to acute py rethroid toxicity (letha lity). The Agency also summarizes the literature that supports age-de pendent 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 FIFR A Science Advisory Panel (SAP) would be meeting

to:

“...discuss experimental studies designed to eva luate 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 re gistrants for input on this topic and are pleased to respond. However, the Agency’s request only addr esses part of the information needed for a robust assessment of potential human risk. Thus, our r esponse necessarily goes beyond the relatively narrow focus of this request and addresses the broad issu e of assessing possible age-related sensitivity in humans. Embedded in this exercise is the developmen t of data that will determine whether an FQPA safety factor is needed to account for pot ential 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 ga ps and a plan to fill them.Pyrethroid/Pyrethrins Technical Working Group

Page 6 of 33 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 Gene ration 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)

TetramethrinPyrethroid/Pyrethrins Technical Working Group

Page 7 of 33 2 BASIS FOR CONCERN ABOUT 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 indicat e that juvenile or neonatal sensitivity as evidenced by acute lethality is limited only to high dose leve ls 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 differe nces 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 c ypermethrin 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 sim ilar age-related sensitivity difference in acute lethality in rats treated with cypermethrin. The oral LD50 in 8- a nd 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) at both ages. Collectively, these re sults 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 limite d (e.g., 0- to maximum 4-fold) difference for pyrethroids that lack this moiety (Typ e 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 u sed 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 neurotoxi c dose levels of representative Type I or Type II pyrethroids compared with adult rats. These inv estigations 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 ju venile was actually less sensitive than the adult to

cismethrin.

Most important, because the acoustic startle respon se is representative of a specific neurological response it should be considered as the endpoint of in terest for use in human risk assessments rather than lethality.Pyrethroid/Pyrethrins Technical Working Group

Page 8 of 33 Table 2. Lethality (LD50) results for pyrethroi d insecticid es 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 LD 50 estimates (mg/kg), based on 6 or 7 males per dose group at each age. The

95% confidence limits, where calculable, a re shown in parentheses (from Sheets, 2000).Pyrethroid/Pyrethrins Technical Working Group

Page 9 of 33 3 FACTORS THAT COULD LEAD TO AGE-RELATED SENSITIVITY

As stated above, the Agency reports their evaluati on of data from developmental neurotoxicity studies and consideration of juvenile susceptibility (Sco llon, 2010) and cites studies from the published literature that indicate juveniles may be more suscep tible 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 mo st 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 devel opmental changes in physiology and metabolism that rats and humans undergo from the fetal period thr ough 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 eliminati on 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 res ponsible for the disposition of the pyrethroids and pyrethrins. Thus, serum esterases exhibit signifi cant activity in the rat, but not the human (Crow et al., 2007) while in the human, intestinal carboxylester ase 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 hydroly tic 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 c onstitutive expression of CYP1A2 is delayed and is absent or expressed at lo w levels through 15 days after birth. Somewhat similarly, CYP2C6 increases slowly after birth, reach ing 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 th e 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 regardi ng 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 adu lt 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 protei n levels, both of these gene produc ts are being expressed at adult levels by 3 months of age.Pyrethroid/Pyrethrins Technical Working Group

Page 10 of 33 Table 3 summarizes the relative activity of the various CYP and h CE isoforms. From the data, it is

clear that:

 Multiple CYP and hCE are involved in pyrethri ns and pyrethroid metabolism; the relative role varies by compound;

 Multilple CYP and hCE that are fully formed in a dults are also available to varying degrees at stages of development;

 The role of a specific CYP or hCE will 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 ava ilable. Thus, even developing children have a significant compliment of enzymes available to me tabolize pyrethrins and pyrethroids. Moreover, given the relatively low exposure levels to be expect ed, 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 nerv ous system will be very small.

Table 3. 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 repres ent 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 va ry significantly across species. Understanding these differences is essential to the estimation of the internal dose, a cr itical factor in risk assessment. Comparisons of several of the changes that occur as rats and humans develop are given below.

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

blood-brain barrier in human infants is re latively undeveloped and displays greaterPyrethroid/Pyrethrins Technical Working Group

Page 11 of 33 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 brainbarrier 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 matu rity 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 th e 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 th e volume of distribution for a water-soluble chemical would be roughly equivalent in adu lt rats and humans, it would be somewhat higher

in neonatal rats than in neonatal infants.

Body fat generally follows a more complex patte rn 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, w ith 3-4% from birth to three weeks, 10% at eight weeks, and 7-18% in adults, depe nding 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 a ll ages, resulting in lower plasma levels than would be predicted using parame ters 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 duri ng 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 cha nges 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 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 liverPyrethroid/Pyrethrins Technical Working Group

Page 12 of 33 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 persis ts during rat development.

 Plasma Protein Binding: Binding of chemicals to plasma proteins can strongly influence their distribution and elimination. Age-relate d 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, reachi ng about 75% of adult le vels by six months of age (Alcorn and McNamara, 2003). Serum concentr ations of albumin are about 75% of adult human levels at birth, rise gradually, and appro ach adult levels after the first year of life (Alcorn and McNamara, 2003). The binding capac ity 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 hu mans, neonatal rats have lower concentrations of plasma binding protein relative to adults. At birth, rats have approximately half of adult tota l 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 difference s 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+ cha nnel development in humans and no studies investigating development of various VGSC isoforms in humans could be located.

Voltage-gated calcium channels (VGCCs) are stru cturally related to th e 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-depe ndent 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 13 of 33 4 ESTIMATION OF INTERNAL DOSE

The assessment of target-tissue concentrations of the compound of interest is essential to reduce the uncertainties in the evaluation of exposure-respon se relationships and to produce the most robustestimate 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

ARTERIAL tissue tissue

tissue P C CQ dt dC V

where V tissue is the volume of the tissue compartment, C tissue is the concentration of chemical in the tissue, Q tissue is blood flow to the tissue, C arterial is the concentration of chemical in arterial blood, and

P tissue:blood is the tissue:blood partition coefficient. PBPK models have been used for extrapolation between test animals and humans (e.g., rat to hum an), within human populations (e.g., gender and age), routes of exposure (e.g., oral to dermal), dur ation adjustment (e.g., acute to chronic), and from high dose to low-dose levels. In recent years, seve ral PBPK models for select pyrethroids have been published (Mirfazaelian et al ., 2006, Tornero-Velez et al ., 2010, Godin et al ., 2010), which allow for assessment of internal dose levels in various organs and tissues. Currently, pyrethroid PBPK models

contain several uncertainties that should be addr essed prior to use in the assessment of juvenile sensitivity in human populations. The following sec tion discusses several of these uncertainties in the PBPK models, and proposes data development and modeling strategi es for improving predictions of target tissue concentrations in human adults and infants. Details of the studies proposed to fill knowledge gaps can be found in Sections 5.2 and 5.3

.

4.1 Uncertainties and Refinements to the Rat PBPK Model

4.1.1 Model structure and physiological parameters

A PBPK model for deltamethrin in the maturing ra t 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 differe nt ages between 10 d and 90 d reasonably well. The model includes compartments representing the br ain, 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 co mpartments while the other compartments are perfusion limited. Some model pa rameters, particularly metabolic rates and tissue volumes, are considered age dependent by using continuous functio ns of age to calculate their values at a certain age. This age dependence is dynamically taken into consideration over the simulation time.

Although the PBPK model as described above gene rates results that show overall a reasonable agreement with observed PK data from rats of di fferent ages, several uncertainties remain in the PBPK description. We identified several issues in the PBPK modeling approach where further refinements may resolve remaining uncertainties.Pyrethroid/Pyrethrins Technical Working Group

Page 14 of 33 Figure 1 PBPK model for the maturing rat (Tornero-Velez et al., 2 010)

 Blood flow rates: In the rat PBPK model, the blood flow rates in different tissues (Q tissue ) 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 Q tissue 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, Q brain , will decrease from birth to adult age. For humans, cerebral perfu sion rates measured in children of different ages support this assumption. It was found that Q brain 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 × su rface area products: No information is given by Tornero-Velez et
al. (2010) about the age-dependence of scaling tissue uptake rates (i.e., permeability × surface area (P×A) products) for diffusion limited tissues. Thus it is suspected that no such scaling was considered in the model, which is an oversimplification of how compounds are taken up into tissues. In actuality, while it may be assu med that permeability is independent of age,

surface area does scale with tissue volume. Unfo rtunately 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 volume 0.75 ) may be appropriate for describing the relationship between surface area and tissue volume.Pyrethroid/Pyrethrins Technical Working Group

Page 15 of 33  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 permeab ility depends on lipophilicity and molecular volume of a substance. This has two imp lications on the use of PBPK models for extrapolations across ages and species that ha ve significantly different body sizes. For extrapolation from smaller organisms to la rger ones, the question whether perfusion limitation is conserved requires evaluation. Th is potential difference in distribution is not a limitation for extrapolations from adults to yo unger animals or humans (i.e., within species). Those cases where tissues are combined requi re careful treatment as well. The tissue specificity of the age/size dependence of dist ribution 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. ti ssues with significant capacity to take up the substance) is preferred.

 Interpretation of results: In Tornero-Velez et al. (2010), the age dependence of toxic effects was correlated to TK-parameters derived from results of the PBPK model. They concluded that treatment-related findings in th e acoustic startle response (i.e., the relevant sub-lethal adverse effect) were correlated to the AUC of deltamethrin in the brain. This correlation, however, needs careful revision. For the youngest rats (PND 10), the model clearly underestimates the concentrations in the investigated tissues at least for the lower, sub-lethal doses, whereas this is not the case for older rats. The experimental data clearly show higher concentration levels for the P ND 10 animals compared to the other ages for which in most cases the concentrations are similar (Godin et al. , 2010). Thus, it may be concluded that, based on experimental concentr ations, the increased sensitivity of the young

rat to deltamethrin is related to increased maximum concentrations in the brain.

The sensitivity of AUC brain on age is determined by changes in the uptake and release kinetics rather than by age-dependent peak levels. Th is is mainly a consequence of changes in the distribution kinetics determined by changing blo od flow rate to volume ratios. On the other hand, elevated concentration levels in the e xperimental data are probably due to reduced metabolic activity in young animals that has pr eviously been shown for deltamethrin and rats (Anand et al., 2006).

At present, there is a lack of clarity on the reasons for significant changes in metabolic rates and in metabolic clearances with age, as considered in the PBPK model; these changes do not

translate into increased brain concentration leve ls simulated using the PBPK model. It is suspected that the deficiencies in the approach taken to extrapolate Q and P×A between different age groupings might lead to a leveling of the respective effect in the present PBPK model. However, from our point of view it is important to clarify this question because the impact of age-dependent clearance on pharmac okinetics needs to be well understood in orderPyrethroid/Pyrethrins Technical Working Group

Page 16 of 33 to achieve a reliable extrapolation to juveniles in humans. Particularly for the pyrethroids and pyrethrins, where it is known that there are si gnificant metabolic differe nces between rats and humans, these differences need to be addressed appropriately in or der to conduct an

informative and accurate risk assessment.

4.1.2 Improving the rat PBPK model

As identified above, the current PBPK model can be improved by using pa rameters that better represent the physiology of the rat. Further, in order to accurately simulate the pharmacokinetic behavior of individual pyrethrins/pyrethroids, biochemical data specific to each compound/material

are needed. These biochemical data ca n be independently measured using in vitro experimental systems. Such parameters include partition coe fficients, absorption rates (gastrointestinal and dermal), metabolic clearance, and protein binding. A list of parameters specific to deltamethrin are

shown in Table 4 below (taken from Tornero-Velez et al., 2 010).

These parameters, while formulating a PBPK model, may be kept constant, allowing other parameters to be examined (Clewell and Clewell, 2008). Several in vitro systems that are useful for experimentally deriving ADME parameters in a PB PK modeling strategy are presented in the section of this document on data gaps (see sections 5.2 and 5.3 ). The ADME parameters obtained from these experi ments, after a ppropriate scaling to in vivo conditions, can be used to refine the rat PBPK model for deltamethrin. This approach can help to better understand the uncertainties in the recently published deltamethrin PBPK models discussed above.

Additionally, in vitro ADME parameters measured in rat cells/tissues may be replaced with parameters derived from experiments conducted us ing human cells/tissues. The results of the in vitro experiments described below 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 should be used to formulate the PBPK model. This strategy allows for appli cation of the deltamethrin PBPK model to other pyrethroids by replacing deltamethrin-specific para meters with parameters specific to other pyrethrins/pyrethroids.

Once the initial rat PBPK model has been formul ated, using physiological parameters from the literature and biochemical pa rameters determined from in vitro experiments, the model needs to be evaluated against in vivo pharmacokinetic data. By using known physiological constants and experimentally-derived ADME parameters in the model, alternative descriptions of the pharmacokinetic behavior (e.g., pe rfusion-limited vs. diffusion-limited tissue compartments) may be investigated with further confidence. Pharmacokinetic in vivo data may be obtained from published and unpublished sources. Altern ative model structures (i.e., changes to the representation of organ/tissue compartments) can be used to comp are simulated tissue concentration time-course profiles under each of the diffusion or perfusion limiting assumptions with different experimental data. Model structures different from previously p ublished work should be ev aluated for consistency with experimental pharmacokinetic measurements of pyrethroids.Pyrethroid/Pyrethrins Technical Working Group

Page 17 of 33 Table 4. Chemical Specific Parameters for PB PK Models of Deltamethrin in Rats (from

Tornero-Velez et al. 2010)

4.2 PBPK Modeling of Early Life Pharmacokinetics in Humans

4.2.1 Rationale

The critical dependence of early life sensitivity on the developmental status of metabolism enzymes presents an additional problem fo r the use of animal studies to assess early life sensitivity in the human. There are often significant species differe nces in the enzymes that contribute to the metabolism of a particular compound. Moreover, as discussed earlier in this document, there are differences in the ontogeny of enzymes in rats and hu mans, so that it is unlikely that clearance of a compound in a neonatal rat would be predictive of cl earance 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 infa nt and child sensitivity cannot account for thesePyrethroid/Pyrethrins Technical Working Group

Page 18 of 33 factors. However, in silico/in vitro/in vivo studies can be used to identify the specific ADME parameters that influence the pharmacokinetic beha vior of pyrethroids in mammalian systems. In summary, the limitations of relative sensitivity studi es in animals indicate that they are unlikely to accurately characterize early-life sensitivity for the infant and childhood exposures of concern under the FQPA.

4.2.2 Proposed approach for characterizing early -life sensitivity in the human

An approach that has the potentia l to overcome the limitations in the animal-based sensitivity studies is to develop a PBPK modeling approach for pr edicting the relationship in the human between internal dose in the adult and internal dose during early life using chemical-specific properties. For pharmaceutical substances, pediatric pharmacokinetic s 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 good PBPK model based on ontogeny data for the elimination (clearance) processes (Johnson, 2003; Hines 2008) is essential for extrapolation to young ages (Björkman, 2006; Edginton et al. 2006a; Alcorn and McNamara, 2008).

A similar approach has been demonstrated for other environmental compounds (Clewell et al ., 2004; Gentry et al ., 2003; Edginton & Ritter, 2009). In order to validate such an approach, proof-of-concept studies with representative pyrethroids could be conducted in the rat. An excellent example of such a study has already been performed with deltamethrin (Mirfazaelian et al . 2006; Anand et al. , 2006).

The literature data on physiological values for the ra t 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 fo r 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 cons tants) were taken from existing PBPK models for adults; partition coefficients were assumed to be independent of ag e, 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 a pproach could be used in the case of pyrethroids. There is considerable information in the literature on the ontogeny of enzyme systems in the rat (Kuo and Hook, 1980; Cresteil, 1987; de Waziers et al ., 1988; Rich and Boobis, 1997; Chanda et al ., 2002) as well as in the human (Augustinsson and Brody, 1962; Augustinsson and Barr, 1963; Ecobichon and Stephens, 1972; Hines and McCarver, 2002; Alcorn and McNamara, 2002a,b; Koukouritaki et
al ., 2004), as summarized previously in this document. 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 tissu e (both adult and non-adult), see section 5.3

.

A first task toward the development of such a capability would be 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 eval uate early life sensitivity to untested pyrethroids by estimating the chemical-specific parameters using QSAR or in vitro studies, and then running the model with the appropriate age-dependent pha rmacokinetic and exposure parameters.Pyrethroid/Pyrethrins Technical Working Group

Page 19 of 33 4.2.3 Implementation Considerations

The parameters in a PBPK model can be categorized into four types: exposure, physiological, partitioning, and metabolism. The exposure paramete rs 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-populatio ns (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-specifi c, 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-sp ecific 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; Poul in and Theil, 2000;

Beliveau et al ., 2003; Rodgers et al ., 2005; Rodgers and Rowland, 2006; Schmitt, 2008), and a variety of in vitro methods are available for estimating pa rameters for absorption and metabolism (reviewed in Clewell et al ., 2007).

A number of software platforms are availabl e to support generic PBPK modeling using in vitro human metabolism data (reviewed in Clewell et al ., 2007); these include Simcyp, GastroPlus, and PK-Sim. However, they currently are designed to support pharmaceutical compounds. Full use of these software platforms for PBPK modeling of pyre throids will require enhancements in two areas: (1) addition of descriptions of dermal and inhala tion exposure, and (2) addition of data on esterase metabolism enzymes. Of these, perhaps the greatest challenge will be the add ition of data on esterase activities, since esterases are present in many tissu es, including the liver, blood, gut, and skin. Fragmented data is available, but there has not been a systematic effort to identify and integrate it into

a useable database. Some additional in vitro data collection will undoubtedly be needed to fill data

gaps.

4.2.4 Human in vitro Experim ents

As dis cussed above, physiological and chemical-sp ecific 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 in clude Caco-2 cells for quantifying intestinal permeability (Dokoumetzidis et al, 2007), protein bi nding using human plasma (Banker and Clark, 2008), and investigation of metabolism using huma n hepatocyptes and/or microsomes (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.Pyrethroid/Pyrethrins Technical Working Group

Page 20 of 33 The metabolic detoxication of pyrethroids and pyrethrins is a critical key event in their toxic action. Therefore, 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 sensitiv ity. Substantial species differences between the rat and human exist in the enzymes responsible for the disposition of the pyrethroids and pyrethrins. Considerable information is available regarding the ontoge ny of some human cytochrome P450 isozymes (Koukouritaki et al., 2004; Stevens et al., 2003). Given that there are known species differences in both the enzymes responsible for pyrethroid metabol ism and the developmental trajectories exhibited by these enzymes, the development of appropriate PBPK models based on direct measurement of

metabolic clearance in vitro using hepatocytes from adults and infants/children would appear to be more informative than developmental studies in an imal models. However, more direct studies of protein levels and/or activity of enzymes involve d in metabolism of pyrethroids in humans would provide increased confidence in such models.Pyrethroid/Pyrethrins Technical Working Group

Page 21 of 33 5 IDENTIFICATION OF KNOWLEDGE GAPS AND PLAN TO FILL THEM

Based on our analysis of the knowledge relati ng 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 – Experimental determina tion of parameters for PBPK models;

3) Study Block #3 – Age-dependent in vitro metabolism of pyrethrins and pyrethroids in human

liver tissue

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 clin ical 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 hy drolytic and oxidative metabolism in the detoxification of pyrethroids and existing know ledge 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 5

.Pyrethroid/Pyrethrins Technical Working GroupPage 22 of 33Table 5. Proposed Pyrethrin andPyrethroid Insecticides for StudiesCharacteristicPyrethrinsBifenthrins-BioallethrinPermethrinDeltamethrinEsfenvalerateα-cyano ornon-α-cyanonon-α-cyanonon-α-cyanonon-α-cyanonon-α-cyanoα-cyanoα-cyanoType I or II ormixedIIIIIII/IIIsomersprimarily a mixture ofpyrethrin I andpyrethrin II100%(Z, 1R cis)92% d-trans-d5.6% d-trans-l1.7% l-trans-d,l40% cis60% trans1:1 of 1R:1S100%1R, 3R,αS86% SS isomer12% SR,RR,RSMW(grams/mole)316 – 374422.9302.4391.3505.2419.9LD50: Adult710 mg/kg55 mg/kg700 mg/kg1200 mg/kg67 mg/kg87 mg/kgLMA ED30(rats)*3.2 mg/kg90 mg/kg43 mg/kg2.5 mg/kg1.2 mg/kgLMA (time ofpeak effect)*4 hr1 hr1.5 hr2 hr2 hrFOB Doselevels (rats)*400-800 mg/kg40-55 mg/kg150-300 mg/kg200-250 mg/kg12.5-35 mg/kg15-50 mg/kgFOB (time ofpeak effect) *4 hr8 hr2 hr4 hr4 hr8 hrUse inAgriculture(0→+++)+++0+++++++Use inResidential (0→+++)++++++++++++++Rationale ofUsePYR are naturalproducts that can beused for comparisonto the pyrethroids.Extensive residentialuse and humanpediculicide use.BIF is a non-αcyano pyrethroidhaving a relativelylongtime to peakeffect based onboth LMA andFOB data.s-BIO is a non-α-cyanopyrethroid having arelativelyshorttime topeak effect based on bothLMA and FOB data.Represents severalresidential usecompounds.PRM is a non-α-cyanopyrethroid having arelativelymoderatetime topeak effect based on bothLMA and FOB data. Widelyused in agriculture andresidential settings. Humanpediculicide use as well.DLM is anα-cyanopyrethroid exhibiting Type IIactivity that has relativelymoderatetime to peak effectbased on both LMA and FOBdata. Data from DLM wasthe driver for this researcheffort.ESF is anα-cyanopyrethroid exhibitingmixed (Type I/II) activitythat has relativelymoderate to longtime topeak effect based on LMAand FOB data,respectively.* Data from Wolanksyet al.,(2006) and Weineret al.,(2009).Pyrethroid/Pyrethrins Technical Working Group

Page 23 of 33 5.1 Study Block #1 - Assessment of Rela tive (Age-Dep endent) Sensitivity

The following studies are proposed to provide qu antitative measures of the relative sensitivity of young and adult animals to commercial pyre throid and pyrethrin insecticides (see Table 5 ), under

circu mstances that are suited for human risk assessments. The a pproach described below involves measuring the concentrations of representative pyreth roids 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 sens itivity 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 prov ide 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 m easured 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 prefe rred for relevance to human exposure, relative to a bolus dose, it does not provide for the management of dose deliv ery (dose level and testing at the time-of-peak effect) that is required for precise comparison. Mo reover, 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 tim e-of-peak effect following treatment.

Dose Selection : Dose levels will be selected to avoid overt toxicity and to provide data suited for benchmark dose analysis (e.g., BMD10). Dose leve ls 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 specifi c to high doses, due to their limited capacity to metabolize a high, bolus dose.

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. While rats have the minimum coordination and strength for evaluatio n of the startle response as early as PND 17, we consider Day 21 as a more appropriate age for quan titative assessment and comparison with the adult.

Toxicokinetics and Dynamics: We propose to measure the concentration of representative (model) pyrethroids in whole-brain tissue 21-day-old and adult rats at the time of peak effect on the ASR, to provide a measure of the relative sensitivity of the ne rvous 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

Page 24 of 33 Table 6. Study Design Elements for Testing Acoustic Startle

Animals 1 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); b ased 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

Table 7. Study Design Elements for Testing Toxicokinetics and Dynamics*

Animals 1 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 (CO 2 /O 2 ) with decapitation Whole-brain collected and snap frozen (liquid CO 2 ) Storage: -70  C (minimum) until assayed Homogenization, methods for analysis, etc.

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

Animals 1 Male SD rats (21-days and 6-8 weeks old) *Design details (e.g., age, time and dose le vels) based on results from the ASR study.

1 Selection of rat strain depends on the specific purpose / circumstancesPyrethroid/Pyrethrins Technical Working Group

Page 25 of 33 5.2 Study Block #2 - Experimental determin ation of parameters for PBPK models

The parameters discussed below are important for th e development of a robust PBPK model for rats and humans. The appropriate studies will be conducte d with pyrethrins and a comprehensive set of pyrethroids (see Table 5 )

Derm al 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 dete rmined from plots of cumulative mass per surface area

vs . time; the diffusivity coefficient is calculated by di viding flux by the concentration of test chemical applied to the donor cell. Existing data on pyrethrins and the pyrethroids will be used when possible.

Gastrointestinal absorption – This can be estimated for the ra t 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 sm all 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 O 2 :CO 2 (95:5) mixture to provide sufficient oxygen tension fo r 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 app earance of the pyrethroid in the receptor compartment. The absorption rate constant ( k

a ) in each GI segment is proportional to

P eff ( 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 perm eability. 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 P eff is calculated in the apical to basolateral and basolateral to apical directions . The k

a is derived from P eff as described above (Dokoumetzidis et al ., 2007)

Partition coefficients - ( P tissue: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:sa line 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 de termined 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 th e free-phase compounds and not those that are tissuebound can partition into the SPME fiber coating. Tiss ue 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 pol ydimethylsiloxane fiber (PDMS, 100 μm thick,Pyrethroid/Pyrethrins Technical Working Group

Page 26 of 33 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 genera lly 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 pl asma 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:salin e partition coefficient measured in the row above.

5.3 Study Block #3 - Age-dependent in vitro m etabolism of pyrethrins and pyrethroids in human

liver tissue

Mic rosomal metabolism assays with the pyrethroids ha ve previously been cond ucted using the parent compound depletion approach (Scollon et al., 2009). Briefly, a mixture of Trizma buffer, pooled microsomal protein, and NADPH is pre-incubated at 37°C for 10 min. Assays are initiated by the addition of pyrethroid in different concentrati ons. Aliquots are removed from the mixture over the time-course of the experiment and quantified by LC/M S. Substrate remaining is converted to product formed and plotted versus time to produce a reacti on velocity. Plotting reaction velocities against initial concentrations yield V

max and K M

estimates for each pyrethroid . The intrinsic clearance is calculated by dividing the V

max by K M . It has long been recognized that the isolated rat hepatocytes generally provide better in vitro grounds for the quantitativ e/qualitative predictions of in vivo metabolism than subcellular fractions (Hewitt et al ., 2007). The advantages of using primary hepatocytes over microsomes have been well documente d in the context of quantitative predictions of

in vivo metabolism. Experiments using hepatocytes can be performed in commercial laboratories such as at CellzDirect (Durham NC). These experi ments may be valuable for confirming results from

Scollon et al. (2009). Additionally, donated hepatocytes a nd/or microsomal fractions from adult and pediatric patients may be used to estimate metabolic clearance in vitro

.Pyrethroid/Pyrethrins Technical Working Group

Page 27 of 33 6 SUMMARY

T he PPTWG has taken a comprehensive and thoughtfu l 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 pyrethro ids in humans particularly at lower doses more

relevant for human health risk assessment. Based on this analys is, 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 rats and in humans, and to evaluate poten tial differential sensitivity between juveniles and adults. The PBPK strategy builds upon previously published models first by identifying the key ADME process that drives the observe d kinetic behavior in rodents. Second, with this improved understanding of pyrethrin/pyreth roid pharmacokinetics in the rat, we propose reformulating the rat 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 version of the 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 Cent ury) and is protective of public health.Pyrethroid/Pyrethrins Technical Working Group

Page 28 of 33 7 REFERENCES

Alcorn J, McNamara, PJ (2002a): Ontogeny of he patic and renal systemic clearance pathways in infants: part I. Clinical Pharmacokinetics

, 41 , 959-  998. Alcorn J, McNamara, PJ (2002b). Ontogeny of he patic and renal systemic clearance pathways in infants: part II. Clinical Pharmacokinetics

, 41 , 1077  -1094. Alcorn J, McNamara PJ (2003): Pharmacokinetics in the newborn. Advances in Drug Delivery

Reviews

, 55, 667-686. Alcorn J, and McNamara, PJ (2008). Using ontogeny information to build predictive models for drug

elimination . Drug Discovery Today

, 13 ,507-512. Altman PL, Dittmer DS eds. (1972–1974): Bi ology Data Book (Volumes I, II, and III) (Second Edition). Federation of American Societies for Experimental Biology, Washington, DC. Anand SS, Kim KB, Padilla S, Muralidhara S, Kim JH, Fisher JW, Bruckner JV (2006): Ontogeny of hepatic and plasma me tabolism of deltamethrin in vitro : Role in age-dependent acute neurotoxicity. Drug Metabolism and Disposition 34

, 389-397. Arms AD, Travis CC (1988): Reference Physiological Parameters in Pharmacokinetic Modeling. Office of Health and Environmental Assessment, Washington DC . EPA/600/6-88/004, US

EPA. Augustinsson KB, Barr M (1963): Age variation in plasma arylesterase activity in children. Clinica

Chimica Acta

, 8 , 568-  573. Augustinsson KB, Brody S (1962): Plasma arylestera se activity in adults and newborn infants.
Clinica Chimica Acta, 7 , 560-  563. Banker MJ, Clark TH (2008): Plasma/ser um protein binding determinations. Current Drug Metabolism, 9 ,854-859. Beliveau M, Tardif R, Krishnan K. (2003): Quantitative structure-property relationships for physiologically based pharmacokinetic modeli ng of volatile organic chemicals in rats. Toxicology and Applied Pharmacology

, 189 , 221-232. Björkman S (2005): Prediction of drug dispos ition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs. British Journal of Clinical Pharmacology, 59 , 691–704. Björkman S (2006): Prediction of Cytochrome P450- Mediated Hepatic Drug Clearance in Neonates,

Infants and Children. Clinical Pharmacokinetics

, 45 , 1-11 Brown RP, Delp MD, Lindstedt SL, Rhomberg LR, Beliles RP (1997): Physiological parameter values for physiologically based pharmacokinetic models. Toxicology and Industrial Health

13 ,4 07–484. Butt AM, Berry M (2000): Oligodendrocytes and the control of myelination in vivo : New insights from the rat anterior medullary velum. Journal of Neuroscience Research, 59 , 477-488. Calabrese EJ (1983): Tissue distribu tion: Interspecies differences. In Principles of Animal Extrapolation. (Ed.: Calabrese, EJ), John Wiley & Sons, New York, p107-201. Cantalamessa F (1993): Acute toxicity of two pyrethroids, permethrin, and cypermethrin in neonatal and adult rats. Archives of Toxicology, 67, 510-513. Chanda SM, Lassiter TL, Moser VC, Barone Jr S, Padilla S (2002): Tissue carboxylesterases and chlorpyrifos toxicity in the developing rat. Human and Ecological Risk Assessment, 8, 75-  90. Clewell HJ, Teeguarden J, McDona ld T, Sarangapani R, Lawrence G, Covington T, Gentry PR, Shipp AM (2002): Review and Evaluation of the Po tential Impact of Age and Gender-SpecificPyrethroid/Pyrethrins Technical Working Group

Page 29 of 33 Pharmacokinetic Differences on Tissue Dosimetry. Critical Reviews in Toxicology

, 32 , 329-

389. Clewell HJ, Gentry PR, Covington TR, Sarangapani R, Teeguarden JG (2004): Evaluation of the potential impact of age- and gender-specific pharmacokinetic differences on tissue dosimetry. Toxicological Science

, 79 ,381-  393. Clewell HJ, Reddy MB, Lave T, Andersen ME ( 2007): Physiologically based pharmacokinetic modeling. in: Gad, SC, ed. Preclinical Development Handbook . John Wiley and Sons, Hoboken, NJ. Clewell RA, Clewell HJ (2008): Development and specification of physiologically based pharmacokinetic models for use in risk assessment. Regulatory Toxicology and Pharmacology 50 ,129-143. Clewell RA, Merrill EA, Yu KO, Mahle DA, Sterner TR, Fisher JW, Gearhart JM (2003): Predicting neonatal perchlorate dose and inhibition of iodide uptake in the rat during lactation using physiologically-based pharmacokinetic modeling. Toxicological Science, 74 , 416-436. Cresteil T (1987): Regulation of drug-metabolizing enzymes during the perinatal period in rat and

human liver. Bioessays, 7 , 120-  124. Crofton KM, Reiter LW (1988): The effects of Type I and Type II pyrethroids on motor activity and the acoustic startle response in the rat. Fundamental and Applied Toxicology, 10 , 624-634. Crow JA, Borazjani A, Potter PM, Ross MK (2007) Hydrolysis of pyrethroids by human and rat tissues: examination of intestinal, liver and serum carboxylesterases. Toxicology and Applied Pharmacology

, 221 , 1-12. de Graaf-Peters VB, Hadders-Algra M (2006): Ontoge ny of the human central nervous system: What is happening when? Early Human Development

, 82 , 257-266. de Waziers I, Boisset M, Atteba S (1988): Pre- and post-weaning developm ent of drug-metabolizing enzyme activities in small intestine and liver of rats. Drug Metabolism Reviews, 16 , 310-  315. de Zwart L, Scholten M, Monbaliu JG, Annaer t PP, Van Houdt JM, Van den Wyngaert I, De Schaepdrijver LM, Bailey GP, Coogan TP, C oussement WC, Mannens GS (2008): The ontogeny of drug metabolizing enzymes and transporters in the rat. Reproductive Toxicology

, 26 , 220-230. Dokoumetzidis A, Kalantzi L, Fotaki N (2007): Predictive models for oral drug absorption: from in

silico methods to integrated dynamical models. Expert Opinion on Drug Metabolism and. Toxicology 3 ,491-505. Downie D, Antipatis C, Delday MI, Maltin CA, Sneddon AA (2005): Moderate maternal vitamin A deficiency alters myogenic regulatory protein expression and perinatal organ growth in the

rat. American Journal of Physiology - Regulatory Integrative Comparative Physiology. 288,

R73-79. Ecobichon, D J, Stephens DS (197 2): Perinatal development of human blood esterases. Clinical Pharmacology and Therapeutics, 14 , 41-  47. Edginton AN, Ritter L (2009): Predicting plasma c oncentrations of bisphenol A in children younger than 2 years of age after typical feedi ng schedules, using a physiologically based

toxicokinetic model. Environmental Health Perspectives, 117 , 645-652 Edginton AN, Schmitt W, Voith B, Willmann S (2006a ): A Mechanistic Approach for the Scaling of

Clearance in Children. Clinical Pharmacokinetics

, 45 , 683-704 Edginton A, Schmitt W, Willmann S (2006b): Development and evaluation of a generic physiologically based pharmacoki netic model for children. Clinical Pharamcokinetics 45

, 1013-1034.Pyrethroid/Pyrethrins Technical Working Group

Page 30 of 33 Ehrnebo M, Agurell S, Jalling B, Boreus LO (1971): Age differences in drug binding by plasma proteins: Studies on human fetuses, neonates a nd adults. European Journal of Clinical Pharmacology, 3, 189-193. Erecinska M, Cherian S, Silver IA.2004. Energy metabolism in mammalian brain during development. Progress in Neurobiology

, 73 , 397-445. Franz TJ (1975): Percutaneous absorption on the relevance of in vitro data. Journal of Investigative Dermatology 64 ,190-195. Gentry PR, Covington TR, Clewell HJ (2003): Evaluation of the potential impact of pharmacokinetic differences on tissue dosimetry in offspring during pregnancy and l actation. Regulatory Toxicology and Pharmacology, 38 , 1-  16. Gentry PR, Haber LT, McDonald TB, Zhao Q, C ovington T, Nance P, Clewell HJ, Lipscomb JC, Barton HA (2004): Data for physiologically based pharmacokinetic modeling in neonatal animals: physiological parameters in mice and Sprague-Dawley rats. Journal of Children’s Health, 2 , 363-411. Ginsberg G, Hattis D, Sonawane B. (2004): In corporating pharmacokinetic differences between children and adults in assessing children' s risks to environmental toxicants. Toxicology and Applied Pharmacolog y, 198 , 164-183.Godin S, DeVito MJ, Hughes MF, Ross DG, Scollon EJ, Starr JM, Setzer RW, Conolly RB, TorneroVelez R (2010): Physiologically based pharmacokinetic modeli ng of deltamethrin: Development of a rat and human diffusion-limited model. Toxicological Sciences accepted for publication. Grabowski CT (1966): Ontogenetic changes in the concentration of serum proteins in chick and mammalian embryos. Journal of Embr yology and Experimental Morphology, 16, 197-202. Hansch C (1979): The fragment method of calculating partition coefficients. in: Hansch C, Leo A., (Eds.), Substituent Constants for Correlation Analysis in Chemistry and Biology , Wiley, New York, NY; pp. 18-43. Hewitt NJ, Lechón MJ, Houston JB, Hallifax D, Br own HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM, Hengstler JG (2007): Primary hepatocytes: current unde rstanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity

studies. Drug Metab Rev

, 39, 159-234. Hines RN (2007): Ontogeny of Human Hepatic Cytochromes P450. Journal of Biochemical and Molecular Toxicology

, 21 , 169-175. Hines RN (2008): The ontogeny of drug metabolism enzymes and implications for adverse drug

events. Pharmacology and Therapeutics

, 118 , 250-267. Hines RN, McCarver DG (2002): The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes. Journal of Pharmacology a nd Experimental Therapeutics

, 300 , 355-  360

. Jepson GW, Hoover DK, Black RK, McCafferty JD, Mahle DA, Gearhart JM (1994): A partition

coefficient determination method for nonvol atile chemicals in biological tissues. Fundamental and Applied Toxicology 22 ,519-524. Johnson TN (2003): The development of drug metabolising enzymes and their influence on the susceptibility to adverse drug reactions in children. Toxicology

. 192 , 37-48. Koukouritaki SB, Manro JR, Marsh SA, Stevens JC , Rettie AE, McCarver DG, Hines RH (2004): Developmental expression of human hepatic CYP2C9 and CYP2C19. Journal of Pharmacology and Experimental Therapeutics, 308 , 965-  974.Pyrethroid/Pyrethrins Technical Working Group

Page 31 of 33 Kuo CH, Hook JB (1980): Postnatal development of renal and hepatic drug-metabolizing enzymes in

male and female Fisher 344 rats. Life Sciences

. 27 , 2433-  2438. Meacham CA, Brodfuehrer PD, Watkins JA and Shafer TJ (2008): Developmentally-regulated sodium channel subunits are differentially sensitive to α -cyano containing pyrethroids. Toxicology and Applied Pharmacology, 231, 273-281. Mirfazaelian A, Kim KB, Sathanandam SA, Kim HJ, Tornero-Velez R, Buckner JV, Fisher JW (2006):. Development of a physiologically-based pharmacokinetic model for deltamethrin in the adult male Sprague-Dawley rat. Toxicological Sciences 93 ,432-442. National Research Council (NRC) (1993) Pesticides in the diets of infants and children. National Academy Press, Washington, DC. Poulin P, Krishnan K (1996a): Molecular structure- based prediction of the partition coefficients of organic chemicals for physiological pharmacokinetic models. Toxicology Methods

, 6 ,117-

137. Poulin P, Krishnan K (1996b): A tissue com position-based algorithm for predicting tissue:air partition coefficients for organic chemicals. Toxicology and Applied Pharmacology

, 136

, 126-130. Poulin P, Theil FP, (2000): A priori prediction of tissue:plasma partition coefficients of drugs to facilitate the use of physiologically-based pharmacokinetic models in drug discovery. Journal of Pharmaceutical Sciences

, 89 , 16-35. Rice D, Barone S (2000): Critical periods of vul nerability for the developing nervous system:

Evidence from humans and animal models. Environmental Health Perspectives

, 108, 511-

533. Rich KJ, Boobis AR (1997): Expression and induci bility of P450 enzymes during liver ontogeny. Microscopy Research and Technique 39 , 424-  435. Rodgers T, Rowland M.(2006): Physiologically based pharmacokinetic modeling II: Predicting the tissue distribution of acids, very weak bases, neutrals and zwitterions. Journal of

Pharmaceutical Sciences

, 95 ,1238-1257. Rodgers T, Leahy D, Rowland M (2005): Physio logically based pharmacokinetic modeling I: Predicting the tissue distribution of moderate-to-strong bases. Journal of Pharmaceutical

Science s, 94 ,1259-1276. Schoeffner DJ, Warren DA, Muralidara S, Bruckner JV, Simmons JE (1999): Organ weights and fat volume in rats as a function of strain and age. Journal of Toxicology and Environmental

Health A

, 56 , 449-62. Schmitt W (2008): General approach for the calculati on of tissue to plasma partition coefficients. Toxicology in Vitro

, 22 , 457 Scollon EJ (2010): Pyrethroids: Evaluation of Data from Developmental Neurotoxicity Studies and Consideration of Comparative Sensitivity. US EPA OPPTS Memorandum, DP Barcode

371723 Scollon EJ, S tarr JM, Godin SJ, DeVito MJ, Hughes MF (2009): In vitro metabolism of pyrethroid pesticides by rat and human hepatic micr osomes and cytochrome P450 isoforms. Drug Metabolism and Disposition 37 ,221-228. Shafer TJ, Meyer DA, Crofton KM (2005): Developmental neurotoxicity of pyrethroid insecticides: Critical review and future research needs. Environmental Health Perspectives, 113, 123-136. Sheets LP (2000): A consideration of age- dependent differences in susceptibility to organophosphorus and pyrethroid insecticides. Neurotoxicology

, 21 , 57-  64.Pyrethroid/Pyrethrins Technical Working Group

Page 32 of 33Sheets LP, Doherty JD, Law MW, Reiter LW, Crofton KM (1994): Short Communication. Agedependent differences in the suscep tibility of rats to deltamethrin. Toxicology and Applied Pharmacology, 126, 186-  190. Soderlund DM, Clark JM, Sheets LP, Mullin LS, Piccirillo VJ, Sargent D, Stevens, JT and Weiner, ML (2002). Mechanisms of pyrethroid neurot oxicity: Implications for cumulative risk

assessment. Toxicology 171 , 3-59. Statz A, Felgenhauer K (1983): Development of the blood-CSF barrier. Developmental Medecine and Child Neurology

, 25 , 152-161. Stevens JC, Hines RN, Gu C, Koukouritaki SB, Manro JR, Tandler PJ (2003): Developmental expression of the major human hepatic CYP3A enzymes. Journal of Pharmacology and Experimental Therapeutics

, 307 , 573-582. Sun H, Chow ECY, Liu S, Du Y, Pang KS (2008): The Caco-2 cell monolayer: usefulness and

limitations. Expert Opinion in Drug Metabolism and Toxicology, 4 , 395-411 Tremblay RT, Kim D, Fisher JW (2010): Determination of Tissue Blood Partition Coefficients for Non-Volatile Herbicides and Fungicides us ing Negligible Depletion Solid Phase Microextraction (nd-SPME), 1567, The Toxicologist CD — An official Journal of the Society of Toxicology

, 114 , Number S-1. Tornero-Velez R, Mirfazaelian A, Kim KB, Anand SS, Kim HJ, Haines WT, Bruckner JV, Fisher JW (2010): Evaluation of deltamethrin kinetics and dosimetry in the maturing rat using a PBPKmodel. Toxicology and Applied Pharmacology, 244 , 208-217. US EPA (2006): Use of physiologically based pha rmacokinetic models to quantify the impact of human age and inter-individual diffe rences in physiology and biochemistry pertinent to risk. Report EPA/600/R-06/014A. National Center fo r Environmental Assessment, Cincinnati,

OH. Ussing HH, Zerahn K (1951): Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiologica Scandinavica, 23 , 110-127. Webster R (2008): Blood brain barrier matura tion: Implications for drug development. Presentation at the European Medicines Agency Workshop on Mo deling in Paediatric Medicines, April 14-

15. Weiner ML, Nemec M, Sheets L, Sargent D, Br eckenridge C (2009:. Comparative functional observational battery study of twelve commercial pyrethroid insecticides in male rats following acute oral exposure. NeuroToxicology, 30, S1-S16. Wolanksy MJ, Gennings C, Crofton KM (2006): Rela tive Potencies for Acute Effects of Pyrethroids

on Motor Function in Rats. Toxicological Sciences, 89

, 271-277

. Yang D, Pearce RE, Wang X, Gaedigk R, Wan YJ, Yan B (2009): Human carboxylesterases HCE1 and HCE2: ontogenic expression, inter-individual variability and differe ntial hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin. Biochemical Pharmacology

, 77 , 238-247. Zhu, H.-J., Appel, D. I. , Jiang, Y. and Markowitz , J. S. (2009). Age- a nd Sex-Related Expression and Activity of Carboxylesterase 1 and 2 in Mouse and Human Liver, Drug M etabolism and Disposition

, 37 , 1819–1825,Pyrethroid/Pyrethrins Technical Working Group

Page 33 of 33 Appendix 1. 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.