Pyrethroid/Pyrethrins Technical Working Group

Page 1 of 34 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 22902Pyrethroid/Pyrethrins Technical Working Group

Page 2 of 34 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

Co mpany Agent: Date: June 18, 2010

Thomas Osimitz

Chair PPTWGPyrethroid/Pyrethrins Technical Working Group

Page 3 of 34 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 - A ssessment 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 characteriz ing 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 D eltamethrin (Tonero, et al., 2010) ........................................................................................................................... 33

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

Page 4 of 34 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. 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) ................................. 21Pyrethroid/Pyrethrins Technical Working Group

Page 5 of 34 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 Corporation 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.

An important outcome from the proposed series of ac tivities will be the determination of the need to maintain the FQPA safety factor for age-related sensitivity based upon the age groupings typicallyPyrethroid/Pyrethrins Technical Working Group

Page 6 of 34 used in aggregate or cumulative exposure assessments. If age-related differences in toxicity were found to occur, both differences in toxicity and exposu re 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 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 34 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 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) 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 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 34 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).

2.2 Knowledge Gaps and Proposed Studies - Assess ment of Age-Dependent Sensiti vity (Study Block #1)

Given the relatively limited dataset, the PPTWG pr oposes to investigate this phenomenon using a broad range of commercial pyrethro id and pyrethrin insecticides (see Table 8 ), under circumstances

that are suite d for human risk assessments. The approach described be low involves measuring the concentrations of representative pyrethroids in brai n tissue that are associatedwith 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 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 me asured for comparison, with the time-to-peak amplitude (msec) used to verify the measured valu e 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 mean s to deliver a specific dose und er controlled conditions. While dietary exposure is generally preferred for releva nce 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.Pyrethroid/Pyrethrins Technical Working Group

Page 9 of 34 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.

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 ar e 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 a nd 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 c onsidered 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 developm ental 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-da y-old and adult rats in order to fully characterize the time-course of pyrethroids in these compartmen ts. Characterization of the concentration-time profiles in plasma and brain will aid in the assessme nt and selection of the dose metric appropriate for evaluation of the toxicodynamics. Plasma and brai n concentration measures will include the time of peak effect on the ASR to provide a measure of th e 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 me asured 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 10 of 34 Table 3. Milestones in the developmen t of Spragu e-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

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 / circumstancesPyrethroid/Pyrethrins Technical Working Group

Page 11 of 34 Table 5. 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 (parent) ; 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 12 of 34 3 EXAMINATION OF 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 13 of 34 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 availabl e. 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 re latively 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 do se 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 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. Some of thesePyrethroid/Pyrethrins Technical Working Group

Page 14 of 34 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 re latively 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 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 microsomalPyrethroid/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 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 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 on togeny 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 ha ve previously been cond ucted 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 ar e 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 form ed and plotted versus time to produce a reaction velocity. Plotting reaction velocities against initial concentrations yield V

max and K M

estimates for the test chemical. The intrinsic clearance is calculated by dividing the V

max by K M

.

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 ha ve 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 Technol ogies (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 patie nts 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 variabilit y in metabolic clearance (personal communication with Dr. Ronald Hines, Medical College of Wisc onsin). For hepatocyte analysis, there are a sufficient number of samples in the 11+ years ag e range to measure clearance for sub-sets of the population (personal communication with Life Technologies, Durham NC).Pyrethroid/Pyrethrins Technical Working Group

Page 17 of 34 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-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, 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 mo st relevant for simulating the age-dependent pharmacokinetic behavior of pyrethroids is the model by Tornero-Velez and coworkers. TheTornero-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 PB PK model revealed that pups (PND 10) have a 3.8 fold higher Area Under the Curve ( AUC) value than mature rats. Wh ether 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 prof iles of pyrethroids in human populations. Godin

et al . (2010) proposed a human PBPK model; this mode l 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 da ta 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 paramete r 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

Page 18 of 34 4.1 PBPK Modeling of Pharmacokinetics in Humans (ad ult 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 documen t, 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 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 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 character izing internal dose in hum ans

An approach that has the poten tial 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-sp ecific properties. For pharmaceutical substances, pediatric pharmacokinetics have been successfully pr edicted using PBPK mode ls based on adults and extrapolated to children using age-appropri ate 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, sp ecies 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 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.

As described in section 3.3 , the PPTWG is proposing a comprehensive block of studies on representativ e pyrethrins and pyrethroids to be c onducted 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 eval uate early life sensitivity to untested pyrethroids byPyrethroid/Pyrethrins Technical Working Group

Page 19 of 34 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.

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 hum ans with high accuracy. Previously published pharmacokinetic data for a dult 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 parame terization may be verified by applying the generic model to simulate the dosimetry of com pounds that have properties similar to those of pyrethroids and pyrethrins for which human PK da ta can be found in the lite rature. This compound specific parameterization will follow common prac tices in order to minimize uncertainty. A sensitivity analysis will be performed for character izing the remaining uncertainty and identifying the most sensitive parameters for determining the tim e-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 a dults and children) followi ng 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 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 aPyrethroid/Pyrethrins Technical Working Group

Page 20 of 34 variety of in vitro methods are available for estimating pa rameters for absorption and metabolism (reviewed in Clewell et al ., 2007).

4.2 Evaluation of the in vitro/silico Human PBPK Modeling Strateg y

4.2.1 Current 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 in to 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 publ ished 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 adu lts 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 a ddition to age-dependent changes in blood flow and permeability cross-product to reliably extrapolate fro m rats to humans and adults to children. Particularly for the pyrethroids and pyrethrins, wh ere 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-s pecific parameters that could inform the agedependent PBPK model with caref ul consideration of known physiol ogical 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 mo st sensitive and important for predicting the pharmacokinetics of pyrethro ids 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 par tition 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., 20 10). 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 ot her pyrethroids by repl acing deltamethrin-specific parameters with parameters specifi c 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 gene rated from rat studies. 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 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 par ticular, the rat PBPK model will be used to identify the key parameters that most influence the dosimet ry 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 e xperimentally using human cells and tissues.

Table 7. Chemical Specific Parameters for PB PK 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 param eters for PBPK models (Study Block #3)

As discussed 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 binding using human plasma (Banker and Clark, 2008), and investigation of metabolism using huma n hepatocyptes and/or mi crosomes 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 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 8 ). Note: because of the varying physical properties of the pyrethrins and

py rethroids 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 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. The data from these studies should allow for re-analy sis to calculate the diffusivity coefficient.

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, for different regions of the GI tract, from P eff as described above (Dokoumetzidis et al ., 2007)Pyrethroid/Pyrethrins Technical Working Group

Page 24 of 34 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, 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.Pyrethroid/Pyrethrins Technical Working Group

Page 25 of 34 5 SUMMARY OF KNOWLEDGE GAPS AND STUDY PLANS

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 – Age-dependent in vitro metabolism of pyrethrins and pyrethroids in human

liver tissue

3) Study Block #3 – Experimental determina tion 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 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 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 fo r other pyrethroid insecticides and the results of the initial group of studies.Pyrethroid/Pyrethrins Technical Working GroupPage 26 of 34Table 8. 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

6 SUMMARY

The 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 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 ev aluate 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 phar macokinetics in the rat, we propose formulating a human PBPK model with human relevant physiol ogical 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 Cent ury) and is protective of public health.

Page 27 of 3 4Pyrethroid/Pyrethrins Technical Working Group

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Appendix 1. Suggested Refinements to the Rat PBPK Model for D eltamethrin (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 (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 perfusion rat es 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: 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 volum e. Unfortunately the scaling law for surface area is not exactly known since the tissue surf ace 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.

 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 larger ones, the question whether perfusion limitation is conserved requires evaluation. This potentia l difference in distribution is not a limitation for extrapolations from adults to younger anim als 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 h as to be considered when extrapolating the parameters of lumped tissues. If possible, th e 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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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.