AGENDA

FIFRA SCIENTIFIC ADVISORY PANEL (SAP)

OPEN MEETING

July 23, 2010

FIFRA SAP WEB SITE http://www.epa.gov/scipoly/sap/

OPP Docket Telephone: (703) 305-5805

Docket Number: EPA-HQ- OPP-2009-0378

U.S. Environmental Protection Agency

Conference Center - Lobby Level

One Potomac Yard (South Bldg.)

2777 S. Crystal Drive, Arlington, VA 22202 

Scientific Issues related to the Comparative Adult and Juvenile Sensitivity 

Toxicity Protocols for Pyrethroids

Please note that all times are approximate 

(See note at the end of the Agenda)

Friday, July 23, 2010

8:30 A.M. Opening of Meeting and Administrative Procedures by Designated 

Federal Official – Sharlene Matten, Ph.D., Designated Federal Official, 

Office of Science Coordination and Policy, EPA

8:35 A.M. Introduction and Identification of Panel Members –

Steven Heeringa, Ph.D., Chair, FIFRA Scientific Advisory Panel

8:40 A.M. Welcome and Opening Remarks – Tina Levine, Ph.D., Director, Health 

Effects Division, Office of Pesticide Programs, EPA 

9:00 A.M. Regulatory Overview and Objectives – Edward Scollon, Ph.D.,

Health Effect Division, Office of Pesticide Programs, EPA

10:00 A.M. BREAK

10:15 A.M. PUBLIC COMMENTS

11:15 A.M. Charge Question 1

1.0 Auditory Startle Response or Acoustic Startle Reflex (ASR)

1.1 The auditory startle is a commonly used technique to assess neurobehavioral 

effects in rats. Auditory startle reflex is a motor reflex characterized by a sequence of 

reflexive muscle movements elicited by sudden and intense acoustic stimuli measured 

by a change in motor output. The proposed reflex path is short, consisting of the 
2

auditory nerve, posteroventral cochlear nucleus, the nucleus reticularis pontis caudalis, 

and motor neurons in the spinal cord (Davis et al. 1982). This mechanism is 

susceptible to a variety of drugs and toxicants making the reflex a useful model of 

sensorimotor reactivity across animal taxa, including rat and human (Lee et al. 1996). 

With regard to pyrethroids, auditory startle data in adult rats have demonstrated differing

response patterns related to pyrethroid structure (Crofton and Reiter 1984; Tilson et al. 

1985; Crofton and Reiter 1988; Hijzen et al. 1988; Hijzen and Slangen 1988); Type I 

pyrethroids produced an increase in startle amplitude and Type II pyrethroids produced 

a decrease in startle amplitude. In addition, ASR has been used to demonstrate agedependent toxicity in rats following high oral doses of pyrethroids (Sheets et al. 1994; 

Sheets 2000). Therefore, ASR is a potentially sensitive measure to evaluate 

differences in neurobehavioral effects between adults and pups. 

Since ASR is a behavioral measurement, it is important to consider development, doseresponse and variability during interpretation of the results. In rats, the onset of ASR 

response corresponds to the development of the external auditory meatus. In the rat, 

this usually occurs between 13 and 16 days of age. Sheets et al. (1988) have shown 

the ability of rats to respond to ASR as early as PND 13, however, the amplitude of 

response continued to increase through PND 21. Pyrethroids modify the voltage gated 

sodium channels in the central nervous system and therefore the brain is considered 

the major target organ for toxicity. Kim et al. (2010) determined the distribution of 

deltamethrin, a Type II pyrethroid, in brain, fat, liver, plasma, and muscle in PND 10, 21, 

40, and 90 rats for up to 510 hours. Brain concentrations in PND 10 pups were 

elevated for a longer time relative to the adults. This suggests that pyrethroid kinetics in 

the brain of pups may not mirror those of adult rats. The Kim et al. (2010) study 

emphasizes the importance of determining the appropriate time course of effects (i.e., 

time-to-peak-effect and/or time-to-tissue-recovery) in both adult and non-adult lifestages 

prior to measuring ASR responses. Additionally, the standard deviation for peak 

amplitude, the ASR measure proposed by the PPTWG, can vary greatly in guideline 

DNT studies (20-125%) and literature reviews (Raffaele et al. 2004). However, this 

variability can be reduced down to 20-30% if the studies are conducted in proven 

laboratories (Sette et al. 2004).

The PPTWG is proposing that the ASR provides a robust and sensitive measure 

of neurotoxicity and is well suited to assess age-dependent sensitivity to 

pyrethroids. Please comment on the appropriateness of the ASR technique as a 

measure of pyrethroid induced toxicity, including suggestions to assure quality

of the study design (i.e., appropriate time-to-peak response, variability of peak 

response, etc.) and resulting data.

1.2 Age-dependent toxicity has been observed in rat studies following high doses 

(i.e., LD50 studies resulting in 50% mortality of test subjects) of Type II pyrethroids 

(Cantalamessa 1993; Sheets et al. 1994; Sheets 2000). However, in sublethal studies 

using the ASR as a measure of toxicity, ED50 (dose at which 50% of the test subjects 

are affected) values were similar between postnatal day (PND) 21 and adult rats. 
3

These findings suggest that age-dependent toxicity may only be observed at high 

doses. Based on in vivo (Cantalamessa 1993) and in vitro (Anand et al. 2006) studies, 

the apparent discrepancy between high- and low-dose age-dependent toxicity is likely 

attributable to incomplete maturation of the enzymes that detoxify pyrethroids in 

immature animals, particularly the carboxylesterases and cytochrome P450s. These 

clearance mechanisms are overwhelmed in younger animals given LD50 doses, leading 

to increased accumulation of the pyrethroids in nervous tissue and ultimately increased 

toxicity. 

Carboxylesterases and P450 enzymes are the two major enzyme families responsible 

for metabolism of pyrethroids. In the rat, it has been shown that carboxylesterase 

activities are below adult levels at weaning (Moser et al. 1998; Karanth and Pope 2000; 

Anand et al. 2006; de Zwart et al. 2008; Yang et al. 2009). Information on the ontogeny 

of carboxylesterase development in the human is more limited. However, increased 

plasma esterase activity during postnatal maturation has been reported (Ecobichon and 

Stephens 1973). In contrast, Pope et al. (2005) found carboxylesterase activity in 

hepatic tissues were similar for humans ranging in age from 2 months to 36 years, 

however, the sample sizes were small and variability among the age groups was high. 

Maturation of the P450 enzymes shows a similar trend. 2C19, a P450 enzyme which 

has shown high pyrethroid metabolic activity (Godin et al. 2006), increases rapidly in the 

human during first 2 years of life, whereas numerous P450s examined in the rat have 

minimal expression levels through gestation and do not approach adult levels of 

expression until PND10 days or later (de Zwart et al. 2008). 

Comparisons between lifestages in the rat and human are difficult because of the 

ontogeny of the brain development and metabolizing enzymes are not an exact match. 

However, PND 11 rats are considered to be close in development to newborn humans 

and PND 17 rats are believed to be closer developmentally to human toddlers (Davision 

and Dobbing 1966; Dobbing and Smart 1974; Benjamins and McKhann 1981). From 

the aspect of exposure, previous experience with developing cumulative risk 

assessments for other insecticide groups, ongoing work on HED's Standard Operating

Procedures for Residential Pesticide Exposure Assessment, and the Agency's 

Guidance on Selecting Age Groups for Monitoring and Assessing Childhood Exposures 

for Environmental Contaminants http://www.epa.gov/raf/publications/guidance-onselecting-age-groups.htm, the Agency believes that children three years old and 

younger, particularly those who are mobile (crawling, walking) and who exhibit hand-tomouth behavior, have the potential for the greatest exposure to pyrethroids. Based on 

1) the current understanding that the two major enzyme families responsible for the 

metabolism of pyrethroids are below adult activity levels at weaning (i.e., PND 21); 2) 

PND 17 rats are approximately comparable to human toddlers in terms of development; 

and 3) children younger than 3 years of age are expected to have the greatest 

exposures to pyrethroids, the Agency is concerned with the PPTWG’s proposal to 

conduct ASR studies in PND 21 rats. Instead, PND 15 to 17 rats, which have been 

shown to respond to ASR stimuli (Sheets et al. 1988), may better represent the most 

susceptible human lifestage.
4

The PPTWG has proposed to use conduct ASR studies on 21-day old rats. Please 

comment on the appropriateness of this age group in regards to i) assessing agedependent toxicity and ii) assessing whether the 21-day old rat will adequately

inform the Agency regarding toxicity as it relates to children three years of age 

and younger. 

12:00 P.M. LUNCH

1:00 P.M. Charge Question 1 cont’d

2:00 P.M. Charge Question 2

2.0 Physiologically-Based Pharmacokinetic (PBPK) Modeling

The PPTWG is proposing to use a model developed collaboratively by EPA’s Office of 

Research and Development (ORD) and the University of Georgia as a starting point in 

their modeling effort. EPA’s ORD has published a series of papers that describe the 

development and enhancement of pyrethroid PBPK models starting with a deltamethrin 

model in rats by Mirfazaelian et al. (2006) , improved by Godin et al.(2010), modified for 

permethrin by Tornero-Velez et al. (in prep.), and finally expanded to include age- and 

chemical-dependent parameters by Tornero-Velez et al. (2010). In 2007, ORD and 

OPP jointly presented an issue paper to the SAP (USEPA 2007) which described an 

approach for using a generic model structure with chemical specific parameters for 

pyrethroids. The “family modeling” approach was endorsed by the SAP and has been 

successfully applied in the above PBPK efforts. The Agency believes that it is both 

reasonable and scientifically sound to use the Tornero-Velez et al. (2010) PBPK model 

as the starting point for the PPTWG effort to build PBPK models for pyrethroids to 

assess young children. Furthermore, the PPTWG is proposing to develop PBPK 

models using in vitro and in vivo rat data, and then using human in vitro data to inform 

model to predict human internal dosimetry, similar to the approach which was previously 

supported by the 2007 SAP.

 

2.1 The PPTWG proposes to increase the complexity of the Tornero-Velez et al. 

(2010) PBPK model by modifying some aspects. For example, the PPTWG is 

proposing to:

a. Predict intestinal permeability through the use of Ussing Chamber 

technique with rat cells and human Caco-2 cells, with the potential to 

increase the number of compartments within the intestinal tract

b. In vitro determination of partition coefficients

c. Obtain estimates of protein binding

Please comment on the proposed modifications to the Tornero-Velez et al. (2010)

model as described in sections 4.3 of the PPTWG proposal. Please include in 
5

your comments consideration for balancing potentially improved performance 

resulting from the increased complexity with model parsimony. 

2.2 Microsomal incubation studies have been used to inform the pyrethroid PBPK 

models developed by ORD (Mirfazaelian et al. 2006; Scollon et al. 2009; Godin et al. 

2010; Tornero-Velez et al. 2010; Tornero-Velez in prep.). The PPTWG has proposed to 

use intact hepatocytes instead because they may provide a better prediction of 

metabolism compared to microcellular fractions (Hewitt et al. 2007). Additionally, the 

PPTWG is proposing to compare the clearance activity of human hepatocytes and 

microsomal fractions for several age groups. However, the Agency notes that there are 

a limited number of human hepatocyte samples available to inform the PBPK model. 

Pooled human microsomes are available representing larger segments of the 

population relative to hepatocyte availability. The Working Group suggests 

characterization of the hepatocytes (i.e., cypP450 and esterase composition) and 

establishing a relationship between hepatocytes and microsomal fraction activities may 

reduce model uncertainty in light of limited data. 

Please comment on the strengths and weaknesses of the PPTWG proposal to use 

hepatocytes in the PBPK effort, including the potential for hepatocytes to 

decrease uncertainty of model predictions in light of limited data.

3:15 P.M. BREAK

3:30 P.M. Charge Question 3

3.0 Alternative Study Design(s) For Evaluating Age Differences in 

Pharmacokinetics

The Agency gives special consideration to the potential pre- and postnatal lifestages 

regarding potential exposure to pesticides. Pre-natal exposure to pyrethroids has been 

evaluated extensively in over 80 developmental toxicity, reproductive toxicity, and DNT 

test guideline studies and no sensitivity from in utero exposure has been observed. As 

previously described, there are gaps in knowledge surrounding the potential for postnatal sensitivity and, as described in Question 1.2, the Agency considers children less 

than 3 years of age to be the most susceptible population. The PPTWG has proposed 

a robust PBPK model development effort to describe pyrethroid dosimetry across 

several lifestages; however, these models will not be ready for use by the Agency until 

approximately 2013. 

Are there alternative approaches using empirical or data generation techniques 

potentially requiring less time than the PBPK effort proposed by the PPTWG for 

evaluating the potential for post-natal sensitivity, particularly with respect to 

differences in pharmacokinetic profiles, that could be used by the Agency?
6

5:00 P.M. Adjourn

Please be advised that agenda times are approximate; when the discussion for 

one topic is completed, discussions for the next topic will begin. For further information, 

please contact the Designated Federal Official for this meeting, Dr. Sharlene Matten, via 

telephone: (202)-564-0130; fax: (202) 564-8382; or email: matten.sharlene@epa.gov.