UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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# f] g:ams d f 'v--

Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: PIPERONYL BUTOXIDE (PBO)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPIPERONYL BUTOXIDE FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................3

II. Sources of Scientific Data and Technical Information......................................4

A. EDSP Tier 1 Screening Assays............................................................4

B. Other Scientifically Rele vant Information (OSRI)......................................4

III. Weight of Evidence (WoE) Evaluation.......................................................4

A. EDSP Tier 1 Screening Assays.............................................................7

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16

IV. Committee’s Assessment of Weight of Evidence..........................................18

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................18

1. Tier 1 in vivo Assays.............................................................19

2. OSRI...............................................................................19

B. Estrogen Pathway...........................................................................22

C. Androgen Pathway.........................................................................22

D. Thyroid Pathway...........................................................................22

E. Conclusions.................................................................................22

V. EDSP Tier 2 Testing Recommendations....................................................23

VI. References.......................................................................................24

LIST OF TABLES: Table 1. Tier 1 Assays for Piperonyl Butoxide......................................................7
Table 2. Estrogenic/Anti-Estrogenic Pathway for Piperonyl Butoxide........................10
Table 3. Androgenic/Anti-Androgenic Pathway for Piperonyl Butoxide.......................14
Table 4. Thyroid Pathway for Piperonyl Butoxide................................................17

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................28
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39

APPENDIX 3. References Not Utilized in the Piperonyl Butoxide WoE Analysis.................53PIPERONYL BUTOXIDE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g enerationPIPERONYL BUTOXIDE FINAL

iv Abbreviation Terminology

Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated Dose
N Ne g ativePIPERONYL BUTOXIDE FINAL

v Abbreviation Terminology

NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tie r 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating HormonePIPERONYL BUTOXIDE FINAL

vi Abbreviation Terminology

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidencePIPERONYL BUTOXIDE FINAL

Page 1 of 54 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether piperony l butoxide (PBO) interacts with E, A or T hormone pathways, the number and type of effects induced, the magn itude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dos e(s) at which endocrine related responses occurred happened concurrently wi th general systemic toxicity.

On October 29, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of PBO with the E, A or T hormone pathwa ys. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For the estrogen pathway, there was no convincin g evidence of an interaction of PBO. It was negative in the estrogen receptor (ER) bindi ng, ER transactivation assay (ERTA), and the aromatase assays. In the ster oidogenesis assay, there was a small (1.11- to 1.31-fold) but significant increase in estradiol concentrations at the highest attainable test concentration (10 μM). PBO was negative in the uterotrophic assay a nd in the female pubertal assay in the absence of overt toxicity. There were no estrogen-related effects in the mammalian Part 158 studies or the avian reproduction study in the ab sence of overt toxicity. In th e fish short-term reproduction assay (FSTRA), while increased fecundity was observed at the middle (14%) and high (19%) treatment concentrations, there was no other female effects observed (lack of complementarity of evidence within the assay), and no other estrogen -related effects observed in the rest of the in

vivo Tier 1 battery in the absence of overt toxicity.

There was no convincing evidence of interacti on of PBO with the androgen pathway. PBO was as negative in the AR binding and steroidogenesi s assays. There were no effects observed in the Hershberger assay and in the male pubertal assay in the absence of overt t oxicity. There were no androgen-related effects in the mammalian Part 158 studies. In the FST RA, while there was a reported increase of minimal to mild testes degeneration in the middle and high treatmentPIPERONYL BUTOXIDE FINAL

Page 2 of 54 concentrations (0.03 and 0.09 mg a.i./L, respectively) and a significant increase in male tubercle score at only the low concentration, there were no other complementarity findings. Additionally, while there were no clinical signs of toxicity observed in the FSTRA, a 17% decrease in male body weight at the highest test concentration wa s observed which may be indicative of potential stress. Additionally, there were no other effects observed in the Tier 1 in vivo battery in the absence of overt toxicity.

There was no convincing evidence of interaction of PBO with the thyroid pathway. There was no complementarity of evidence for the histopathologi cal changes seen in the male and female rats in the pubertal assays since no treatment-relat ed effects were seen in thyroid hormones (T 4 or TSH) or thyroid weights. No thyroid-related effects were seen in the mammalian Part 158 studies, except for a histopatholog ical effect in the thyroid in the chronic rat study. Furthermore, there was no evidence of thyroid-rela ted effects observed in the AMA.

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not
recommended for PBO since there was no convinci ng evidence of potential interaction with the estrogen, androgen or thyroid pathways.PIPERONYL BUTOXIDE FINAL

Page 3 of 54 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether piperony l butoxide (PBO) interacts with E, A or T hormone pathways, the number and type of effects induced, the magn itude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dos e(s) at which endocrine related responses occurred happened concurrently wi th general systemic toxicity.

On October 29, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence analysis of the potential interaction of PBO with the E, A or T hormone pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Piperonyl butoxide is a pesticide active ingredient that acts as a synergist. As a synergist, it inhibits the mixed function oxidase system of insects and reduces the oxidative breakdown of other pesticides. PBO has a vapor pressure of <1 × 10

− 7 mm Hg (25°C) with a water solubility of 14 mg/L at 25°C (USEPA, 2006). In water bodies, it is slowly metabolized (75-day half-life), or in shallow, clear waters, rapidly degraded by photolysis. PBO reaching anaerobic sediments will
tend to be stable and available for desorption to the water column. In soil, metabolism appears to be relatively quick (parent half-life 14 days in one soil tested). If present in the air as a vapor or spray drift, it is expected to react rapidly with hydroxyl radicals. A fish bioaccumulation study in bluegill sunfish had bioconcentration factors of 260, 91, and 380 for whole fish, edible tissue, and nonedible tissue, respectively.

The available information considered in dete rmining the potential inte raction of piperonyl butoxide with the E, A, or T pathways incl ude submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP Tier 2 study recommendations are presented in Section V.PIPERONYL BUTOXIDE FINAL

Page 4 of 54 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). However, the Test Order recipient elected to conduct all eleven Tier 1 screening assays, so no OSRI were submitte d in lieu of the Tier 1 assays. The Agency has
also conducted a more recent search of available scientific litera ture for any additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally,
literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays werePIPERONYL BUTOXIDE FINAL

Page 5 of 54 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.PIPERONYL BUTOXIDE FINAL

Page 6 of 54 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.PIPERONYL BUTOXIDE FINAL

Page 7 of 54 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and biol ogically relevant with regard to a treatment- related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP T1 Screening Assays

The Tier 1 assays submitted in response to the agency’s the test order for PBO are shown below
in Table 1

.

Table 1. Tier 1 Screening Assay for Piperonyl Butoxide

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID 48618803)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID 48618804)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID 48618801)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID 48618807)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48618802)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID 48618808)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID 48618805)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID 48618806a)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID 48618806b)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID 48687701)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID 48673101)PIPERONYL BUTOXIDE FINAL

Page 8 of 54 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for PBO to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

PBO was negative in the Tier 1 in vitro estrogen receptor (ER) binding, ERTA, and the aromatase assays. PBO was positive in the steroidogenesis assay, where a small but significant increase in estradiol producti on (1.11- to 1.31-fold) was observed in all runs at the highest attainable test concentration (10 μM).

In the FSTRA, there were significant increases of 14 and 19% in fecundity at the mid and high treatment concentration (0.03 a nd 0.09 mg/L), respectively. Ho wever, there were no other
treatment-related effects observed in females.

PBO was negative in the uterotr ophic assay. In the female pubertal rat assay, a 16% decrease in ovarian weights was noted in the presence of ove rt toxicity (moribund sa crifice and clinical signs); no other estrogen-related effects were observed. There were no estrogen-related effects
noted in the other Part 158 mammalian studies.

In an OSRI developmental t oxicity study in rats (Tanaka et al ., 1995), the total number of resorptions was increased, with two complete litte r resorptions, and the mean number of viable
fetuses was decreased in dams treated with 1800 mg/kg/day. These dams also displayed decreased body weight gains, and mean decreased feta l weights at this dose level. An increase in fetal limb deformities was also observed at this dose. Finally, in a developmental toxicity study in mice (Tanaka et al ., 1994), doses of 1385 and 1800 mg/kg/ day caused abortions, increased numbers of resorptions/dam and increased post- implantation loss. These doses also caused decreased mean fetal weights and increased incidence of oligodactyly of the forelimbs.

In a Part 158 avian reproduction toxicity study in bobwhite quail, no treatment-related reproductive effects were noted at dietary levels up to 3000 mg/ kg-diet. However, in an avian reproduction toxicity study in mallard duck, dietar y treatment with 1200 mg/ kg-diet (the highest treatment concentration) resulted in a reducti on in the number of eggs laid, reduced eggshell thickness, and an increased propor tion of cracked eggs. This treatment group was also associated with overt toxicity as indicated by mortality, significan t reductions in body weight and clinical signs of toxicity that included weakness, subdued behavior and inability to stand.PIPERONYL BUTOXIDE FINAL

Page 9 of 54 In an early life-stage toxicity study on fath ead minnows, treatment with PBO at 0.42 and 0.80 mg a.i/L (the two highest treatment concentratio ns) caused reductions in the percentage of hatch, but there were also significant decreases (p <0.05) in body weight and body length at these concentrations. In a fish life-cycle st udy with fathead minnow tested at 0.0077, 0.0273, and 0.106 mg/L, based on a preliminary review of the study (MRID 49031801), the study author reported a significant decrease in parental female body weight at the low and high-concentration and decrease in male body length at the mid and high-treatment. There were no effects on fecundity, GSI, VTG, tubercle scores or gonada l histopathology. In the F1 generation, larvaejuvenile survival was significantl y decreased at all test concentr ations (9.5-14.5%), F1 fecundity ( ↑ 32%) was increased at 0.106 mg/L (F1 male body we ight and length was also increased at high concentration); F2 4-week post hatch body weightand length were reduced at the mid and hightreatment.PIPERONYL BUTOXIDEFINALPage 10 of 54Table 2: Estrogenic/Anti-Estrogenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-EstrogenicPathway for Piperonyl Butoxide1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48618803)NERTA(MRID 48618804)NAromatase(MRID 48618802)NSteroidogenesis(MRID 48618807)PUterotrophic(MRID 48618808)NX(L)X(H)Female Pubertal Rat(MRID 48618806a)NNNNNNX4(L,H)X5FSTRA(MRID 48687701)NENNNFcd↑14%,↑19%(M, H)NNOSRIDevelopmentalToxicity (Rat; MRID42380801)NX(M, H)NDevelopmentalToxicity (Rat; Tanakaet al., 1995)P6(H)X(M, H)NDevelopmentalToxicity (Mouse;Tanakaet al., 1994)P7(M, H)X(L, M,H)NPIPERONYL BUTOXIDEFINALPage 11 of 54Table 2: Estrogenic/Anti-Estrogenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-EstrogenicPathway for Piperonyl Butoxide1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3DevelopmentalToxicity (Rabbit;MRID 00026192)NNX(M, H)Chronic toxicity/Carcinogenicity (Rat;MRID 40323701)NENNNEX(L, M,H)X(H)Carcinogenicity(Mouse; MRID42903701)NENENNEX(M, H)NChronic Toxicity(Dog; MRID42926002)NENNNX(M, H)X(H)Subchronic Toxicity(Rat; Fujitaniet al.,1992)NNNENEX(M, H)X(H)Subchronic InhalationToxicity (Rat; MRID42477101)NENNNEX(MH, H)NAvian Reproduction(Quail; MRID43876301)NN N8Avian Reproduction(Duck; MRID43876302)NNX(H)Fish Life-cycle(fathead minnow;MRID 49031801,preliminary review)NNX(L,M,H)PIPERONYL BUTOXIDEFINALPage 12 of 541.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.. Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Increases in liver (34-44%)and adrenal (21-29%) weights.5. Moribund sacrifice and clinical signs.6. At 1800 mg/kg/day, the total number of resorptions was increased resulting in an increase (p<0.05) in the percent total resorption rate7. Increases in abortions and resorptions and number of dams with viable fetuses was reduced at 1385 and 1800 mg/kg/day.8. Male body weights were decreased 6 and 2% in a non-treatment responsive manner at 1500 and 3000 ppm.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedPIPERONYL BUTOXIDE FINAL

Page 13 of 54 2. Effects on Androgen Pathway

The potential for PBO to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

PBO was negative in the Tier 1 in vitro AR binding and steroidogenesis assays. PBO was negative for both androgenic and anti-androgenic activity in the He rshberger assay. In the male pubertal assay, the decreases in serum testostero ne levels (67%) and in the pituitary (17%), seminal vesicle (26-35%) and LABC (25%) weights occurred in the presence of overt toxicity (14% decrease in body weight and clinical signs) at the limit dose (1000 mg/kg/day).

In the FSTRA, there was a significant increase in median male tubercle score at the lowest treatment concentration (0.01 mg a.i/L) but this effect was not observed at the mid and high treatment concentrations (0.03 and 0.09 mg a.i/L, respectively). Additiona lly, increased minimal to mild testes degeneration was observed in the high treatment and a marginal increase of the same finding was observed in the mid treatment. Th e pathologist report stated that it is unlikely that this minimal to mild testicular degenera tion observation would have a measureable impact
on testicular function. It is noted that male VT G levels could not be measured due insensitive methodology (relatively high LOQ). Additionally, at highest treatment concentration, there was a 17% decrease in male body weight although this was not observed with any mortality or any other clinical signs of toxicity.

In a Part 158 chronic toxicity /carcinogenicity study in rats, significantly increased bilateral atrophy, increased interstitial ce lls and increased inte rstitial cell tumors we re observed in the testes at 500 mg/kg/day. Ther e were no androgen-related eff ects noted in other Part 158 mammalian studies or in the Part 158 avian studies. In a fish life-cycle study with fathead based on a preliminary review of the study (MRID 49031801), F1 male body weight, length, and GSI was increased at the high treatment concentra tion (0.106 mg/L); effects on F1 larvae-juvenile
survival also decreased at this concentrations. Eff ects on parental and F2 growth were also reported.PIPERONYL BUTOXIDEFINALPage 14 of 54Table 3: Androgenic/Anti-Androgenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Piperonyl Butoxide1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48618801)NSteroidogenesis(MRID 48618807)NHershberger(MRID 48618805)NNX(L, H)NMale Pubertal Rat(MRID 48618806b)NNNNNNNNX(L, H)X(H)FSTRA(MRID 48687701)NENP4(M, H)↑tuberclescore (L)NNE5NOSRIChronic toxicity/carcinogenicity (Rat;MRID 40323701)NNNENNEX(H)X(H)Carcinogenicity(Mouse; MRID42903701)NNNENNEX(M, H)NChronic toxicity(Dog; MRID42926002)NNNENNX(M, H)X(H)Subchronic toxicity(Rat; Fujitaniet al.,1992)NNENENENEX(M, H)X(H)SubchronicInhalation Toxicity(Rat; MRID42477101)NNNNNEX(MH, H)NPIPERONYL BUTOXIDEFINALPage 15 of 54Table 3: Androgenic/Anti-Androgenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Piperonyl Butoxide1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 321-Day Dermaltoxicity (Rabbit;MRID 42218201)NNNNNEN NAvian Reproduction(Quail; MRID43876301)NN N6Avian Reproduction(Duck; MRID43876302)NNX(H)Fish Life-cycle(Fathead minnow;MRID 49031808(preliminary review)NNX(L,M,H)1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weightare absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate(VP), Dorsal prostate (DP),Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. Increased testes degeneration (minimal to mild) was higher thancontrols in the 0.0944 mg ai/L group, but only marginally higher in the 0.0348 mg ai/L group.5. Male vitellogenin (Vtg) was notsuccessfully measured due to insufficiently sensitive methodology.6. Male body weights were decreased 6 and 2% in a non-treatment responsive manner at 1500 and 3000 ppm.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedPIPERONYL FINAL

Page 16 of 54 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal male and female (rat) assays, and the AMA (frog).

The potential for PBO to interact with the thyroid pathway is summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

In the male and female pubertal assays, there was a dose-dependent increase in thyroid follicular cell height with a concomitant decrease in follic ular cell colloid area at both doses tested (500 and 1000 mg/kg/day). However, the changes at the high dose was seen in the presence of overt toxicity (decreased body weight in males and clinical signs in both sexes). Furthermore, there were no effects of treatment on thyroid weights, or T 4 or TSH levels.

In the AMA, the only significant effect was a 28% increase in normalized Day 21 HLL at the highest concentration tested (0.0741 mg/L). There were no treatment-related effects on developmental stage (NF stage) or thyroid histopathology, or growth parameters. Day 7 body weight was significantly reduced at the lowest treatment concentra tion (0.001 mg a.i/L) but this effect was not observed at higher treatment concentrations nor was it observed at Day 21

measurements.

In a Part 158 chronic toxicity/c arcinogenicity study in rats, incr eased incidences of pigment in thyroid follicles were observ ed in the high-dose group (500 mg /kg/day), and thyroid follicle hyperplasia was increased in the mid-dose (100 mg /kg/day) females and the high dose males and females; at the high dose only, this effect occurred in the presence of overt toxicity (decreased body weight). In a Part 158 chronic toxicity st udy in dogs, thyroid weight was increased in females at the highest dose tested (71.0 mg/kg/day), however t hyroid histopathology was unaffected by treatment, and the effect co-occurred with overt toxicity (d ecreased body weights). There were no thyroid-related effects observed in the other Part 158 mammalian studies.PIPERONYL FINAL

Page 17 of 54 Table 4. Thyroid Pathway for Piperonyl Butoxide.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Piperonyl Butoxide 1

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48618806a) N P 4 (L) N N N X (L, H) X 5 (H)
Male Pubertal Rat (MRID 48618806b) N P 4 (L) N N N X (L, H) X 6 (H)

AMA (MRID 48673101) N Day 21: ↑ 28%
HLL (H) N N

OSRI Chronic toxicity/ carcinogenicity (Rat; MRID 40323701) NE P 7 (M) NE X (L, M,

H) X (H)
Carcinogenicity (Mouse; MRID 42903701) NE N NE X (M, H) N
Chronic toxicity (Dog; MRID 42926002) N N N X (M, H) X (H) Subchronic inhalation toxicity (Rat; MRID 42477101) NE N NE X (MH,

H) N

21-Day Dermal toxicity (Rabbit; MRID 42218201) NE N NE N N

1 Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the re sponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Increase in thyroid follicular cell height with a conc omitant decrease in follicular cell colloid area at 500 mg/kg/day. 5. At the high dose of 1000 mg/kg-day, clinical signs including moribund sacrifice. 6. At the high dose of 1000 mg/kg/day decreased body weight and clinical signs.
7. Increased follicular hyperplasia at 100 mg/kg/day in females P Positive findings N Negative findings NE Not examinedPIPERONYL FINAL

Page 18 of 54 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of PBO to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) were sufficient for a dose/conc entration to be consid ered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).PIPERONYL FINAL

Page 19 of 54 1. Tier 1 in vivo Assays

In the androgen agonist portion of the Hers hberger assay, at 1000 mg/kg/day, terminal body weights were decreased (NS) by 7%, but overall (Days 1-11) body weight gains were decreased (p<0.05) by 96%. In the anti-androgen portion, te rminal body weights were decreased (NS) by 4% at 320 mg/kg/day and 9% at 1000 mg/kg/day. The overall body weight gains were decreased (p<0.05) by 36% at 320 mg/kg/day and decrea sed (p<0.05) by 62% at 1000 mg/kg/day.

In the pubertal assays, final adjusted body weight s were decreased (p<0.05) by 5% (both sexes; NS in males) and 7-9%, and the adjusted body weight gains were decreased (p<0.05) by 6-9% (NS in males) and 10-11%, respectively, at 500 and 1000 mg/kg/day. In the females, the adjusted and relative (to body) liver weight s were increased (p<0.05) by 15 and 35%, respectively, at 500 mg/kg/day and 22 and 44% at 1000 mg/kg/day, and the unadjusted liv er weights were increased (p<0.05) by 36% at 1000 mg/kg/day. In the males, relative liver weights we re increased (p<0.05) by 15 and 34% at 500 and 1000 mg/kg/day, respec tively, and unadjusted and adjusted liver weights were increased (p<0.05) by 16 and 20% , respectively, at 1000 mg/kg/day. Increases (p<0.05) were also noted in the relative kidney weight ( ↑ 8% in the 500 mg/kg/day males; ↑ 6- 11% in both sexes at 1000 mg/kg/ day) and unadjusted, adjusted, and relative adrenal weights ( ↑ 15-31%) in both sexes at 1000 mg/kg/day. The adju sted and unadjusted pituitary weights were decreased by 16-17% (p<0.05 or NS) at both dose le vels in the males. In the females, at 500 and 1000 mg/kg/day, serum levels of alkaline phosphatas e were decreased, and total protein levels were increased. At 1000 mg/kg/day, increases we re observed in serum levels of sodium, phosphorous, and blood urea nitrogen.

In the uterotrophic assay, final body weight s were decreased (p<0.05) by 10% at 1000 mg/kg/day, resulting in a 240% decrease (p<0.05) in overall body weight gain.

In the FSTRA, at 0.09 mg/L, male body weight was significantly decreased 17%. There was no mortality or any other clinical signs of t oxicity observed.

In the AMA, there were no significa nt effects on mortality and no othe r clinical signs of toxicity. Day 7 body weight was significantly reduced at the lowest trea tment concentration (0.001 mg a.i./L) but this effect was not observed at higher treatment concen trations nor was it observed at Day 21 measurements.

2. OSRI

In a prenatal developmental t oxicity study in rats, at 500 and 1000 mg/kg/day, treatment-related clinical signs were observed following PBO ad ministration which increased in frequency and severity with dose. Body weight gains were decreased (p<0.05) by 51% in the 1000 mg/kg/dayPIPERONYL FINAL

Page 20 of 54 group during GD 6-9, and by 21-22% in the 500 and 1000 mg/kg/day gr oups during GD 12-15, resulting in decreased (p<0.05) body weight gains of 18-22%, at 500 and 1000 mg/kg/day for the treatment period (GD 6-15). Food consumption (g /rat/day) was also decreased (p<0.05) by 15- 21% during GD 6-9 at 500 and 1000 mg/kg/day, and by 8-9% for the treatment period. Increased absolute ( ↑ 8%; NS) and relative to body ( ↑ 11%; p<0.05) liver weight s were noted at 1000 mg/kg/day. In a published, developmenta l toxicity study in rats (Tanaka et al., 1995), PBO decreased (p<0.01) maternal body weight ga ins by 24 and 37% at 1065 and 1800 mg/kg/day, respectively, during GD 11-20. At 1800 mg/kg/da y, the total number of resorptions was increased (54 treated vs. 1 cont rol), resulting in an increase (p<0.05) in th e percent total resorption rate (35.3% treated vs. 0.56% controls). Two litters we re totally resorbed. The mean
number of viable fetuses/dam was decreased (not analyzed statisti cally) at 1800 mg/kg/day ( ↓ 16%). Mean fetal weights were decreased (p <0.05) by 5% in the 1065 mg/kg/day males and by 14% each in the 1800 mg/kg/day males and females. In a published prenatal developmental toxicity study in mice (Tanaka et al., 1994), abortions were observ ed in one dam at 1385 mg/kg and two dams at 1800 mg/kg/day PBO. The number of dams with viable fetuses was reduced and increased numbers of resorptions/dam were noted in the 1385 and 1800 mg/kg/day groups. Additionally, 1 and 3 litters were totally resorbed at 1385 and 1800 mg/kg/day, respectively. The increased resorptions/dam were reflected in an increase in post-implantation loss at these doses. Mean fetal weights were decreased (p<0.05) by 3-7% in the males at 1385 and 1800 mg/kg/day and by 4-6% in all treated groups in the females. In an prenatal developm ental toxicity study in rabbits, body weight losses of

− 19 g at 100 mg/kg/day and

− 159 g at 200 mg/kg/day were observed during treatment (GD 7-19) with PBO.

In a combined chronic toxicity/carcinogenici ty study, male body weights at 500 mg/kg/day PBO were decreased by 3-4% (p<0.05) for the first 10 weeks of the study and by 21% at termination (Week 104). In the 500 mg/kg/day females, body we ights were decreased by 6% at Week 5 and by 21% at termination. Food consumption was si gnificantly decreased in the high dose group on
some occasions. Serum cholesterol was increase d (p<0.05) in the 500 mg/kg/day females at all assay times. A bsolute liver weights were increased by 21% in males at 500 mg/kg/day, and by 20-27% in females at 30, 100, and 500 mg/kg/day. Microscopic lesions in the liver included hepatocyte hyperplasia in the high dose groups of both sexes and focal mixed cells in the mid and high dose females and high dose males

.
In a carcinogenicity study in mice, male body we ights were decreased (p<0.05) by 7% at Week 74 and by 6% at test terminati on at 300 mg/kg/day. It was stated that body weight gains were reduced (p<0.05) in the 100 and 300 mg/kg/da y group males for most of the study when compared to the second control group, and occas ionally reduced (p<0.05) in the 300 mg/kg/day group when compared to the first control group. Relative liver weights we re increased (p<0.05) at 100 and 300 mg/kg/day in males by 16 and 67% , respectively, and in f emales by 8 and 20%. Increased (p<0.05) incidences of hepatocellu lar adenomas were observed in the 100 and 300PIPERONYL FINAL

Page 21 of 54 mg/kg/day males and in the 300 mg/kg/day females. In the 300 mg/kg/day males, incidences of hepatocellular carcinomas were increased (NS).

In a chronic toxicity study in dogs, PBO decr eased final body weights by 8 and 19% in the 600 and 2000 ppm males and by 5 and 25% in the 600 and 2000 ppm females. Food consumption was decreased by 15 and 20% in the 600 and 2000 ppm males. Alkali ne phosphatase was increased in the 600 ppm males ( ↑ 60-64%; NS) and in the 2000 ppm males ( ↑ 340-539%; p<0.05) and females ( ↑ 368-612%; NS) at both intervals. Abso lute liver weights were increased (NS) in the 2000 ppm males ( ↑ 22%) and in the 600 and 2000 ppm females ( ↑ 12 and 36%), and mild hepatocellular hypertrophy wa s observed in the 2000 ppm males (3/4) and females (4/4).

In a subchronic inhalation toxicity study in rats , an increased incidence of secretory signs, such as dried red and brown material on the face, dried nasal discharge, and matted coat, were noted at 0.155 mg/L PBO, and the incidence of these findings increased at 0.512 mg/L. At 0.512 mg/L, columnar epithelium-squamous/squamoid metaplasia /hyperplasia in the vent ral diverticulum was observed. In addition, mucosal stratified squamo us epithelium hyperplasia and hyperkeratosis of the larynx was also noted. Absolute liver wei ghts were increased (NS) at 0.150 and 0.512 mg/L in the males ( ↑ 6 and 23%; p<0.05 at 0.512 mg/L) and females ( ↑ 5 and 20%). Absolute kidney weights were increased (NS) in the 0.512 mg/L males ( ↑ 7%) and females ( ↑ 6%).

Systemic toxicity was not observed in the 21-day dermal toxicity study in rabbits dosed with PBO at up to 1000 mg/kg/day.

In an avian reproduction toxicity study with qua il, it was stated that male body weights were decreased (p<0.05) at 1500 and 3000 ppm PBO; however, the decreases were slight ( ↓ 6% and ↓ 2%, respectively) and not dose-dependent. In an avian reproduction toxi city study in ducks, treatment related signs including weakness, inab ility to stand, subdued behavior and several mortalities, in addition to reductions (p<0.05) in male ( ↓ 20%) and female ( ↓ 18%) body weights, and reductions (p<0.05) in food consumption ( ↓ 19%) were observed at 1200 ppm PBO.

In a fish early life stage toxi city study with fathead minnow s, at the two highest test concentrations (0.42, and 0.80 mg ai/L), decreases of 11 and 20% in body length were reposted compared to the control. In the fish multi-gen eration life-cycle with fathead minnow, based on a preliminary review of the st udy, the study author reported a si gnificant decrease in parental female body weight at the low and high-concentr ation and decrease in male body length at the mid and high-treatment. In the F1 generation, larvae-juvenile survival was significantly
decreased at all test concen trations (9.5-14.5%), F1 male body weight, length, and GSI was increased at 0.106 mg/L, and F2 4-week post hatc h body weight and length were reduced at the mid and high-treatment.PIPERONYL FINAL

Page 22 of 54 B. Estrogen Pathway

For the estrogen pathway, there was no convincin g evidence of an interaction of PBO. It was negative in the estrogen receptor (ER) bindi ng, ER transactivation assay (ERTA), and the aromatase assays. In the ster oidogenesis assay, there was a small (1.11- to 1.31-fold) but significant increase in estradiol concentrations at the highest attainable test concentration (10 μM). PBO was negative in the uterotrophic assay a nd in the female pubertal assay in the absence of overt toxicity. There were no estrogen-related effects in the mammalian Part 158 studies or the avian reproduction study in the ab sence of overt toxicity. In th e fish short-term reproduction assay (FSTRA), while increased fecundity was observed at the middle (14%) and high (19%) treatment concentrations, there was no other female effects observed (lack of complementarity of evidence within the assay), and no other estrogen -related effects observed in the rest of the in

vivo Tier 1 battery in the absence of overt toxicity.

C. Androgen Pathway

There was no convincing evidence of interacti on of PBO with the androgen pathway. PBO was as negative in the AR binding and steroidogenesi s assays. There were no effects observed in the Hershberger assay and in the male pubertal assay in the absence of overt t oxicity. There were no androgen-related effects in the mammalian Part 158 studies. In the FST RA, while there was a reported increase of minimal to mild testes degeneration in the middle and high treatment concentrations (0.03 and 0.09 mg a. i/L, respectively) and a signifi cant increase in male tubercle score at only the low concentration, there were no other complementarity findings. Additionally, while there were no clinical signs of toxicity observed in the FSTRA, a 17% decrease in male body weight at the highest test concentration wa s observed which may be indicative of potential stress. Additionally, there were no other effects observed in the Tier 1 in vivo battery in the absence of overt toxicity.

D. Thyroid Pathway

There was no convincing evidence of interaction of PBO with the thyroid pathway. There was no complementarity of evidence for the histopathologi cal changes seen in the male and female rats in the pubertal assays since no treatment-relat ed effects were seen in thyroid hormones (T 4 or TSH) or thyroid weights. No thyroid-related effects were seen in the mammalian Part 158 studies, except for a histopatholog ical effect in the thyroid in the chronic rat study. Furthermore, there was no evidence of thyroid-rela ted effects observed in the AMA.

E. Conclusions

The conclusion of the WoE evaluation is that PBO demonstrates no convincing evidence of potential interaction with the es trogen, androgen or thyroid pathways in mammals or wildlife.PIPERONYL FINAL

Page 23 of 54 V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not
recommended for PBO since there was no convinci ng evidence of potential interaction with the estrogen, androgen or thyroid pathways.PIPERONYL FINAL

Page 24 of 54 VI. References

Chun, J, and Neeper-Bradley, T (1991) Developmen tal toxicity evaluation of piperonyl butoxide administered by gavage to CD ® (Sprague Dawley) rats. Union Carbide, Bushy Run Research Center, Export, PA. Laboratory Study No. 54-586, December 20, 1991. Unpublished. (MRID 42380801)

Fort, D (2012) 21-Day amphibian metamorphosis assay. Fort Environmental Laboratories, Inc., Stillwater, OK 74074. Sponsored by Consumer Speci alty Products Associ ation, Inc. for the Piperonyl Butoxide Task For ce II, Washington, DC. Laborator y Report No. PBTF01-00256, July 6, 2012. Unpublished. (MRID 48673101)

Fort, D (2012) 21-Day fish reproduction assay. Fort Environmental Laborator ies, Inc., Stillwater, OK, Golden Pacific Laboratori es, Inc., Fresno, CA, and Experi mental Pathology Laboratories, Inc., Sterling, VA. Sponsored by Consumer Sp ecialty Products Association, Inc. for the Piperonyl Butoxide Task Force II, Washi ngton, DC. Laboratory Re port No.: PBTF01-00257, November 8, 2012. Unpublished. (MRID 48687701)

Fujitani, T, Tada, Y, and Yoneyama, M (1993) He patotoxicity of piperonyl butoxide in male

F344 rats. Toxicology 84 : 171-183.

Fujitani, T, Tanaka, T, Hashimoto, Y, et al . (1992) Subacute toxicity of piperonyl butoxide in
ICR mice. Toxicology 83 : 93-100.

Goldenthal, E (1992) 21-Day repeated dose dermal toxicity study with piperonyl butoxide in rabbits. International Research and Developmen t Corporation, Mattawan, MI. Sponsored by the Piperonyl Butoxide Task Force II. Laboratory Study No. 542-007, February 11, 1992. Unpublished. (MRID 42218201)

Goldenthal, E (1993) Evaluation of piperonyl butoxi de in a one year chro nic dietary toxicity study in dogs. International Research and De velopment Corporation, Mattawan, MI. Sponsored by the Piperonyl Butoxide Task Force. Laboratory Study No. 542-005, September 9, 1993. Unpublished. (MRID 42926002)

Graham, C (1987) 24-Month dietar y toxicity study and carcin ogenicity study of piperonyl
butoxide in the albino rat. Bi o-Research Ltd., Senneville, Quebec, Canada. Sponsored by the Piperonyl Butoxide Task Force. Laboratory Study No. 81690, August 27, 1987. Unpublished. (MRID 40323701)

Hermanski, S, and Wagner, C (1993) Chronic dietary oncogenicity study with piperonyl
butoxide in CD-1 ® mice. Union Carbide, Bushy Run Res earch Center, Export, PA. Laboratory Study No. 91NO134, August 27, 1993. Unpublished. (MRID 42903701)PIPERONYL FINAL

Page 25 of 54 Kennedy, G, Smith, S, Kinoshita, F, et al . (1977) Teratogenic evalua tion of piperonyl butoxide
in the rat. Food Cosmet. Toxicol

. 15 : 337-339.

Machado, M (1994) Piperonyl Butoxide Technical Task Force Blend PB200 – the toxicity to fathead minnow ( Pimephales promelas ) during an early life-stage exposure. Springborn Laboratories, Inc., Wareham, MA. submitted by the Piperonyl Butoxide Task Force IT, c/o McLaughlin Gormley King Co., Minneapolis , MN. Laboratory Report ID: 94-5-5264. Unpublished. (MRID 43300301)

Newton, P (1992) A subchronic (3-mont h) inhalation toxicity study of piperonyl butoxide in the rat via whole body exposures. Bio/dynamics, Inc ., East Millstone, NJ. Sponsored by Piperonyl Butoxide Task Force II, c/o McKenna and C uneo, Washington, DC. Laboratory Study No. 91- 8333. Unpublished. (MRID 42477101)

Robinson, K, Pinsonneault, L, and Proctor, B (1 986) A-two generation (t wo-litter) reproduction study of piperonyl butoxide administered in the diet to the rat. Bio-Re search Laboratories Ltd., Canada. Sponsored by the Piperonyl Butoxi de Task Force. Laboratory Study No. 81689. Unpublished. No MRID (Accession No. 263635, -6, -7, -8, -9,-40)

Rodgers, M, Cameron, D, Healey, G, et al . (1995) Piperonyl butoxide : effects on reproduction in bobwhite quail after dietary administration. Huntingdon Res earch Centre Ltd., Huntingdon, Cambridgeshire, UK. Sponsored by the Piperonyl Butoxide Task Force II, c/o S.C. Johnson & Son, Inc., Racine, WI. Laboratory Repor t ID: PBT 2/950167. Unpublished. (MRID 43876301)

Rodgers, M, Cameron, D, Healey, G, et al . (1995) Piperonyl butoxide : effects on reproduction in mallard duck after dietary administration. Huntingdon Reserch Centre Ltd., Huntingdon, Cambridgeshire, UK. Sponsored by the Piperonyl Butoxide Task Force II, c/o S.C. Johnson & Son, Inc., Racine, WI. Laboratory Repor t ID: PBT 2/950168. Unpublished. (MRID 43876302)

Schardein, J (1986) Teratology study in rabbits . International Resear ch and Development Corporation, Mattawan, MI. Laboratory Study No. 542-002, February 7, 1986. Unpublished. (MRID 00026192)

Tanaka, T, Fujitani, T, Takahashi, O, et al . (1994) Developmental t oxicity evaluation of piperonyl butoxide in CD-1 mice. Toxicology Letters 71 : 123-129.

Tanaka, T, Fujitani, T, Takahashi, O, et al . (1995) Developmental toxi city study of piperonyl
butoxide in CD rats. Toxicol. Ind. Health 11 (2) : 175-184.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy. U.S. Environmental Protection Agency, Washington, DC.PIPERONYL FINAL

Page 26 of 54 USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H (2011) Piperonyl butoxide: H295R st eroidogenesis assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9146V-100377STER, November 17, 2011. Unpublished. (MRID 48618807)

Weight of Evidence Evaluation for EDSP Tier 1 Screening of Piperonyl Butoxide The Piperonyl Butoxide Task Force II. MRID No.48702501

Wilga P (2011) Piperonyl butoxide: human r ecombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Report N o.: 9146V-100377AROM, November 17, 2011. Unpublished. (MRID 48618802)

Willoughby, J (2011). Piperonyl butoxide: androgen receptor binding (rat prostate cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9146V-100377ARB, November 17, 2011. Unpublished. (MRID 48618801)

Willoughby, J (2011). Piperonyl butoxide: estrogen receptor binding (rat uterine cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9146V-100377ERB, November 17, 2011. Unpublished. (MRID 48618803)

Willoughby, J (2011) Piperonyl butoxide: estrogen receptor transcriptional activation (human cell line (HeLa-9903)). CeeTox, Inc., Kala mazoo, MI. Laboratory Report No.: 9146V100377ERTA, November 17, 2011. Unpublished. (MRID 48618804)

Willoughby, J (2012). Supplemental information – saturation binding proficiency information provided by CeeTox. CeeTox, Inc., Kalamaz oo, MI. July 19, 2012. Unpublished. (MRID 48843501)

Zorilla, L (2011). The Hershberger bioassay fo r piperonyl butoxide. In tegrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C192-200, November 10, 2011. Unpublished. (MRID 48618805)

Zorilla, L (2011). Pubertal development and thyr oid function in intact juvenile/peripubertal female and male rats with pipe ronyl butoxide. Integrated Laborato ry Systems, Inc., Durham, NC. Laboratory Project Study ID: C192-300, N ovember 10, 2011. Unpublished. (MRID 48618806)PIPERONYL FINAL

Page 27 of 54 Zorilla, L (2011). The uterotrophic assay for piperonyl butoxide (PBO). Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No.: C192-100, November 18, 2011 (revised report date). Unpublished. (MRID 48618808)PIPERONYL FINAL

Page 28 of 54 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRIDs 48673101 and 49606701) of piperonyl butoxide (PBO; 93.7% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/group) were exposed to nominal concentrations of 0 (n egative control), 0.011, 0.033, and 0.100 mg a.i./L; time-weighted average (TWA) concentrati ons were <0.0010 (<LOQ; control), 0.00599, 0.0263, and 0.0741 mg a.i./L. The test system was mainta ined at 21.4 to 21.9°C and a pH of 7.3 to 7.9.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. The survival of tadpoles exposed to PBO was not significa ntly affected (p>0.05) as survival was 100% in the low treatment group w ith one mortality each in the control and mid and high treatment groups. There were no clinical si gns of toxicity or observations of abnormal
behavior.

PBO caused no significant accelerati on or delay of median NF deve lopmental stage at Day 7 or 21 in any treatment group. No asynchronous development was observed. By test termination on Day 21, the median NF stage was Stage 62 for the control group and 61 for each of the treatment groups. All but four test organisms we re considered to be late stage ( i.e. , above Stage 60) at test termination. Therefore, Day 21 analyses were conducted using all ta dpoles (there were insufficient non-late-stage tadpol es for separate analyses).

Day 7 wet weight and snout-vent length (SVL) we re significantly decreased across all treatment groups compared to the negative control. There were no significant incr eases or decreases for Day 21 wet weight or SVL, or for Day 7 or 21 hind limb length (HLL) or normalized (for snoutvent length) HLL.

Effects on thyroid gland histopat hology were observed in the c ontrol and all treatment groups, and included mild follicular cell hyperplasia and hypertrophy. There were no treatment-related
patterns.

All performance and validity cr iteria were met in this st udy. Although all but four of the surviving frogs were ≥ NF stage 60 at test termination, which may confound the ability to adequately evaluate potential thyroid-related effects in the AMA, there was no significant acceleration or delay of median NF developmental stage at any treatment concentration throughout the test or effect s on thyroid histopathology co mpared to the control.PIPERONYL FINAL

Page 29 of 54 Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48618801), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of piperonyl butoxide (93.8% a.i., Batch #E104/10).

DMSO was used as the vehicle at a final assay concentration of approximately 3.2%. A total of three independent runs were performed, and th e assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference androgen (R 1881). Saturation binding data we re not originally provided in the study report; however, summar ized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three K d values was within the range repor ted in the EPA validation program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein). Confidence in these numbers is high based on the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, precipitat ion of piperonyl butoxide was visually observed at the highest concentration (10

− 3 M) tested in all three runs, th erefore the highest concentration
used for data evaluation was 10

− 4 M. The estimated average log IC 50 was

− 9.3 M for R1881 and

− 4.4 M for the weak positive control (dexamethasone ). Compared to R1881, the mean RBA for
dexamethasone was 0.0022% for the two valid runs (excluding the first run). All performance
criteria were met for two of the three runs. The concentr ations prepared for R1881 and dexamethasone were lower than the targeted (guideline) levels in the first run because of an error in the stock preparation. Therefore, the curve for dexamethasone was not fully defined (bottom percent binding was not achieved). The range of concentrations for R1881 in the first run fully defined the curve, but the concentration range wa s off by more than an order of magnitude. An

IC 50 and relative binding affinity (RBA) could not be calculated for piperonyl butoxide as it did not competitively bind to the AR.

The percent labeled R1881 bound to the AR in the presence of pi peronyl butoxide was >75% at
concentrations ≤ 10

− 4 M in all three runs. As speci fic binding was not <50% at any concentration, an IC 50 and RBA could not be calculated for piperonyl butoxide. Based on the results from the three runs, piperonyl butoxide is classified as a Non-Binder in the Androgen Receptor Binding Assay.PIPERONYL FINAL

Page 30 of 54 Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48618802), piperonyl butoxide (93.8% a.i. Lot # E104/10) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

- 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in ≤ 1% dimethyl sulfoxide (DMSO) at log
concentrations of 10

− 10 to 10

− 3 M for 15 minutes to assess the poten tial of piperony l butoxide to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counti ng (LSC). Three runs were co nducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandroste nedione; 4-OH ASDN) in duplicate, and the piperonyl butoxide series (10

− 10 to 10

− 3 M) with three replicat es per concentration.

Aromatase activity in the full activity controls was 0.207±0.025 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity c ontrol within a run was between 90 to 110% of the average full activity. Activity in the backgr ound controls ranged from 0.52 to 0.78% and averaged 0.63% of the full activity controls.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.13 M and the Hill slope was

− 1.02.

For piperonyl butoxide, the two high est tested concentrations (10

− 3 and 10

− 3.5 M) showed precipitation, and the results for the lowest test concentration (10

− 10 M) were considered by the study author to be a statistical outlier ( ≤ 51% of the control); therefore, the 10

− 4 and 10

− 9 M test
concentrations were selected as the highest and lowest test con centrations to be used for study interpretation. For piperonyl butoxide, the lowest portion of the response curve across runs was greater than 75% activity at all soluble con centrations. Aromatase activity was 95.9 % and
85.9% of the control at 10

− 9 and 10

− 4 M, respectively.

Based on the data from the average response curv e, piperonyl butoxide is classified as a Noninhibitor of aromatase ac tivity in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48618803), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiment s. A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present inPIPERONYL FINAL

Page 31 of 54 reasonable numbers and was functioning with appropr iate affinity for the radiolabeled reference estrogen prior to routinely conducting ER comp etitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β

estradiol (1 nM) in the presence of increasing concentrations (10

− 11 M to 10

− 3 M) of piperonyl butoxide (93.8% a.i., Batch #E104/10 ). DMSO was used as the ve hicle at a final concentration of approximately 2%. A total of three runs were performed, and each run included 19-norethindrone as a weak positive control, oc tyltriethoxysilane as a negative control, and 17 β

estradiol as the natural ligand reference material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. Data from two separate saturation binding assays were reported due to the use of two separate cytosol preparations during this assay for piperonyl butoxide. For the batch of cytoso l prepared on September 27, 2010, the K d for [ 3 H]-17 β

estradiol was 0.081±0.006 nM, and the estimated B max was 52.70±1.05 fmol/100 μg protein. For the batch of cytosol prepared on July 10, 2011, the K d for [ 3 H]-17 β -estradiol was 0.303±0.090 nM, and the estimated B max was 33.59±0.70 fmol/100 μg protein. The K d for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard pl ots fit straight lines to the data, the concavity observed with the data sets from the July 2011 cytosol preparations may
indicate issues with ligand depletion.

In the competitive binding experiment, the estimated mean log IC 50 was

− 9.2 M for the natural ligand (17 β -estradiol) and

− 5.6 M for the weak positive control (19-norethindrone). Compared to the natural ligand, the mean RBA was 0.026% for 19-norethindrone.

Precipitate was observed at 10

− 3 M piperonyl butoxide in the firs t two runs; therefore, data from
the 10 –3 M concentration were not evaluated. The hi ghest concentration test ed in the third run

was 10 –4 M. The mean specific binding in the pr esence of piperonyl butoxide was >77% at
concentrations ≤ 10

− 4 M in all three runs. . The lowest average percent [ 3 H]-17 β -estradiol binding in the presence of pi peronyl butoxide was >75% at concentrations up to 10

− 4 M, and therefore a mean log IC 50 and Relative Binding Affinity (RBA) were not calculated.

Based on the three runs, piperony l butoxide is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48618804), hER α -HeLa-9903
cells cultured in vitro were exposed to piperonyl butoxide (93.8% a.i., Lot# E104/10 ) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M in 0.1% DMSO forPIPERONYL FINAL

Page 32 of 54 approximately 24 hours in two independent runs . The experiments were performed using 96- well plates and each piperonyl butoxide concentrati on was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to indu ce reporter (luciferase) gene products. Luciferase expression in res ponse to activation of the estrogen receptor was measured using a proprieta ry luciferase assay.

Piperonyl butoxide was tested up to the limit of cytotoxicity, which was noted at 10

− 4 M in one run. At concentrations up to 10

− 5 M, the relative transcriptiona l activation (RTA) of piperonyl
butoxide was ≤ 2.8%.

In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, piperonyl butoxi de responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to
reach a maximum of 16.5-24.3% PC . Piperonyl butoxide was only able to reach a maximum of 1.9-2.8% PC when tested up to the highest c oncentration possible ba sed on cytotoxicity.
Because the RPC Max < PC 10 in both assay runs, piperonyl buto xide was considered negative for estrogen receptor transcriptional ac tivation in this test system.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (MRI D 48687701) of piperonyl butoxide (PBO) with fathead minnows ( Pimephales promelas) was conducted under c ontinuous flow-through conditions. Adult fish, 16 spawning groups (2 male s and 4 females in each group; 4.5-6 months old), were exposed to PBO (93.7% purity) at nominal concentrations of 0 (control), 0.011, 0.033, and 0.100 mg a.i./L; time-weighted average (T WA) measured concentrations were <LOQ (<0.0010 negative control), 0.0101, 0.0348, and 0.0944 mg a.i./L. The test system was
maintained at 24.4 to 25.5 o C and a pH of 7.3 to 8.1.

Survival was 100% in all treatment groups throug hout the study. Body weight of male fish was
decreased in a concentration -response pattern by 3%, 8% and 17% at the low, mid, and high treatment concentrations, respectively. Only the d ecrease at the high treatment concentration was significantly different from the negative contro l. No statistically significant effects were observed on female body weight or on male or female length. There were no notable observations with regards to behavior or chan ges in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups, or clinical signs of toxicity for any treatment group compared to the controls.PIPERONYL FINAL

Page 33 of 54 Spawning frequency was at least two times in 7 da ys and mean fecundity in the negative control was 33.5 eggs/female/day/replicate (range 33.0-34.3); fertilization success in the negative control group averaged 99% (range 98.8-99.1 %). There was a significant increa se in fecundity at the mid and high treatment levels of 14 and 19%, resp ectively, compared to the negative control. Additionally, median male tubercle score at the low treatment leve l was significantly greater than
the median male tubercle score in the cont rol; higher levels were unaffected by treatment (p>0.05). There was no other signi ficant difference between the treatment levels and negative controls for any other endpoint. Male vitellogeni n (VTG) was not successfully measured due to insufficiently sensitive methodology. The sole hist opathology finding that appeared related to PBO exposure was increased testicular degenerati on (minimal to mild) in males. The prevalence of this finding was higher than controls in th e high treatment group, but only marginally higher in the mid treatment group. There were no apparent differences between hi stopathology severity scores or gonadal stages in the PBO-treated groups compared to the negative controls. Sex steroids (17 β -estradiol and testos terone) were not meas ured in this study.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger assay (MRID 48618805) screeni ng for androgenic activit y, piperonyl butoxide (93.8% a.i., Lot# E104-10) in corn oil was administ ered daily via oral gava ge to groups of 58- to 59-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 0 (vehicle), 320, or 1000 (limit dose) mg/kg/day. An androgenic positiv e control group consisting of eight castrated rats exposed to 0.4 mg/kg/day testosterone propio nate (TP) in corn oil by subcutaneous (s.c.) injection was also included. To screen for pot ential anti-androgenic ac tivity, piperonyl butoxide in corn oil was administered daily via oral gavage to 58- to 59-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 100, 320, or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The negative control group consisted of eight castrated male rats dosed dail y with TP (0.4 mg/kg/day) by s.c. injection, and the anti-androgenic positive cont rol group consisted of eight cast rated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day).

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

In the androgen agonist assay, no adverse, treatm ent-related findings were observed on mortality, clinical signs, or acce ssory sex organ weight. Body weight s and overall body weight gains for the 320 mg/kg/day treatment group were comparable to controls throughout the duration of the assay. At 1000 mg/kg/day, termin al body weight was decreased (not statistically significant) byPIPERONYL FINAL

Page 34 of 54 only 7%, but overall (Days 1-11) body weight gains were decr eased (p<0.05) by 96%. No
treatment-related increases in th e accessory sex tissue weight we re seen in the animals dosed with piperonyl butoxide.

Animals dosed with TP in the androgen agoni st assay had increased (p<0.05) body weights on Day 11 (increased 9%) and increased (p<0.05) ov erall body weight gains (increased 110%). Animals in the TP group had increases (p ≤ 0.05) in accessory sex organ weights as follows: 1799% in seminal vesicles; 997% in ventral pr ostate; 242% in levato r ani-bulbocavernosus (LABC); 571% in Cowper’s gland; and 53% in glans penis. For the vehicle control, the maximum guideline-specified %CV values were ex ceeded for the ventral prostate (58% vs. 45%) and glans penis (26% vs. 22%). The maximum guideline-specifie d %CV was slightly exceeded for the Cowper’s gland in the 1000 mg/kg/day piperonyl butoxide group (58% vs. 55%). All
other %CV values were less than the maximum recommended values.

In the anti-androgen assay, no adverse, treatment -related findings were observed on mortality, clinical signs, or acce ssory sex organ weight. Terminal body weight was decreased (NS) by 4% at 320 mg/kg/day and 9% at 1000 mg/kg/day. Th e overall (Days 1-11) body weight gains were decreased (p<0.05) by 36% at 320 mg/kg/da y and decreased (p< 0.05) by 62% at 1000 mg/kg/day. The only significant decrease in androgen-sensitive tissue weights observed in animals dosed with piperonyl butoxide + TP, was a 21% (p ≤ 0.05) decrease in LABC weight at 1000 mg/kg/day.

Animals dosed with TP +FT had decreased (p<0.05) overall b ody weight gains ( ↓ 40%). Animals dosed with 0.4 mg/kg/day TP + 3 mg/ kg/day FT in the anti-androgen assay had decreased (p ≤ 0.05) accessory sex organ weights as follo ws: 90% in seminal vesicles; 85% in ventral prostate; 56% in LABC; and 66% in Cowper’s gland. The maximum guideline-specified %CV was exceeded for the glans penis in the negativ e control and for the ventral prostate in the TP + FT positive control. All other %CV values were less than the maximum recommended
values.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Piperonyl butoxide was negative for androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay (OCSPP 890.1450)

In a female pubertal assay (MRID 48618806), 16 fe male Sprague Dawley rats/dose group were treated daily via oral gavage with piperonyl butoxide ( 93.8% a.i., Batch/lot# E104-10) in corn oil at doses of 0 (vehicle), 500, or 1000 (limit dos e) mg/kg/day from post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, andPIPERONYL FINAL

Page 35 of 54 weight at day of attainment was recorded. Following sacrifice on PND 42 or 43, total serum levels of thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay; serum was also analyzed for leve ls of selected clinical chemistry parameters. Adrenal, kidneys, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the ovaries, uterus, t hyroid, and kidneys.

No treatment-related effects were observed on morta lity or clinical signs of toxicity. There were also no treatment-related effects on the weights of pituitary, uterus, or thyroid. With regards to reproductive development, no treatment-related e ffects were observed on the age at VO, weight at VO, and number of animals attaining VO, mean age at first vaginal es trus, mean cycle length, percent cycling, and percent regularly cycling, nu mber of ovary follicles, follicular cysts or corpora lutea.

At 500 and 1000 mg/kg/day, the final adjusted bo dy weight was decreased (p<0.05) by 5 and 7%, respectively, and the adjusted body weight gain was decreased (p<0.05) by 9 and 10%, respectively. In addition, the adjusted and relative liver weight s were increased (p<0.05) dosedependently by 15-35% and 22- 44% at 500 and 1000 mg/kg/ day, respectively, and the unadjusted liver weight was incr eased (p<0.05) by 36% at 1000 mg/kg/day. Compared to the controls, increases (p<0.05) were also noted in the relative kidney weight ( ↑ 6%) and unadjusted, adjusted, and relativ e adrenal weights ( ↑ 20-29%) at 1000 mg/kg/day, a nd the adjusted weights for ovaries were decreased (p<0.05) by 16%. Although significant weight changes were observed in the kidneys and ovaries, no treatment -related histopathological effects were observed in these organs.

Although uterine weights were not affected by treatment, selected histological findings were
noted in uteri of rats in the high dose group. At 1000 mg/kg/ day, moderate atrophy of the myometrium, uterine stroma, and endometrial gla nds was noted in 2 of 14 rats, mild atrophy of the endometrial gland was observed in another tw o rats, and mild luminal dilation was observed in 2 of 14 rats; compared to 0 of 16 rats in the control group.

Although no treatment-related effects were noted on serum levels of T 4 or TSH and thyroid weights were not affected, ther e was a dose-dependent increase in the thyroid gland follicular epithelial cell height with a conc omitant decrease in the colloid area. Follicular cell height was Grade 1 in controls compared to Grades 1–3 at 500 mg/kg/day and Grades 3–5 at 1000 mg/kg/day. Follicular colloid area was Grades 4 –5 in the controls compared to Grades 3–4 at 500 mg/kg/day and Grades 1–3 at 1000 mg/kg/day.

The doses tested were adequate since the hi gh dose was the Limit Dose (1000 mg/kg/day).PIPERONYL FINAL

Page 36 of 54 Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48618806), 16 male Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gava ge with piperonyl butoxide (93.8% a.i., Lot #: E104-10) in corn oil at doses of 0, 500, or 1000 (limit dose) mg/kg/day from post-natal day (PND) 23 to 53/54. The animals were examined for preputial separati on (PPS) daily beginning on PND 30, and body weight at day of attainment was recorded. Following sacrifice on PND 53/54, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were determined using radioimm unoassays. Adrenal, liver, t hyroid, pituitary, and urogenital organ weights were recorded, and microscopic exam inations of the testes, epididymides, thyroid, and kidneys were performed.

Although four rats died during th e study (1 control, 2 low-dose, and 1 high-dose), there were no
treatment-related effects on mortalit y as the deaths were attributed to gavage errors. There were no clinical signs of toxicity in any group, and no treatment-rela ted effects were observed on age at PPS, weight at PPS, and serum levels of T 4 and TSH. There were also no treatment-related histopathological findings noted in the testes, epididymides, or kidneys at either dose.

General growth was decreased in a dose dependent manner. The final body weight (adjusted for body weight on PND 23) was decreased by 5% (not statistically signi ficant [NS]) and 9% (p<0.05) at 500 and 1000 mg/kg/day, respectively. The adjusted overall body weight gain was also decreased by 6% (NS) and 11% (p<0.05) at 500 and 1000 mg/kg/day, respectively.

Relative liver weight was increased (p<0.05) in a dose-dependent manner, by 15% and 34% at 500 and 1000 mg/kg/day, respectively, and unadjusted and adjusted liver we ights were increased (p<0.05) by 16% and 20%, respectively, at 1000 mg/kg/day. Relative ki dney weights were increased (p<0.05) by 8% and 11% at 500 and 1 000 mg/kg/day, respectively, and adjusted and unadjusted pituitary weights were decreased by 16 -17% (p<0.05 or NS) at both dose levels. In addition, adjusted and relative adrenal weight s were increased (p< 0.05) by 15% and 31%, respectively, at 1000 mg/kg/day.

Although thyroid weight was not affected by trea tment, there was a dose-dependent increase in follicular cell height with a concomitant decreas e in colloid area Follicular cell height was shortest in the control (15/15 ra ts at Grades 1-2) and tallest at 1000 mg/kg/day (14/15 at Grades 3-5), and colloid area was largest in the control (15/15 at Grades 4-5) a nd smallest at 1000 mg (12/15 at Grades 1-3), with cell height and coll oid area being intermediate in the 500 mg/kg/day group. There were no significant differe nces in serum thyroid hormone (T 4 and TSH) levels among treated and control groups.

Serum testosterone levels were decrease d by 36% (NS) and 67% ( p<0.05) at 500 and 1000 mg/kg/day, respectively. At 500 mg/kg/day, ad justed LABC weight wa s decreased by 14%PIPERONYL FINAL

Page 37 of 54 (p<0.05). At 1000 mg/kg/day, adjusted and una djusted LABC weights were decreased by 20- 26% (p<0.05), and adjusted and unadjusted weights of the semina l vesicles with coagulating gland (with/without fluid) were decreased by 21-35% (p<0.05).

The dose concentrations used in the study are considered adequate based on the 11% decrease in adjusted body weight grain observed at 1000 mg/kg/day, which also is the dose limit for the

assay.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48618807), H295R cells cultured in vitro in 24-well plates were incubated with piperonyl butoxide

, (PBO; 93.8% purity, Batch # E104/10) at log
concentrations from 0.0001 to 100 μ M in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%.

Testosterone and estradiol concentrations we re measured by HPLC/MS-MS with positive ion multiple reaction monitoring. Four independent experiments were performed. A Quality Control (QC) plate was run concur rently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels; positive controls include d the known inhibitor (p rochloraz) and indu cer (forskolin) of
estradiol and testos terone production.

Laboratory proficiency testing was not conducted in the current study. The amendment stated that the laboratory proficiency data was previous ly determined for this batch of cells; however, details of a previous proficiency de termination, including estimates of EC 50 s for prochloraz and
forskolin were not included in the study report. Data from the highest tested PBO concentration, 100 μM, were not evaluated due to precipitation that was observed post-exposure.

All guideline acceptability requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate
induction and inhibition with positive controls.

No statistically significant effects on testosterone pr oduction were observed in three out of four
test runs after incubation with PB O. However, four of the six PB O concentrations in the fourth run exhibited statistically signi ficant decreases (0.87- to 0.91-fol d) in testosterone production,
but these decreases were not dose related and are not considered an effect of PBO exposure. Statistically significant increases in estradiol production ( 1.11- to 1.31-fold) were observed in three test runs (with a non-signi ficant increase of 1.45-fold in a fourth test run) at the PBO concentration of 10 μM. The remaining PB O concentrations in Run 4 (0.0001 to 1 μM) exhibited statistically signifi cant decreases (0.78- to 0.89-fold) in estradiol production, but thesePIPERONYL FINAL

Page 38 of 54 decreases are not considered an effect of PBO exposure since no effect was observed in the previous three runs.

Based on the hormone responses in at least thre e independent runs, PBO treatment resulted in statistically significant and repr oducible increases in estradiol production, but did not result in statistically significant and reproducible alterations in testosterone production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48618808) conducted to screen for potential estrogenic activity, piperonyl butoxide (93.8% a.i., Batch #E104/10) in corn oil was administered daily via oral gavage to groups of eight ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 320 or 1000 (limit dose) mg/kg/da y on post-natal days (PND) 58-60. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 0.1 mg/kg/day by oral gavage. Body weights were determined dail y. All animals were terminated and necropsied on PND 61 approximately 24 hours after the fina l dose administration to determine wet and blotted uterine weights.

All animals survived until sche duled termination. Two animals administered piperonyl butoxide at 320 mg/kg exhibited abnormal clinical signs (uncoordinated movements or lethargy) during dosing, but all animals were noted as normal prio r to termination. All animals dosed with 1000 mg/kg piperonyl butoxide group exhi bited abnormal clinical signs (decreased or uncoordinated movements, hunched posture, lethargy, and/or nasal discharge) by Day 3 of dosing. At termination, all high dose animals were noted as normal, except for one rat which exhibited multiple signs of toxicity.

Body weights were decreased (p<0.05) on Day 4 ( ↓ 10%) in the high dose group compared to the vehicle controls, resulting in a decrease (p<0.05) in overall body weight gain (BWG; ↓ 240%). Body weights in the low-dose group were comparab le to the vehicle c ontrols throughout the duration of the assay. Statistical significance was not determined for the body weights and BWG of animals in the positive control (EE) group. However, body weights were decreased by Day 4 ( ↓ 7%) in the EE group compared to the vehicle cont rols, with a decrease in overall body weight gain ( ↓ 148).

Uterine weights in all piperonyl bu toxide treated groups were compar able to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 348% and 173%, respectively, as expected.

No statistically significant changes were seen in uterine weight in this assay. Piperonyl butoxide is negative in the ut erotrophic assay.PIPERONYL FINAL

Page 39 of 54 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a multi-generation reproduction toxicity study ( Robinson et al ., 1986 ), 26 Sprague Dawley
rats/sex/dose were administered PBO (purity not reported) in th e diet at dose levels of 0, 300, 1000, or 5000 ppm (equivalent to 0, 15, 50, and 250 mg/kg/day, calculated by reviewer). Dosing with test diet started at 42 days of age for the P generation during the pre-mating period; with exposure continuing in dams through gestation, lactation and weaning. The P generation rats were mated to produce F 1a and F 1b litters, and selected F 1b rats were mated to produce F 2a and F 2b litters.

There were no treatment related effects on mortality or clinical signs in adults or pups. In the 5000 ppm group, decreases (p<0.05) in body weight gain were observed for the adults and pups. Occasional significant body weight decreases were observed in the 1000 ppm group, but this
effect was not considered treatment related. Corresponding decreases in food consumption were
observed. There were no treatment related eff ects based on reproductive performance (mating, fertility, or conception indices), pup development (viability, surviv al, or lactation indices), or gross pathology. No data was provided re garding organ weight or pathology.

The LOAEL for parental and offspring toxic ity is 5000 ppm (equivale nt to 250 mg/kg/day), based on decreased body weight gain and f ood consumption. The NOAEL for parental and offspring toxicity is 1000 ppm (equivalent to 50 mg/kg/day).

Two-Generation Reproduction and Neur obehavioral Toxicity Study in Mice

In a non-guideline behavioral, reproductive, and developmental toxicity study in mice ( Tanaka
et al ., 1992 ), 10 CD-1 (Crj:CD-1) mice/sex/dose group were administered PBO (purity not provided) in the diet at dose levels of 0, 1000, 2000, 4000, or 8000 ppm (equivalent to 0/0, 174/214, 344/387, 650/752, and 1150/1275 in males/fema les during pre-mating). The purpose of this study was to provide data related to the reproductive toxici ty effects on the P generation mice and the behavioral and developm ental toxicity effects on the F 1 generation mice. Dosing began at 5 weeks of age and continued through pr e-mating (4 weeks), mati ng (5 days), gestation, and lactation. Food consumption was measured in the parental animals. For the F 1 and F 2 generations, the numbers of litters and pups, litter size and weight, and sex ratio were measured at birth (PND 0), and the pups were weighed on PND 0, 4, 7, 14, and 21. The F 1 pups were removed from their dams on PND 28, and 10 pups/s ex/dose group were mated at 9 weeks of age (avoiding sibling matings) to produce the F 2 pups. The following neurobehavioral tests were performed on the F 1 and F 2 pups: surface righting and negative geotaxis at PND 4 and 7, cliff avoidance at PND 7, swimming beha vior at PND 4 and 14, and olf actory orientati on at PND 14.PIPERONYL FINAL

Page 40 of 54 Food consumption was decreased in the 8000 pp m P males and females during pre-mating, and in the 4000 and 8000 ppm P and F 1 females during lactation. No additional data was provided for the parental animals.

In the F 1 pups, the 8000 ppm litter weights were decrea sed (p<0.05) by 38%; litter size and sex ratio were similar to controls. Male pup weights were decreased (p<0.05) as follows: ↓ 9-21% during PND 0-21 in the 1000 ppm group; ↓ 19-25% during PND 7-21 in the 4000 ppm group;
and ↓ 13-52% during PND 0-21 in the 8000 ppm group. Female pup weights were decreased (p<0.05) as follows: ↓ 11-21% during PND 4-21 in the 1000 ppm group; ↓ 15-16% during PND 14-21 in the 2000 ppm group; ↓ 17-26% during PND 7-21 in the 4000 ppm group; and ↓ 16-45% during PND 0-21 in the 8000 ppm group. The PN D 21 survival index was decreased at 8000 ppm in both males (63.0% treated vs. 90.6% c ontrols) and females (78.9% treated vs. 88.7% controls). Neurobehavioral parameters were unaffected by treatment.

In the F 2 pups, litter size was decr eased (p<0.05) in the 4000 ( ↓ 21%) and 8000 ( ↓ 47%) ppm groups; litter weights were de creased (p<0.05) in the 1000 ( ↓ 15%), 4000 ( ↓ 25%), and 8000 ( ↓ 57%) ppm groups. Sex ratios were similar to controls. Male pup weights were decreased (p<0.05) as follows: ↓ 7-8% during PND 4-7 in the 1000 ppm group; ↓ 9-16% during PND 4-21 in the 2000 ppm group, ↓ 12-19% during PND 4-21 in the 4000 ppm group; and ↓ 20-41% during PND 0-21 in the 8000 ppm group. Female pup weight s were decreased (p<0.05) as follows: ↓ 8% during PND 7 in the 1000 ppm group; ↓ 10-14% during PND 4-21 in the 2000 ppm group; ↓ 9- 15% during PND 4-21 in the 4000 ppm group; and ↓ 20-32% during PND 0-21 in the 8000 ppm group. The PND 21 survival index was decreased at 8000 ppm in both males (59.4% treated vs. 100% controls) and females (79.2% treated vs. 100% controls). Deficits were noted in surface righting and cliff avoidance in all treated groups, and in olfact ory orientation in the 2000, 4000, and 8000 ppm groups.

One-Generation Reproduction and Neur obehavioral Toxicity Study in Mice

In a non-guideline behavioral, reproductive, and developmental toxicity study in mice ( Tanaka,
1992 ), PBO (purity not provi ded) was administered in the di et to 10 CD-1 mice/sex/dose group at doses of 0, 1500, 3000, or 6000 ppm (equivalent to 0, 225, 450, and 900 mg/kg/day, calculated by reviewer). The purpose of this study was to provide data related to the behavioral and reproductive toxicity effects on the P generation mice and the be havioral and developmental toxicity effects on the F 1 generation mice. Treatment of the P generation began at 5 weeks of age. At 8 weeks of age, the animals were observe d in the open-field. At 9 weeks of age, they were mated (1 male:1 female). Litter observation s (number of litters, number of pups, litter size, pup weight, and litter weight) were made, and the offspring were weaned at 4 weeks of age. Survival and pup weight were assessed on postnatal day (PND) 4, 7, 14, and 21. Functional developmental parameters (surface righting, nega tive geotaxis, swimming, cliff avoidance, and olfactory orientation) were ev aluated on PND 4, 7, and/or 14. Th e offspring were evaluated inPIPERONYL FINAL

Page 41 of 54 the open-field at 3 and 8 weeks of age. At 6 w eeks of age, the pups were evaluated in a water

maze.

Parental behavioral effects were noted at 6000 ppm in the males and included decreased ambulation ( ↓ 41%; p<0.01) and rearing ( ↓ 45%; NS).

The LOAEL for parental toxicity is 6000 ppm, based on decreased ambulation and rearing. The NOAEL is 3000 ppm.

Offspring systemic toxicity was indicated. Decr eased (p<0.001) pup weight s were observed at all doses, but were considered adverse at 6000 ppm ( ↓ 13%). In the 6000 ppm group, decreased (NS) number of pups ( ↓ 14%) and litter weight were noted ( ↓ 16%). The survival indices at PND 4, 7, and 14 were 62.1-67.8% at 6000 ppm compared to 79.2% in the controls. Th e survival index at PND 21 was 51.7% at 6000 ppm compared to 79.2% in the controls. De creased (p<0.05) pup body weights were noted at 3000 ppm on PND 7, 14, and 21 ( ↓ 7-12%) and at 6000 ppm on PND 4, 7, 14, and 21 ( ↓ 24-41%).

Developmental and behavioral e ffects were observed in the offs pring. The olfactory orientation
test resulted in decreased (p<0.01) values in the 3000 and 6000 ppm groups (0.93-0.96) compared to controls (1.58) on PND 14, indicating an increased latency to enter the home wood shavings compartment. At 3 weeks of age, decreased ambulation was observed in the 3000 ( ↓ 33%; p<0.05) and 6000 ppm males ( ↓ 29%; NS).

Rearing in the open-field test was decreased in all male dose groups by 36-38% (p<0.05; except at 3000 ppm), without an apparent dose-response relations hip. Rearing was also decreased (NS) by 43% in the 6000 ppm females. Because the eff ect on rearing was unrelated to dose and/or without statistical significance, this effect was considered eq uivocal. At 8 weeks of age, increased (NS) preening was obs erved in the 6000 ppm males ( ↑ 219%). As the effect on preening was not statistically signi ficant and the biological significan ce is unclear, this effect was considered equivocal.

The LOAEL for offspring toxicity is 3000 pp m, based on decreased pup weight, olfactory orientation, and ambulation. The NOAEL is 1500 ppm.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 42380801 ), groups of 25 pregnant Sprague Dawley (Crl:CD BR) rats were administered undiluted PBO (90.8% purity) by oral gavage at dose levels of 0, 200, 500, or 1000 mg/kg/day du ring gestation days (GD) 6-15. Dams were euthanized on GD 21, and their fetuses were removed by caesarean section. Examinations at sacrifice consisted of evaluation of body weight, liver and gravid uterine weight, number of corpora lutea, and number and stat us of implantation sites (incl uding early and late resorptions,PIPERONYL FINAL

Page 42 of 54 dead fetuses and live fetuses). All live fetuses were counted, weighed, sexed and examined for
external malformations. About one -half of the fetuses were exam ined for thoracic and abdominal visceral abnormalities. These fetuses were decap itated and the heads were fixed in Bouin's
solution and examined for soft tissue craniofaci al malformations and variations. The remaining
half of the fetuses were fi xed in ethanol, stained with alizarin red S and examined for skeletal
malformations and variations.

There were no maternal deaths during the st udy, and no treatment-rela ted observations were noted at necropsy. At 1000 mg/ kg/day, treatment-related clini cal signs included urogenital wetness, urine stains, red discharge, and pe rinasal encrustation. At 500 mg/kg/day, treatmentrelated clinical signs included re d urogenital discharge (2 animals) and perinasal encrustation (1 animals). Body weight gains were decreased (p<0.05) by 51% in the 1000 mg/kg/day group during GD 6-9, and by 21-22% in the 500 and 1000 mg/kg/day groups during GD 12-15, resulting in decreased (p<0.05) body weight gains of 18-22%, at 500 and 1000 mg/kg/day for the treatment period (GD 6-15). Food consumption (g /rat/day) was also decreased (p<0.05) by 15- 21% during GD 6-9 at 500 and 1000 mg/kg/day, and by 8-9% for the treatment period. Increased absolute ( ↑ 8%; NS) and relative to body ( ↑ 11%; p<0.05) liver weight s were noted at 1000 mg/kg/day. At 200 mg/kg/da y, decreases (p<-0.05) in body weight gains ( ↓ 23%) and food consumption ( ↓ 10%) were observed during GD 6-9; however , these transient decreases were not
considered adverse. The maternal LOAEL was 500 mg/kg/day, based on decreased body weight gains and food consumption. The ma ternal NOAEL was 200 mg/kg/day.

No treatment-related changes were observed for percent pre-implantation loss, number of total and viable implants, early resorptions, late re sorption, percent live fetuse s, fetal weights, sex ratios, or external/visceral malformations at any dose. For the 500 and 1000 mg/kg/day groups, the skeletal examination resulted in increases (p <0.05) in the incidences of unossified cervical centrum; however, these incidences fell within the range of historical cont rols. Therefore, they
were not considered treatment related. The developmental LOAEL was not determined. The developmental NOAEL was 1000 mg/kg/day.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a published, developmental toxicity study ( Tanaka et al., 1995 ), PBO (>95% purity) was administered via oral gavage (dose volume not re ported) to 10 presumed pregnant CD rats/dose group at doses of 0 (15 dams), 630, 1065, or 1800 mg/kg/day on GD 11-12. On GD 20, all dams
were euthanized and the fetuses were removed by caesarean section. Fetuses were examined for
external malformations and variations.

No treatment-related effects on mortality or clinical signs of t oxicity were observed at any dose in the dams. Maternal body weight gains we re decreased (p<0.01) by 24 and 37% at 1065 and 1800 mg/kg/day, respectively, during GD 11-20.PIPERONYL FINAL

Page 43 of 54 The maternal LOAEL is 1065 mg/kg/day, based on decreased body weight gain during gestation. The maternal NOAEL is 630 mg/kg/day.

The number of implantation sites/dam and th e sex ratio (% male) were not significantly decreased at any dose level compared to contro ls. Additionally, no aborti ons were reported in any dam. At 1800 mg/kg/day, the total number of resorptions was increased (54 treated vs. 1 control), resulting in an increase (p<0.05) in the percent total re sorption rate (35.3% treated vs. 0.56% controls). Two litters were totally resorbed. The mean numb er of viable fetuses/dam was decreased (not analyzed stat istically) at 1800 mg/kg/day ( ↓ 16%). Mean fetal weights were decreased (p<0.05) by 5% in the 1065 mg/kg/da y males and by 14% each in the 1800 mg/kg/day
males and females.

Treatment-related external findings were limited to a dose-dependent increase (p<0.05) in limb deformities (both in number of fetuses affect ed and % incidence) at 1065 and 1800 mg/kg/day (27-44 fetuses affected vs. 0 cont rols and 19-44% incidence treated vs. 0% controls). The percent incidence (% treated vs. 0% controls) of the following limb deformities was increased (p<0.05) at 1065 and 1800 mg/kg/day: oli godactyly (19-35%) and syndactyl y (4-6%). Additionally, the incidence of polydactyly was increased (p< 0.001) at 1800 mg/kg/day (10% treated vs. 0% controls).

The developmental LOAEL is 1065 mg/kg/day base d on increased incidence of limb deformities (oligodactyly and syndactyly) in fetuses and decreased fetal body weight in males. The developmental NOAEL is 630 mg/kg/day.

Although evidence of developmenta l toxicity was observed in this study, it occurred after exposure to dose levels higher th an the limit dose (1000 mg/kg/day).

Developmental Toxicity (Rat, OCSPP 870.3700)

In a published, developmental toxicity study ( Kennedy et al., 1977 ), groups of 20 presumed pregnant COBS random-bred albino rats/dose le vel were administered PBO (technical grade specified) in corn oil via daily oral gavage (dose volume 1 mL/kg) at doses of 0, 300, or 1000 mg/kg/day (limit dose) from GD 6- 15. On GD 20, all dams were euthanized; each dam’s uterus
was removed via cesarean section and its contents examined. Fetuses were examined for external, visceral, and skeletal malformations and variations.

No treatment-related effects were observed on mortality or clinical signs of toxicity. Maternal body weight gains were unaffected during the tr eatment period (GD 6-15); however, maternal body weight gains were decreased (p<0.05) by 10% and 15% in the 300 and 1000 mg/kg/day groups, respectively, during GD 6-20.PIPERONYL FINAL

Page 44 of 54 The maternal LOAEL is 300 mg/kg/day, based on decreased maternal body weight gains. The
maternal NOAEL was not determined.

There were no abortions, premature deliveries, or dead fetuses, a nd no effects of treatment on the numbers of litters or on the growth or developmen t of the fetuses. Fetal weights of the treated groups were comparable to controls. There were no treatment-related ex ternal, visceral, or
skeletal malformations or variations.

Increased numbers of resorptions/dam were not ed in the 300 (0.6 resorptions/dam) and 1000 (0.7 resorptions/dam) mg/kg/day groups compared to conc urrent controls (0.2 resorptions). However, these findings were within the range of histor ical controls (0.3-0.7 resorptions/dam). The
increases were attributed to one 300 mg/kg/day female that reso rbed 8 of 11 fetuses, and one 1000 mg/kg/day female that had a total litter loss (7 fetuses resorbed). The increased resorptions/dam were also refl ected in an increase in post-implantation loss (6.3% and 8.0% at 300 and 1000 mg/kg/day, respectively, compared to 1.5% controls), and resulte d in a decrease in the number of live fetuses/dam at 300 (8.9 fetu ses/dam) and 1000 (8.5 fetuses/dam) mg/kg/day compared to 10.2 fetuses/dam in controls.

The developmental LOAEL was not observed. The developmental NOAEL is 1000 mg/kg/day (limit dose).

Developmental Toxicity (Mice, OCSPP 870.3700)

In a published prenatal deve lopmental toxicity study ( Tanaka et al., 1994 ), groups of 20 presumed pregnant CD-1 mice/dose level were ad ministered PBO (>95% purity) in olive oil via oral gavage at doses of 0, 1065, 1385, or 1800 mg/kg/day on GD 9 only. On GD 18, all dams were euthanized; each dam’s uterus was exposed and examined in situ for the presence and position of resorption sites, fetuses (dead or alive), and the number of implantation sites. Fetuses
were examined for external malformations and variations.

No treatment-related effects on mortality, clinical signs of toxicity, or body weight gains were observed at any dose in the dams.

The maternal LOAEL was not observed. The maternal NOAEL is 1800 mg/kg/day.

Abortions were observed in one dam at 1385 mg/kg and two dams at 1800 mg/kg/day. The number of live fetuses/dam was unaffected by tr eatment; however, the number of dams with viable fetuses was reduced at 1385 and 1800 mg/ kg/day (18 and 14 litters, respectively vs. 19 litters in controls). Increased numbers of re sorptions/dam were noted in the 1385 (3.5) and 1800 (4.3) mg/kg/day groups compared to concurrent controls (0.74). Additionally, 1 and 3 litters were totally resorbed at 1385 and 1800 mg/kg/da y, respectively. The increased resorptions/damPIPERONYL FINAL

Page 45 of 54 were also reflected in an increase in post-implantation loss at 1385 (26%) and 1800 (32%) mg/kg/day compared to controls (6%).

Evidence of treatment-related developmental toxicity was observed at 1385 and 1800 mg/kg/day. Mean fetal weights were decreased (p<0.05) by 3-7% in the males at 1385 and 1800 mg/kg/day and by 4-6% in all treated groups in the females. It was stated that the numbers of viable male and female fetuses/dam were similar in all groups, and therefore there was no effect on sex ratio.

Treatment-related external findings were limited to a dose-dependent increase (p<0.05) in the incidence of oligodactyly of the forelimbs at 1385 and 1800 mg/kg/day (2-16% treated vs. 0% controls). There were no treatment-related external variations.

The developmental LOAEL is 1065 mg/kg/day, based on decreased fetal body weights. The developmental NOAEL was not determined.

Although evidence of developmental toxicity was obs erved in this study, it occurred after acute exposure to dose levels substantially hi gher than the limit dose (1000 mg/kg/day).

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 00026192 ), groups of 16 pregnant New Zealand White rabbits were administered PBO (100% purity) in corn oil by oral gavage at dose levels of 0, 50, 100, or 200 mg/kg/day during GD 7-19. Surviving rabbits were euthanized on

GD 29.

Body weight losses of

− 19 g at 100 mg/kg/day and

− 159 g at 200 mg/kg/day were observed during treatment (GD 7-19). The maternal LOAEL is 100 mg/kg/day, based on body weight losses. The maternal NOAEL is 50 mg/kg/day.

No treatment-related changes were observed fo r viable fetus/doe, pos t-implantation loss/doe, total implantation loss/doe, corpora lutea/doe, group mean post-implantation loss, mean fetal body weight, or sex ratio at any dose. No fetal effects were observed based on external, visceral or

skeletal examinations. The developmental LOAEL was not observed. The developmental NOAEL is 200 mg/kg/day.

Chronic Toxicity/Carcin ogenicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 40323701 ), groups of 60 SpragueDawley Crl-CDR rats/sex were administered PB O (87.67-89.7% purity) in th e diet at dose levels of 0 (two control groups), 15, 30, 100, or 500 mg/kg/day (mean achieved intakes of 0/0, 29.8/29.9, 99.2/99.7, and 495.4/497.6 mg/kg/day in ma les/females) for 24 months. Ten
additional rats/sex/dose in the control, 15 and 30 mg/kg/day groups were randomly allocated for 1-month interim sacrifice, and th e remainder of the 15 mg/kg/day ra ts were euthanized at Week 8PIPERONYL FINAL

Page 46 of 54 as there were no observed effects of treatment on this group. For clinical chemistry examinations, additional groups of 10 rats/sex we re sacrificed at 25-, 51-, 79-, and 97/98-week intervals. Organs that were weighed include d the liver, kidneys, test es, ovaries, brain, and adrenals. Gross and microscopic pathological examinations were conducted on these tissues as well as the epididymides, prostate, seminal ve sicles, uterus, and thyroids/parathyroids.

No treatment related clinical si gns or differences in mortality were observed during the study. At 500 mg/kg/day, male body weight gains were 3-4% less (p<0.05) than control Group 1 for the first 10 weeks of the study. After that time the de creases in body weight ga ins grew in magnitude to 8% at Week 20, 10% at Week 40, 13% at Week 70, 17% at Week 90, and 22% at Week 100. At termination (Week 104), the decrease in male body weight gains was 21%. In the 500 mg/kg/day females, body weight gains were decreased by 6% at Week 5, growing in magnitude to 9% at Week 9, 13% at Week 20, 17% at W eek 40, 23% at Week 70, 28% at Week 90, and at termination, the difference was 21%. No consistent body weight differences were observed in the low or mid dose groups. Food consumption was si gnificantly decreased in the high dose group
on some occasions.

No consistent significant or tr eatment-related effects were ob served for the ophthalmological, hematological, or urinalysis parameters. Seru m cholesterol was increased (p<0.05) by 71-128% in the 500 mg/kg/day females at all assay times. No other clear treatment-related responses were observed in significant clinical chemistry results. A bsolute liver weights were increased in males by 21% at the 500 mg/kg/day, and in female s by 20-27% at 30, 100, and 500 mg/kg/day. No effects of treatment were reported for the ovary or adrenal weights.

For the liver, the microscopic lesions included hepatocyte hyperplasia in the high dose groups of
both sexes and focal mixed cells in the mi d and high dose females and high dose males

. For the testis, significantly increased bilateral atrophy was observed in all treatment groups, increased interstitial cells in the high dos e group, and increased interstiti al cell tumors in the high dose group. These observed testis effect s were not considered treatmen t related. Increased incidences
of small seminal vesicles were observed in all treatment groups (9-12 incidences) compared to the controls (3-4 incidences). For the adrenals , significantly decreased incidences of enlarged cortical cells were observed in the mid and high dose males but were not considered treatment related. For the thyroid, increased incidences of pigment in folli cles were observed in the high dose group of both sexes. Follicle hyperplasia wa s significantly increased in the high dose group
of both sexes and the mid dose females. No tr eatment-related histopat hological lesions were observed in the ovaries or mammary gland.

Based on increased liver wei ghts, the NOAEL is <30 mg/kg/day and the LOAEL is 30 mg/kg/day.PIPERONYL FINAL

Page 47 of 54 Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 42903701 ), CD-1 mice (60/sex/dose group) were fed diets containing PBO (90.4% purity) at nominal concentrations equivalent to dosing at 0 (two control groups), 30, 100, or 300 mg/kg/day for 78 weeks. The organ weights for liver, kidneys, brain, heart, spleen and testes were determined. The tissues subj ected to histological examination included these organs, as well as epididymides, prostate, semi nal vesicle, ovaries, uterus, pituitary, adrenals, and thyroids/parathyroids.

No treatment-related effects were observed on mo rtality or clinical signs. At 300 mg/kg/day, male body weights were decreased (p<0.05) by 7% at Week 74 and by 6% at test termination. It was stated that body weight gains were redu ced (p<0.05) in the 100 and 300 mg/kg/day group males for most of the study when compared to the second control group, and occasionally reduced (p<0.05) in the 300 mg/kg/day group when compared to the first control group. No treatment-related differences in body weights we re observed for females. No treatment related
effects were observed for food c onsumption or hematology parame ters during the study. Relative liver weights were increased (p<0.05) in the 100 and 300 mg/kg/day males ( ↑ 16 and 67%) and females ( ↑ 8 and 20%). No effects of treatment were reported for testes weights, or on histopathology of the testes, epididymides, prosta te, seminal vesicle, ovar y, or uterus. Increased (p<0.05) incidences of hepato cellular adenomas were observed in the 100 and 300 mg/kg/day males and in the 300 mg/kg/day females. In the 300 mg/kg/day male s, incidences of hepatocellular carcinomas were increased (NS). No treatment related effects were observed for thyroid pathology.

Based on reduced body weights and liver tumors , the LOAEL is 100 mg/kg/day and the NOAEL is 30 mg/kg/day.

Chronic Toxicity (Dog, OCSPP 870.4300)

In a chronic toxicity study ( MRID 42926002 ), groups of 4 beagle dogs/sex were administered PBO (90.8% purity) in the diet at dose levels of 0, 100, 600, or 2000 ppm (equivalent to 0/0, 2.9/2.7, 15.5/16.3, and 52.8/71.0 mg/kg/day in males/fema les) for one year. Organs that were weighed included the adrenals, hear t, liver, pituitary, thyroid/para thyroids, brain, kidneys, ovary, and testis. Gross and microscopic pathological ex aminations were conducted on these tissues as well as the epididymides, prostate, uterus, and mammary gland.

There were no clinical signs of toxicity. All dogs survived to scheduled euthanasia. Final body weights were decreased by 8 and 19% in the 600 and 2000 ppm males and by 5 and 25% in the 600 and 2000 ppm females. Food consumption wa s decreased by 15 and 20% in the 600 and 2000 ppm males; however, decreases in food consumption in the females did not parallel the final body weight decreases. No treatment relate d effects were observed at the ophthalmological examinations or for the hematological or ur inalysis parameters. Alkaline phosphatase wasPIPERONYL FINAL

Page 48 of 54 increased in the 600 ppm males ( ↑ 60-64%; NS) and in the 2000 ppm males ( ↑ 340-539%; p<0.05) and females ( ↑ 368-612%; NS) at both intervals. Ther e were no treatment-related lesions observed at necropsy. Absolute liver weights were increased (NS) in the 2000 ppm males ( ↑ 22%) and in the 600 and 2000 ppm females ( ↑ 12 and 36%), and mild hepatocellular hypertrophy was observed in the 2000 ppm ma les (3/4) and females (4/4). Absolute thyroid/parathyroid weights were also increased (NS) in the 2000 ppm females ( ↑ 34% left; ↑ 35% right), but there were no corr oborating microscopic findings.

The LOAEL is 600 ppm (equivalent to 15.5/16.3 mg/kg/day in males/females), based on decreases in body weight gain and liver eff ects. The NOAEL is 100 ppm (equivalent to 2.9/2.7 mg/kg/day in males/females).

Subchronic Toxicity (Rat, OCSPP 870.3100)

In a published subchronic toxicity study ( Fujitani et al., 1992 ), groups of 10 F344 rats/sex were administered PBO (technical gr ade) in the diet at dose le vels of 0, 6000, 12,000, or 24,000 ppm (equivalent to 0, 300, 600, and 1200 mg/kg/day, calculated by revi ewer) for 13 weeks. Organs that were weighed included the brain, liver, hear t, kidney, spleen, lung, test is, prostate, adrenal, thymus, ovary and uterus. Histological examin ations were conducted on the liver and kidneys.

Clinical signs in all treatment groups included no se bleeding and abdominal distention. Terminal body weights were decreased (p< 0.05) in the 24,000 ppm males ( ↓ 36%) and females ( ↓ 24%). Hemoglobin was decreased (p<0.05) in the 24,000 ppm males ( ↓ 11%) and in the 12,000 and 24,000 ppm females ( ↓ 7-10%). It was stated that the mean corpuscular hemoglobin volume was significantly lower in the 24,000 ppm females (d ata not provided). The following clinical chemistry increases (p<0.05) were observed in the 24,000 ppm male s and females, respectively: albumin ( ↑ 16% and ↑ 23%); cholesterol ( ↑ 93% and ↑ 88%); urea nitrogen ( ↑ 24% and ↑ 28% [NS]); and GGT ( ↑ 500% and ↑ 425%). Absolute liver weights we re increased (p<0.05) in the 12,000 and 24,000 ppm males ( ↑ 47 and 57%) and females ( ↑ 58 and 94%; NS at 1200 ppm), while absolute kidney weights were d ecreased (p<0.05) in the 24,000 ppm males ( ↓ 10%) and females ( ↓ 8%). Examination of livers of the male 24,000 ppm group by light m icroscopy showed enlarged hepatocytes with glassy cytoplas m and fatty deposition. On occasion, there was coagulative necrosis of a few hepatocytes in th e periportal area and oval cell proliferation. The kidney of treated rats showed atrophy of the epithe lium in the proximal convoluted tubules.

For the 24,000 ppm males, significa nt decreases were reported in the absolute weight of brain, heart, spleen, lung, prostate, thymus and in the re lative weight of the pr ostate, and the relative weights of the brain, testes and adrenal were increased; however, no data were presented. In the 24,000 ppm females, significant decreases were repor ted in the absolute we ight of lung and the absolute and relative weights of uterus.PIPERONYL FINAL

Page 49 of 54 The LOAEL is 12,000 ppm (equivalent to 600 mg /kg/day, calculated by reviewers), based on increased liver weights in both sexes and decreased hemoglobin in the females. The NOAEL was 6000 ppm (equivalent to 300 mg/kg/ day, calculated by reviewers).

90-Day Inhalation Toxicity (Rat; OCSPP 870.3465)

In a subchronic inhalation toxicity study ( MRID 42477101 ), Sprague-Dawley rats (15/sex/dose) were exposed by whole body inhalation to PBO (90.8% purity) at concen trations of 0, 0.015, 0.074, 0.155, or 0.512 mg/L for 6 hours/day, 5 consecutive days/week for 13 weeks (MMADs of 0, 1.6, 1.4, 1.9, and 2.0 microns). The organ weights for adrenals, lungs, liver, kidneys, brain, ovaries, testes, and epididymides were determ ined. The tissues subjected to histological examination included these organs, as well as uterus and thyroids/parathyroids.

Mortality, body weight, food c onsumption, ophthalmoscopic observations, hematological parameters, clinical chemistry parameters, a nd gross pathology were unaffected by the test
substance.

At 0.015, 0.074, 0.155, and 0.512 mg/L, changes in the larynx included inflammatory cells, congestion, edema, dilated sero-mucous glands a nd lymphoid cell aggregates in the mucosa, and eosinophilic material and inflam matory cells in the lumen. In addition, pseudostratified ciliated/non-ciliated columnar epithelium-squa mous/squamoid metaplasia/hyperplasia in the ventral seromucous glands of the larynx was observe d in all treated groups.

At 0.155 mg/L, an increased incidence of secretor y signs, such as dried red and brown material on the face, dried nasal discharge, and matted co at, were noted. The incidence of these findings increased at 0.512 mg/L.

At 0.512 mg/L, columnar epithelium-squamous/squamo id metaplasia/hyperpl asia in the ventral diverticulum was observed. In addition, mucosal stratified squamous epithelium hyperplasia and hyperkeratosis of the larynx was also noted.

Absolute liver weights were increased (N S) at 0.150 and 0.512 mg/L in the males ( ↑ 6 and 23%; p<0.05 at 0.512 mg/L) and females ( ↑ 5 and 20%). Absolute kidney weights were increased (NS) in the 0.512 mg/L males ( ↑ 7%) and females ( ↑ 6%).

No treatment-related changes were seen in the weights of the adrenals , ovaries, testes, and epididymides, and no treatment-related histopathol ogical findings were obse rved in the adrenals, ovaries, testes, and epididymides, ut erus and thyroids/parathyroids.

The inhalation toxicity LOAEL for this study is 0.015 mg/L base d on histopathological changes in the larynx. The inhalation toxicity NOAEL for this study was not established.PIPERONYL FINAL

Page 50 of 54 The systemic toxicity LOAEL for this study is 0.155 mg/L based on increased secretory activity and slightly increased liver wei ghts. The systemic toxicity N OAEL for this study is 0.074 mg/L.

21-Day Dermal Toxicity (Rabbit; OCSPP 870.3200)

In a 21-day dermal toxicity study ( MRID 42218201 ), groups of five New Zealand rabbits/sex were administered PBO (89.1% purity) by dermal application at dose levels of 0, 100, 300, or 1000 mg/kg/day for 6 h/day, 5 days/week, for 3 week s. Individual doses were adjusted weekly, based on the most recent body weights. The rabbit s were inspected twice daily for signs of toxicity and mortality; detailed dermal evaluations were perf ormed daily. Organ weights were determined for the liver, kidney, testes, epididym ides, and adrenals. Microscopic examinations were performed on the uterus , thyroid and above tissues.

No animals died during the study. Clinical si gns, body weight gains, food consumption and efficiency, hematological parameters, clinical chemistry parameters, and organ weights were unaffected by the test substance.

At 100, 300, and 1000 mg/kg, very slight to slight erythema, very slight edema, and fissuring
were observed at the treated site s. Mild to moderate acanthosis and hyperkeratosis and chronic inflammation of the epidermis were also noted in these animals. Desquamation and red raised areas were observed in the 300 and 1000 mg/kg animals only.

The dermal LOAEL for this study is 100 mg/kg/day based on dermal irritation noted at all dose levels during the gross and hi stopathological examinations. Th e dermal NOAEL for this study
was not established.

The systemic LOAEL for this study was not observed. The systemic NOAEL for this study is 1000 mg/kg/day (limit dose).

Avian Reproduction (Quail; OCSPP 850.2300)

In an avian reproduction study ( MRID 43876301 ), Bobwhite quail ( Colinus virginianus; 11- months old) were administered PBO (92.4% purity) in the feed at nominal concentrations of 0, 300, 1500, or 3000 ppm for 24 weeks (11 weeks prio r to the start of egg production and 12 weeks of egg production). Each treatment group wa s comprised of twenty mating pairs, with one pair per pen. Individual adult body weights were recorded at study ini tiation, biweekly through Week 8, and at study termination. Food consumpti on in each replicate wa s measured at least biweekly. Individual chick body weights were recorded after hatchi ng and on Day 14 after hatching; egg shell thickness was measured weekly during egg produc tion. Adult birds and chicks were observed routinely fo r mortality and clinical signs.

No treatment related effects were observed for adult mortality, signs of toxicity, or food consumption. It was stated that male body we ights were decreased (p<0.05) at 1500 and 3000PIPERONYL FINAL

Page 51 of 54 ppm; however, the decreases were slight ( ↓ 6% and ↓ 2%, respectively) an d not dose-dependent. The LOAEC was 1500 ppm, based on reduced a dult body weights, and the NOAEC is 300 ppm.

Avian Reproduction (Duck; OCSPP 850.2300)

In an avian reproduction study ( MRID 43876302 ), Mallard ducks ( Anas platyrhynchos; 7.5 months old) were administered PBO (92.4% purity) in the feed at nominal concentrations of 0, 18.75, 75, 300, or 1200 ppm for 22 weeks (10 weeks pr ior to the start of egg production and 12 weeks of egg production). The bi rds were housed with two male and five females per pen, with six pens per dose group. Indivi dual adult body weights were recorded at study initiation, biweekly through Week 8, and at study termin ation. Food consumption in each replicate was measured at least biweekly. Individual chick body weights were recorded after hatching and on Day 14 after hatching; egg she ll thickness was measured weekly during egg production. Adult
birds and chicks were observed routin ely for mortality and clinical signs.

No treatment related effects were observed for adult mortality, signs of toxicity, or food consumption in the ≤ 300 ppm groups. In the 1200 ppm group, tr eatment related signs of toxicity included weakness, inability to stand, and s ubdued behavior. Treatment-r elated effects at 1200 ppm included several mortalities, reductions (p<0.05) in male ( ↓ 20%) and female ( ↓ 18%) body weights, and reductions (p<0.05) in food consumption ( ↓ 19%).

No significant effects were observed for reproduction paramete rs, egg shell thickness, or offspring body weight in the ≤ 300 ppm groups. In the 1200 pp m group, significant effects included reduction in number of eggs laid, reduction in eggshell th ickness, and increased proportion of cracked eggs.

The LOAEC is 1200 ppm and the NOAEC is 300 ppm.

Fish Early Life-Cycle Toxicity (Fathead minnow, OCSPP 850.1500)

In a fish early life st age toxicity study ( MRID 43300301 ), groups of fathead minnows ( Pimphales promelas ) embryos (<24 hours old) were exposed to PBO (92.4% purity) at nominal concentrations of 0.047, 0.094, 0.19, 0.38, and 0.75 mg ai/L from egg stage to 31 days post-hatch in a flow-through system. A negative control gr oup (dilution water) and solvent control group (acetone, ≤ 0.018 mL/L) were included in the study. Mean measured concentrations were 0.040, 0.11, 0.18, 0.42, and 0.80 mg ai/L. Groups cons isted of 120 embryos/treatment (60 embryos/replicate; 2 replicates/treatment). The embryos were evaluated for percent hatch, and time to hatch. The fry were evaluated for clin ical signs and survival daily. Growth was determined as standard length and wet weight at 31 days post-hatch.

Mean percent hatch was 95, 90, 96, 86, and 73% in the 0.040, 0.11, 0.18, 0.42, and 0.80 mg ai/L treatment groups, respectively, comp ared to 90-93% in the controls. Post ha tch survival at 31PIPERONYL FINAL

Page 52 of 54 days was 98, 94, 96, 93, and 91% in the 0.040, 0.11, 0.18, 0.42, and 0.80 mg ai/L treatment groups, respectively, compared to 94-100% in the controls. Total body lengths were 33.4, 32.9, 32.5, 30.2, and 27.0 mm in the 0.040, 0.11, 0.18, 0.42, a nd 0.80 mg ai/L treatment groups, respectively, compared to 33.3-33.6 mm in the controls. Wet weights were 0.38, 0.36, 0.35, 0.27, and 0.19 g in the 0.040, 0.11, 0.18, 0.42, and 0.80 mg ai/L treatment groups, respectively, compared to 0.38-0.39 g in the controls. Compared to the control, the decreases in body weight and length were 31 and 11%, resp ectively, at the 0.42 mg ai/L a nd 51 and 20% at 0.80 mg ai/L.
The overall LOAEC and NOEAC were 0.42 and 0.18 mg ai/L, respectively, based on decreases in percent hatch and larval growth (total length and wet weight).

Fish Life-Cycle Toxicity Test (multi-generational)

In a fish life-cycle study with fathead minnow tested PBO (93.7%) at 0.0077, 0.0273, and 0.106 mg/L (MRID 49031801). The study was initiated with adult fish that were shown to be actively spawning and reproduction monito red for 21 days. F1 embryos were exposed thru sexual maturation and fecundity monitored. F2 embr yos were exposed for 4 weeks post hatch and monitored for growth and survival. In addition to fecundity in parent al and F1 generation, survival, body weight and length, VTG, GSI, tube rcle scores, gonadal staging and histopathology were also measured. Based on a preliminary re view of the study, the study author reported a significant decrease in parental female body we ight at the low and high-concentration and decrease in male body length at the mid and high- treatment. There were no effects on fecundity, GSI, VTG, tubercle scores or gonadal histopat hology. In the F1 gene ration, larvae-juvenile survival was significantly decrease d at all test concen trations (9.5-14.5%), F1 fecundity and male body weight, length, and GSI was increased at 0.106 mg/L, and F2 4-week post hatch body weight and length were reduced at the mid and high-treatment.PIPERONYL FINAL

Page 53 of 54 APPENDIX 3: References Not Utilized in the Piperonyl Butoxide WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for Piperonyl Butoxide but were not utilized due to one or more of the following reasons: 1) the article was not available in Englis h; 2) the compound of interest was not used in the study; 3) the test material was not adequately described; 4) a formulated end-use product or
mixture of chemicals was utilized as the test material; 5) only acu te mortality toxicity data were provided; 6) the experimental c onditions were not adequately desc ribed; 7) only an abstract of the study was available; 8) the re ference is a review article or book chapter and does not contain primary study data; 9) insufficient information wa s available to adequately assess the validity of the study results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) the study dealt only with non-EDSP assay development; 12) no specific endocrine-related endpoint s were assessed in the study; and 13) the study contained only
data on invertebrates.

Fujimoto, N, Inoue, K, Yoshida, M, et al . (2012) Estrogen and androge n receptor status in hepatocellular hypertrophy induced by phenobarbital, clofibrate, a nd piperonyl butoxide in F344

rats. J. Toxicol. Sci. 37(2) : 281-286.

Nemoto, K, Tanaka, T, Ikeda, A, et al . (2011) Super-induced ge ne expression of the N-methylD-aspartate receptor 2C subunit in chemi cal-induced hypertrophic liver in rats. J. Toxicol. Sci

. 36(5) : 507-514.PIPERONYL FINAL

Page 54 of 54 Tanaka, T, Takahashi, O, and Oishi, S (1992) Re productive and neurobehavi oral effects in threegeneration toxicity study of piper onyl butoxide administered to mice. Food Chem. Toxicol. 30 : 1015-1019.

Tanaka, T (1992) Effects of Piperonyl Butoxide on F 1 Generation Mice. Toxicology Letters 60 : 83-90.

Tanaka, T (1993) Behavioral effects of piperonyl butoxide in male mice. Toxicol. Lett. 69 : 155-
161.