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 Assessment Type: 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 & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 62
Page 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 

Page 2 of 62
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: PIPERONYL BUTOXIDE (PBO) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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PIPERONYL 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 Relevant 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.................53 

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PIPERONYL 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

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PIPERONYL BUTOXIDE FINAL 

iv 

Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

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PIPERONYL BUTOXIDE FINAL 

v 

Abbreviation Terminology 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

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PIPERONYL BUTOXIDE FINAL 

vi 

Abbreviation Terminology 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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PIPERONYL BUTOXIDE FINAL 

Page 1 of 54 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 piperonyl butoxide (PBO) interacts with E, A or T hormone pathways, 

the number and type of effects induced, the magnitude 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 dose(s) at which endocrine related responses 

occurred happened concurrently with 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 weight-of-evidence (WoE) analysis of the potential interaction 

of PBO with the E, A or T hormone pathways. 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 convincing evidence of an interaction of PBO. It was 

negative in the estrogen receptor (ER) binding, ER transactivation assay (ERTA), and the 

aromatase assays. In the steroidogenesis 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 and 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 absence of overt toxicity. In the 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 interaction of PBO with the androgen pathway. PBO was 

as negative in the AR binding and steroidogenesis assays. There were no effects observed in the 

Hershberger assay and in the male pubertal assay in the absence of overt toxicity. There were no 

androgen-related effects in the mammalian Part 158 studies. In the FSTRA, while there was a 

reported increase of minimal to mild testes degeneration in the middle and high treatment 

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PIPERONYL 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 was 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 histopathological changes seen in the male and female rats 

in the pubertal assays since no treatment-related effects were seen in thyroid hormones (T4 or 

TSH) or thyroid weights. No thyroid-related effects were seen in the mammalian Part 158 

studies, except for a histopathological effect in the thyroid in the chronic rat study. Furthermore, 

there was no evidence of thyroid-related effects observed in the AMA. 

Based on weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is not 

recommended for PBO since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

 

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PIPERONYL BUTOXIDE FINAL 

Page 3 of 54 

I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 piperonyl butoxide (PBO) interacts with E, A or T hormone pathways, 

the number and type of effects induced, the magnitude 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 dose(s) at which endocrine related responses 

occurred happened concurrently with 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 weight-of-evidence analysis of the potential interaction of PBO 

with the E, A or T hormone pathways. 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).

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 determining the potential interaction of piperonyl 

butoxide with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other 

scientifically relevant information (OSRI) such as general toxicity studies 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 approach outlined by the Agency (USEPA, 2011), is 

presented in Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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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 believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order 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 submitted in lieu of the Tier 1 assays. The Agency has 

also conducted a more recent search of available scientific literature 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 presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual 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 endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing 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 were 

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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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, 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 interact with E, A, and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development 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 relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological 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 consideration 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 effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

 

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PIPERONYL BUTOXIDE FINAL 

Page 6 of 54 

This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally 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 included, but were not limited to: mortality; 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, especially 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/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular 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/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

 

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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 biologically 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) 

ERα 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) 

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Page 8 of 54 

B. Effects on Hypothalamic-Pituitary-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 assays 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 reported 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 production (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 and 0.09 mg/L), respectively. However, there were no other 

treatment-related effects observed in females. 

PBO was negative in the uterotrophic assay. In the female pubertal rat assay, a 16% decrease in 

ovarian weights was noted in the presence of overt toxicity (moribund sacrifice 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 toxicity study in rats (Tanaka et al., 1995), the total number of 

resorptions was increased, with two complete litter 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 fetal 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, dietary treatment with 1200 mg/kg-diet (the highest 

treatment concentration) resulted in a reduction in the number of eggs laid, reduced eggshell 

thickness, and an increased proportion of cracked eggs. This treatment group was also associated 

with overt toxicity as indicated by mortality, significant reductions in body weight and clinical 

signs of toxicity that included weakness, subdued behavior and inability to stand. 

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Page 9 of 54 

In an early life-stage toxicity study on fathead minnows, treatment with PBO at 0.42 and 0.80 

mg a.i/L (the two highest treatment concentrations) 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 study 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 gonadal histopathology. In the F1 generation, larvaejuvenile survival was significantly decreased at all test concentrations (9.5-14.5%), F1 fecundity 

(↑32%) was increased at 0.106 mg/L (F1 male body weight and length was also increased at high 

concentration); F2 4-week post hatch body weight and length were reduced at the mid and hightreatment. 

Page 17 of 62
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Page 10 of 54 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Piperonyl Butoxide 1Study Type/ Literature Citation 

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 

EDSP Tier 1 Assay 

ER Binding (MRID 48618803) N ERTA (MRID 48618804) N Aromatase (MRID 48618802) N Steroidogenesis (MRID 48618807) P Uterotrophic (MRID 48618808) N Female Pubertal Rat (MRID 48618806a) N N N N N N FSTRA (MRID 48687701) NE N N N 

Fcd 

↑14%, 

↑19% 

(M, H) 

N 

OSRI 

Developmental Toxicity (Rat; MRID 42380801) 

N 

Developmental Toxicity (Rat; Tanaka et al., 1995) 

P6

 (H) 

Developmental Toxicity (Mouse; Tanaka et al., 1994) 

P7

(M, H) 
PIPERONYL BUTOXIDE FINAL 

Page 11 of 54 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Piperonyl Butoxide 1Study Type/ Literature Citation 

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 

Developmental Toxicity (Rabbit; MRID 00026192) 

N 

Chronic toxicity/ Carcinogenicity (Rat; MRID 40323701) 

NE N N NE 

Carcinogenicity (Mouse; MRID 42903701) 

NE NE N NE 

Chronic Toxicity (Dog; MRID 42926002) 

NE N N N 

Subchronic Toxicity (Rat; Fujitani et al., 1992) 

N N NE NE 

Subchronic Inhalation Toxicity (Rat; MRID 42477101) 

NE N N NE 

Avian Reproduction (Quail; MRID 43876301) 

N 

Avian Reproduction (Duck; MRID 43876302) 

N 

Fish Life-cycle (fathead minnow; MRID 49031801, preliminary review) 

N 
PIPERONYL BUTOXIDE FINAL 

Page 12 of 54 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High 

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in t

weight 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 OS

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 

details see Section IV. A 

4. 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 r

7. 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 findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
PIPERONYL 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 androgenic, anti-androgenic, 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 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 Hershberger assay. In the male 

pubertal assay, the decreases in serum testosterone 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). Additionally, 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. The pathologist report stated that it is unlikely 

that this minimal to mild testicular degeneration observation would have a measureable impact 

on testicular function. It is noted that male VTG 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 cells and increased interstitial cell tumors were observed in the 

testes at 500 mg/kg/day. There were no androgen-related effects 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 concentration (0.106 mg/L); effects on F1 larvae-juvenile 

survival also decreased at this concentrations. Effects on parental and F2 growth were also 

reported. 

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Page 14 of 54 

Table 3: Androgenic/Anti-Androgenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Piperonyl Butoxide 1Study 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 

EDSP Tier 1 Assay 

AR Binding (MRID 48618801) N Steroidogenesis (MRID 48618807) N Hershberger (MRID 48618805) N N Male Pubertal Rat (MRID 48618806b) N N N N N N N N FSTRA (MRID 48687701) NE N P4 (M, H) ↑ tubercle score (L) N NE5 

OSRI 

Chronic toxicity/ carcinogenicity (Rat; MRID 40323701) 

N N NE N NE 

Carcinogenicity (Mouse; MRID 42903701) 

N N NE N NE 

Chronic toxicity (Dog; MRID 42926002) 

N N NE N N 

Subchronic toxicity (Rat; Fujitani et al., 1992) 

N NE NE NE NE 

Subchronic Inhalation Toxicity (Rat; MRID 42477101) 

N N N N NE 
PIPERONYL BUTOXIDE FINAL 

Page 15 of 54 

Table 3: Androgenic/Anti-Androgenic Pathway for Piperonyl ButoxideLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Piperonyl Butoxide 1Study 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 

21-Day Dermal toxicity (Rabbit; MRID 42218201) 

N N N N NE 

Avian Reproduction (Quail; MRID 43876301) 

N 

Avian Reproduction (Duck; MRID 43876302) 

N 

Fish Life-cycle (Fathead minnow; MRID 49031808 (preliminary review) 

N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High tre

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this aare absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), D

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 OSR

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 i

see Section IV. A 

4. Increased testes degeneration (minimal to mild) was higher than controls in the 0.0944 mg ai/L group, but only marginally higher in t

5. Male vitellogenin (Vtg) was not successfully 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 findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
PIPERONYL FINAL 

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

The current EDSP Tier 1 battery 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 detect 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 thyroid 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 are not reported in the table 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 follicular 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 T4 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 concentration (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/carcinogenicity study in rats, increased incidences of pigment in 

thyroid follicles were observed 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 study in dogs, thyroid weight was increased in 

females at the highest dose tested (71.0 mg/kg/day), however thyroid histopathology was 

unaffected by treatment, and the effect co-occurred with overt toxicity (decreased body weights). 

There were no thyroid-related effects observed in the other Part 158 mammalian studies. 

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Table 4. Thyroid Pathway for Piperonyl Butoxide.

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

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48618806a) N P4 

(L) N N N X 

(L, H) 

X5 

(H) 

Male Pubertal Rat 

(MRID 48618806b) N P4

(L) N N N X 

(L, H) 

X6

(H) 

AMA 

(MRID 48673101) N 

Day 21: 

↑28% 

HLL 

 (H) 

N N 

OSRI

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 40323701) 

 NE P7

 

(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 response. 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 concomitant 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 examined 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of PBO to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, 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 evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those 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 are 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 evaluation of the results of the Tire 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall 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 below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) were sufficient for a dose/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry).

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1. Tier 1 in vivo Assays 

In the androgen agonist portion of the Hershberger 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, terminal 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 decreased (p<0.05) by 62% at 1000 mg/kg/day. 

In the pubertal assays, final adjusted body weights 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 weights 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 liver weights were increased 

(p<0.05) by 36% at 1000 mg/kg/day. In the males, relative liver weights were increased (p<0.05) 

by 15 and 34% at 500 and 1000 mg/kg/day, respectively, 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 adjusted and unadjusted pituitary weights were 

decreased by 16-17% (p<0.05 or NS) at both dose levels in the males. In the females, at 500 and 

1000 mg/kg/day, serum levels of alkaline phosphatase were decreased, and total protein levels 

were increased. At 1000 mg/kg/day, increases were observed in serum levels of sodium, 

phosphorous, and blood urea nitrogen. 

In the uterotrophic assay, final body weights 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 toxicity observed. 

In the AMA, there were no significant effects on mortality and no other clinical signs of toxicity. 

Day 7 body weight was significantly reduced at the lowest treatment concentration (0.001 mg 

a.i./L) but this effect was not observed at higher treatment concentrations nor was it observed at 

Day 21 measurements. 

2. OSRI 

In a prenatal developmental toxicity study in rats, at 500 and 1000 mg/kg/day, treatment-related 

clinical signs were observed following PBO administration which increased in frequency and 

severity with dose. Body weight gains were decreased (p<0.05) by 51% in the 1000 mg/kg/day 

Page 27 of 62
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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 weights were noted at 1000 

mg/kg/day. In a published, developmental toxicity study in rats (Tanaka et al., 1995), PBO 

decreased (p<0.01) maternal body weight gains by 24 and 37% at 1065 and 1800 mg/kg/day, 

respectively, during GD 11-20. 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 

resorption rate (35.3% treated vs. 0.56% controls). Two litters were totally resorbed. The mean 

number of viable fetuses/dam was decreased (not analyzed statistically) 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 observed 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 developmental 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/carcinogenicity 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 weights were decreased by 6% at Week 5 and 

by 21% at termination. Food consumption was significantly decreased in the high dose group on 

some occasions. Serum cholesterol was increased (p<0.05) in the 500 mg/kg/day females at all 

assay times. Absolute 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 weights were decreased (p<0.05) by 7% at Week 

74 and by 6% at test termination at 300 mg/kg/day. It was stated that body weight gains were 

reduced (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. Relative liver weights were increased (p<0.05) 

at 100 and 300 mg/kg/day in males by 16 and 67%, respectively, and in females by 8 and 20%. 

Increased (p<0.05) incidences of hepatocellular adenomas were observed in the 100 and 300 

Page 28 of 62
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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 decreased 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. Alkaline 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. 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 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 ventral diverticulum was 

observed. In addition, mucosal stratified squamous epithelium hyperplasia and hyperkeratosis of 

the larynx was also noted. Absolute liver weights 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 quail, 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 toxicity study in ducks, 

treatment related signs including weakness, inability 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 toxicity study with fathead minnows, 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-generation life-cycle with fathead minnow, based on a 

preliminary review of the study, 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. In the F1 generation, larvae-juvenile survival was significantly 

decreased at all test concentrations (9.5-14.5%), F1 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. 

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B. Estrogen Pathway 

For the estrogen pathway, there was no convincing evidence of an interaction of PBO. It was 

negative in the estrogen receptor (ER) binding, ER transactivation assay (ERTA), and the 

aromatase assays. In the steroidogenesis 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 and 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 absence of overt toxicity. In the 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 interaction of PBO with the androgen pathway. PBO was 

as negative in the AR binding and steroidogenesis assays. There were no effects observed in the 

Hershberger assay and in the male pubertal assay in the absence of overt toxicity. There were no 

androgen-related effects in the mammalian Part 158 studies. In the FSTRA, 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 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 was 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 histopathological changes seen in the male and female rats 

in the pubertal assays since no treatment-related effects were seen in thyroid hormones (T4 or 

TSH) or thyroid weights. No thyroid-related effects were seen in the mammalian Part 158 

studies, except for a histopathological effect in the thyroid in the chronic rat study. Furthermore, 

there was no evidence of thyroid-related 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 estrogen, androgen or thyroid pathways in mammals or wildlife. 

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PIPERONYL FINAL 

Page 23 of 54 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is not 

recommended for PBO since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

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PIPERONYL FINAL 

Page 24 of 54 

VI. References 

Chun, J, and Neeper-Bradley, T (1991) Developmental 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 Specialty Products Association, Inc. for the 

Piperonyl Butoxide Task Force II, Washington, DC. Laboratory Report No. PBTF01-00256, July 

6, 2012. Unpublished. (MRID 48673101) 

Fort, D (2012) 21-Day fish reproduction assay. Fort Environmental Laboratories, Inc., Stillwater, 

OK, Golden Pacific Laboratories, Inc., Fresno, CA, and Experimental Pathology Laboratories, 

Inc., Sterling, VA. Sponsored by Consumer Specialty Products Association, Inc. for the 

Piperonyl Butoxide Task Force II, Washington, DC. Laboratory Report No.: PBTF01-00257, 

November 8, 2012. Unpublished. (MRID 48687701) 

Fujitani, T, Tada, Y, and Yoneyama, M (1993) Hepatotoxicity 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 Development 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 butoxide in a one year chronic dietary toxicity 

study in dogs. International Research and Development 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 dietary toxicity study and carcinogenicity study of piperonyl 

butoxide in the albino rat. Bio-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 Research Center, Export, PA. Laboratory 

Study No. 91NO134, August 27, 1993. Unpublished. (MRID 42903701) 

Page 32 of 62
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Page 25 of 54 

Kennedy, G, Smith, S, Kinoshita, F, et al. (1977) Teratogenic evaluation 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-month) 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 Cuneo, Washington, DC. Laboratory Study No. 91-

8333. Unpublished. (MRID 42477101) 

Robinson, K, Pinsonneault, L, and Proctor, B (1986) A-two generation (two-litter) reproduction 

study of piperonyl butoxide administered in the diet to the rat. Bio-Research Laboratories Ltd., 

Canada. Sponsored by the Piperonyl Butoxide 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 Research Centre Ltd., Huntingdon, 

Cambridgeshire, UK. Sponsored by the Piperonyl Butoxide Task Force II, c/o S.C. Johnson & 

Son, Inc., Racine, WI. Laboratory Report 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 Report ID: PBT 2/950168. Unpublished. (MRID 43876302) 

Schardein, J (1986) Teratology study in rabbits. International Research 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 toxicity evaluation of 

piperonyl butoxide in CD-1 mice. Toxicology Letters 71: 123-129. 

Tanaka, T, Fujitani, T, Takahashi, O, et al. (1995) Developmental toxicity study of piperonyl 

butoxide in CD rats. Toxicol. Ind. Health 11 (2): 175-184. 

USEPA (2011) Endocrine Disruptor Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

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Page 26 of 54 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Wagner, H (2011) Piperonyl butoxide: H295R steroidogenesis 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 recombinant aromatase assay. CeeTox, Inc., 

Kalamazoo, MI. Laboratory Report No.: 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., Kalamazoo, 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., Kalamazoo, MI. July 19, 2012. Unpublished. (MRID 

48843501) 

Zorilla, L (2011). The Hershberger bioassay for piperonyl butoxide. Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Project Study ID: C192-200, November 10, 2011. 

Unpublished. (MRID 48618805) 

Zorilla, L (2011). Pubertal development and thyroid function in intact juvenile/peripubertal 

female and male rats with piperonyl butoxide. Integrated Laboratory Systems, Inc., Durham, NC. 

Laboratory Project Study ID: C192-300, November 10, 2011. Unpublished. (MRID 48618806) 

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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) 

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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 (negative control), 0.011, 0.033, and 0.100 mg a.i./L; 

time-weighted average (TWA) concentrations were <0.0010 (<LOQ; control), 0.00599, 0.0263, 

and 0.0741 mg a.i./L. The test system was maintained at 21.4 to 21.9°C and a pH of 7.3 to 7.9. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. The survival of tadpoles exposed to PBO was not significantly affected (p>0.05) 

as survival was 100% in the low treatment group with one mortality each in the control and mid 

and high treatment groups. There were no clinical signs of toxicity or observations of abnormal 

behavior. 

PBO caused no significant acceleration or delay of median NF developmental 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 were considered to be late stage (i.e., above Stage 60) at test 

termination. Therefore, Day 21 analyses were conducted using all tadpoles (there were 

insufficient non-late-stage tadpoles for separate analyses). 

Day 7 wet weight and snout-vent length (SVL) were significantly decreased across all treatment 

groups compared to the negative control. There were no significant increases 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 histopathology were observed in the control and all treatment groups, 

and included mild follicular cell hyperplasia and hypertrophy. There were no treatment-related 

patterns. 

All performance and validity criteria were met in this study. 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 effects on thyroid histopathology compared to the control. 

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Androgen Receptor (AR) Binding 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 the 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 was functioning with appropriate affinity for the 

radiolabeled reference androgen (R1881). Saturation binding data were not originally provided in 

the study report; however, summarized saturation binding data (MRID 48843501) from the 

performing laboratory were submitted following a request by the Agency. The mean 

dissociation constant (Kd) for [3

H]-R1881 was 1.080±0.690 nM and the estimated Bmax was 

1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. 

Only one of the three Kd values was within the range reported in the EPA validation program 

(0.685 to 1.57 nM), and all Bmax 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 (R2 = 0.957-0.984) 

and the small variation among runs. 

 

In the competitive binding experiment, precipitation of piperonyl butoxide was visually observed 

at the highest concentration (10−3

 M) tested in all three runs, therefore the highest concentration 

used for data evaluation was 10−4 M. The estimated average log IC50 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 concentrations 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 was off by more than an order of magnitude. An 

IC50 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 piperonyl butoxide was >75% at 

concentrations ≤10−4 M in all three runs. As specific binding was not <50% at any 

concentration, an IC50 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. 

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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 ([1β- 3

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

concentrations of 10−10 to 10−3 M for 15 minutes to assess the potential of piperonyl 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. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three runs were conducted and each run included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5 M) 

using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN) in duplicate, and the 

piperonyl butoxide series (10−10 to 10−3

 M) with three replicates 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 control within a run was between 90 to 110% of the average full 

activity. Activity in the background 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 IC50 for 4-OH ASDN 

averaged −7.13 M and the Hill slope was −1.02. 

For piperonyl butoxide, the two highest 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 concentrations 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 concentrations. 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 curve, piperonyl butoxide is classified as a Noninhibitor of aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48618803), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A saturation binding 

experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in 

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reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference 

estrogen prior to routinely conducting ER competitive binding experiments. The competitive 

binding experiment was conducted to measure the binding of a single concentration of [3

H]-17βestradiol (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 vehicle 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, octyltriethoxysilane as a negative control, and 17βestradiol as the natural ligand reference material. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the 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 cytosol prepared on September 27, 2010, the Kd for [3H]-17βestradiol was 0.081±0.006 nM, and the estimated Bmax was 52.70±1.05 fmol/100 μg protein. For 

the batch of cytosol prepared on July 10, 2011, the Kd for [3H]-17β-estradiol was 0.303±0.090 

nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd for each run was 

within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots 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 IC50 was −9.2 M for the natural 

ligand (17β-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 first two runs; therefore, data from 

the 10–3 M concentration were not evaluated. The highest concentration tested in the third run 

was 10–4 M. The mean specific binding in the presence of piperonyl butoxide was >77% at 

concentrations ≤10−4 M in all three runs. . The lowest average percent [3

H]-17β-estradiol 

binding in the presence of piperonyl butoxide was >75% at concentrations up to 10−4

 M, and 

therefore a mean log IC50 and Relative Binding Affinity (RBA) were not calculated. 

Based on the three runs, piperonyl butoxide is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48618804), hERα-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 for 

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approximately 24 hours in two independent runs. The experiments were performed using 96-

well plates and each piperonyl butoxide concentration 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 induce reporter (luciferase) 

gene products. Luciferase expression in response to activation of the estrogen receptor was 

measured using a proprietary 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 transcriptional activation (RTA) of piperonyl 

butoxide was ≤2.8%. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, piperonyl butoxide responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-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 concentration possible based on cytotoxicity. 

Because the RPCMax < PC10 in both assay runs, piperonyl butoxide was considered negative for 

estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48687701) of piperonyl butoxide (PBO) with 

fathead minnows (Pimephales promelas) was conducted under continuous flow-through 

conditions. Adult fish, 16 spawning groups (2 males 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 (TWA) 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 25.5oC and a pH of 7.3 to 8.1. 

Survival was 100% in all treatment groups throughout 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 decrease at the high treatment concentration was 

significantly different from the negative control. 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 changes 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. 

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Spawning frequency was at least two times in 7 days 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 increase in fecundity at the mid 

and high treatment levels of 14 and 19%, respectively, compared to the negative control. 

Additionally, median male tubercle score at the low treatment level was significantly greater than 

the median male tubercle score in the control; higher levels were unaffected by treatment 

(p>0.05). There was no other significant difference between the treatment levels and negative 

controls for any other endpoint. Male vitellogenin (VTG) was not successfully measured due to 

insufficiently sensitive methodology. The sole histopathology finding that appeared related to 

PBO exposure was increased testicular degeneration (minimal to mild) in males. The prevalence 

of this finding was higher than controls in the high treatment group, but only marginally higher 

in the mid treatment group. There were no apparent differences between histopathology severity 

scores or gonadal stages in the PBO-treated groups compared to the negative controls. Sex 

steroids (17β-estradiol and testosterone) were not measured in this study. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger assay (MRID 48618805) screening for androgenic activity, piperonyl butoxide 

(93.8% a.i., Lot# E104-10) in corn oil was administered daily via oral gavage 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 positive control group consisting of eight castrated 

rats exposed to 0.4 mg/kg/day testosterone propionate (TP) in corn oil by subcutaneous (s.c.) 

injection was also included. To screen for potential anti-androgenic activity, 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 daily with TP (0.4 mg/kg/day) by s.c. injection, and 

the anti-androgenic positive control group consisted of eight castrated 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 animals 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, treatment-related findings were observed on mortality, 

clinical signs, or accessory sex organ weight. Body weights 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, terminal body weight was decreased (not statistically significant) by 

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only 7%, but overall (Days 1-11) body weight gains were decreased (p<0.05) by 96%. No 

treatment-related increases in the accessory sex tissue weight were seen in the animals dosed 

with piperonyl butoxide. 

Animals dosed with TP in the androgen agonist assay had increased (p<0.05) body weights on 

Day 11 (increased 9%) and increased (p<0.05) overall 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 prostate; 242% in levator ani-bulbocavernosus 

(LABC); 571% in Cowper’s gland; and 53% in glans penis. For the vehicle control, the 

maximum guideline-specified %CV values were exceeded for the ventral prostate (58% vs. 45%) 

and glans penis (26% vs. 22%). The maximum guideline-specified %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 accessory sex organ weight. Terminal body weight was decreased (NS) by 4% 

at 320 mg/kg/day and 9% at 1000 mg/kg/day. The overall (Days 1-11) body weight gains were 

decreased (p<0.05) by 36% at 320 mg/kg/day 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 body 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 follows: 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 negative 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 female 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 dose) mg/kg/day from post-natal day (PND) 22 to 

42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and 

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weight at day of attainment was recorded. Following sacrifice on PND 42 or 43, total serum 

levels of thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using a 

radioimmunoassay; serum was also analyzed for levels of selected clinical chemistry parameters. 

Adrenal, kidneys, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic 

examinations were performed on the ovaries, uterus, thyroid, and kidneys. 

No treatment-related effects were observed on mortality 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 effects were observed on the age at VO, weight 

at VO, and number of animals attaining VO, mean age at first vaginal estrus, mean cycle length, 

percent cycling, and percent regularly cycling, number of ovary follicles, follicular cysts or 

corpora lutea. 

At 500 and 1000 mg/kg/day, the final adjusted body 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 weights 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 increased (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 relative adrenal weights (↑20-29%) at 1000 mg/kg/day, and 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 glands was noted in 2 of 14 rats, mild atrophy of 

the endometrial gland was observed in another two 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 T4 or TSH and thyroid 

weights were not affected, there was a dose-dependent increase in the thyroid gland follicular 

epithelial cell height with a concomitant 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 high dose was the Limit Dose (1000 mg/kg/day). 

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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 gavage 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 separation (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 (T4), and thyroid stimulating hormone (TSH) levels 

were determined using radioimmunoassays. Adrenal, liver, thyroid, pituitary, and urogenital 

organ weights were recorded, and microscopic examinations of the testes, epididymides, thyroid, 

and kidneys were performed. 

Although four rats died during the study (1 control, 2 low-dose, and 1 high-dose), there were no 

treatment-related effects on mortality as the deaths were attributed to gavage errors. There were 

no clinical signs of toxicity in any group, and no treatment-related effects were observed on age 

at PPS, weight at PPS, and serum levels of T4 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 significant [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 weights were increased 

(p<0.05) by 16% and 20%, respectively, at 1000 mg/kg/day. Relative kidney weights were 

increased (p<0.05) by 8% and 11% at 500 and 1000 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 weights were increased (p<0.05) by 15% and 31%, 

respectively, at 1000 mg/kg/day. 

Although thyroid weight was not affected by treatment, there was a dose-dependent increase in 

follicular cell height with a concomitant decrease in colloid area Follicular cell height was 

shortest in the control (15/15 rats 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) and smallest at 1000 mg 

(12/15 at Grades 1-3), with cell height and colloid area being intermediate in the 500 mg/kg/day 

group. There were no significant differences in serum thyroid hormone (T4 and TSH) levels 

among treated and control groups. 

Serum testosterone levels were decreased by 36% (NS) and 67% ( p<0.05) at 500 and 1000 

mg/kg/day, respectively. At 500 mg/kg/day, adjusted LABC weight was decreased by 14% 

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(p<0.05). At 1000 mg/kg/day, adjusted and unadjusted LABC weights were decreased by 20-

26% (p<0.05), and adjusted and unadjusted weights of the seminal 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 (MRID 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 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) 

was used as the vehicle, at a final concentration of 0.05%. 

Testosterone and estradiol concentrations were measured by HPLC/MS-MS with positive ion 

multiple reaction monitoring. Four independent experiments were performed. A Quality 

Control (QC) plate was run concurrently with each independent run of a test chemical plate to 

demonstrate that the assay responded properly to positive control agents at two concentration 

levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of 

estradiol and testosterone production. 

Laboratory proficiency testing was not conducted in the current study. The amendment stated 

that the laboratory proficiency data was previously determined for this batch of cells; however, 

details of a previous proficiency determination, including estimates of EC50s 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, acceptable reproducibility (low %CV), and appropriate 

induction and inhibition with positive controls. 

No statistically significant effects on testosterone production were observed in three out of four 

test runs after incubation with PBO. However, four of the six PBO concentrations in the fourth 

run exhibited statistically significant decreases (0.87- to 0.91-fold) 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-significant increase of 1.45-fold in a fourth test run) at the PBO 

concentration of 10 μM. The remaining PBO concentrations in Run 4 (0.0001 to 1 μM) 

exhibited statistically significant decreases (0.78- to 0.89-fold) in estradiol production, but these 

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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 three independent runs, PBO treatment resulted in 

statistically significant and reproducible 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/day on post-natal days (PND) 58-60. A positive 

control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 0.1 mg/kg/day by 

oral gavage. Body weights were determined daily. All animals were terminated and necropsied 

on PND 61 approximately 24 hours after the final dose administration to determine wet and 

blotted uterine weights. 

All animals survived until scheduled 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 prior to termination. All animals dosed with 1000 

mg/kg piperonyl butoxide group exhibited 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 comparable to the vehicle controls 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 controls, with a decrease in overall body weight 

gain (↓148). 

Uterine weights in all piperonyl butoxide treated groups were comparable 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 uterotrophic assay. 

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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 the 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 F1a and F1b litters, and selected F1b rats were mated to produce F2a and F2b 

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 effects based on reproductive performance (mating, 

fertility, or conception indices), pup development (viability, survival, or lactation indices), or 

gross pathology. No data was provided regarding organ weight or pathology. 

The LOAEL for parental and offspring toxicity is 5000 ppm (equivalent to 250 mg/kg/day), 

based on decreased body weight gain and food consumption. The NOAEL for parental and 

offspring toxicity is 1000 ppm (equivalent to 50 mg/kg/day). 

Two-Generation Reproduction and Neurobehavioral 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/females during pre-mating). The purpose of 

this study was to provide data related to the reproductive toxicity effects on the P generation 

mice and the behavioral and developmental toxicity effects on the F1 generation mice. Dosing 

began at 5 weeks of age and continued through pre-mating (4 weeks), mating (5 days), gestation, 

and lactation. Food consumption was measured in the parental animals. For the F1 and F2

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 F1 pups were 

removed from their dams on PND 28, and 10 pups/sex/dose group were mated at 9 weeks of age 

(avoiding sibling matings) to produce the F2 pups. The following neurobehavioral tests were 

performed on the F1 and F2 pups: surface righting and negative geotaxis at PND 4 and 7, cliff 

avoidance at PND 7, swimming behavior at PND 4 and 14, and olfactory orientation at PND 14. 

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Food consumption was decreased in the 8000 ppm P males and females during pre-mating, and 

in the 4000 and 8000 ppm P and F1 females during lactation. No additional data was provided for 

the parental animals. 

In the F1 pups, the 8000 ppm litter weights were decreased (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 PND 21 survival index was decreased at 8000 

ppm in both males (63.0% treated vs. 90.6% controls) and females (78.9% treated vs. 88.7% 

controls). Neurobehavioral parameters were unaffected by treatment. 

In the F2 pups, litter size was decreased (p<0.05) in the 4000 (↓21%) and 8000 (↓47%) ppm 

groups; litter weights were decreased (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 weights 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 olfactory orientation in the 2000, 4000, 

and 8000 ppm groups. 

One-Generation Reproduction and Neurobehavioral Toxicity Study in Mice 

In a non-guideline behavioral, reproductive, and developmental toxicity study in mice (Tanaka, 

1992), PBO (purity not provided) was administered in the diet 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 behavioral and developmental 

toxicity effects on the F1 generation mice. Treatment of the P generation began at 5 weeks of 

age. At 8 weeks of age, the animals were observed in the open-field. At 9 weeks of age, they 

were mated (1 male:1 female). Litter observations (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, negative geotaxis, swimming, cliff avoidance, and 

olfactory orientation) were evaluated on PND 4, 7, and/or 14. The offspring were evaluated in 

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the open-field at 3 and 8 weeks of age. At 6 weeks 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. Decreased (p<0.001) pup weights 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. The survival index at 

PND 21 was 51.7% at 6000 ppm compared to 79.2% in the controls. Decreased (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 effects were observed in the offspring. 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 relationship. Rearing was also decreased (NS) 

by 43% in the 6000 ppm females. Because the effect on rearing was unrelated to dose and/or 

without statistical significance, this effect was considered equivocal. At 8 weeks of age, 

increased (NS) preening was observed in the 6000 ppm males (↑219%). As the effect on 

preening was not statistically significant and the biological significance is unclear, this effect was 

considered equivocal. 

The LOAEL for offspring toxicity is 3000 ppm, based on decreased pup weight, olfactory 

orientation, and ambulation. The NOAEL is 1500 ppm. 

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developmental 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 during 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 status of implantation sites (including early and late resorptions, 

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dead fetuses and live fetuses). All live fetuses were counted, weighed, sexed and examined for 

external malformations. About one-half of the fetuses were examined for thoracic and abdominal 

visceral abnormalities. These fetuses were decapitated and the heads were fixed in Bouin's 

solution and examined for soft tissue craniofacial malformations and variations. The remaining 

half of the fetuses were fixed in ethanol, stained with alizarin red S and examined for skeletal 

malformations and variations. 

There were no maternal deaths during the study, and no treatment-related observations were 

noted at necropsy. At 1000 mg/kg/day, treatment-related clinical signs included urogenital 

wetness, urine stains, red discharge, and perinasal encrustation. At 500 mg/kg/day, treatmentrelated clinical signs included red 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 weights were noted at 1000 

mg/kg/day. At 200 mg/kg/day, 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 maternal 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 resorption, percent live fetuses, 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 controls. 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 reported) 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 toxicity were observed at any dose 

in the dams. Maternal body weight gains were decreased (p<0.01) by 24 and 37% at 1065 and 

1800 mg/kg/day, respectively, during GD 11-20. 

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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 the sex ratio (% male) were not significantly 

decreased at any dose level compared to controls. Additionally, no abortions 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 resorption rate (35.3% treated vs. 

0.56% controls). Two litters were totally resorbed. The mean number of viable fetuses/dam was 

decreased (not analyzed statistically) 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. 

Treatment-related external findings were limited to a dose-dependent increase (p<0.05) in limb 

deformities (both in number of fetuses affected and % incidence) at 1065 and 1800 mg/kg/day 

(27-44 fetuses affected vs. 0 controls 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: oligodactyly (19-35%) and syndactyly (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 based 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 developmental toxicity was observed in this study, it occurred after 

exposure to dose levels higher than 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 level 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 treatment 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. 

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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, and no effects of treatment on the 

numbers of litters or on the growth or development of the fetuses. Fetal weights of the treated 

groups were comparable to controls. There were no treatment-related external, visceral, or 

skeletal malformations or variations. 

Increased numbers of resorptions/dam were noted in the 300 (0.6 resorptions/dam) and 1000 (0.7 

resorptions/dam) mg/kg/day groups compared to concurrent controls (0.2 resorptions). However, 

these findings were within the range of historical controls (0.3-0.7 resorptions/dam). The 

increases were attributed to one 300 mg/kg/day female that resorbed 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 reflected 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 resulted in a decrease in 

the number of live fetuses/dam at 300 (8.9 fetuses/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 developmental toxicity study (Tanaka et al., 1994), groups of 20 

presumed pregnant CD-1 mice/dose level were administered 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 treatment; 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 resorptions/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/day, respectively. The increased resorptions/dam 

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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 observed in this study, it occurred after acute 

exposure to dose levels substantially higher than the limit dose (1000 mg/kg/day). 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental 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 for viable fetus/doe, post-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/Carcinogenicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study (MRID 40323701), groups of 60 SpragueDawley Crl-CDR rats/sex were administered PBO (87.67-89.7% purity) in the 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 males/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 the remainder of the 15 mg/kg/day rats were euthanized at Week 8 

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as there were no observed effects of treatment on this group. For clinical chemistry 

examinations, additional groups of 10 rats/sex were sacrificed at 25-, 51-, 79-, and 97/98-week 

intervals. Organs that were weighed included the liver, kidneys, testes, ovaries, brain, and 

adrenals. Gross and microscopic pathological examinations were conducted on these tissues as 

well as the epididymides, prostate, seminal vesicles, uterus, and thyroids/parathyroids. 

No treatment related clinical signs 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 decreases in body weight gains 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 Week 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 significantly decreased in the high dose group 

on some occasions. 

No consistent significant or treatment-related effects were observed for the ophthalmological, 

hematological, or urinalysis parameters. Serum 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. Absolute liver weights were increased in males 

by 21% at the 500 mg/kg/day, and in females 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 mid 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 dose group, and increased interstitial cell tumors in the high dose 

group. These observed testis effects were not considered treatment 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 follicles were observed in the high 

dose group of both sexes. Follicle hyperplasia was significantly increased in the high dose group 

of both sexes and the mid dose females. No treatment-related histopathological lesions were 

observed in the ovaries or mammary gland. 

Based on increased liver weights, the NOAEL is <30 mg/kg/day and the LOAEL is 30 

mg/kg/day. 

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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 subjected to histological examination 

included these organs, as well as epididymides, prostate, seminal vesicle, ovaries, uterus, 

pituitary, adrenals, and thyroids/parathyroids. 

No treatment-related effects were observed on mortality 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 reduced (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 were observed for females. No treatment related 

effects were observed for food consumption or hematology parameters 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, prostate, seminal vesicle, ovary, or uterus. Increased 

(p<0.05) incidences of hepatocellular 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 males, 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/females) for one year. Organs that were 

weighed included the adrenals, heart, liver, pituitary, thyroid/parathyroids, brain, kidneys, ovary, 

and testis. Gross and microscopic pathological examinations 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 was 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 related effects were observed at the ophthalmological 

examinations or for the hematological or urinalysis parameters. Alkaline phosphatase was 

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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. There 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 males (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 corroborating 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 effects. 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 grade) in the diet at dose levels of 0, 6000, 12,000, or 24,000 ppm 

(equivalent to 0, 300, 600, and 1200 mg/kg/day, calculated by reviewer) for 13 weeks. Organs 

that were weighed included the brain, liver, heart, kidney, spleen, lung, testis, prostate, adrenal, 

thymus, ovary and uterus. Histological examinations were conducted on the liver and kidneys. 

Clinical signs in all treatment groups included nose 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 (data not provided). The following clinical 

chemistry increases (p<0.05) were observed in the 24,000 ppm males 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 were 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 decreased (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 microscopy showed 

enlarged hepatocytes with glassy cytoplasm and fatty deposition. On occasion, there was 

coagulative necrosis of a few hepatocytes in the periportal area and oval cell proliferation. The 

kidney of treated rats showed atrophy of the epithelium in the proximal convoluted tubules. 

For the 24,000 ppm males, significant decreases were reported in the absolute weight of brain, 

heart, spleen, lung, prostate, thymus and in the relative weight of the prostate, 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 reported in the absolute weight of lung and the 

absolute and relative weights of uterus.

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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 concentrations 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 determined. The tissues subjected to histological 

examination included these organs, as well as uterus and thyroids/parathyroids. 

Mortality, body weight, food consumption, ophthalmoscopic observations, hematological 

parameters, clinical chemistry parameters, and 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 and lymphoid cell aggregates in the mucosa, and 

eosinophilic material and inflammatory cells in the lumen. In addition, pseudostratified 

ciliated/non-ciliated columnar epithelium-squamous/squamoid metaplasia/hyperplasia in the 

ventral seromucous glands of the larynx was observed in all treated groups. 

At 0.155 mg/L, an increased incidence of secretory signs, such as dried red and brown material 

on the face, dried nasal discharge, and matted coat, were noted. The incidence of these findings 

increased at 0.512 mg/L. 

At 0.512 mg/L, columnar epithelium-squamous/squamoid metaplasia/hyperplasia 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 (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%). 

No treatment-related changes were seen in the weights of the adrenals, ovaries, testes, and 

epididymides, and no treatment-related histopathological findings were observed in the adrenals, 

ovaries, testes, and epididymides, uterus and thyroids/parathyroids. 

The inhalation toxicity LOAEL for this study is 0.015 mg/L based on histopathological changes 

in the larynx. The inhalation toxicity NOAEL for this study was not established. 

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The systemic toxicity LOAEL for this study is 0.155 mg/L based on increased secretory activity 

and slightly increased liver weights. The systemic toxicity NOAEL 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 weeks. Individual doses were adjusted weekly, 

based on the most recent body weights. The rabbits were inspected twice daily for signs of 

toxicity and mortality; detailed dermal evaluations were performed daily. Organ weights were 

determined for the liver, kidney, testes, epididymides, and adrenals. Microscopic examinations 

were performed on the uterus, thyroid and above tissues. 

No animals died during the study. Clinical signs, 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 sites. 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 histopathological examinations. The 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 prior to the start of egg production and 12 

weeks of egg production). Each treatment group was comprised of twenty mating pairs, with one 

pair per pen. Individual adult body weights were recorded at study initiation, biweekly through 

Week 8, and at study termination. 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 shell thickness was measured weekly during egg production. Adult birds and 

chicks were observed routinely for 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 weights were decreased (p<0.05) at 1500 and 3000 

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ppm; however, the decreases were slight (↓6% and ↓2%, respectively) and not dose-dependent. 

The LOAEC was 1500 ppm, based on reduced adult 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 prior to the start of egg production and 12 

weeks of egg production). The birds were housed with two male and five females per pen, with 

six pens per dose group. Individual adult body weights were recorded at study initiation, 

biweekly through Week 8, and at study termination. 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 shell thickness was measured weekly during egg production. Adult 

birds and chicks were observed routinely 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, treatment related signs of toxicity 

included weakness, inability to stand, and subdued behavior. Treatment-related 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 parameters, egg shell thickness, or 

offspring body weight in the ≤ 300 ppm groups. In the 1200 ppm group, significant effects 

included reduction in number of eggs laid, reduction in eggshell thickness, 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 stage 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 group (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 consisted 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 clinical 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, compared to 90-93% in the controls. Post hatch survival at 31 

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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, and 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%, respectively, at the 0.42 mg ai/L and 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 monitored for 21 days. F1 embryos were exposed thru sexual 

maturation and fecundity monitored. F2 embryos were exposed for 4 weeks post hatch and 

monitored for growth and survival. In addition to fecundity in parental and F1 generation, 

survival, body weight and length, VTG, GSI, tubercle scores, gonadal staging and histopathology 

were also measured. Based on a preliminary review of the study, 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 gonadal histopathology. In the F1 generation, larvae-juvenile 

survival was significantly decreased at all test concentrations (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. 

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APPENDIX 3: References Not Utilized in the Piperonyl Butoxide WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support 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 English; 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 acute mortality toxicity data were 

provided; 6) the experimental conditions were not adequately described; 7) only an abstract of 

the study was available; 8) the reference is a review article or book chapter and does not contain 

primary study data; 9) insufficient information was 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 endpoints 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 androgen receptor status in 

hepatocellular hypertrophy induced by phenobarbital, clofibrate, and piperonyl butoxide in F344 

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

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

36(5): 507-514. 

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Tanaka, T, Takahashi, O, and Oishi, S (1992) Reproductive and neurobehavioral effects in threegeneration toxicity study of piperonyl butoxide administered to mice. Food Chem. Toxicol. 30: 

1015-1019. 

Tanaka, T (1992) Effects of Piperonyl Butoxide on F1 Generation Mice. Toxicology Letters 60: 

83-90. 

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

161. 

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