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

OFFICE OF CHEMICAL SAFETY AND

POLLUTION PREVENTION



## MEMORANDUM

Date : 6/29/2015

SUBJECT: Piperony l butoxide: Data Evaluation Records (DERs) 

## for EDSP Tier 1 

## Assays

PC Code: 067501



## Decision No.: 460709



## Petition No.: NA



## Risk Assessment Type: NA

TXR 

## No

. : 0056259



## MRID No.: See Tabl e 

## DP Barcode : D398558 

## Registration No.: NA 

## Regulatory Action: NA



## Case No

. : NA



## CAS No. : 5 l-03-6



## 40 

## CFR: NA



## FROM: Greg Akerman

, Ph.D.

# ~ 

# A-

- --

Imm ediate Office

Health Effects Di vision (7509P)



## THROUGH: JessRowland

# ~~~~ Deputy Director

Health Effects Di vis ion



## TO: Jolene Trujillo Biologist/Chemical R eview Manager

Risk Management and Implement at i on Branch V

Pesticide Re- evaluat i on Di v i sion (7505 P )



## I. ACTION REQUESTED Ver.Apr. 20 I 0



## The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects

Division (HED) review the Endocrine Disruptor Sc reening Program (E DSP ) Tier 1 assays
submitted in response to the agency's Test Order for piperonyl butoxide: Test Order # CON067501-9.

Page I of2

### Page 2 of 2 

## II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for piperonyl butoxide.

III

. MRID Table

Chemical: Piperonyl butoxide PC Code: 067501

Guideline Assay MRID

890.1100 Amphibian Metamorphosis Assay (Frog) 48673101, 49606701

890.1150 Androgen Receptor Binding (Rat Prostate) 48618801

890.1200 Aromatase Assay (Human Recombinant) 48618802

890.1250 Estrogen Receptor Binding 48618803

890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa

- 9903) 48618804

890.1350 Fish Short

- Term Reproduction 48687701

890.1400 Hershberger (Rat) 48 618805

890.1450 Female Pubertal (Rat) 48618806

890.1500 Male Pubertal (Rat) 48618806

890.1550 Steroidogenesis (Human Cell Line

– H295R) 48618807

890.1600 Uterotrophic (Rat) 48618808

### Data Evaluation Record on the Toxicity of Piperony l Butoxide (PBO) to Amphibians, Metamorphosis Assay

EPA MRID Numbers 48673101, 49606701

Data Requirement: EPA DP Barcode 404227 (original report), 426891 (amended report)

OECD Data Point 231

EPA MRID 48673101 (original report), 49606701 (amended report)

EPA Guideline 890.1100

Amphibian Metamorphosis Assay (Frog)

Test Material: Piperonyl Butoxide Purity (%): 93.7%

Common Name: PBO

Chemical Name: IUPAC: 5-[[2-(2-buto xyethoxy)ethoxy]methyl]-6-propyl-1,3-benzodioxole

or 2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

CAS Name: 5-[[2-(2-butoxyethoxy)ethoxy]methyl]-6-propyl-1,3-benzodioxole

CAS No.: 51-03-6

Synonyms: 4,5-Methylenedioxy-2-propylbenzyldiethyloneglycol butyl ether

(Butylcarbityl) (6-propyl-piperonyl) ether, Piperonyl butoxide

technical grade

EPA PC Code 067501

Primary Reviewer: John M. Marton, Ph.D. Date: 3/25/13

Environmental Scientist, CDM Smith

Secondary Reviewer(s): Teri S. Myers, Ph.D. Date: 3/27/13

Program Manager, CDM Smith

Primary Reviewer: Donna R. Judkins, Ph.D.

Biologist/OPP/EFED/ERB2 Date : 05/28/13

Additional Reviewer: Justin Housenger

Biologist/OPP/EFED/ERB5 Date: 3/20/14

Additional Reviewer: Elyssa Arnold

Biologist/OPP/EFED/ERB2 Date: 5/28/14

Date Evaluation Completed: 28-05-2015

Page 1 of 38

DER Template Version: 22 September 2011 Digitally signed by JUSTIN HOUSENGER
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN
HOUSENGER, dnQualifier=0000044455

Date: 2015.05.29 08:51:49 -04'00'

### Data Evaluation Record on the Toxicity of Piperony l Butoxide (PBO) to Amphibians, Metamorphosis Assay

EPA MRID Numbers 48673101, 49606701

CITATION: Fort, D.J. 2012 (Amended 2015). OPPTS 890.1100 21-d Amphibian Metamorphosis Assay.

Unpublished study performed by Fort Environmental Laboratories, Inc., Stillwater, OK 74074. Laboratory

report number PBTF01-00256. Study sponsored by Consumer Specialty Products Association, Inc., for the

Piperonyl Butoxide Task Force II, Washington, DC 20006. Study completed July 6, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of

eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to

interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. Th e robustness of the Tier 1 battery

is based on the strengths of each individual assa y to identify potential endocrine bioactivity with

complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the

results of each individual assay should not be considered in isolation but rather should be considered in the

context of other assays in the battery as well as Othe r Scientifically Relevant Information (OSRI). In order

to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence

(WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken

(refer to the WoE Document).

D

isclaimer: The guideline recommendations in this DER template are offered as a general reference to aid

in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test

Guidelines, nor to provide any guidance on how the study should be conducted.

Page 2 of 38

DER Template Version: 22 September 2011

### Data Evaluation Record on the Toxicity of Piperony l Butoxide (PBO) to Amphibians, Metamorphosis Assay

EPA MRID Numbers 48673101, 49606701

EXECUTIVE SUMMARY

The 21-day assay 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 snout-vent 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-mediated

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.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP

Guideline 890.1100).

Page 3 of 38

DER Template Version: 22 September 2011

### Data Evaluation Record on the Toxicity of Piperony l Butoxide (PBO) to Amphibians, Metamorphosis Assay

EPA MRID Numbers 48673101, 49606701

Results Synopsis:

Test organism NF stage at test initiation: 51

Test organism total length at test initiation (optional): Not reported

Test type: flow-through

Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 

### in the Amphibian

Metamorphosis Assay (AMA) with Piperonyl Butoxide.

Treatment

(mg a.i./L)

[TWA-measured] NF Developmental

Stage Hind Limb

Length 3 

### Asynchronous

Development Thyroid Gross and

Histopathology

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.00599 No No No No No No No

0.0263 No No No No No No No

0.0741 No No No No No No No

1 

### A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 

### The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of

the DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a

board-certified pathologist.

3 

### Hind-limb length is normalized to snout-vent length (SVL).

I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in U.S. EPA, Endocrine

Disruptor Screening Program Test Guidelines, OPPTS 890.1100 Amphibian

Metamorphosis (Frog). The following deviations were noted:

1. Dilution water hardness (132-144 mg/L as CaCO 3 

### ) was greater than recommended (40-43 mg/L

as CaCO 3 

### ).

2. All surviving tadpoles were late stage (NF  61) which may have confounded the ability to adequately

evaluate potential thyroid-mediated effects in non-late stage Xenopus laevis tadpoles.

These deviations do not impact the results of the study.

Page 4 of 38

DER Template Version: 22 September 2011

### Data Evaluation Record on the Toxicity of Piperony l Butoxide (PBO) to Amphibians, Metamorphosis Assay

EPA MRID Numbers 48673101, 49606701

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements

were provided. This study was conducted in compliance with 40 CFR 160.

A. Test Material Piperonyl Butoxide (PBO)

Description: Viscous liquid

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow

or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : E 104/10

Purity: 93.7%

Impurities: None listed

Stability of Compound: Mean-measured concentrations yielded recoveries of 56.2, 79.6, and 76.4%

of nominal in the low, middle, and high treatment groups, with coefficients of

variation (CV) of 11.1, 12.9, and 12.9%. Using time-weighted averages, the CVs

were 12.9, 12.9, and 13.3%.

Storage Conditions of

Test Chemicals: Stored at ambient conditions

Page 5 of 38

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be identified.Species scientific name: Xenopus laevisSpecies strain (if stated): Not reportedWere parents maintained asin-house stock?Yes Adult X. laevisfrogs were initially obtainedfrom XenopusI, Dexter, MI. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitive test?Yes1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Page6of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsAcclimation period for parentalfrogs (if applicable):Not specifiedDetails on parental feeding: Parental frogs were fedapproximately 1Tablespoon of #3 frogfood pellets per tanktwice a day.Food source is from Zeigler Bros., Inc.Details on parental health: Parental frogs were ingood health, displayingno abnormalities or signsof illness.Page7of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest singlespawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn (i.e., the spawns are not co-mixed)Number of spawnsevaluated (ifapplicable):3Number of eggssampled perspawn:All specimens on a givendayEmbryonic viability checked dailyNF stage at testinitiation51Study authors state that the developmental stage of X. laevistadpoles was determined by using the staging criteria ofNieuwkoop and Faber. EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 17 days post-fertilization).Page8of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsHandling at stagingIndividual larvae were randomly removed from the pooling tankby scooping with a small strainer. Animals were carefullyhandled during this transfer in order to minimize handling stressand to avoid any injury. The developmental stage was thendetermined by using a binocular dissection microscope.Selected larvae were randomly distributed to exposuretreatment tanks and inspected for animals with abnormalappearance (e.g., injuries, abnormal swimming behavior, etc.).Overtly unhealthy looking tadpoles were removed from thetreatment tanks and replaced with larvae newly selected fromthe holding tank. The treatment tanks were randomly assignedto a position in the exposure system. EPA recommends that to reduce variability associated with measurement (staging) error, it is important that staging be conducted as accurately as possible. It is preferred not to use anesthesia; however, it is possible to individually anesthetize the tadpoles using an appropriate method [e.g., 100 mg/L tricaine methanesulfonate (MS-222), appropriately buffered with sodium bicarbonate (pH 7.0)], prior to initial staging. Animals are carefully handled during transfer in order to minimize handling stress and to avoid any injury. If anesthesia is used, the DER should document the method.Page9of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsOrganismSamplingOn Day 7 of the study, 5 randomly chosen tadpoles perreplicate were removed from each test tank and humanelyeuthanized in 150 to 200 mg/L MS-222, appropriatelybuffered with sodium bicarbonate. Tadpoles were rinsed inwater and blotted dry, followed by body weight determinationto the nearest mg. Hind limb length and SVL, along withdevelopmental stage (using a binocular dissectionmicroscope), were determined for each tadpole. At testtermination (Day 21), the remaining tadpoles were removedand similarly euthanized. Tadpoles were rinsed in water andblotted dry, followed by body weight determination to thenearest mg. Developmental stage, hind limb length, and SVLwere measured for each tadpole. Prior to fixing tadpole tissuefor histopathological analyses, blood samples were collectedfrom each buffered MS-222 anesthetized larvae, plasmaisolated, and stored for possible analysis of thyroid hormones(thyroxine [T4] and triiodothyronine [T3]). In addition, tail finsamples from each specimen were collected and preserved inRNAlater for possible thyroid receptor beta (TRȕ) geneexpression analysis. Lab SOPs were referenced for thesemethods but information not provided with study. EPA recommends that the DER identify which measurements and observations were recorded on Day 7 of the assay and briefly describe the method used to subsample tadpoles (i.e., whether the selection method was consistent with the randomization procedure recommended in the guideline).Page10of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsAge at testinitiation:14 days post-fertilization(dpf)Mean total lengthat test initiation (ifreported):Not reportedSnout to vent length was the first of two endpoints used toassess tadpole growth. SVL (millimeters) was used to helpestablish generalized toxicity of the test article. All lengthmeasurements were based on digital photographs of thesurviving organisms from each treatment. EPA recommends that the DER briefly describe the method of measurement.Range of totallength at testinitiation (ifreported):Not reportedWas the optionalsize selectionmethod used?NoDetails on larvalselection:Normal appearing NFstage 51In order to guarantee at least 400 stage 51 acceptable larvaeat test setup, clutch sizes of ~1,500 embryos were givenpreference.Page11of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsLoading rate(rearing density):5 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for

flow- through systems or 4 tadpoles/L in static-renewal exposure systems.Type of food: Sera Micron® EPA recommends Sera Micron ® throughout pre- exposure (after NF stage 45/46) and during the entire 21-d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food: Sera Micron® USA,Montgomeryville, PAIodideconcentration indiet (if known):Not providedFrequency offeeding:2 times/day EPA recommends that feeding occur at least twice per day.Page12of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feedingregime:Feeding regime followedEPA guidelinerecommendations. It is recommended that food rations during the pre-exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study Day 0-4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure: Flow-through EPA recommends the use of a flow-through

system.Type of flow-throughdilution system (ifapplicable):Continuous flow serial diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 22Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Page13of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsFlow-through rate (ifapplicable):25 mL/min Flow rate provided complete volumereplacement every 2.7 hours. Recommended flow-through rate is 25 mL/min (complete volume replacement ca.

every 2.7 hrs).Details on toxicantmixing for flow-throughsystems (if applicable):Modified Benoit Mini-Diluter Mixing chamber was a continuous mix chamberin which stock solution and dilution water weresupplied. Baffle ensured complete mixing sincethe concentration desired was at or below limitof water solubility. Concentration prepared inmixing chamber represents high testconcentration. Recommended toxicant mixing for

flow- through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within

10%.Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72 hours.Aeration? No EPA recommends maintaining dissolved oxygen concentrations >40% air saturation (>3.5 mg/L). Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on

the tadpoles.Page14of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water: Treated municipal tap water Prepared by passing tap water through a 4 filtersystem: a multimedia filter to removesuspended solids; a 10” pre-treatment filter(5μm) to remove any solids; a 3.6ft3activatedvirgin carbon treatment filter to remove chlorine,ammonia, and higher molecular weightorganics; and a 5μm polishing filter to removeany carbon particles from the carbon treatmentphase. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland

(e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution wateranalyzed for pesticides,heavy metals, and othercontaminants?Yes. See Reviewer’sCommentsAnalyzed on January 28, 2011.Iodide supplementationin water?No If reconstituted water is used or if background levels of iodide in natural water are less than

0.5 μ g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Page15of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsTest vesseltype/materials:Glass aquaria EPA and OECD recommend that water-contact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size: 22.5 x 14.0 x 16.5 cmFill volume:4 LAdditional details onexposure system:NATable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3) 132 144 138.25 Weekly EPA recommends hardness 40 to 48 mg/L

as CaCO 3.pH 7.37.97.53xWeekly EPA recommends pH 7.5 ± 1, inter-replicate and inter-treatment differentials should not

exceed 0.5.Page16of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsDissolved oxygen (mg/L) 6.19.97.63x Weekly EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature (°C)21.421.921.6Daily EPA recommends temperature 22±1 o C; inter-replicate and inter- treatment differentials should not exceed 0.5 o C.Iodide (μg/L) 2 5.1 3.6 Semi-annually EPA recommends aquatic iodide range 0.5 –

10 μ g/L (supplemental iodide should not

exceed 2 μ g/L).Page17of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAmmonia (mg/L)0.010.060.04Bi-monthly General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia,

should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously. OECD recommends that DO and temperature be measured at least weekly and that pH and hardness be measured at least at the beginning and end of the test.Fluoride NotReportedPerchlorate NotReportedChlorate NotReportedOther --------Page18of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in the range-finder?Historical toxicity dataprovided in a similarspecies by the Sponsor EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Species: Xenopus laevisLife stage:51Test duration:4 daysPage19of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:Nominal concentrationswere 0 (negativecontrol), 0.0001, 0.001,0.01, 0.1, 1.0, 10.0, and25.0 mg/LData from the rangefinder showed that the4-day NOAEC, EC50, and LC50were 0.1,0.22, and 3.16 mg/L, respectively. Nomorbidity was observed at 0.1 mg/L PBOmg/L test concentration was the highestconcentration evaluated in the rangefinding study that induced <10% mortalityand morbidity, 0.1 mg/L PBO was set asthe maximum tolerated concentration(MTC).Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Page20of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:Range-finder EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of test concentrations:0.33 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?NoIndicate the type of valuespresented for measuredconcentrations:Time-weighted average TWA- 0.00599, 0.0263, and 0.0741 mga.i./LPage21of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsLimit of quantification (LOQ): 0.001 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD): Not reportedFrequency of measurement: Samples collected foranalysis on Days 0, 7,14, and 21. It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80Was a solvent used?NoSolvent type (if applicable): NAPage22of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration (ifapplicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used? NoPositive control (if applicable): NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’s surface: 0.6-2.0 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:None3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Page23of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Piperonyl Butoxide (PBO).Treatment IDNominalConcentration(mg a.i./L)TWA MeasuredConcentration(mg a.i./L)Mean CV(%)Details or RemarksGuideline RecommendationsNegative Control 0.00<0.001NA NA EPA and OECD recommend that test item concentrations be maintained at a coefficient
of variation (CV) 20%.Treatment 1 0.0110.0059912.9 NATreatment 2 0.0330.026312.9 NATreatment 3 0.1000.074113.3 NAAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Day 7: NF Stage, normalized HLL, SVL, wet weightDay 21: NF Stage, normalized HLL, SVL, wet weight, thyroid histopathologyDaily: mortality, clinical signsWere raw (individual) data provided?YesPage24of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701 EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development , indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-base d larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also incl udes thyroid gross

pathology observations.Page25of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701II. RESULTS AND DISCUSSIONA. ResultsBy test termination, a single mortality was observed in the each of the control, middle, and high concentration treatment groups (Table 9).Table 9: Larval Mortality in X enopus laevis.Treatment (mg a.i./L) [TWA-measured]Larval MortalityDay 71Day 21nMortality #Mortality %nMortality #Mortality %Negative Control80006011.70.0059980 0 0 60 0 00.0263 80006011.70.0741 80006011.71Sample size and cumulative mortality values at Day 7 prior to interim sacrifice.On Day 7, all replicate and treatment median values for developmental stage were 54 (Table 10). By Day 21, all replicate and treatment medianvalues were between 61 and 62, though there were a few non-late stage tadpoles in the control and treatment groups. No asynchronous developmentwas observed.Page26of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 10: Larval Development in X enopus laevis– Developmental Stage and Asynchronous Development.Treatment (mg a.i./L) [TWA-measured]Developmental StageDay 7Day 21nMedianStage# AsynchronousnMedianStage# AsynchronousNegative Control4 54 0 4 62 00.00599 454046100.0263 454046100.0741 45404610Day 7 normalized HLL averaged 0.153 in the negative control and 0.171, 0.180, and 0.163 in the mean-measured 0.00599, 0.0263, and 0.0741mg a.i./L treatment groups, respectively (Table 11). The study author reported that mean normalized HLL on Day 21 was 0.941 inthe negativecontrol and 0.826, 0.916, and 0.959 in the mean-measured 0.00599, 0.0263, and 0.0741 mg a.i./L treatment groups, respectively.Page27of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 11: Larval Development in X enopus laevis– Hind Limb Length.Treatment(mg a.i./L)[TWA-measured]Hind Limb Length (HLL)Day 7Day 21nMean(mm)±SDHLL:SVL1nMean(mm)±SDHLL:SVL1Negative Control4 1.77 0.417 0.153 416.33 5.5 0.9410.00599 41.790.3250.171415.076.30.8260.02634 1.84 0.441 0.180 4 16.25 5.8 0.9160.0741 41.700.4130.163416.916.40.959Abbreviations:SDStandard deviation.1Summary results for snout-vent length (SVL) are presented in the next table (Table 12).Day 7 SVL ranged from 10.28 mm in the mean-measured 0.0263 mg a.i./L treatment group to 11.50 mm in the negative control (Table12). Attest termination, mean SVL ranged from 17.7 mm in the negative control to 18.5 mm in the mean-measured 0.00599 mg a.i./L treatment group.Mean Day 7 body weight ranged from 0.144 g in the mean-measured 0.0263 mg a.i./L treatment group to 0.196 g in the negative control (Table12). By Day 21, , mean body weight ranged from 0.677 g in the negative control to 0.769 g in the mean-measured 0.00599 mg a.i./L treatmentgroup.Page28of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 12: Larval Growth in X enopus laevis.Treatment(mg a.i./L)[TWAmeasured]Snout-Vent Length (SVL)Body Weight1Day 7Day 21Day 7Day 21nMean(mm)±SDnMean(mm)±SDnMean(g)±SDnMean(g)±SDNegative Control 4 11.50 0.96 4 17.62 1.5 4 0.196 0.04 4 0.677 0.170.00599 410.470.934 18.501.840.1500.0440.7690.250.02634 10.28 1.11 4 18.14 1.9 4 0.144 0.05 4 0.756 0.230.07414 10.35 1.02 4 18.09 1.7 4 0.144 0.04 4 0.730 0.22Abbreviations:SDStandard deviation.1Also referred to as “wet weight” in the test guideline.No thyroid gland hypertrophy or atrophy were observed in any control or treatment tadpoles (Table 13). Mild follicular cell hypertrophy was observedin the control and all treatment groups and mild follicular cell hyperplasia was observed in the mean-measured 0.00599 and 0.0263 mg a.i./Ltreatment groups. There were no apparent treatment-related patterns as the incidence in the control and treatment groups was similar. The samepattern of mild incidence across the control and all treatment groups was apparent for follicular cell height increase and follicular cell shape.Page29of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 13: Gross Pathology and Histopathology of the Thyroid Gland in X enopus laevis.Treatment(mg a.i./L)[TWAmeasured]Diagnostic Observations1SeverityThyroid GlandHypertrophyThyroid Gland AtrophyFollicular CellHypertrophyFollicular Cell HyperplasianIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 20 20 20 20 15 20 201 20 0 20 0 20 5 20 02 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 00.005990 20 20 20 20 20 16 20 191 20 0 20 0 20 4 20 12 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 00.02630 20 20 20 20 20 13 20 191 20 0 20 0 20 7 20 12 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 00.07410 20 20 20 20 20 15 20 201 20 0 20 0 20 5 20 02 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 0Page30of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 496067011Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECDNo. 82 for reference.Table 14: Additional Thyroid Gland Histopathology Observations in X enopus laevis.Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 20 20 20 20 15 20 20 20 151 20 0 20 0 20 5 20 0 20 52 20 0 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 0 20 00.005990 20 20 20 20 20 16 20 20 20 161 20 0 20 0 20 4 20 0 20 42 20 0 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 0 20 00.02630 20 20 20 20 20 13 20 20 20 131 20 0 20 0 20 7 20 0 20 72 20 0 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 0 20 0Page31of38 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.07410 20 20 20 20 20 15 20 20 20 151 20 0 20 0 20 5 20 0 20 52 20 0 20 0 20 0 20 0 20 03 20 0 20 0 20 0 20 0 20 01Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.Page32of38 DER Template Version: 22 September 2011

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EPA MRID Numbers 48673101, 49606701

At test termination, no adverse clinical signs were observed in control or treatment tadpoles (Table 15).

Table 15: Clinical Signs in X enopus laevis

.

Treatment

(mg a.i./L)

[TWA-measured] Clinical Signs 1



### Type n Incidence

Negative Control None 59 0

0.00599 None 60 0

0.0263 None 59 0

0.0741 None 59 0

1 

### Note that asynchronous development (unable to stage) is reported previously in Table 10

B. Study Author’s Analysis and Conclusions

Concentration-response monotonicity was assessed visually from the replicate and treatment medians or

means. The statistical significance of all tests indicated was assessed at p = 0.05. None of the continuous

quantitative endpoints (HLL, SVL, wet weight) followed a monotonic concentration-response; therefore,

the data were evaluated for normality (Shapiro-Wilk’s test) and homogeneity (Levene’s test). Data sets

that failed either assumption were considered non-parametric. Data sets that were parametric were

evaluated using one-way ANOVA. For non-parametric data sets, a ranked ANOVA (Kruskal-Wallis

ANOVA) was used.

A consistent concentration-response was not observed with mortality; therefore, the data were evaluated

for normality (Shapiro-Wilk’s test) and homogeneity (Levene’s test) and a ranked ANOVA (Kruskal-Wallis

ANOVA) was used to analyze the endpoint. A significant treatment effect for developmental stage was

determined on the replicate median values using the Mann-Whitney U test.

Tadpoles with developmental stages lower or equal to NF stage 60 were not present at the conclusion

of the study. A two-factor ANOVA with a nested variance structure to account for the effect of late stage

development on growth was not performed.

The No Observable Effect Concentration (NOEC) was defined as the highest measured concentration

at or below which no statistically significant effect was observed. The Lowest Observed Effect

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EPA MRID Numbers 48673101, 49606701

Concentration (LOEC) was the lowest measured concentration at which a statistically significant effect

occurred relative to the dilution water control. The maximum acceptable toxicant concentration (MATC)

was defined as the geometric mean of the NOEC and LOEC.

Larval growth was reduced in all treatment groups on Day 7, though SVL and wet weight were comparable

between control and treatment tadpoles on Day 21. There were no significant effects on larval development

throughout the test and there were no apparent treatment-related effects on thyroid histopathology.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Mortality was less than 2% in the control and treatment groups and was therefore

not statistically analyzed by the reviewer. If endpoints exhibited a monotonic trend, the

Jonckheere-Terpstra Trend test was used to compare treatment data to the negative control. If the data

were non-monotonic, they were tested for normality using the Shapiro-Wilk test and for homogeneity of

variance using the Bartlett test ( Į = 0.01). If the data met these assumptions, the data were analyzed

using Dunnett’s test. If these assumptions were not met, the Mann-Whitney U Two-Sample test was used.

Day 7 and Day 21 developmental stage were analyzed using the Mann-Whitney U Two-Sample test. Day

7 SVL and wet weight were analyzed using the Jonckheere-Terpstra Step-Down Test, and the remaining

endpoints were analyzed using the Dunnett Multiple Comparison Test. By Day 21, late-stage tadpoles

(NF  61) accounted for 97, 97, 100, and 100% of tadpoles in the negative control and TWA 0.00059,

0.00263, and 0.00741 mg a.i./L treatment groups, respectively. EDSP statistical guidance recommends

running separate analyses for late-stage and non-late-stage tadpoles when late-stage tadpoles account

for at least 20% of the surviving tadpoles. However, the negative control and the lowest treatment group

only had two non-late-stage tadpoles each, which is not sufficient for statistical analysis. Therefore, the

Day 21 analyses were conducted using all tadpoles.

No asynchronous development was observed. Unless otherwise indicated, effects were considered

statistically significant at p < 0.05.

Conclusions:

On Day 7 wet weight and SVL were significantly decreased at all treatment levels (approx. 10% decrease

for SVL and approx.. 25% decrease for wet weight across all treatment levels, Jonckheere-Terpstra, p

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EPA MRID Numbers 48673101, 49606701

< 0.05) relative to the negative control. All other endpoints on Day 7 were comparable between the control

and treatment levels. On Day 21, there were no treatment effects observed for any endpoint.

Conclusions derived from statistical output are advised to be based on non-late stage tadpoles because

tadpoles typically exhibit test material independent reductions in size and weight after NF Stage 60. In

the current study, there were inadequate tadpoles at NF stage < 61 to conduct analyses on non-late stage

tadpoles. Results for Day 21 should therefore be interpreted cautiously.

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Piperonyl Butoxide (PBO) to Amphibians, Metamorphosis AssayEPA MRID Numbers 48673101, 49606701Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Piperonyl Butoxide.Treatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control 54 -- 62 -- -- -- -- -- -- -- -- -- --0.0059954 1.0 61 0.43 33.3 0.08 -13.2 0.14 -- -- -- -- No0.026354 1.0 61 1.0 33.3 0.08 -5.26 0.74 -- -- -- -- No0.074154 1.0 61 1.0 33.3 0.08 2.63 0.95 -- -- -- -- NoStatistical Test Mann-Whitney Mann-Whitney Dunnett Dunnett -- ----Abbreviations:Diff.Difference.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).Page36of38 DER Template Version: 22 September 2011

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EPA MRID Numbers 48673101, 49606701

Table 17: Growth Endpoints 1,2 

### in the AMA with Piperonyl Butoxide.

Treatment

(mg a.i./L)

[TWA-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. p % Diff. p % Diff. p %

Diff. p

Negative Control -- -- -- -- -- -- -- --

0.00599 -9.33 0.01 4.82 0.14 -23.6 0.01 13.6 0.17

0.0263 -10.8 0.02 2.83 0.50 -26.7 0.05 11.7 0.26

0.0741 -9.98 0.03 2.41 0.61 -26.8 0.03 7.9 0.54

Statistical Test Jonckheere-Terpstra Dunnett Jonckheere-Terpstra Dunnett

Abbreviations: Diff. 

### Difference.

1 

### Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.

2 

### Unless otherwise specified, effects are considered statistically significant at p < 0.05.

E. Study Deficiencies

Although there were deviations from the guideline, as noted in the Materials and Methods section of the

DER, the study met all validity and performance criteria.

F. Reviewer’s Comments

All but four surviving tadpoles in the control and treatment groups were late-stage (NF  61). Guidance

recommends that late-stage tadpoles be excluded from calculation of replicate means of growth endpoints.

When there are 20% or more in each treatment group, with at least one late-stage tadpole in each of

three replicates, separate analyses should be conducted for late-stage and non-late-stage tadpoles.

However, there were insufficient non-late-stage tadpoles in this study to conduct separate analyses.

Although almost all of the surviving frogs were beyond NF stage 60 at test termination, which may

confound the ability to adequately evaluate potential thyroid-mediated effects in the AMA, there was no

significant acceleration or delay of median NF developmental stage at any treatment concentration

throughout the test nor effects on thyroid histopathology compared to the control. The reviewer’s results

agreed with those of the study author.

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EPA MRID Numbers 48673101, 49606701

The most recent analysis of the dilution water for pesticides, PAHs, and metals on January 28, 2011 and

all water quality measurements met the U.S. EPA criteria for aquatic toxicity test culture/dilution water.

Measured concentrations were based on time-weighted averages.

III. REFERENCES

Nieuwkoop, P.D. and Faber, J. Normal Tables of Xenopus laevis (Daudin). Garland Publishing, London,

1994.

Grim, KC et al. Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect

Thyroid Active Substances. Toxicol. Pathol. 37:415-424, 2009.

Hamilton, M.A., Russo, R.C. and Thurston, R.V. Trimmed spearman-karber method for estimating median

lethal concentration in toxicity bioassays. Environ. Sci. Technol. 11(7):714-719, 1997.

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DER Template Version: 22 September 2011

### CETIS Summary Report Report Date: 28 May-15 14:29 (p 1 of 5)

Test Code: 067501 48673101 | 16-9668-8968

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Fort Evironmental Laboratories, Inc.

Batch ID: 08-1345-7463

Start Date: 15 Sep-11

Ending Date: Test Type: EDSP AMA Tier 1

Duration: NA Protocol: OCSPP 890.1100 Tier I AMA Diluent: Dechlorinated Tap Water

Brine: Species: Xenopus laevis

Source: Xenopus 1 Analyst: D. Judkins

Age: NF51

Sample ID: 10-9371-5354

Sample Date: 15 Sep-11

Receive Date: Code: 48673101

Sample Age: NA Source: Consumer Specialty Products (CONTASKF

Station: Client: EPA OCSPP EFED

Project: Synergist Material: Piperonyl butoxide

067501 4863101

067501 48673101 Batch Note:

Sample Note:

Comparison Summary

NOEL LOEL TOEL Endpoint Analysis ID Method PMSD TU

74.1 >74.1 NA 01-5334-4077 Mann-Whitney U Two-Sample Test NA 07d Developmental Stage

74.1 >74.1 NA 00-3175-4618 Dunnett Multiple Comparison Test 32.7% 07d HLL

74.1 >74.1 NA 13-4844-8105 Dunnett Multiple Comparison Test 36.5% 07d Normalized HLL

<5.99 5.99 NA 02-1444-7915 Jonckheere-Terpstra Step-Down Test NA 07d SVL

74.1 >74.1 NA 05-5064-2869 Dunnett Multiple Comparison Test 12.1% 07d SVL

<5.99 5.99 NA 15-2502-4532 Jonckheere-Terpstra Step-Down Test NA 07d Wet Weight

74.1 >74.1 NA 08-7049-2554 Dunnett Multiple Comparison Test 29.2% 07d Wet Weight

74.1 >74.1 NA 07-1725-8004 Mann-Whitney U Two-Sample Test 1.35% 21d Developmental Stage

74.1 >74.1 NA 04-2481-9849 Dunnett Multiple Comparison Test 15.9% 21d Only LS HLL

74.1 >74.1 NA 04-7392-1985 Dunnett Multiple Comparison Test 16.8% 21d Only LS Normalized HL

74.1 >74.1 NA 21-1535-1713 Dunnett Multiple Comparison Test 6.21% 21d Only LS SVL

74.1 >74.1 NA 02-3867-3812 Dunnett Multiple Comparison Test 18.5% 21d Only LS Wet Weight

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 28 May-15 14:29 (p 2 of 5)

Test Code: 067501 48673101 | 16-9668-8968

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Fort Evironmental Laboratories, Inc.

Control Type Mean Min Max Std Dev Count CV% Std Err Group 07d Developmental Stage Summary

95% LCL 95% UCL %Effect

0 Negative Control 54 54 54 0 4 0.0% 0 54 54 0.0%

5.99 54 54 54 0 4 0.0% 0 54 54 0.0%

26.3 54 54 54 0 4 0.0% 0 54 54 0.0%

74.1 54 54 54 0 4 0.0% 0 54 54 0.0%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 07d HLL Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.75 1.4 2.2 0.412 4 23.6% 0.206 1.09 2.41 0.0%

5.99 1.75 1.6 2 0.191 4 10.9% 0.0957 1.45 2.05 0.0%

26.3 1.9 1.8 2.2 0.2 4 10.5% 0.1 1.58 2.22 -8.57%

74.1 1.65 1.2 2 0.342 4 20.7% 0.171 1.11 2.19 5.71%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 07d Normalized HLL Summary

95% LCL 95% UCL %Effect

0 Negative Control 0.15 0.1 0.2 0.0577 4 38.5% 0.0289 0.0581 0.242 0.0%

5.99 0.2 0.2 0.2 0 4 0.0% 0 0.2 0.2 -33.3%

26.3 0.2 0.2 0.2 0 4 0.0% 0 0.2 0.2 -33.3%

74.1 0.2 0.2 0.2 0 4 0.0% 0 0.2 0.2 -33.3%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 07d SVL Summary

95% LCL 95% UCL %Effect

0 Negative Control 11.5 11 12.2 0.538 4 4.67% 0.269 10.7 12.4 0.0%

5.99 10.5 10.1 10.8 0.351 4 3.36% 0.176 9.89 11 9.33%

26.3 10.3 9.2 11.3 0.95 4 9.25% 0.475 8.76 11.8 10.8%

74.1 10.4 9 10.9 0.918 4 8.85% 0.459 8.91 11.8 9.98%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 07d Wet Weight Summary

95% LCL 95% UCL %Effect

0 Negative Control 0.196 0.168 0.23 0.0317 4 16.2% 0.0159 0.146 0.246 0.0%

5.99 0.15 0.14 0.161 0.00991 4 6.62% 0.00496 0.134 0.166 23.6%

26.3 0.144 0.099 0.18 0.04 4 27.9% 0.02 0.08 0.207 26.7%

74.1 0.144 0.099 0.164 0.0306 4 21.3% 0.0153 0.0949 0.192 26.8%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 21d Developmental Stage Summary

95% LCL 95% UCL %Effect

0 Negative Control 61.5 61 62 0.577 4 0.94% 0.289 60.6 62.4 0.0%

5.99 61 61 61 0 4 0.0% 0 61 61 0.81%

26.3 61.5 61 62 0.577 4 0.94% 0.289 60.6 62.4 0.0%

74.1 61.3 61 62 0.5 4 0.82% 0.25 60.5 62 0.41%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 21d Only LS HLL Summary

95% LCL 95% UCL %Effect

0 Negative Control 16.1 14.7 17.5 1.15 4 7.13% 0.575 14.3 18 0.0%

5.99 15 12.8 16.5 1.59 4 10.6% 0.796 12.5 17.6 6.67%

26.3 16.3 15 17.7 1.13 4 6.96% 0.566 14.5 18.1 -0.93%

74.1 17 15.5 18.7 1.47 4 8.62% 0.733 14.7 19.3 -5.43%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 21d Only LS Normalized HLL Summary

95% LCL 95% UCL %Effect

0 Negative Control 0.95 0.9 1 0.0577 4 6.08% 0.0289 0.858 1.04 0.0%

5.99 0.825 0.7 0.9 0.0957 4 11.6% 0.0479 0.673 0.977 13.2%

26.3 0.9 0.8 1 0.0816 4 9.07% 0.0408 0.77 1.03 5.26%

74.1 0.975 0.9 1.1 0.0957 4 9.82% 0.0479 0.823 1.13 -2.63%

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 28 May-15 14:29 (p 3 of 5)

Test Code: 067501 48673101 | 16-9668-8968

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Fort Evironmental Laboratories, Inc.

Control Type Mean Min Max Std Dev Count CV% Std Err Group 21d Only LS SVL Summary

95% LCL 95% UCL %Effect

0 Negative Control 17.6 16.8 18.3 0.686 4 3.88% 0.343 16.6 18.7 0.0%

5.99 18.5 17.5 19.1 0.698 4 3.77% 0.349 17.4 19.6 -4.82%

26.3 18.2 17.9 18.4 0.208 4 1.15% 0.104 17.8 18.5 -2.83%

74.1 18.1 17.3 18.7 0.58 4 3.21% 0.29 17.2 19 -2.41%

Control Type Mean Min Max Std Dev Count CV% Std Err Group 21d Only LS Wet Weight Summary

95% LCL 95% UCL %Effect

0 Negative Control 0.677 0.606 0.784 0.0802 4 11.9% 0.0401 0.549 0.805 0.0%

5.99 0.769 0.675 0.884 0.0908 4 11.8% 0.0454 0.624 0.913 -13.6%

26.3 0.756 0.705 0.796 0.0386 4 5.11% 0.0193 0.695 0.818 -11.7%

74.1 0.73 0.682 0.769 0.0363 4 4.96% 0.0181 0.673 0.788 -7.9%

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 28 May-15 14:29 (p 4 of 5)

Test Code: 067501 48673101 | 16-9668-8968

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Fort Evironmental Laboratories, Inc.

Control Type Group 07d Developmental Stage Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 54 54 54 54

5.99 54 54 54 54

26.3 54 54 54 54

74.1 54 54 54 54

Control Type Group 07d HLL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 2 1.4 1.4 2.2

5.99 1.6 1.8 1.6 2

26.3 1.8 1.8 1.8 2.2

74.1 1.2 1.6 1.8 2

Control Type Group 07d Normalized HLL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.2 0.1 0.1 0.2

5.99 0.2 0.2 0.2 0.2

26.3 0.2 0.2 0.2 0.2

74.1 0.2 0.2 0.2 0.2

Control Type Group 07d SVL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 11.7 11.2 11 12.2

5.99 10.2 10.7 10.1 10.8

26.3 9.8 10.8 9.2 11.3

74.1 9 10.8 10.9 10.8

Control Type Group 07d Wet Weight Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.216 0.168 0.17 0.23

5.99 0.14 0.161 0.143 0.155

26.3 0.121 0.18 0.099 0.175

74.1 0.099 0.148 0.163 0.164

Control Type Group 21d Developmental Stage Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 61 62 61 62

5.99 61 61 61 61

26.3 62 62 61 61

74.1 62 61 61 61

Control Type Group 21d Only LS HLL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 16 14.7 17.5 16.3

5.99 15.7 15.2 12.8 16.5

26.3 16.5 17.7 15 15.9

74.1 16.1 15.5 18.7 17.7

Control Type Group 21d Only LS Normalized HLL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.9 0.9 1 1

5.99 0.9 0.8 0.7 0.9

26.3 0.9 1 0.8 0.9

74.1 0.9 0.9 1.1 1

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 28 May-15 14:29 (p 5 of 5)

Test Code: 067501 48673101 | 16-9668-8968

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Fort Evironmental Laboratories, Inc.

Control Type Group 21d Only LS SVL Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 18.3 17.4 18.1 16.8

5.99 18.8 18.6 17.5 19.1

26.3 18.1 17.9 18.2 18.4

74.1 18.1 17.3 18.2 18.7

Control Type Group 21d Only LS Wet Weight Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.784 0.626 0.692 0.606

5.99 0.723 0.793 0.675 0.884

26.3 0.752 0.705 0.796 0.772

74.1 0.741 0.682 0.769 0.73

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 890.1150, Androgen Receptor Binding (Rat Prostate Cytosol)

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48618801 to include laboratory proficiency data; Main study was originally reviewed under TA 2

- 32 -2012)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Sandra Hastings Date: 3 / 29 /2012

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/16/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7 / 27 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7 / 27 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each in dividual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather shoul d be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIP E RO NY L BUTO X IDE I 067501 Androgen Rec ep tor Bindin g (Rat Pro s tate Cytoso l ) (201 1 ) I P age I of 17

OCSPP 890. 11 50/ OE C D None



## Primary Reviewer: Deborah Smega l



## Health E ffect s Division, Office of Pesticide Programs

Seco nd ary Reviewer: Greg Ake rm an, Ph.D

. 

## Health Effects Divi sion, Office of Pesticide Programs

# gn~y).,**~~** Date :**~** 

## Sig natur e:
, Date: G 

## IL 'f/ld""' Template ve r s i o n 0 8/ 2011



## DATA EVALUATION RECORD

STUDY TYPE: Androgen Receptor Bi nd i n g ( R at Prostate Cytoso l ); OCSPP 890. l 150



## PC COD E : 06750 1 DP 

## BARCODE: D398558



## TXR #: 0056259 

## CAS No.: 51-03-6



## TEST MATERIAL (P U RITY) : Piperonyl b ut oxide (93.8% a.i.)



## S YNONYM S : 5 -[ [2

- (2 -b utoxye th oxy)ethoxy] methy l ]-6-propy l-1

, 3 -b enzodioxo l e



## CITATION: Willoughby

, 

## J. A. (20 11 ). Piperonyl b ut oxide: androgen recepto r binding (rat

prostate cytoso l )

. CeeTox, In c

. , Kalamazoo

, Ml. Laboratory Study Number:

9146V- 1 00377ARB, Novembe r 17

, 2011. MRID 486 18801. Un p ubl i s h ed.



## S PONSOR: W ill oughby

, J.A. 

### (2012). Su p plementa l In formatio n

- Laborato r y Profic i ency

Dat a for ERTA assays and Sat u ra t io n Bind in g Data for AR and E R Binding Assays for Asso 1t ed Chemicals

. CeeTox

, In c.
, Kalamazoo

, M I. September 24,

20 11. MR ID 48843501. Unpub li shed.

Consumer Specia lt y Products Assoc i at i o n
, I nc. Fo r Piperonyl But ox i de T ask

Force II

, 1900 17 1 11 

### Street

, Was hin gto n
, D.C.



## T ES T ORDER#: CON-067501-9



## EXECUT IVE SUMMARY: In an androgen receptor (AR) binding assay (M RID 486 1 8801),

ventral prostate cytoso l from Sp r ague D awley rats was u sed as the so ur ce of AR to co ndu ct a competi tive binding experiment, which meas u red the binding of a s in g l e concentration of [3 H]

- Rl 881 (l nM) in t h e presence of i ncreasing concentra ti ons ( 1 0

- 1 0 

### to 10

- 3 

### M) ofp ip e r o n y l

butoxide (93.8% a .i

. , Batch # El 04110)

. DMSO was used as the ve hi cle at a final assay
concentration of approximately 3.2%. A tota l of three independent run s were performed

, and th e

assay included d examet hasone as a weak positive con trol

, and RI 881 as the li ga nd r eference

standa rd.

A saturation binding experime nt was co ndu c t ed t o demo n strate tha t the A R in the rat prostate cytosol was p r ese n t in r easonable numbers and was fu n ctioni n g wi th appropria t e affin it y for the

radiolabeled r eference androgen (R l 881 ). Sa tur a ti on binding data were not origina ll y provided in

the s tud y report ; how eve r
, s ummarized saturat i o n binding data (M RID 48843501) from the perfo rmin g l aboratory were s ubmitted follow in g a r eq u est by the Agency. The mean
dissociation constant (Kct) for [3 T

- I ]-R 1 881 was 1.080±0.690 nM a nd the est im ated Bm i tx 

### wasAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 2 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of t he three K d 

## values was within the range reported in the EPA validation program (0.685 to 1.57 nM), and all B
max 

## values were below the recommended range (7 to 16 fmol/100 μg protein). Confidence in these numbers is high based on the goodness of fit (R 2 

## = 0.957- 0.984) and the small variation among runs.

In the competitive binding experiment, 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 l og IC 50 

## was −9.3 M for R1881 and

−4.4 M for the weak positive control (dexamethasone). Compared to R1881, t he 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 fo r R1881 and dexamethasone were lower than the targeted (guideline) levels in the first run because of an error in the stock preparation. The refore, 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

IC 50 

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

The percent labeled R1881 bound to the AR in the presence of piperonyl butoxide was >75% at
concentrations ≤ 10 −4 

## M in all three runs. As specific binding was not < 50% at any

concentration

, an IC 50 

## and RBA could not be calculated for piperonyl butoxide. Based on the results from the three runs, p iperonyl butoxide is classified as a Non-Binder in the Androgen Receptor Binding Assay.

Th e assay satisf ies the EDSP Tier 1 Test Order requirements for an Androgen R eceptor Binding Assay (OCSPP 890.1150).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 3 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: Willoughby, J.A.

Other Personnel: Rutherford, K. (Director Laboratory Operations); Blakeman, D. (Senior Scientist); Haines, C. (Scientist); McColley, S . (Scientist); Toole, C. (Director of Project Management) Study Pe riod: May 23, 2011 to November 17, 2011



## 2

. Test substance : Piperonyl Butoxide

Description: T echnical ; oily yellow liquid

Source: Endura Fine Chemicals, Italy

Batch #: E104/10 ( expir y date : July 2012) Purity: 93.8 % Solubility : Not reported Volatility : Not reported Stability: Not reported Storage conditions: Ambient (room temperature)

CAS #: 51

- 03

- 6 Molecular weight: 338.44 g/mol

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Non

- labeled ligand : R1881

Supplier : Sigma Aldrich (St. Louis, MO) Catalog # : Batch #: R0908

060M4638 Purity: 98 %

CAS #: 965

- 93

- 5



## 4

. Radioactive ligand : [ 3 

## H]

- R1881

Supplier : Perkin

- Elmer (Boston, MA) Catalog :
Lot #: NET590

653698 Date of production: February 24, 2011

Date of use: September 10, 2011 to September 17, 2011 Radiochemical p urity: >97 % Specific activity : 85.1 Ci/ m mol

Concentration of stock : 10 nM



## 5

. Positive control : Dexamethasone

Supplier : Sigma Aldrich (St. Louis, MO) Catalog #

Lot #: D1756

077K1050 Purity: 99%

CAS # : 50

- 02

- 2



## 6

. Solvent/vehicle control : D imethyl sulfoxide (D MSO )

Justification for choice of Piperonyl butoxide solutions up to 10

− 4 M (limit concentration for the assay) can beAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 4 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None

solvent : prepared while limiting the final concentration of DMSO to ~3.2 %

Final Concentration : ~ 3.2% (v/v)



## B. METHODS

1. Preparation of R at Ventral Prostate Cytosol : The rat prostate tissue was purchased from Charles River Laboratories (location not specified; Batch #210007463). M ale Sprague Dawley rats (number not reported) were castrated at approximately 90 days of age and were

euthanized <24 hours later and t he ventral prostate tissues collected.

The ventral prostates w ere received at the laboratory frozen and inspected for healthy

appearance, weighed, and placed in ice -cold low- salt TEDG (pH 7.4) buffer (inclusion of protease inhibitor not reported), homogenized, and centrifuged for 30 min at 30,000 × g at
4oC. The s upernatants w ere pooled, discarding the resulting pellets. Protein concentration of the cytosol was determined to be 6.2 mg/mL using a commercially available protein kit compatible with DTT in the TEDG buffer (Bradford Method; Bio- Rad Dye Reagent

Concentrate ). Cytosol was divided into aliquots (amounts not specified ) and stored frozen

until use.

2. Saturation Radioligand B inding Experiment : A saturation binding e xperiment measuring total and non-specific binding of [ 3 

## H] -R1881 was performed to demonstrate that the AR was present in reasonable concentrations and had the appropriate affinity for the R1881 ligand ( MRID 48843501). The conditions for the saturation binding e xperiment are

summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a


### Source of receptor Rat ventral prostate cytosol
Concentration of radioligand (as serial dilutions) 0.25 to 10 nM

Concentration of non

- labeled ligand (100X [radioligand]) 2 to 1000 nM
Optimization of receptor concentration Sufficient to bind 25 to 35 % of radioligand at 0.25 nM
Temperature 4o C

Incubation time 16 to 20 hours
Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10%
Phenylmethylsulfonyl fluoride (PMSF) 1 mM

DTT 1 mM
Sodium Molybdate 1 mM

a Data were obtained from page 2 4 of the study report (MRID 48843501 ).



## On the day of the assay, the specific activity of the stock solution [ 3 

## H] -R1881 (originally 85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and serial dilutions in low- salt

TEDG+ PMSF buffer were prepared to achieve the final concentrations in cytosol of 0.25,0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonspecific binding, solutions of non-labeled R1881 were prepared in a similar manner toAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 5 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## achieve concentrations that were 100

- fold greater than each respective radiolabeled concentration, resulting in final concentrations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, the radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

## H] -R1881 and 7.5, 15, or 30 μL of 100 n M [ 3 

## H] -R1881 was measured.

For each batch of cytosol, the optimal protein concentration was determined by calculating specific binding to differing amounts of protein per tube with 0.25 nM radiolabeled R1881. The optimal protein concentration was determined to be 1.86 mg protein/assay tube, which resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic protein used in this assay was thawed fresh for this experiment at 4°C, and maintained at 4°C during the binding assa y. Each saturation b inding experiment consisted of three noncurrent runs (conducted on September 9, 11, and 16, 2011, respectively). Each run contained three replicates at each concentration, resulting in the 72 samples depicted in

Table 2.

TABLE 2

. Saturation Binding Experiment Run a ,b



### Total Binding Non

- Specific Binding Radioligand alone

Tubes 1

- 24 c 

### Tubes 25

- 48 d 

### Tubes 49

- 72 e


### [ 3 

### H]

- R1881 Final conc. (nM) [ 3 

### H]

- R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 

### H]

- R1881 Initial conc. (nM) [ 3 

### H]

- R1881 (μL)

0.25 0.25 25 10 7.5

0.50 0.50 50 10 15

0.70 0.70 70 10 21

1.0 1.0 100 10 30

1.5 1.5 150 10 45

2.5 2.5 250 100 7.5

5.0 5.0 500 100 15

10 10 1000 100 30

a Data were obtained from page 25 of the study report ( MRID 48843501 ).

b Each concentration was run in triplicate for a total of 72 samples.

c Tubes 1

- 24 contained 50 μL of triamcinolone acetonide and 7.5

- 45 μL [ 3 

### H]

- R1881. Samples were dried, and 300 μl of prostate cytosol were added.

d Tubes 25

- 48 contained 50 μL of triamcinolone acetonide and 7.5

- 45 μL [ 3 

### H]

- R1881. R1881 was added in a

100

- fold molar excess of [ 3 

### H]

- R1881 in a volume of 7.5

- 45 μL. Samples were dried, and 300 μl of prostate cytosol were added.

e Tubes 49

- 72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

### H]

- R1881 or 7.5, 15, 21 , or 30 μL of 100
nM [ 3 

### H]

- R1881 without cytosol or other components to determine the total counts added.



## Following addition of triamcinolone acetonide, [ 3 

## H] -R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 or 21 hours at approximately 4°C. Samples were maintained at temperatures of approximately 4°C except during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed 5 times with 4

- minute intervals over 20 minutes. The samples were then centrifuged, and the supernatant was aspirated and discarded. The samples were washed 3 times in 2 mL of ice cold

TEDG+ PMSF buffer, followed by vortexing and centrifugation for 3 min utes at 600 x g. Following the last wash and decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. The samples were vortexed and centrifuged for 10 minutes atAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 6 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## 1000 x g. Samples were maintained on ice at all times between vortexing. After the final centrifugation, the ethanol supernatants were decanted into scintillation vials that each contained 14-mL portions of scintillation cocktail, and the radiation was quantified by liquid scintillation counting. A total of th ree runs were performed.

3. Competitive B inding Experiment : A summary of the assay conditions for the competitive binding experiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a


### Source of receptor Rat prostate cytosol
Concentration of radioligand 1 nM Optimization of receptor c oncentration Sufficient to bind 8.7

- 9.7 % of 1.0 nM radioligand b



### Concentration of test substance (as serial dilutions) 10

− 1 0 

### to 10

− 3 

### M c



### Incubation Temperature 4 ± 2 o C

Incubation time 16

- 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4 )

EDTA 1.5 mM Glycerol 10 % (v/v)
Protease Inhibitor (PI) d 

### 0.5% (v/v)

DTT 1 mM Sodium Molybdate 1 mM

a Data were obtained from page 16 of the study report.

b Values were calculated by the reviewer; data obtained from pages 35

- 37 of the study report. Protein concentrations (μg/tube) were not reported.

c Data for 10 −3 

### M were not assessed due to precipitation at the higher concentration

. d The recommended PI in the Guideline is 1 mM PMSF. The PI was identified in the study protocol as Calbiochem Protease Inhibitor Cocktail, Set III, EDTA free. Although it was described as containing PMSF, it is a mixture of six PIs that includes 4

- (2

- aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF has similar specificity to PMSF with greater stability at lower pH.



## The competitive binding experiment was performed according to the protocol provided in

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experimen t measures the binding of a single concentration of [ 3 

## H] -R1881 ( adjusted specific activity of 82.6 Ci/mmol for Run 1; 8 2.5 Ci/mmol for Runs 2 and 3) to the AR in the presence of increasing

concentrations of a test substance.

DMSO was used as the vehicle, and precipitation was visually observed at 10

− 3 

## M piperonyl

butoxide in all three run s. Therefore, the highest concentration of piperonyl butoxide used in evaluating the data was 10 −4 

## M.

Only limited information was provided for the saturatio n binding experiment; however, the

reviewer was able to calculate from the competitive binding data that the amount of cytosolic protein used in the assay contain ed enough receptor to bind 9-10 % of the [ 3 

## H]

- R1881.

Dilutions of the test substance, reference standard (R1881), weak positive control (dexamethasone), and solvent control ( DMSO ) were prepared to achieve the concentrationsAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 7 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## shown in Table 4. E ach assay consisted of three independent runs on three different days,

and each run contained thre e replicates at each concentration plus six replicates of the [ 3 

## H]

- R1881, NSB, and solvent controls, resulting in a total of 8 4 samples per run

. It is noted that in Runs 2 and 3 only three replicates of the [ 3 

## H] -R1881 were analyzed.

TABLE 4 . Competitor Final Molar (M) Concentrations in Competitive Binding Assay a , b



### Solvent Control Reference standard c 

### Weak positive control c 

### Test Chemical None (Hot Mix) DMSO R1881 Dexamethasone Piperonyl Butoxide

Tubes 7

- 12 Tubes 13

- 18 and 19

- 33 d 

### Tubes 37

- 60 Tubes 6 1

- 84 e 

### Tubes 1

- 6

1×10

− 6 

### (3.3 ×10

− 8 

### ) 1×10

− 3 

### (1 ×10

− 4 

### ) 1×10

− 3



### 1×10

− 7 

### (3.3 ×10

− 9 

### ) 1×10

− 4 

### (1 ×10

− 5 

### ) 1×10

− 4



### 1×10

− 8 

### (3.3 ×10

− 10 

### ) 1×10

− 5 

### (1 ×10

− 6 

### ) 1×10

− 5



### 1×10

− 9 

### (3.3 ×10

− 11 

### ) 1×10

− 6 

### (1 ×10

− 7 

### ) 1×10

− 6



### 1×10

− 10 

### (3.3 ×10

− 12 

### ) 1×10

− 7 

### (1 ×10

− 8 

### ) 1×10

− 7



### 1×10

− 11 

### (3.3 ×10

− 13 

### ) 1×10

− 8 

### (1 ×10

− 9 

### ) 1×10

− 8



### -- 1×10

− 9 

### (1 ×10

− 10 

### ) 1×10

− 9



### -- 1×10

− 1 0 

### (1 ×10

− 11 

### ) 1×10

− 1 0



### a Data were obtained from page s 35

- 37 of the study report.

b The [ 3 

### H]

- R1881 , NSB, and solvent controls were run in 6 replicates and e ach concentration of each chemical was run in triplicate for a total of 8 4 tubes per run. Tubes 1

- 84 contained 50 μL of triamc inolone acet onide and 30 μL [ 3 

### H]

- R1881 . Samples were dried, and 300 μ L of prostate cytosol were added. Tubes 7- 84 also contained

10 μL of the solvent control, reference standard (non

- radiolabeled R

- 1881), weak positive control, or test substance, with the exception of Tubes 13

- 18 that contained 30 μL of non

- radiolabeled R1881 (used to evaluate

non

- specific binding). Tubes 1-6 contained only 30 μ L of [ 3 

### H]

- R1881 ; Runs 2 and 3 only analyzed triplicate samples of [ 3 

### H]

- R1881 (Tubes 1

- 3), but tube numbering was maintained as for Run 1

.

c Because of an error in the stock preparation, concentrations of R1881 and dexamethasone deviated from the targeted concentration in the first run. Actual concentrations for R1881 and dexamethasone for the first run are reported in parentheses.

d Tubes 13

- 18 were used to evaluate non

- specific binding by adding 100X of cold (non

- radiolabeled) R1881

, except for Run #1 in which 30X of cold (non

- radiolabeled) R1881 was added due to an error in the stock preparation.

e P recipitation was observed at 10

− 3 

### M, therefore the h ighest concentration used for data evaluation was 10

− 4 

### M.



## Sample tubes were stored 16-20 hours at 4°C to allow the reaction to reach equilibrium

, and bound R1881 was separated from free R1881 by washing with an HAP slurry and extraction with ethanol, followed by scintillation counting of bound [ 3 

## H] -R1881. A total of three runs were performed as described.

4. Data Analysis : For the saturation binding experiment, total binding and non- specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 ( GraphPad Software, Inc., La Jolla, CA)] , incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

## implemented by using the ROUT procedure in Prism v. 5 

## with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

## Swillens (1995) 2 

## . For the competitive binding assay,

similar methods of nonlinear regression were used to fit a curve (for R1881, the positive

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressi on

-  a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123

- 142

. 2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197

- 1203.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 8 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## control, and the test substance) to the Hill equation formula which incorporated l og IC 50 

## as a

p arameter to be estimated. For parameters reported from the saturation b inding experiment
( 

## K d 

## and B
max 

## ) and c ompetitive binding e xperiment ( l og IC 50 

## and RBA), m ean and standard

deviation were calculated for each run and mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the composite three runs with

Microsoft Excel 2010.



## 5. Definitions

a. Classification of test material

If the data fit a 4 -parameter nonlinear regression model , the test chemical is classified as :

Binder : The a verage curve for the test chemical across runs crosses 50% of radioligand

bound.

Equivocal : The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at least 25% but less than 50%), or the curve falls outside the range for the weak positive control (

− 0.6 to

− 1.4).

Non

- Binder : The average lowest portion of curves across runs is greater than 75% activity ( i.e. less than 25% displacement of radioligand), or the data do not fit the model.

Untestable: If the test compound is not soluble above 1×10

− 6 

## M and the binding curve does not cross 50%, the chemical is judged to be untestable.

b. Descriptors for receptor binding

B max 

## : maximal binding capacity

K d 

## : dissociation constant

IC 50 

## : Concentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor 

## Relative Binding Affinity (RBA) : ( IC 50 

## of R1881 ÷ IC 50 

## of test substance) × 100 Log RBA: Log 10 

## (IC 50 

## of R1881 ÷ IC 50 

## of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Saturation binding e xperiment parameters are presented in Table 5. The dissociation constant (K d 

## ) for [ 3 

## H] -R1881 was 1.080±0.690 nM, and the estimated B
max 

## was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of t he three K d 

## values was within the range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was in the acceptable range . Confidence in these numbers is based on large variation in K d 

## estimates among runs.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 9 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



### TABLE 5 . Saturation Binding Experiment of [ 3 

### H]

- R1881 with Androgen Receptor from Rat Prostate Cytosol a



### Parameter Run 1 Run 2 Run 3 Mean ± SE b



### R 2 

### (unweighted) 0.880 0.986 0.893 0.880

- 0.986

B
max 

### (nM) 0.010 0.008 0.011 0.010 ± 0.002

B
max 

### (fmol/100 μg protein) 1.087 0.896 1.229 1.071 ± 0.167

K d 

### (nM) 0.855 0.531 1.855 1.080 ± 0.690

a Data were obtained from page 34 of the study report (MRID 48843501 ).

b The range of R 2 

### is reported and the mean ± SE is reported for the other parameters.

R 2 

### Goodness of fit for curve calculated for specific binding



## Figure 1 illustrates the non -specific, specific, and total binding curves for [ 3 

## H] -R1881 to the androgen receptor for the three independent runs. The specific binding reached a plateau in each run, and non-specific binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

## H] -R1881 to the androgen receptor.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 10 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## F IGURE 1. Binding of [ 3 

## H] -R1881 to the Androgen Receptor during the Saturation Binding Experiment.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 11 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## F IGURE 2. Scatchard Plots of the Binding of [ 3 

## H] -R1881 to the Androgen Recepto r.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 12 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## B. COMPETITIVE BINDING EXPERIMENT : The results from the three c ompetitive binding e xperiment s are summarized in Table 6 and shown graphically in Figures 3-5. Precipitation of piperonyl butoxide was visually observed at the highest concentration (10

− 3 

## M) in all three run s; therefore , the highest concentration of piperonyl butoxide used for

data evaluation was 10

− 4 

## M. The specific binding 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 IC 50 

## and RBA could not be calculated for piperonyl butoxide.

The estimated average log IC 50 

## was −9.0 M for R1881 and −4.5 M for the weak positive control (dexamethasone). Compared to R1881, t he mean RBA for dexamethasone was 0.0032% for the two valid runs (excluding the first run). Confidence in these numbers is high due to the small variation between the two runs. Ho we ver, it is noted that variations between Run 1 compared to Runs 2 and 3 are likely due to the concentr ation differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the first run. 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. In addition, all solvent control tubes were run at the beginning of each experiment, preventing the reviewer from determining drift in the assay from the beginning to the end of the run.

TABLE 6. Competitive Binding Assay of Piperonyl Butoxide with AR from Rat Prostate Cytosol a



### Parameter Run 1 Run 2 Run 3 Mean ± SE

R 2 

### (unweighted) R1881 NR NR NR NA

Dexamethasone NR NR NR NA Piperonyl butoxide NR NR NR NA
Log IC 50 

### (M) R1881

- 9.9

- 9.0

- 9.0

- 9.3±0.3 0 b



### Dexamethasone

- 4.3

- 4.4

- 4.6

- 4.4±0.09 b


### Piperonyl Butoxide NA NA NA NA

IC 50 

### ( M) d 

### R1881 1.26x10

- 10 

### 1.00x10

- 9 

### 1.00x10

- 9 

### 7.09x10

- 10 

### (±2.91)

Dexamethasone 5.01x10

- 5 

### 3.98x10

- 5 

### 2.51x10

- 5 

### 3.83x10

- 5 

### (±0.73) Piperonyl Butoxide NA NA NA N A
Log RBA d 

### Dexamethasone

- 5.6

- 4.6

- 4.4

- 4.9 ±0.37 Piperonyl Butoxide NA NA NA N A
RBA (%) d 

### Dexamethasone 0.0003 0.0025 0.0040 0.0022±0.0011 Piperonyl Butoxide NA NA NA NA

a Data were obtained from the results discussion, page 21

- 22 of the study report. The study report erroneously reported the l og IC 50 

### for dexamethasone from the first run as

- 4.4 M, but three other study reports that include the reference compound data from September 10, 2011 report the l og IC 50 

### for dexamethasone from the first

run as

- 4.3 M.

b Values are reviewer

- calculated from the l og IC 50 

### values reported in the study report. For means expressed in

scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean

value.

NR Not reported

NA Not applicable

R 2 

### Goodness of fit

RBA Relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 13 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## FIGURE 3. Percentage [ 3 

## H]

- R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Piperonyl Butoxide (Run 1 ).Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 14 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## FIGURE 4. Percentage [ 3 

## H]

- R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Piperonyl Butoxide (Run 2 ).Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 15 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## FIGURE 5. Percentage [ 3 

## H]

- R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Piperonyl B utoxide (Run 3 ).Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 16 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## C. PERFORMANCE CRITERIA : To ensure that the competitive binding assay was functioning properly, each run was evaluate d using the criteria shown in Table 7:

T ABLE 7. Criterion a 

### Tolerance Limit(s) b 

### Value Yes No Ligand depletion is minimal. The recommended r atio of total binding in the absence of competitor to total

amount of [ 3 

### H]

- R1881 added per assay tube. ≤15% 8.7 to 9.7 % X

Piperonyl butoxide Top (% binding) 80 to 115 98 to 108 X

R1881 fitted curve parameters
Top (% binding) 82 to 114 96 to 103 X Bottom (% binding)

− 2.0 to 2.0 0 X Hill Slope

− 1.2 to

− 0.8

− 1.0 X Weak positive control (dexam e thasone ) fitted curve parameters
Top (% binding) 87 to 106 101 to 105 X Bottom (% binding)

− 12 to 1 2 NA;

− 1 to 7 c 

### X Hill Slope

− 1. 4 to

− 0. 6 −1.0 to

− 0.6 X
Saturation Binding Experiment K d 

### (nM) 0.685 to 1.57 0.531 to 1.855 X d



### Non

- specific binding (%) ≤ 10.0 0.28 to 0.50 e 

### X

a Data were obtained from page s 24

- 29 of the study report.

b These values represent ranges from the validation study.

c The bottom plateau level for dexamethasone in Run 1 was reported as NA (not applicable) because lower concentrations were used due to an error in the stock preparation.

d MRID 48843501

e Calculate d by the reviewers using the data from pages 35

- 37 of the study report.

NR Not reported



## In the first run, the concentrations for R1881 and dexamethasone were lower than the targeted (guideline) levels because of an error in the stock preparation. Therefore, non

specific binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold R1881, and the resulting curve for dexamethasone fit two of the three criteria (Hill slope and Top plateau) but the Bottom plateau was not achiev ed for the first run. All performance criteria were met for the other two runs

.

Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [ 3 

## H] -R1881 in a manner consistent with one-site binding, as

indicated by a Hill slope of

− 1.0. Examination across the runs indicated consistency of the Hill slope, placement along the X -axis, and top and bottom p lateaus for R1881.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Piperonyl butoxide is classified as a “non

- binder” in all three valid independent runs and thus has a final classification of “non-binder” to the

AR.

B. AGENCY C OMMENTS : In the saturation binding assay, t he mean K d 

## for [ 3 

## H] -R1881

was 1.080 nM, and the mean estimated B
max 

## was 1.071 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three K d 

## values was within theAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 17 of 17

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1150 / OECD None



## range reported in the EPA validation program, and the mean value was in the acceptable range. Confidence in these numbers is low based on the large variation in K d 

## estimates

among runs.

In the c ompetitive bindin g e xperiment , p recipitation of piperonyl butoxide was visually

observed at the highest concentration (10

− 3 

## M) in all three run s , therefore the highest

concentration used for data evaluation was 10

− 4 

## M. T he estimated average log IC 50 

## was −9.3 M for R1881 and −4.4 M for the weak positive control (dexamethasone). Compared to
R1881, t he mean RBA for dexamethasone was 0.0022%. Confidence in these numbers is generally high due to the small variation between the runs. However, it is noted that variations between Run 1 compared to Runs 2 and 3 are likely due to the concentration

differences used for R1881 and dexamethasone. The bottom of the curve for dexamethasone was not fully defined in the first run. 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. The concentrations prepared for R1881 and dexamethasone were lower than the targeted (guideline) levels because of an error in the stock preparation. Therefore, non-specific binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold R1881, and the resulting curve for dexamethasone fit only two of the three criteria (Hill slope and Top plateau ) in the first run

. All performance criteria were met for

the other two runs.

The bottom percent binding for dexamethasone was not achieved in the first run, leaving only two valid runs instead of the required three. However, as the performance was consistent between the other two runs, it is unlikely that information from an additional run would change the interpretation of this study.

Piperonyl butoxide is classified as a “non

- binder” as radiolabeled R1881 binding was >75%

at concentrations ≤10 −4 

## M in all three runs. An IC 50 

## and RBA could not be calculated for piperonyl butoxide as it did not result in 50% inhibition at any concentration.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• The bottom percent binding for dexamethasone was not achieved in the first run. T he concentrations prepared for R1881 and dexamethasone were lower than the targeted (guideline) levels because of an error in the stock preparation.

• The protein concentration used in the assay was not reported.

• All solvent control tubes were run at the beginning of the experiment, preventing the reviewer from determining drift in the assay from the beginning to the end of the run.

# D ATA EVALUATION RECOR D

P IPERONYL BUTOXIDE Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618802)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature:

Joan Harlin , M.S. Date: 4/7 / 2012

Secondary Reviewer Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 4/18/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/ 23 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/ 23 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIPERONYL BUTOX ID E I 06750 1



## Primary Reviewer : Chris Sch l osser, M

. F.S

Health E ff ects Divi sio n Aromatase ( H uman Recomb in a n t) Assay (20 1 I) I P age 1of14

OCSPP 890. 1200 I OEC D No n e**.**



## Secondary Re v iewer : Greg Akerman, Ph.D.

___ _ S i g natur e:

# ~-**--** Date:**ff** 

# I I 

# >

- Signature:

## ~**~** Date : ' <;;' /; ( 

# ltrr: Health Effec t s Di v i s i o n
Template ve r s ion 08 / 20 11



## DA TA EVALUA TIO N RECORD

STU D Y TYPE: Aromatase ( Hum an Recombinant); OCSPP 890. 1 200



## PC CODE: 06750 I 

## DP BARCODE: 0 398558



## TXR#: 0056259 

## CAS No.: 51-03-6



## TEST MATERIAL (PURITY): Piperonyl butoxide (93

. 8% a.i

. )



## SYN O NYMS: 5-[[2-(2-butoxyethoxy)ethoxy ]methy l ]-6-propyl- 1 ,3-be n zod i oxole



## C ITATION: Wilga, P .C. (2011) P i peronyl butoxide: human r eco mbin ant aromatase assay

. CeeTox, Inc

., Kalamazoo

, MI. Laboratory Report No

. : 9146V-100377AROM

, November 17, 2011. MRID 48618802. Unpublished.



## SPONSOR: Consumer Specialty Produ cts Associa tion

, 1900 17 111 

## Stree t NW

, Wash i ngton

, D

. C

.



## TEST ORDER#: CON-067501-9



## EXECUTIVE SUMMA RY : In an 

## in vitro aromatase (CYP 19) assay (M RID 48618802),

p ip e r onyl butoxide (93.8% a.i. Lot # £104110) was incubated with human recombinant

aromatase and tritiated androstenedione 

## ([1B

3 

## H(N)]-androst-4-ene-3

, 17-dion e; [31

- I )AS D N) in

:S l % dimethyl sulfoxide (DMSO) at lo g concen tration s of 10

- 1 0 

## to 10

- 3 

## M for 15 minutes to

assess the potential of piperonyl butoxide to inhibit aromatase activity.

Aromatase activ ity was detennined by measur i ng the amoun t of tritiated water produced at the

end of a 15-minute incubation for each concentration of chemica l. Tritiated water was quantified

u s ing liquid scintillat i on counting (LSC). Three runs were conducted and each run included a full activity contro l

, a background act i vity contro l

, a positive co ntr ol ser ie s (10

- 1 0 

## to 

## io

- 5 

## M)

u si ng a known inhibitor (4-hydroxyandrostened ion e; 4-0H ASDN) in dup l icate, and the piperon y l butoxide ser i es ( 

## 10

- 1 0 

## to I 0

- 3 

## M) wit h three replicates per concentration

.

Aromatase activity in the f ull act i v it y contro l s was 0

. 207 ± 0

. 025 nmo l· mg-protein

- 1 

## ·min

- 1

. 

## The

r es pons e of each full activity control within a run was between 90 to 110 % of the average full activity. Activity in the background contro l s ranged from 0.52 to 0

. 78% and averaged 0

. 63% of

the full activity controls

.

For the positive contro l substance (4-0H ASDN)

, aromatase activity r esu l ts were wi thin the

re co mmended ranges for the performance criteria. The estimated log IC so for 4-0H ASDN

ave raged

- 7.13 Mand th e Hill slope was -1.02.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 2 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## For piperonyl butoxide, t he two highest test ed 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. A romatase activity was 95.9 % and

85.9% of the control at 10

− 9 

## and 10

− 4 

## M, respectiv e ly

.

B ased on the data from the average response curve , piperonyl butoxide is classified as a Noninhibitor of aromatase activity in this assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Aromatase a ssay (OCSPP 890.1200).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 3 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : P iperonyl butoxide

Description: Technical; oily yellow liquid

Source: Endura Fine Chemicals, Bologna, Italy

Lot # (expiration date): E104/10 (July 2012) Purity: 9 3

. 8 % Volatility: N ot reported Storage conditions: Room temperature (e.g. ambient) Stability: N ot reported

Solvent: DMSO Solubility (in test solvent): 10

− 1 M Highest Concentration Tested: 10

− 3 M Stock Solution Preparation: Not reported Molecular weight: 338.43 g/mol

CAS #: 5 1

- 03

- 6

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS # : 63

- 05

- 8

Source: Steraloids, Inc. (Catalog # A6030

- 100)

Batch # (expiration date) : L1712 (April 2016) Purity: 99. 8 %



## 3

. Radiolabeled S ubstrate : 1

- β [ 3 

## H(N)]

- Androst

- 4

- ene

- 3,17

- dione ; ( [ 3 

## H] ASDN)

Source: Perkin Elmer (Catalog # NET

- 926)

Batch # (expiration date) : 619344 ( January 10, 2012 )

Radiochemical Purity (Supplier): >97 % Specific activity: 26.3 mCi/mmol Radiochemical Purity (In

- lab determination): Not determined



## 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN)

CAS # 566

- 48

- 3

Source: Sigma ( Cat alog # F2552 )

Batch # (expiration date) : 081K2133 (March 2015) Purity: 99. 6 %



## 5. Solvent (V ehicle C ontrol ) : Dimethyl sulfoxide ( DMSO )

Source:

Batch # (expiration date) : Not reported Not reported

Justification for choice of solvent One of the acceptable vehicles detailed in the EPA guideline ; less volatile than ethanol; and more accurate to pipette because of density and volatility

. Concentration
(% of total volume in assays) ≤ 1%Aromatase (Human Recombinant) Assay ( 2011 ) / Page 4 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## 6. Test M icrosomes : Human recombinant aromatase ( CYP19 ) microsomes

Source : BD Gentest TM , Woburn, MA ( Cat alog # 456260 )

Lot # (expiration date) : 03897 ( February 201 4 )

Protein concentration : 7.4 mg / mL
Cytochrome C reductase activity: 290 nmol∙mg

- protein −1 ∙min −1

Aromatase activity: 6.0 p mol/ pmol P450 /min



## B. METHODS

1. Assay C omponent s and Preparations : A mixture of non- labeled and radiolabeled
[ 3 

## H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 0.1 μM, and the amount of tritium added to each incubation tube was

0.1 μCi.

The test chemical(s) were formulated in DMSO such that the volume of DMSO used per assay was no more than 1% v/v of the total assay volume. DMSO was selected because it is listed as one of the solvents of choice detailed in the EPA guideline ; it not as volatil e as ethanol and so evaporation was less of a concern in the assay, and is more accurate to pipette because of its density and viscosity.

A stock solution of the positive control substance, 4- OH ASDN, was formulat ed in DMSO

. Dilutions wer e prepared such that the target concent rations of the positive control substance (10

− 10 

## to 10

− 5 

## M; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from BD Gentest TM 

## , and stored at

− 80±10oC. Before use of the microsomes, the batch was thawed and sub-aliquoted into individual vials before being refrozen to minimize freeze-thaw cycles to no more than one.

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

T ABLE 1. Assay Components and Conditions

Assay Factor Values
0.1M sodium phosphate buffer (pH 7.4)

Microsomal Protein 0.00 4 mg/mL

NADPH 0.3 mM

[ 3 H]ASDN 100 nM Propylene Glycol 5%
Temperature 37°C

Incubation Time 15 min



## 2. Suitability Ass es s ments : The protein concentration of the microsomes (Lot #19701 ) was supplied by the vendor as 7.4 mg/m L . Aromatase activity of the microsomes was also provided by the vendor as 6.0 pmol/min/ pmol P450. The m inimum aromatase activity in the full activity control samples was determined to be 0. 173 nmol

∙ mg -protein −1



## ∙ min −1 

## , which was greater than the minimum recommended aromatase activity of 0.1 nmol

∙ mg

protein −1



## ∙ min −1



## .Aromatase (Human Recombinant) Assay ( 2011 ) / Page 5 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## 3. Aromatase A ssay : Each assay run contained 4 tubes for the full enzyme activity and 4 tubes for the background activity controls. Two tubes of each control were run at the beginning and en d of each assay run . A full concentration curve in duplicate for the positive control, and a full concentration curve in triplicate for the test substance were established. The aromatase assay was generally conducted according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

## H 2 

## O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of p mol∙mg

- protein

− 1 

## ∙min

− 1



## .

4. Demonstration of P roficiency : It was stated that p roficiency testing of th e CYP19 aromatase assay was conducted. Each of the proficiency chemicals (atrazine, econazole, fenarimol and nitrofen) were incubated alongside the positive control inhibitor (4- OH

ASDN ) on three separate occasions using the same methods employed in the current study. The data were not included in the study report.

Positive Control

(1) Initial Demonstration of Laboratory P roficiency : The positive control data for laboratory met the following criteria:

• Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mg

protein/min.

• Mean background control activity was ≤ 15 % of the full activity control.

• Coefficient of variation (CV) for replicates within each sample type and concentration

of 4

- OH ASDN was <15%.

• Performance criteria (Table 2) were met, and served as guidance in identifying runs that provided parameters in the preferred ranges.

(2) Demonstration o f P roficiency of N ew T echnician for C onducting Assay (when applicable) : The positive control data for slope, top and bottom percent met the

criteria as listed in section (i ) of OCSPP 890.1200.

TABLE 2. Performance Criteria for the Positive Control a

Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit
Slope

− 1.2

− 0.8

− 1. 17

− 0. 9 2
Top (%) 90 110 9 7

. 9 10 4

. 1
Bottom (%)

− 5 +6 0. 2 2.4
Log IC 50 (M)

− 7.3

− 7.0

− 7. 2

− 7. 0

a Data were obtained from pages 18 and 23 of the study report.



## b. Proficiency C hemicals : It was stated that t he proficiency of the laboratory was demonstrated prior to running any test chemicals by conducting three full scale test runs on each of the proficiency chemical s as listed in Table 3. The data were not included in the study report. It was stated that t he four proficiency chemicals were correctly classified as

non-inhibitor or inhibitor.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 6 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



### TABLE 3

. Proficiency Chemicals
Compound CAS# Class Concentrations

Econazole 24169

- 02

- 6 Inhibitor No Data

Fenarimol 60168

- 88

- 9 Inhibitor No Data

Nitrofen 1836

- 75

- 5 Inhibitor No Data

Atrazine 1912

- 24

- 9 Non

- inhibitor No Data



## 5. Determination of Aromatase A ctivity with T est C hemical(s) : T he response of aromatase activity to the presence of eight concentrations of piperonyl butoxide per run, in triplicate, was tested during three independent runs (Table 4)

. Solubility was assessed visually (presence of cloudiness or a precipitate ). If insolubility was observed at the highest test concentration (10

− 3 

## M) for the first run, then the highest test concentration would be adjusted for the second and third runs at the highest test concentration that appeared soluble using log or half-log concentrations.

The full enzymatic activity was obtained at the two lowest concentrations of piperony l butoxide, defining the top of the concentration-response curve.

T ABLE 4. Test Chemical Study Design for each Test Run a

Sample Type Repe titions (Tubes) Description Reference or Chemical (M)
Full Activity Control 4 All test components b plus solvent vehicle N/A
Bkgd Activity Control 4 Same as above without NADPH N/A

4

- OH ASDN Conc 1 2 All test components plus 4

- OH ASDN 1×10

− 5

4

- OH ASDN Conc 2 2 All test components plus 4

- OH ASDN 1×10

− 6

4

- OH ASDN Conc 3 2 All test components plus 4

- OH ASDN 1×10

− 6.5

4

- OH ASDN Conc 4 2 All test components plus 4

- OH ASDN 1×10

− 7

4

- OH ASDN Conc 5 2 All test components plus 4

- OH ASDN 1×10

− 7.5

4

- OH ASDN Conc 6 2 All test components plus 4

- OH ASDN 1×10

− 8

4

- OH ASDN Conc 7 2 All test components plus 4

- OH ASDN 1×10

− 9

4

- OH ASDN Conc 8 2 All test components plus 4

- OH ASDN 1×10

− 10
Piperonyl butoxide Conc 1 c , d 3 All test components plus Piperonyl butoxide 1×10

− 3
Piperonyl butoxide Conc 2 c , e 3 All test components plus Piperonyl butoxide 1×10

− 3.5

Piperonyl butoxide Conc 3 c 3 All test components plus Piperonyl butoxide 1×10 −4

Piperonyl butoxide Conc 4 c , f 3 All test components plus Piperonyl butoxide 1×10

− 4. 5
Piperonyl butoxide Conc 5 c 3 All test components plus Piperonyl butoxide 1×10

− 5
Piperonyl butoxide Conc 6 c 3 All test components plus Piperonyl butoxide 1×10

− 6
Piperonyl butoxide Conc 7 c 3 All test components plus Piperonyl butoxide 1×10

− 7
Piperonyl butoxide Conc 8 c 3 All test components plus Piperonyl butoxide 1×10

− 8
Piperonyl butoxide Conc 9 c , g 3 All test components plus Piperonyl butoxide 1×10 −9
Piperonyl butoxide Conc 10 c , h 3 All test components plus Piperonyl butoxide 1×10 −10

a Data were obtained from page 19 of the study report.

b The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH

. c T est chemical

. d Hig h est concentration in Run#1; not used in Runs #2 and 3 due to precipitation.

e Highest concentration in Runs #2 and 3.

f Concentration added to Runs #2 and 3.

g Lowest concentration in Run s # 2 and 3

. h Lowest concentration in Run # 1.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 7 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## C. D ATA ANALYSIS

1. Raw D ata : Raw data were converted to aromatase activity (nmol

∙ mg

- protein −1



## ∙ min −1 

## ) and percent control for the positive control and test chemical. The following raw data and calculated endpoints for each run were included in the report (Table 5).

T ABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X)
DPM/mL for each portion of extracted aqueous incubation mixture X
Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X

Total DPM after extraction corrected for background X Aromatase activity expressed in nmol∙mg

- protein −1 ∙min −1 X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X

DPM Disintegrations per minute



## 2. S tatistical M ethods : For data generated at CeeTox, basic statistical analysis was performed on the data, which included means of replicates, standard deviation of the mean, standard error of the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed.



## The response curve was fitted by weighted nonlinear regression analysis using a 4-parameter regression model (XLfit; IDBS; Version 5.2.0.0; Fit Model 208) and Tukey’s BiWeight statistical analysis for out lier analysis.

For each independent run of the test substance, the individual percent of control values were plotted versus logarithm of the test chemical concentration

. The fitted concentration response curves were superimposed on the plot, with individual plots prepared for each run, and with plotted means. The fitted concentration response curves for each run were superimposed on the plots. On separate plots, the average percent of control values for each run were plotted versus logarithm of test s ubstance concentration. The average concentration response curve across runs was superimposed on the same plot.



## Assay drift was assessed by comparing the % full activity and background activity at values at the beginning of each run to the end of each run.

3. Interpretation of Results : Interpretation of the assay results was based on the average of three runs, using the categories presented in Table 6.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 8 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



### TABLE 6

. Interpretation of Results

Criteria Interpretation

Data fit 4

- parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor
Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

Average lowest portion of curves across runs is greater than 75% activity b Non

- inhibitor

Data do not fit model

---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

− 1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than

− 2.0, the result is classified as equivocal.

b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cross 50%, the chemical is typically

determined to be untestable in the aromatase assay

.



## II

. RESULTS

A. CONTROL ACTIVITY : Aromatase activity in the full activity controls for the three

successful test runs ranged from 0.173-0.247 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## , with a mean and

standard deviation of 0.207±0.025 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## . Activity in the background controls ranged from 0.52 to 0.78% and averaged 0.63%% of the full activity controls. The response of each full activity control within a run was between 90 to 110% of the average full activity

.

B. POSITIVE CONTROL : For the positive control substance (4- OH ASDN ), aromatase activity averaged 0.203±0.040 nmol∙mg -protein −1 

## ∙min −1 

## at the lowest tested concentration

10

− 10 

## M and 0.002±0.000 nmol∙mg -protein −1 

## ∙min −1 

## at the highest tested concentration

10 −5 

## M. The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across all 3 runs is presented in Table 7, along with

the overall standard deviation , SEM, and % CV. As a percent of the control, aromatase activity for 4

- OH ASDN was 1.05% at 10 −5 

## M and 97.58% at 10 −10 

## M. Inhibition response curves for the positive control from each run are shown in Figures 1- 3, the average inhibition response curve across all runs is shown in Figure 4, and the combined response of

mean (s) and all runs is shown in Figur e 5

.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 9 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



### TABLE 7

. Effect of P iperonyl butoxide on Aromatase Activity (as percent of control) from Independent

Runs a

Chemical Concen. ( Log M ) # Runs Overall Mean b Overall SD b Overall SEM b Overall % CV b

4

- OH ASDN (positive control) −5 3 1.05 0.09 0.05 8 .1

−6 3 8.15 0.60 0.35 7 .4

−6.5 3 20.85 0. 98 0. 57 4.7

−7 3 42.29 2.86 1.65 6.8

−7.5 3 71.05 4.46 2.58 6 .3

−8 3 89.7 1 3.79 2.19 4 .2

−9 3 102.29 2.67 1.54 2.6

−10 3 97.58 6.83 3.94 7 .0
Piperonyl butoxide

− 4 3 85.90 4.31 2.49 5.0

− 4. 5 2 93.64 5.22 3.69 5.6

− 5 3 95. 94 1. 76 1.02 1.8

− 6 3 95.80 1.11 0.64 1 .2

− 7 3 96.03 3.43 1.98 3.6

− 8 3 95.88 3. 89 2.25 4 .1

− 9 3 95.93 2. 29 1.62 2 .4

a Data were obtained from Appendix 1, pp. 4 2 , 4 4 , and 4 6 of the study report

b Calculated by the reviewers from data presented in this table.

c Data obtained for Runs 2 and 3; test concentration was not used for Run 1.

SD Standard Deviation

SEM Standard error of the mean

CV Coefficient of Variance



## C. TEST SUBSTANCE : For piperonyl butoxide , aromatase activity averaged 0.210 

## ± 0.024 

## nmol∙mg -protein

− 1 

## ∙min

− 1 

## at the lowest successfully tested concentration of 10

− 9 

## M and 0.179



## ± 0.028 

## nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## at the highest successfully tested concentration of 10

− 4 

## M.

The 10

− 3 

## M and 10

− 3.5 

## M test concentrations from Runs 1-3 were not used by the study author for interpretation because of observed precipitation. In the first test run of the assay, piperonyl butoxide was found to be soluble at concentrations of ≤ 10

− 4 

## M; therefore, for Runs 2 and 3 of the assay, the test concentration 10

− 4 

## M was the highest tested concentration used for study interpretation. Also in Run 1, the two lowest concentrations (10

− 9 

## and 10

− 10


## M) were removed from analysis because they were ≤ 51% of the control and wer e considered by the study author to be statistical outliers. In Runs 2 and 3, 10

− 9 

## M was the

lowest tested concentration. The mean aromatase activity of piperonyl butoxide (expressed

as % full control activity) for each concentration tested across all 3 runs is presented in

Table 7 (presented above) , along with the overall standard deviation, SEM, and % CV. As a percent of control, aromatase activity for piperonyl butoxide was ≥ 92.9 % at all

concentrations from 10

− 9 

## to 10

− 4 

## M. Inhibition response curves for piperonyl butoxide f rom each run are shown in Figures 1-3, the average inhibition response curve across all runs is shown in Figure 4, and the combined response of mean(s) and all runs is shown in Figure 5.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 10 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## F IGURE 1. Inhibition R esponse C urves for P iperonyl butoxide and 4

- OH ASDN f rom Run 1.

F IGURE 2. Inhibition Response Curves for P iperonyl butoxide and 4

- OH ASDN f rom Run 2.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 11 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## F IGURE 3. Inhibition Response Curves for P iperonyl butoxide and 4

- OH ASDN f rom Run 3.

F IGURE 4. Mean Inhibition Response Curves for P iperonyl butoxide and 4

- OH ASDN.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 12 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## FIGURE 5. Combined Response of Means and Runs 1 -3 for P iperonyl butoxide and

4- OH ASDN.

The average dose- response curve indicated that the aromatase activity in the presence of piperonyl butoxide at concentrations of 10 −9 

## to 10 −4 

## M was essentially equivalent to the activity observed in the full activity control. These data could not be fit to the 4- parameter nonlinear regression model; therefore, log IC 50 

## and Hill slope estimates were not determi ned for piperonyl butoxide. For 4

- OH ASDN, the estimated log IC 50 

## average d

− 7.13 M and the slope was

− 1.02 (Table 8) . The variation in the positive control values was acceptable ( <1 5% CV).

TABLE 8

. Effect of P iperonyl butoxide on Aromatase Activity (as P ercent of C ontrol) F rom Independent

Runs a

Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b

Log IC 50 ( M )
Piperonyl butoxide N A NA NA NA NA NA

4

- OH ASDN

− 7. 0

− 7. 2

− 7.2

− 7. 1 3 0. 12

− 2

Hill Slope
Piperonyl butoxide NA NA NA NA NA NA

4

- OH ASDN

− 1

. 17

− 0.9 2

− 0.96

− 1.0 2 0. 1 3

− 1 3

a D ata were obtained from Table 13, page 30 of the study report

b Calculated by the reviewers from data presented in this table.

SD Standard Deviation

CV Coefficient of Variance

NA N ot applicableAromatase (Human Recombinant) Assay ( 2011 ) / Page 13 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS : Piperonyl butoxide was determined to be a noninhibitor of aromatase activity in this validated assay as defined by EDSP guideline OPPTS (OCSPP) 890.1200.

B. AGENCY COMMENTS : Although data demonstrating proficiency with the positive control or recommended proficiency chemicals were not included, the report included a statement indicating that proficiency of the laboratory was demonstrated prior to analysis of piperonyl butoxid e by the conduct of three full scale test runs on each of the recommended proficiency chemicals. The report stated that the proficiency chemicals were correctly

classified as non -inhibitor or inh ibitor.

Aromatase activity in the full activity controls wa s 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 results were within the recommended ranges for the top of the curve, bottom curve, H ill slope, log IC 50 

## , and %CV for replicates of each concentration within runs. The estimated log IC 50 

## for 4

- OH ASDN averaged

− 7.13 M and the Hill slope was

− 1.02.

For piperonyl butoxide, aromatase activity at the lowest and highest successfully tested

concentration s, 10

− 9 

## and 10

− 4 

## M, w as 95.93 % and 85.90 % of the control , respectively . In Run 1-3, the highest tested concentrations of piperonyl butoxide (10 −3 

## and 10 −3.5 

## M) had observed precipitation ; therefore, the results of th ese concentration s were not used for interpretation. Also in Run 1, the two lowest tested concentrations, 10

− 9 

## and 10

− 10 

## M, were removed from analysis because they were ≤ 51% of the control and considered statistical outliers by the study author.

The average dose- response curve indicated that the aromatase activity in the presence of piperonyl butoxide at 10 −9 

## to 10

− 4 

## M was essentially equivalent to the activity observed in the full activity control. Therefore, these data could not be fit to the 4- parameter nonlinear regression model. Because mean aromatase activity for piperonyl butoxide was >75% at all successfully tested concentration up to 10 −4 

## M, piperonyl butoxide is classified as a noninhibitor of aromatase activity in this assay.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• Data for the proficiency chemical testing were not provided.

• For piperonyl butoxide, eight test concentrations were not used for interpretation in the aromatase assay results. Instead, only six test concent rations for Run 1 and seven test concentrations for Runs 2 and 3 were used by the study author for interpretation because the highest tested concentrations (10

− 3 

## and 10

− 3.5 

## M) caused precipitation, and

because the lowest tested concentration 10

− 10 

## M was considered a statistical outlier. AAromatase (Human Recombinant) Assay ( 2011 ) / Page 14 of 14

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0. 1200 / OECD None



## second mid-log concentration (i.e. 10

− 6.5 

## M) should have been added in order to obtain the required eight test concentrations, as noted in section (j) of OCSPP 890.1200. In addition, the 10

− 10 

## M test concentratio n for Runs 2 and 3 should have been included in data analyses.

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48618803 to include Saturation binding data; Main study was originally reviewed under TA 2

- 32 -2012)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlingt on, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Sandra Hastings Date: 3 / 30 /2012

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph .D., D.A.B.T. Date: 7/20/ 2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7 / 30 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7 / 30 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each in dividual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather shoul d be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).P I P E RO N Y L B U TO X ID E / 06750 1 Es tr oge n R ecc 1H o r Bindin g ( R a t U t e ri n e Cy t oso l ) ( 2 0 1 1 ) I P age I o r 1 8

O CS PP 890. 1250 / OE C D No n e



### Pri m ary Reviewer: C h ris Sc hl osse r
, M.F.S.

# Sig**natur ~~** **~-** Hca lth Effects Divi s i o n Date:**~LI** 

# (

Seco nda ry Reviewe r :

__

## G_**e__.g _.__** **e_ nn** **--"'-a n**  .....

, '"""

P

.....

h ._.=-

D

...... ______ S i g n a turc: 

# ,4;; '}'-

-

H ca Ith Effects Di vi s ion Date: 

## <:;;;**r/t~** T e mplat e ver s i o n 0 8/ 20 11



### D ATA EVALUATION RECORD

S TUDY TYPE : Estrogen Recep t o r Bin d in g Assay Usi n g R a t U t e rin e Cytoso l (E R-R UC);

OCS P P 890. 1 250

P C CO D E: 0 6750 l DP BARCODE: 0 398558

TXR # : 0056259 CAS No. : 5 1- 03

- 6

TEST MATERIAL (PUR I TY) : P i peronyl butoxide (93.8% a

. i.)

S YNONYMS: 5

- [[2

- (2 -bu t oxye th oxy)e th oxy ]met h y l] -6 -p ro p y l

- 1 ,3-be n zod i oxo l e

C ITATION: W ill oug h by

, J .A

. (2 011 )

. Pip e r o n y l b ut ox ide : e s tro ge n r ece pt o r bindin g ( r a t

ut e rin e cy t oso l ). Cee T o x
, In c., Ka l a m azoo, MI. L a b o rat o r y S tud y Numb e r:
9 1 46Y- 1 00377E RB

, Novembe r 1 7

, 20 11. M RID 4 8 618803. Unpubli s he d.

Willou g hby

, J.A. (2 012 )

. Supp l ementa l lnfonnat i on -Sa tu r at i o n B i n ding

P roficiency Informatio n provided b y CeeTox. CeeTox, I nc., Ka l amazoo
, M I.

Jul y 19

, 2012. M R I D 4884350 

## I. Unpub li s h ed

.

S PONSOR: Co n su m er Spec i a l ty P ro du c t s Assoc i a ti o n
, In c. Fo r Pi pe r o n y l B u tox i de Tas k

Fo r ce II

, 1 900 I 7 1 h 

### S tr ee t

, W as hin g t o n D .C

.

TEST ORDER#: CON-06750 1- 9

EXECUT I VE SUMMARY: I n an es t rogen receptor (E R ) b in d in g assay (M RID 486 1 8803)

, uterine cytosol from Sp r ague-Daw l ey rats was used as t he so u rce of ER to co n duct bi n d in g

ex pe r iments

. A saturation bindi n g experimen t was co n d u cted to de m ons t ra t e t hat t h e E R i n the

rat ute ri ne cytoso l wa s present in reaso n able numbe r s a n d was funct i o nin g w i t h a p propriate affinity for th e radio l abe l ed refe r e n ce estroge n prio r t o ro utin e l y co n d u c tin g E R co mpeti t i ve
b in d in g ex p e rim e nt s. T h e com p e titiv e bind i n g expe r i m e nt w a s co ndu c t ed l o m eas ur e th e bindin g
of a s in g l e co n ce nt ra ti o n of [3 H } l 7 P- est radi o l ( 1 nM ) in th e pr ese n ce o f in c r eas in g

conce n tra ti o n s ( 1 0

- 11 

### M t o 1 0

- 3 

### M) of pi pe r o n y l b ut ox id e (93 .8 % a. i

., Batc h # E 1 04 / I 0). O MSO

was used as the veh i c l e at a final co n ce n tration of approx i ma t e l y 2%. A tota l of t h ree run s were performed, and each run inc l uded 19

- no r et h indrone as a weak pos i tive contro l

,

oc t y ltriethox ys ilane as a negative contro l

, a n d I 7P-es tr adiol as the natura l ligand refere n ce

ma t erial.

Saturat i o n b indin g data we r e n o t o ri g in a ll y p rovided in th e s tud y r epo rt ; h oweve r
, s umm a ri ze d

sa tur at i o n bindin g da t a (M RID 4884 3 5 0 1 ) fro m th e p e r fo rmin g l a b ora t o r y we r e s ubmitted

fo ll owing a r e q ues t b y th e Age n cy. Dat a fr o m t wo se p a rate s atur a ti o n bin d i ng assays we r eEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 2 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## 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, t he K d 

## for [ 3 

## H]

- 17β

estradiol wa s 0.081±0.006 nM , and the estimated B
max 

## was 52.70±1.05 fmol/100 μg protein. For

the batch of cytosol prepared on July 10, 2011, t he K d 

## for [ 3 

## H]

- 17β

- estradiol was 0.303±0.090

nM , and the estimated B
max 

## was 33.59±0.70 fmol/100 μg protein. The K d 

## for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard 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, t he estimated mean log IC 50 

## 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.

P re cipitate 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. . T he 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 l og IC 50 

## and Relative Binding Affinity ( RBA ) w ere not calculated

.

Based on the three runs, piperonyl butoxide is classified as Not I nteractive in th e Estrogen Receptor Binding Assay.

The assay satisf ies the EDSP Tier 1 Test Order requirements for an Estrogen R eceptor Binding

A ssay (OCSPP 890.1250 ).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statement s were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 3 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: Willoughby, J.A.

Other Personnel: Rutherford, K. (Director Laboratory Operations); Blakeman, D. (Senior Scientist); Haines, C. (Scientist); McColley, S . (Scientist); Toole, C. (Director of Project Management) Study Period: May 23, 2011 to November 17, 2011



## 2

. Test substance : Piperonyl Butoxide

Description: Technical ; oily yellow liquid

Source: Endura Fine Chemicals, Italy

Lot/Batch #: E104/10 ( expir y date : July 2012) Purity: 93.8 % Solubility : Not reported Volatility : Not reported Stability: Not reported Storage conditions: Ambient (room temperature)

CAS #: 51

- 03

- 6 Molecular weight: 338.44 g/mol

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Non

- labeled ligand : 17 β

- estradiol

Supplier : Sigma

- Aldrich (St. Louis, MO) Catalog # : E8875

Lot #: 110M0138V Purity: 100 %

CAS #: 50

- 28

- 2



## 4

. Radioactive ligand : [ 3 

## H]

- 17 β

- estradiol

Supplier : Not reported Catalog #: Not reported

Batch#: Not reported Radiochemical p urity: Not reported Specific activity : 130.2 Ci/mmol (certification date: May 6, 2011)

Concentration of stock : 1 nM



## 5

. Positive control : 19

- norethindrone

Supplier : Sigma

- Aldrich (St. Louis, MO) Catalog # : N4128

Lot #: 030M1359V Purity: 99%

CAS #: 66

- 22

- 4Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 4 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## 6

. Negative control : Octyltriethoxysilane

Supplier : Sigma

- Aldrich (St. Louis, MO) Catalog # : 440213

Lot #: 24996KK Purity: 99.34%

CAS #: 2943

- 75

- 1



## 7

. Solvent/vehicle control : Dimethyl sulfoxide (DMSO)

Justification for choice of

solvent : Piperonyl butoxide solutions up to 10

− 4 M (limit concentration for the assay) can be prepared while limiting the final concentration of DMSO to 2 %

Final Concentration : 2 %



## B. METHODS

1. Preparation of R at U terine Cytosol (RUC) : Frozen Sprague Dawley uteri were purchased from Harlan Laboratories (Batch 210007463) . Female Sprague Dawley rats were ovariectomized 7 days prior to being euthanized. Animals were 12-13 weeks old at the time of euthanasia. The uteri were weighed, placed in ice

- cold TEDG (Tris, EDTA, DTT, glycerol) + P rotease Inhibitor (PI, unspecified ) buffer and used immediately. Uteri were homogenized in buffer and then centrifuged for 10 min at 2500 × g at 4oC. The resulting supernatant was transferred and centrifuged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein concentration of the cytosol was determined to be 1.10 mg/mL using a protein kit compatible with DTT in the TEDG buffer (Bradford Method; Bio- Rad Dye Reagent Concentrate ). Cytosol was divided into aliquots (1 to 6 mL) for immediate use

or storage at

− 80oC. Two separate preparations of RUC were used for this assay, one isolated September 27, 2010 and one isolated July 10, 2011

2. Saturation (radioligand) B inding E xperiment : A saturation binding e xperiment measuring total and non-specific binding of [ 3 

## H] -17 β

- estradiol was performed for each preparation of RUC, to demonstrate that the ER was present in reasonable concentrations and had the appropriate affinity for the native ligand (MRID 48843501). The conditions for the saturation binding are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a


### Source of receptor Rat uterine cytosol
Concentration of radioligand (as serial dilutions) 0.03 to 3 nM

Concentration of non

- labeled ligand (100X [radioligand]) 3 to 300 nM
Concentration of receptor Sufficient to bind approximately 25

to 35 % of radioligand at 0.03 nM
Temperature 4oC

Incubation time 16 to 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10%
Phenylmethylsulfonyl fluoride (PMSF) 1 mM

DTT 1 mM

a Data were obtained from page 1 of the study report ( MRID 48843501 ).Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 5 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## On the day of the assay, the specific activity of the stock solution [ 3 

## H] -17 β

- estradiol (originally 162.0 Ci/mmol as manufactured on January 28, 2009 or 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and serial dilutions in TEDG + PMSF buffer were prepared to ach ievethe final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of nonlabeled 17 β

- estradiol were prepared in a similar manner to achieve concentrations that were

100- fold greater than each respective radiolabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM.

For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was reached that bound approximately 25 to 35% of the total radioactivity added. The protein concentration was 35 μg per assay tube for the three saturation binding experiments conducted in October 2010 and 127.5 μg per assay tube for the three saturat ion binding experiments conducted in July 2011 ( Note: typically 50 ± 10 μg protein per tube) . E ach assay consisted of three non-concurrent runs (conducted on October 19, 21, and 25, 2010 or July 15, 16, and 18, 2011, respectively). Each run included thre e replicates of each test substance at each concentration, resulting in the 72 samples depicted in Table 2. It should be noted that the cytosolic preparation created on September 27, 2010 was more than the guideline 90 days old at the beginning of the competitive binding assay, and the cytosolic

prep was not re- tested for adequate performance.

TABLE 2. Saturation Binding Experiment Run a


### Total binding b 

### Non

- specific binding c 

### Radioligand alone d 

### Assay Components

Tubes 1

- 24 Tubes 25

- 48 Tubes 49

- 72
350 μL 300 μL

--- TEDG + PMSF buffer
50 μL 50 μL 50 μL [ 3 

### H]

- 17β

- estradiol (8 serial dilutions) e



### --- 50 μL

--- Non

- labeled 17β

- estradiol (8 serial dilutions, 100x each respective labeled concentration) f



### 100 μL 100 μL

--- Uterine cytosol (diluted to appropriate concentration)
500 μL 500 μL 50 μL Total volume in each assay tube

a Data were obtained from page 2 of the study report ( MRID 48843501 ).

b Total binding = [ 3 

### H]

- 17β

- estradiol bound to ER

c Non

- specific binding = [ 3 

### H]

- 17β

- estradiol and 100

- fold greater non

- labeled bound to ER

d Total [ 3 

### H]

- 17β

- estradiol alone for dpm determination at each concentration

e Final concentrations of [ 3 

### H]

- 17β

- estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non

- labeled 17β

- estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



## Tubes were incubated with gentle vortexing for 18-19.5 hours (October 2010) or 17-21 hours (July 2011) , respectively, at approximately 4 ° C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed (3 times with

5- min ute intervals). Subsequently, the contents of each tube were washed three times as

follows: 2 -mL portions of TEDG+PMSF buffer were added, vortexed, centrifuged for 10

min utes at 1000 x g , and the supernatant decanted and discarded. After the final centrifugation , ethanol (1.5 mL) was added to the HAP pellet remaining in each tube to

extract the [ 3 

## H] -17 β -estradiol, followed by vortexing, and centrifugation for 10 min utes at
1000 x g . Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. TheEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 6 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## temperature was maintained at approximately 4oC throughout the assay prior to extraction

with ethanol.

3. Competitive B inding E xperiment : A summary of the experimental conditions for the competitive binding experiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a



### Source of receptor Rat u terine c ytosol

Concentration of radioligand 1 nM

Concentration of receptor Sufficient to bind 11.5

- 13.3% of radioligand b



### Concentration of test substance (as serial dilutions) 10

− 1 1 

### to 10

− 3 

### M c



### Temperature 4±2 ° C

Incubation time 16

- 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10% (v/v)

PI (unspecified) d 

### 0.5% (v/v)

DTT 1 mM

a Data were obtained from page 16 of the study report.

b Calculated by reviewer from data on pages 3 6- 38 of the study report.

c The first and second runs tested the test substance at 10

− 10 

### M to 10

− 3 

### M, but the highest concentration precipitated, therefore only data for 10

− 10 

### M to 10

− 4 

### M were evaluated

. The third run tested the test

substance at 10

− 11 

### M to 10 −4 

### M.

d The recommended p rotease inhibitor in the Guideline is PMSF, but the PI was not identified.



## The limit of test chemical solubility in DMSO was determined by visual observation. In addition, the solutions were observed closely for precipitation during addition to the experiment tube. On t he day of the assay, the specific activity of the stock solution [ 3 

## H]

- 17 β -estradiol was adjusted for decay over time (129.2 Ci/mmol for the first run; 129.1 Ci/mmol for the second run; and 128.3 Ci/mmol for the third run), and diluted in TEDG+P I buffer to achieve the final appropriate concentrations. Serial dilutions of piperonyl butoxide, the weak positive control (19-norethindrone), the negative control (octyltriethoxysilane), and the reference material (non

- labeled 17β

- estradiol) were prepare d to achieve the concentrations shown in Table 4. Each assay consisted of three runs, and each run contained three replicates at each concentration, plus six replicates measuring total activity (50μL master mix) and three replicates measuring total bindin g (solvent control) resulting in a total of 306 samples. It is noted that in Run 1 only three replicates of the [ 3 

## H]

- 17β

- estradiol (master mix) were analyzed , but the tube numbering was maintained as for the

other runs.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 7 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



### TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a , b



### Piperonyl Butoxide c 

### Weak Positive control Negative control Reference Chemical

19

- norethindrone Octyltriethoxysilane Non

- labeled 17β

- estradiol

Tubes 79

- 102 Tubes 31

- 54 Tubes 55

- 78 Tubes 1

- 30

10

− 11 

### 10

− 8.5 

### 10

− 10 

### Solvent control / master mix d



### 10

− 10 

### 10

− 7.5 

### 10

− 9 

### 10

− 11



### 10

− 9 

### 10

− 7 

### 10

− 8 

### 10

− 10



### 10

− 8 

### 10

− 6.5 

### 10

− 7 

### 10

− 9.5



### 10

− 7 

### 10

− 6 

### 10

− 6 

### 10

− 9



### 10

− 6 

### 10

− 5.5 

### 10

− 5 

### 10

− 8.5



### 10

− 5 

### 10

− 4.5 

### 10

− 4 

### 10

− 8



### 10

− 4 

### 10

− 4 

### 10

− 3 

### 10

− 7



### 10

− 3



### -- -- --

a Data were obtained from page s 25, 27, 29, and 36

- 38 of the study report.

b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control,

or non

- labele d 17β

- estradiol; 390 μL of TEDG+ PI buffer with [ 3 

### H]

- 17β

- estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL.

c Runs 1 and 2 tested 10

− 10 

### to 10

− 3 

### M piperonyl butoxide; p recipitate was observed at the highest

concentration and data for 10

− 3 

### M were not included in the data evaluation. Run 3 tested 10

− 11 

### to 10 −4 

### M piperonyl butoxide.

d Tubes 1

- 6 contained the master mix ([ 3 

### H]

- 17β

- estradiol ); Tubes 7

- 9 contained the s olvent (DMSO).



## Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 ° C. To separate bound from free estradiol, HAP slurry was added to each tube and the tubes were vortexed (3 times

with 5 -minute intervals). Subsequently, the contents of each tube were washed three times

as follows: TEDG+P I buffer was added, vortexed, centrifuged for 10 min at 1000× g , and the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in

each tube to extract the [ 3 

## H] -17 β

- estradiol , allowed to sit at room temperature for 15-20 min with vortexing, and centrifugation for 10 min at 1000× g . An aliquot of supernatant was radioassayed by scintillation counting. The temperature was maintained at 4±2 °C throughout the assay prior to extraction with ethanol.

C. D ATA ANALYSIS : For the saturation binding experiment, total binding and non- specific binding data were modeled via non- linear reg ression by using Graph Pad Prism v. 5 ( GraphPad Software, Inc., La Jolla, CA)] , incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

## implemented by using the ROUT procedure in Prism v. 5 

## with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

## Swillens (1995) 2 

## . For the competitive binding assay,

similar methods of nonlinear regression were used to fit a curve (for 17β -estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated l og IC 50 

## as a parameter to be estimated. For parameters reported from the saturation b inding experiment ( 

## K d 

## and B
max 

## ) and c ompetitive binding e xperiment ( l og IC 50 

## and RBA), m ean and standard deviation were calculated for each run, and mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000;

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressi on: a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123

- 142

. 2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197

- 1203.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 8 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the composite three runs with Microsoft Excel 2010.



## 1. Definitions

a. Classification of test material : Classification of the test material is based on the average of

three runs. Each run wa s first individually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estradiol has been displaced from the ER).

Not interactive = there are usable data points at or above 10

− 6 

## M and either the lowest point on the fitted respon se curve within the range of the data is above 75 % (i.e.,

<25 % of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Eq uivocal up to the limit of concentrations tested = there are no data points at or above a

test chemical concentration of 10

− 6 

## M and either a binding curve can be fit but ≤50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be fit and the lowest average percent binding among concentration groups in the data is >50%.

Equivocal = A run is classified as equivocal if it does not fall into any of the categories

above.

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value

Interactive 2 Equivocal 1

Not interactive 0 Equivocal up to the limit of concentrations tested “missing”



## The values for each run were then averaged across runs and the chemical classified using the following ranges:

Test Material Classification Numerical Range

Interactive average ≥1.5 Equivocal 0.5 ≥ average <1.5

Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 9 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## b. Descriptors for receptor binding :

B max 

## : maximum specific binding number (fmol ER/100 μg cytosol protein) measures the concentration of active receptor sites

K d 

## : dissociation constant ( nM), measures the affinity of the receptor for its natural ligand

IC 50 

## : concentration of the test substance (M) at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity ( RBA % ): ( IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance) × 100 Log RBA: Log 10 

## ( IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Figure 1 illustrates the non- specific, specific, and total binding curves for [ 3 

## H] -17 β

- estradiol to the estrogen receptor for representative runs from the two saturation binding assays. The specific binding reached a plateau in each run, and non-specific binding was less than 20% of total binding. Figure 2 contains representative Scatchar d plots that illustrate the binding of [ 3 

## H] -17 β -estradiol to the estrogen receptor for the two saturation binding assays.

Saturation binding e xperiment parameters are presented in Table 5. For the Septenber 27, 2010 batch of cytosol, t he K d 

## for [ 3 

## H]

- 17β

- estradiol was 0.081±0.006 nM, and the estimated

B max 

## was 52.70±1.05 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

## for each run was within the expected range of 0.03 to 1.5 nM.

For the July 10, 2011 batch of cytosol, t he K d 

## for [ 3 

## H]

- 17β

- estradiol was 0.303±0.090 nM,

and the estimated B
max 

## was 33.59±0.70 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

## for each run was within the expected range of 0.03 to 1.5 nM. Confidence in these numbers is high due to the goodness of fit and the small variation among runs. Although the Scatchard plots fit straight lines to the data, the concavity observed in the data sets may indicate issues with ligand depletion.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 10 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



### TABLE 5. Saturation Binding Experiment of [ 3 

### H]

- 17 β

- e stradiol with Estrogen Receptor from Rat Uterine Cytosol a



### Parameter Run 1 Run 2 Run 3 Mean ± SE b



### Cytosol Prep Sept 27, 2010

R 2 

### (unweighted) 0.9 91 0.986 0.979 0.97 9

- 0.9 91

B
max 

### (nM) 0.0 6 3 0.0 61 0.0 61 0.0 61 ± 0.00 1

B
max 

### (fmol/100 μg protein) 53.91 52.07 52.12 52.70 ± 1.05

K d 

### (nM) 0.082 0.087 0.074 0.081 ± 0.006
Cytosol Prep Jul 10, 2011

R 2 

### (unweighted) 0.992 0.993 0.955 0.955

- 0.992

B
max 

### (nM) 0.029 0.029 0.027 0.029 ± 0.001

B
max 

### (fmol/100 μg protein) 34.09 34.46 32.21 33.59 ± 0.70

K d 

### (nM) 0.265 0.475 0.169 0.303 ± 0.090

a Data were obtained from page 3 of the study report ( MRID 48843501 ).

b The range of R 2 

### is reported and the mean ± SEM is reported for the other parameters.

R 2 

### Goodness of fit for curve calculated for specific binding

.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 11 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## FIGURE 1. Binding of [ 3 

## H] -17 β

- estradiol to the Estrogen Receptor during the Saturation Binding Experiment

. Representative of the runs conducted in October 2010 and July 2011Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 12 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## FIGURE 2. Scatchard Plot s of the Binding of [ 3 

## H] -17 β

- estradiol to the Estrogen Receptor. Representative of the runs conducted in October 2010 and July 2011.

B. COMPETITIVE BINDING EXPERIMENT : The results from the three competitive binding ex periments conducted on June 26, July 2, and August 8, 2011 are summarized in

Table 6 and presented graphically in Figures 3-5 . 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, and therefore t he estimated mean l og IC 50 

## and RBA w ere not calculated for piperonyl butoxide.

The estimated mean l og IC 50 

## was

− 9.2 M for the natural ligand (17 β

- estradiol ) and

− 5.6 M

for the weak positive control (19-norethindrone). Compared to the natural ligand , t he mean

RBA was 0.0 26 % for 19-norethindrone. Confidence in these numbers is high due to the

small variation

. As the lowest average percent binding in the presence of piperonylEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 13 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## butoxide was >7 5% at concentrations up to 10

− 4 

## M, piperonyl butoxide is classified as not
interactive (0) in this assay (Table 7)

.

TABLE 6

. Competitive Binding Assay of P iperonyl Butoxide with Estrogen Receptor from Rat Uterine Cytosol a



### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c



### R 2 

### (unweighted) 1 7 β

- estradiol NR NR NR NA

19

- norethindrone NR NR NR NA Piperonyl butoxide NR NR NR NA
Log IC 50 

### (M) 17 β

- estradiol

− 9.0

− 9.6

− 9.1

− 9.2 ± 0.19

19

- norethindrone

− 5.6

− 5.5

− 5.6

− 5.6 ± 0.03 Piperonyl butoxide NA NA NA NA

IC 50 

### (M) c 

### 17 β

- estradiol 1.00 x 10

- 9 

### 2.15 x 10

- 10 

### 7.94 x 10

- 10 

### 6.82 x 10

- 10 

### (±2.23)

19

- norethindrone 2.51 x 10

- 6 

### 3.16 x 10

- 6 

### 2.51 x 10

- 6 

### 2.73 x 10

- 6 

### (±0.22) Piperonyl butoxide NA NA NA NA
Log RBA (%) c 

### 19

- norethindrone

− 3.4

− 4.1

− 3.5

− 3.67 ±0.22 Piperonyl butoxide NA NA NA NA
RBA (%) c 

### 19

- norethindrone 0.040 0.008 0.032 0.026 ±0.010 Piperonyl butoxide NA NA NA NA

a Data were obtained from results discussion page 22 of the study report.

b The mean is reported for the concurrent replicates within each run.

c Calculated by the reviewers ; for means expressed in scientific notation, the standard error (SE) values in parentheses are reported in the same order of magnitude as the mean value

. R 2 

### Goodness of fit

RBA R elative binding affinity

NR Not reported

NA Not applicable

TABLE 7 . Binding Classification of P iperonyl Butoxide with Estrogen Receptor a



### Run 1 2 3 Mean Binding Classification c


### Classification category value b 

### 0 0 0 0 Not interactive

a Data were obtained from page 22 of the study report.

b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of

concentrations tested (“missing”, i.e., not included in calculation of mean).

c Interactive = mean ≥1.5 ; Equivocal = 0.5≤ mean <1.5 ; Not interactive = mean <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 14 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## F IGURE 3. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of Piperonyl Butoxide, Unlabeled E2, 19- Norethindrone or Octyltriethoxysilane. Run 1Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 15 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## FIGURE 4. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of Piperonyl Butoxide, Unlabeled E2, 19- Norethindrone or Octyltriethoxysilane. Run 2Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 16 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## FIGURE 5. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of Piperonyl Butoxide, Unlabeled E2, 19- Norethindrone or Octyltriethoxysilane. Run 3Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 17 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## C. P ERFORMANCE CRITERIA : To ensure that the competitive binding assay function ed properly, each run was evaluate d using the criteria shown in Table 8 :

TABLE 8. Criterion a 

### Tolerance Limit(s) Value Yes No

17 β

- estradiol fitted curve parameters
Log
e 

### residual SD ≤2.35 0.55 to 1.64 X Top (% binding) 94 to 111 98 to 102 X Bottom (% binding)

− 4 to 1

− 1 to 0 X Hill Slope ( Log 10 

### (M)

− 1 

### )

− 1.1 to

− 0.7

− 1.0 X

Weak Positive control ( 19

- norethindrone ) fitted curve parameters b



### Log
e 

### residual SD NA 0.95 to 1.28 X Top (% binding) NA 97 to 101 X Bottom (% binding) NA

− 3 to 2 X Hill Slope ( Log 10 

### (M)

− 1 

### ) NA

− 1.3 to

− 0.9 X

Solvent concentration

DMSO ≤10%

~ 2% X Negative control (octyltriethoxysilane) does not displace

more than 25% of [ 3 

### H]

- 17 β

- estradiol from the ER on average across all concentrations ≤25% ≤ 19.6% X

a Data were obtained from page s 2 6, 28, and 30 of the study report.

b The EPA Guideline does not define a set of tolerance limits for 19

- norethindrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19

- norethindrone to be an acceptable weak positive control.

NA Not applicable



## T he curve for the reference material showed that increasing concentrations of unlabeled 17β

- estradiol displaced [ 3 

## H]- 17β -estradiol in a manner consistent with one-site binding, as indicated by Hill slopes of

− 1.0 in the three runs. For 19-norethindrone in the first run, the study reported that the bottom plateau was slightly high (2% compared to

− 5 to 1%) and the

Hill slope was slightly low (

− 1.3% compared to

− 1.1 to

− 0.7%). The ranges cited in the study are acceptance criteria in the guidance for norethynodrel not 19-norethindrone. The fitted curve parameters demonstrate that 19 -norethindrone is an acceptable weak p ositive

control in the subject assay.

Piperonyl butoxide was tested over a concentration range that fully defined the top of the

curve. The highest percent binding (95.5-100%) for piperonyl butoxide was within 25 percentage points of the percent binding at the lowest concentration of the estradiol standard (97 -98%).

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Piperonyl butoxide was classified as “non

interactive” in all three valid independent runs and thus has a final classification of “non

interactive” with the ER.

B. AGENCY COMMENTS : Data from two separate saturation binding assays were reported due to the use of two separate cytosol preparations during this assay with piperonylEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 18 of 18

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1250 / OECD None



## butoxide. For the September 27, 2010 batch of cytosol, t he K d 

## for [ 3 

## H]

- 17β

- estradiol was 0.081 nM, and the estimated B
max 

## was 52.70 fmol/100 μg protein for the prepared rat uterine cytosol. For the July 10, 2011 cytosol, t he K d 

## for [ 3 

## H]

- 17β

- estradiol was 0.303 nM,

and the estimated B
m ax 

## was 33.59 fmol/100 μg protein. The K d 

## for each run was within the expected range of 0.03 to 1.5 nM. Confidence in these numbers is high based on t he goodness of fit and the small variation among runs. Although the Scatchard plots fit straight

lines t o all of 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, p recipitate 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, th erefore the mean l og IC 50 

## and RBA w ere not calculated.

The estimated mean l og IC 50 

## was

− 9.2 M for the natural ligand (17 β

- estradiol ) and

− 5.6 M

for the weak positive control (19-norethindrone). Compared to the natural ligand, t he mean

RBA was 0.0 26 % for 19-norethindrone. All performance criteria were met for 17 β

estradiol , 19-norethindrone, and the negative control, octyltriethoxysilane

.

Piperonyl butoxide was tested over a concentration range that fully defined the top of the

curve. The highest percent binding (95.5-100%) for piperonyl butoxide was within 25 percentage points of the percent binding at the lowest concentration of the estradiol standard (97 -98%). As the lowest average percent [ 3 

## H]

- 17β

- estradiol binding in the presence of piperonyl butoxide was >7 5% at concentrations up to 10

− 4 

## M, piperonyl butoxide is

classified as not interactive in this assay

.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• Curves were not provided showing the average binding of each test substance across all

three runs.

• The cytosolic preparation created on September 27, 2010 was more than the Guidelinespecified 90 days old at the beginning of the competitive binding assay, and the cytosolic prep was not re- tested for adequate performance.

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48618804 to include laboratory proficiency data; Main study was o riginally reviewed under T A 2- 32 -2012)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Michelle Sharpe

- Kass, M.S Date: 7 / 13 /2012

Secondary Reviewer : Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 7 /1 6 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7 / 25 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/25 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIPERO NYL B TO X ID E / 06750 1 Es tr oge n Rec ep t o r Transcr ipti o n a l Activation (2011) Page I o f 1 5

OCSPP 890.1300 / O EC D 455



## Primary Reviewer: Ch ri s Sch l osse r, M.F .S

. Health Effects Division Secondary Reviewer: M in e rva Mercado, Ph .D.

Health Effects Divi s ion

DATA EVALUATION RECORD

STU DY TYP E: Estrogen Receptor Transcriptiona l Activation ( Hum a n ce ll Line, HeLa-990 3);

OCSP P 890. 1 300; OECD 455.

PC C ODE: 06750 I 

## DP BARCODE : D 398558

TXR # : 0056259 CAS No. : 5 1- 03-6

TEST MATERIAL (P U RITY) : Piperon y l But oxide (93.8% a.i.)

SYN ONYMS: S

- [[2-(2-butoxyethoxy)ethoxy ]me th y l ]-6 -prop yl -1

, 3-benzodioxo l e

C ITATION: Willoughby, J.A. (2011) Pip cro n y l butoxide: es tr oge n r ece pt o r transcripti o n al

ac ti vation ( hum an ce ll lin e ( l l c La -9903))

. CeeTox

, In c.
, K a l a ma zoo, Ml. Laboratory Repo11 No .: 9146V- 1 00377E R TA, November 17

, 20 1 1. MRID

48618804. Unpub li shed.

SP ONSOR: W i lloughby

, J.A. (20 12 ). Supp l emen tal Informati o n

- Laboratory Proficienc y Data for ERTA assays and Sa 1ura1ion Binding Data fo r AR an d E R Binding Assays for Assorted Chemicals. CceTox, In c

. , Kalama zoo, MI. July 19, 20 1 2.

MRID 4884350 1

. Unpublis h ed.

Cons um e r S pe c ialt y Produ cts Assoc iati o n
, In c.
, 1900

- 1 7 111 

## S tr ee t NW, Suite

300

, Wa s hington

, D C

TEST ORDER #: CON-06750 1- 9

E XEC U TIV E SUMMARY: In an estrog e n re cepto r transcriptional ac ti vation assay ( MRID 48618804), hERa-H eLa

- 9903 ce ll s c ul tured in vitro 

## were exposed to piperonyl butox i de ( 93.8 % a.i., Lot # El 04/10) al l oga ri th mi ca ll y increasing conce ntrati ons from 10

- 11 

## to l o

- .i 

## Min 0.1 %



## DMSO fo r approx im ately 24 hours in two independent run s

. The experi m e nt s we r e perform ed

u s in g 96-we ll plates a n d eac h pip ero n y l butoxide co n cen trati o n was t este d in 6 wells / plate in

eac h run

. Ce ll s were ex po se d to the t est agent for 24 ± 2 h ou r s at 37±1 °C to indu ce r e p o rt er ( luciferase ) gene product s. Luciferase expressio n in response to activa ti o n of the est rogen receptor was measured u s ing a proprietary lucif erase assay.

Piperonyl butoxide was t ested up to the l imit of cytotoxicity

, which was n o t e d at 10

- .i 

## M in one

run. At concentrations up to 10

- 5 

## M

, th e r e lativ e tran sc riptional activat i on (RTA) of piperon y l

butox i de was :::; 2.8%.Estrogen Receptor Transcriptional Activation ( 2011) Page 2 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



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

methyltestosterone was lower than the expected values, indicating a decreased sensitivi ty 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 17 α

- methyltestosterone, which was able to

reach a maximum of 16.5-24.3 % PC. P iperonyl 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 RPC Max 

## < PC 10 

## in both assay runs, piperonyl butoxide was considered negative for estrogen receptor transcriptional activation in this test system.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE : Signed and dated GLP, Quality Assurance and Data Confidentiality

statements were provided.Estrogen Receptor Transcriptional Activation ( 2011) Page 3 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: Kalamazoo, MI Study Director: J.A. Willoughby

Other Personnel: K. Rutherford (Director Laboratory Operations); D. Blakeman (Senior Scientist); C. Haines (Scientist); S. McColley (Scientist); B. Wallace (Lead Cell Culture Scientist); and C. Toole (Director of Project Management) Study Period: May 23, 2011 to November 17, 2011



## 2

. Test Substance : Piperonyl Butoxide

Description: Technical, oily yellow liquid, M W 338.44 g/mol

Source (cat #) : Endura Fine Chemicals, Bologna, Italy (Cat# not reported)

Lot/Batch # (Exp Date) : E104/10 (July 2012) Purity: 93.8 % Solubility : N ot reported (N R ) Volatility : NR Stability: ~2 years Storage conditions: Ambient

CAS #: 51

- 03

- 6

Structure :



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Reference substances

17 β

- estradiol (strong estrogen; positive control)

Supplier : NR Catalogue and Batch #: NR Purity: NR

CAS # : 50

- 28

- 2



## 17α

- estradiol (weak estrogen)

Supplier: NR Catalogue and Batch #: NR Purity: NR

CAS # : 57

- 91

- 0



## Corticosterone (negative compound)

Supplier: NR Catalogue and Batch #: NR Purity: NR

CAS # : 50

- 22

- 6



## 17α

- methyltestosterone (very weak agonist)

Supplier: NR Catalogue and Batch #: NR Purity: NR

CAS # : 58

- 18

- 4



## 4

. Vehicle(s)

Solvent: DMSO

Solvent control (final conc e n tration ): 0.1%Estrogen Receptor Transcriptional Activation ( 2011) Page 4 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## B. METHODS

1. C ell C ulture : Stably

- transfected hER α

- HeLa

- 9903 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma infection by a DNA fluorochrome assay. Cells were maintained in E agles Minimum

Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10%

dextran

- coated charcoal

- treated fetal bovine serum ( so urce not reported), in an incubator

under 5% CO 2 

## at 37 oC. Upon reaching 75-90% confluence , cells were s ubcultured 32 times prior to exposure to the test material. The cells used in this study were passage 35 (range -finder), and passage 17 (first and second run).

2. Transcriptional Activation Assay s : For each test, cells were plated in a 96

- well plate (estrogenic activity of plastic not reported) at a density of about 1×10 4 

## cells/100 μL

medium/well and allowed to attach for 3 hours. G rowth media was replaced with media containing serial log dilutions of piperonyl butoxide in DMSO (0.1% final concentration ).

Cells were incubated for 24 ±2 hours at 37±2 °C. Cytotoxicity was determined by two- read propidium iodide uptake. M ain assay runs 1 and 2 were conducted on June 14 and June 16, 2011, respectively. T ranscriptional activation of the estrogen receptor was determined as described in CeeTox Standard Operating Protocol (SOP) 2041 (not provided) . A list of reagents was provided, but assay reagent was classified as proprietary information.

a. Preliminary Test : A preliminary test evaluating concentrations ranging from 10 −1 0 

## to
10

− 3 

## M was conducted to determine the appropriate concentration range and to determine concentrations resulting in insolubility and/or cytotoxicity.

b. Proficiency Chemicals : Responsiveness of the test system was tested on March 5, April 12 and April 28, 2011 (MRID 48843501), using c ells at passage 15, 25 and 28, respectively

. Based on assay dates and passage numbers, it is un likely that the cell used for proficiency testing we re from the same frozen stock as cells used in th e main assay. E ach of the following chemical s was tested in duplicate on separate days:

Compound CAS No. Concentration Range (M) Expected Response a Notes

Diethylstilbestrol (DES) 56

- 53

- 1 10

− 14 to 10

− 8 Positive

--- 17 α

- Ethynyl estradiol (EE) 57

- 63

- 6 10

− 14 to 10

− 8 Positive

--- Hexestrol 84

- 16

- 2 10

− 13 to 10

− 7 Positive

--- Genistein 446

- 72

- 0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM

Estrone 53

- 16

- 7 10

− 12 to 10

− 6 Positive

--- Butyl paraben 94

- 26

- 8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM

1,3,5

- Tris(4

- hydroxyphenyl)benzene c 15797

- 52

- 1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC.

Binds to hER α and has ER antagonistic activity
Dibutyl phthalate (DBP) 84

- 74

- 2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM

Atrazine 1912

- 24

- 9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b

Corticosterone 50

- 22

- 6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration

a Positive = RPC max ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs

b Cytotoxicity is expected to be close to 80% at this concentration.

c Compound selected to challenge solubility and cytotoxicity

. d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non

- ER β mediated transcriptional activation. A positive result would indicate that the system is detecting activity other than that due to pure ER α, and is therefore unacceptable.Estrogen Receptor Transcriptional Activation ( 2011) Page 5 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## c. Reference Chemicals : To ensure the stability of the response from the cell line, eight concentrations of each of the following reference chemicals were included in each plate in the current assay, along with the test chemical:

Reference Chemical CAS No. Concentration Range Class 17β

- estradiol 50

- 28

- 2 10

− 1 5 to 10 −8 Strong estrogen

17α

- estradiol 57

- 91

- 0 10 −1 3 to 10 −6 Weak estrogen

Corticosterone 50

- 22

- 6 10 −1 1 to 10 −4 Negative compound

17α

- methyltestosterone 58

- 18

- 4 10 −1 2 to 10 −5 Very weak agonist



## 3. Data analysis : To obtain the relative transcriptional activity to the 1 nM E2 positive control (PC), the luminescence signals from the concurrent plate were analyzed by subtracting the mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to express relative transcriptional activity as a percentage of the PC. The test material was defined as negative for inducing estro gen receptor transcriptional activation if the RPC Max 

## < PC 10 

## in at least 2 of 2 runs. Log EC 50 

## and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the luminescence data triplica tes. Concentrations showing >20% cytotoxicity or evidence of insolubility were excluded from analyses.

4. Definitions

EC 50 

## = concentration of agonist that induces a response halfway between the baseline (bottom) and maximum (top) response

PC 10 

## = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

## = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

## = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

## = concentration of a test chemical inducing the RPC Max



## II. RESULTS

A. PRELIMINARY TEST : In order to identify a suitable top concentration for use in the transcriptional activation assays, a preliminary cytotoxicity and precipitation assay w as conducted. Precipitation was observed at 10 –3 

## M piperonyl butoxide, the results of the cytotoxicity test are presented in Table 1. Concentrations of 10 −4 

## M showed cell viability of 74%, but no cytotoxicity was observed at 10

− 3 

## M. Based on these results, logarithmically increasing concentrations from 10

− 11 

## to 10

− 4 

## M were selected for the assay.Estrogen Receptor Transcriptional Activation ( 2011) Page 6 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



### TABLE 1

. Preliminary T est for Solubility , C ytotoxicity , and Concentration

- Selection for Piperonyl

butoxide a

Concentration (M) % Viability Comments

10

− 3 82 Precipitation observed

10

− 4 74 Cytotoxic

10

− 5 91

10

− 6 89

10

− 7 89

10

− 8 94

10

− 9 93

10

− 10 93

E2 1nM NR

VC b NR

a Data were obtained from page 20 of the study report.

b VC = Vehicle control



## B. P OSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals : The laboratory proficiency assays using the required reference compounds were not included in the original study report, but were provided to the Agency at a later date (MRID 48843501). The responsiveness of cells to the required proficiency

chemicals was performed in duplicate on different days for each chemical. The reported

responses are in Table 2 a . In the proficiency test s , the reference chemicals 17β -estradiol,

17 α -estradiol and 17 α -methyltestosterone were tested concurrently with each run of the

assay (Table 2b). In the first run, the responsiveness of 17 β

- estradiol indicated decreased sensitivity to strong agonists, and the response to 17 α -methyltestosterone showed an increased responsiveness to very weak agonists; despite the minor deviations this run is considered acceptable. Run 2 was inadequate as the PC 50 

## could not be calculated for 17 α

methyltestosterone indicating a decreased sensitivity to very weak agoni sts. Run 3 was acceptable as 17 β -estradiol and 17 α -methyltestosterone performed wi thin the expected range, but the Hill Slope for 17 α -estradiol was higher than expected. The PC -induced fold

induction for the three reference chemicals was within the Guideline

- recommended historical range of 4 - to 30-fold in Runs 1 and 3, but fold induction was 75.1- to 84.7-fold in Run 2 with no explanation given for this 3- to 4-four increase. Although reportedly performed, t he results of the cytotoxicity assay were not provided for review

. Raw data pertaining to the RTA of each chemical were not reported, but the scales of the graphs provided indicate Genistein and Butyl paraben had maximum RTAs well above 400%. The responses do not demonstrate proficiency as the assay cannot be fully evaluated by the

reviewer.Estrogen Receptor Transcriptional Activation ( 2011) Page 7 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



### TABLE 2 a . Proficiency Chemicals

Compound Expected Response Lab Response

Run 1 Run 2 Run 3
Diethylstilbestrol Positive Positive Positive NA

17 α

- Ethynyl estradiol Positive Positive Positive NA

Hexestrol Positive Positive Positive NA

Genistein Positive Positive Positive NA

Estrone Positive Positive Positive NA Butyl paraben Positive Positive Positive NA

1, 3, 5

- Tris(4

- hydroxyphenyl)benzene Positive NA Positive Positive
Dibutyl phthalate Negative Negative Negative NA

Atrazine Negative Negative NA Negative

Corticosterone Negative Negative NA Negative
NA = not applicable. The chemical was not tested at this time.



### Table 2 b. Performance Criteria for Reference Chemicals in the Proficiency test

Reference Chemical

Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Yes No
17β

- estradiol Log PC 50 − 11.4 to

− 10.1

− 9.6

− 11.3

− 10.6 Run 1 Log PC 10 <

− 11

− 11.5

− 12.5

− 12.1 X Log EC 50 − 11.3 to

− 10.1

− 9.0

− 11.3

− 10.6 Run 1 Hill Slope 0.7 to 1.5 1.2 0.9 0.8 Test range (M) 10

− 14 to 10

− 8 10

− 14 to 10

− 8 10

− 14 to 10

− 8 10

− 14 to 10

− 8 X

17α

- estradiol Log PC 50 − 9.6 to

− 8.1

− 8.3

− 9.4

− 8.7 X Log PC 10 − 10.7 to

− 9.3

− 9.3

− 10.5

− 9.9 X Log EC 50 − 9.6 to

− 8.4

− 8.2

− 9.3

− 8.9 Run 1 Hill Slope 0.9 to 2.0 0.9 0.9 2.9 Run 3 Test range (M) 10

− 12 to 10

− 6 10

− 12 to 10

− 6 10

− 12 to 10

− 6 10

− 12 to 10

− 6 X

17α

- methyltestosterone Log PC 50 − 6.0 to

− 5.1

− 6.2 NC

− 5.2 Run 1, 2 Log PC 10 − 8.0 to

− 6.2

− 8.1

− 6.3

− 7.7 Run 1 Test range (M) 10

− 11 to 10

− 5 10

− 11 to 10

− 5 10

− 11 to 10

− 5 10

− 11 to 10

− 5 X



## 2. Reference Chemicals : The response curves for the reference chemicals are shown in Figures 1 -3, and the values derived from the concentration response curves ( e.g ., log PC 50 

## , log PC 10 

## , log EC 50 

## , Hill slope) are presented in Table 3 . Although all performance criteria were met for 17β -estradiol and corticosterone, and three of four criteria were met for 17α

estradiol

, there was variability in the response curves for 17β

- estradiol and 17α

- estradiol and

the lack of responsiveness of the cells to 17α

- methyltestos terone.

In Run 2, the maximum response of both 17β

- estradiol and 17α

- estradiol exceeded (~120%) the response of the PC. In addition, the coefficients of variation (%CV) were 25 -53 % for 17β

- estradiol at 10 −11 

## to 10 −8 

## M, 35-42 % for 17α

- estradiol at 10

− 6 

## to 10

− 9 

## M, and 61% for

17α

- methyltestosterone at 10

− 5 

## M. T he normalized luminescence data indicates that the response of the cells in Run 2 was approximately 85-90% lower than in Run 1 at

concentration s eliciting a positive response (10 −11 

## to 10 −8 

## M for 17β -estradiol and 10 −9 

## to
10 −6 

## M for 17α

- estradiol ). In both assay runs, Log PC 50 

## values could not be determined for the very weak agonist, 17α

- methyltestosterone , as it did not reach 50% activation at up to

10 −5 

## M. For 17α

- methyltestosterone, the mean R PC Max 

## was 16.5% for the first run andEstrogen Receptor Transcriptional Activation ( 2011) Page 8 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## 24.3% for the second run. C ytotoxicity was observed at the highest concentrations tested of

corticosterone and 17α

- methyltestosterone

.

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical

Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Yes No
17β

- estradiol
Log PC 50 −11.4 to −10.1

− 10.2

− 10.7 X
Log PC 10 <−11

− 11.7

− 11.9 X
Log EC 50 −11.3 to −10.1

− 10.3

− 10.8 X Hill Slope 0.7 to 1.5 0.8 1.1 X
Test range 10 −14 to 10 −8 M 10 −15 to 10 −8 M 10 −15 to 10 −8 M X

17α

- estradiol
Log PC 50 −9.6 to −8.1

− 8.6

− 8.7 X
Log PC 10 −10.7 to −9.3

− 9.8

− 9.7 X
Log EC 50 −9.6 to −8.4

− 8.7

− 8.9 X Hill Slope 0.9 to 2.0 1.2 5.1 X
Test range 10 −12 to 10 −6 M 10 −13 to 10 −6 M 10 −13 to 10 −6 M X

Corticosterone
Test range 10 −10 to 10 −4 M 10 −11 to 10 −4 M 10 −11 to 10 −4 M X

17α

- methyltestosterone
Log PC 50 −6.0 to −5.1 NA b NA X
Log PC 10 −8.0 to −6.2

− 6.6

− 7.2 X
Test range 10 −11 to 10 −5 M 10 −12 to 10 −5 M 10 −12 to 10 −5 M X

a Data were obtained from page s 12 and 23 of the study report

. b NA = not applicable; 17α

- methyltestosterone did not achieve 50% activation at up to 10 −5 M.Estrogen Receptor Transcriptional Activation ( 2011) Page 9 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## FIGURE 1. Relative Transcription Activation (RTA) of the Natural Ligand,

17 β -estradiol, Compared to the Positive Control, 1 nM 17β

- estradiol.

Mean ± SD are present ed (n=6).Estrogen Receptor Transcriptional Activation ( 2011) Page 10 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## FIGURE 2. Relative Transcription Activation (RTA) of the Weak Estrogen,

17 α

- estradiol, Compared to the Positive Control, 1 nM 17β

- estradiol.

Mean ± SD are present ed (n=6).Estrogen Receptor Transcriptional Activation ( 2011) Page 11 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## FIGURE 3. Relative Transcription Activation (RTA) of the Very Weak Estrogen,

17 α

- methyltestosterone , Compared to the Positive Control, 1 nM 17β

- estradiol.

Mean ± SD are present ed (n=6).Estrogen Receptor Transcriptional Activation ( 2011) Page 12 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls : Data for the vehicle and positive controls are included

in Table 4. The overall mean TA value for the vehicle control was 17483 for the first run and 850 for the second run (expressed as arbitrary light units). T he overall mean TA value for the positive control was 1251383 for the first run and 54983 for the second run. The induction for the positive control was 72-fold for the first run and 65- fold for the

second run. The mean normalized value for the positive control was 1233900 for the first

run and 54133 for the second run. The PC 50 

## (50% of the maximum response) for 1nM E2 in this assay was 616950 and 27067 in Runs 1 and 2, respectively, and the PC 10 

## (10% of the maximum response) was 123390 and 5413 in Runs 1 and 2, respectively. Although the fold induction for the PC was acceptable in both runs, the overall reduced responsiveness of the cells in the Run 2 suggests problems with the cells in this run. The average response of the cells in Run 2 for both the vehicle control and PC was 4- 5% of the response observed in Run 1. The study authors provided no explanation difference in the assays.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a

Sample Vehicle Control Positive Control b Normalized Positive Control b

Runs Mean SD Mean SD Fold Induction c Mean SD

1 17483 4627 1251383 170 6 20 72 1233900 170620

2 850 319 54983 21606 65 54133 21606

a Data were obtained from page s 31

- 32 of the study report.

b Positive control was 17 β

- estradiol (E2) at 1 nM.

c Fold

- induction = (mean TA of PC)/(mean TA of VC)



## 2. Test Material : Relative (to the PC) transcriptional activation at each concentration of piperonyl butoxide during the two assay runs is presented in Table 5, and the concentrationresponse curves depicting RTA is presented in Figure 4. The mean RPC Max 

## was 1.9% for the first run and 2.8% for the second run, and the associated PC Max 

## was 10

− 4 

## M for the firs t
run and 10

− 7 

## M for the second run. Because the RPC Max 

## < PC 10 

## in both runs, piperonyl butoxide was considered negative for estrogen receptor transcriptional activation in this test

system

.Estrogen Receptor Transcriptional Activation ( 2011) Page 13 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



### TABLE 5. Relative Transcriptional Activation (RTA) of Piperonyl Butoxide a

Parameter RTA (mean ± SD); % of Positive Control (PC)

Run 1 Run 2
Conc. (M) Mean SD Mean SD

10 −4 1.9 1.1

-- *

-- 10 −5 0.8 0.4 2.3 1.5

10 −6 0.5 0.3 2.2 1.0

10 −7 0.6 0.3 2.8 1.6

10 −8 0. 0 0.4 0.9 1.1

10 −9 0. 1 0.4 1.1 1.3

10 −10 0. 0 0.4 0.1 0.8

10 −11 0.3 0.2 0.8 1.0

Log EC 50 NA NA

Hill Slope NA NA

RPC Max 1.9 2.8

PC Max 10 −4 10 −7

PC 50 NA NA

PC 10 NA NA

a Data were obtained from page s 21

- 22 of the study report.

* Cytotoxicity observed (55.6% viability)

NA = Not ApplicableEstrogen Receptor Transcriptional Activation ( 2011) Page 14 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## F IGURE 4. Relative Transcription Activation (RTA) of Piperonyl Butoxide Compared to

th e Positive Control , 1 nM 17 β

- estradiol.

3. Performance C riteria : While the L og PC 50 

## , Log PC 10 

## , Log EC 50, 

## and Hill slope values for the concurrent reference chemicals fell within or near the acceptable ranges (Table 3), theEstrogen Receptor Transcriptional Activation ( 2011) Page 15 of 15

PIPERONYL BUTOXIDE / 067501 O CSPP 89 0.1300 / OECD 455



## full response of the cells to the reference chemicals was not satisfactory , as detailed above in

Section B.2.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS ’ CONCLUSIONS : The suitable top concentration of piperonyl butoxide for use in the transcriptional activation assays was 10

− 4 

## M , as cytotoxicity was observed at the highest concentration of piperonyl butoxide tested in the second independent run. In two independent runs of the transcriptional activation assay, piperonyl butoxide did not result in an increase in luciferase activity at any of the concen tration s tested.

Piperonyl butoxide is not an agonist of the human estrogen receptor alpha (hER α ) in the

HeLa 9903- model system.

B. AGENCY COMMENTS : Piperonyl butoxide was tested up to the limit of cytotoxicity,

which was observed at 10

− 4 

## M in the second run. At concentrations up to 10 −5 

## M, the RTA of piperonyl butoxide was ≤ 2.8%. In the main assays, the responsiveness of the cells to the very weak positive control 17α

- methyltestosterone was lower than the expected values, indicating a decr eased 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 RPC Max 

## <
PC 10 

## in both assay runs, piperonyl butoxide was considered negative for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES : The following deficiencies were noted:

• C ytotoxicity was observed in cells treated with two of the reference compounds ( corticosterone an d 17 α

- methyltestosterone ). Cytotoxicity should not be observed at any level for the reference chemicals.

• The Hill slope for 17α

- estradiol in the second run (5.1) was out of the expected range

.

• The maximum response of 17β

- estradiol and 17α estradiol in Run 2 exceeded (~120%) the response of the PC (1 nM 17β -estradiol). The maximum responsiveness should be

observed in the PC.

• Th e cells responded inadequately (<50% RTA) to the very weak estrogen

, 17α

- methyltestosterone.

• Certificates of Analyses were not provided for the Reference Chemicals.

• The source of the fetal bovine serum was not provided.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

Data Requirement: EPA DP Barcode 404227

OECD Data Point 229

EPA MRID 48687701

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Piperonyl Butoxide (PBO) Purity: 93.7%

Common name: PBO

Chemical name: IUPAC: 5-[2-(2-butoxyethoxy)ethoxymethyl]-6-propyl-1,3-benzodioxole or

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

CAS name: 5-[[2-(2-butoxyethoxy)ethoxy]methyl]-6-propyl-1,3-benzodioxole

CAS No.: 51-03-6

Synonyms: 4,5-Methylenedioxy-2-propylbenzyldiethyloneglycol butyl ether (Butylcarbityl-0-

(6-propyl-piperonyl) ether, Piperonyl butoxide technical grade

EPA PC Code: 067501

Primary Reviewer: Joan Gaidos Signature:

Senior Scientist, CDM Smith Date: 4/16/13

Secondary Reviewer: Teri S. Myers Signature:

Senior Scientist, CDM Smith Date: 4/29/13

Primary Reviewer: Donna R. Judkins, Ph.D. Signature:

Biologist, EPA/OPP/EFED/ERB2 Date: 5/19/13

Additional Reviewer: Elyssa Arnold

Biologist, EPA/OPP/EFED/ERB2 Date: 6/1/15

Date Evaluation Completed: 01-06-2015

Page 1 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

CITATION: Fort, D.J. 2012. OPPTS 890.1350 21-d Fish Reproduction Assay. Performed by Fort

Environmental Laboratories, Inc., Stillwater, Oklahoma, Golden Pacific Laboratories, Inc., Fresno,

California, and Experimental Pathology Laboratories, Inc., Sterling, Virginia, Report No.: PBTF01-00257.

Submitted by Consumer Specialty Products Association, Inc. for Piperonyl Butoxide Task Force II,

Washington, DC. Completion date November 8, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of

eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to

interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1

battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with

complementary endpoints within the assay, where available, and redundancy across the battery. Thus,

the results of each individual assay should not be considered in isolation but rather should be considered

in the context of other assays in the battery as well as Other Scientifically Relevant Information

(OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a

Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the

OSRI, should be undertaken (refer to the WoE Document).

D

isclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in

preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test

Guidelines, nor to provide any guidance on how the study should be conducted.

Page 2 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay 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 25.5 o 

### C 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.

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 (17 ȕ -estradiol and testosterone) were

not measured in this study.

All validity and performance criteria were met in this study.

Page 3 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

This assay satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction Assay

(OCSPP Guideline 890.1350).

Results Synopsis

Test Organism age at test initiation: ca . 6 months

Mean body weight at test initiation: Not reported.

Mean length at test initiation: Not reported.

Test Type: Continuous flow-through

Table 1: Summary of Reproductive and HPG Effects 1,2 

### in the Fish Short-Term Reproduction Assay

(FSTRA) with PBO

.

Treatment

(mg a.i./L)

[TWA

measured] Fecundity Fert.

Success Tubercle

Score GSI Gonadal

Histo. Plasma

VTG Plasma T Plasma

E2

M F M F M F M F M F M F

0.0101 No No Yes No No No No No No No NA NA NA NA

0.0348 Yes No No No No No Yes 3 No No No NA NA NA NA

0.0944 Yes No No No No No Yes 3 No No No NA NA NA NA

Abbreviations: E2 17 ȕ -estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology.

M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER.

Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

3 Increased testes degeneration at mid (marginally significant) and high concentrations.



### I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish

Short-Term Reproductive Assay” and OECD 229 (2009). The following

deviations were noted:

Page 4 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

1. The study author’s analysis of male VTG resulted in <0.1 x 10 6 

### P g/mL for all treatment

groups, indicating that the methodology may not have been sufficiently sensitive to

accurately detect male VTG. The measurement of VTG in plasma samples was

performed using a commercial ELISA kit (Biosense, Bergen, Norway).

2. Five female fish weighed more than 20% of the mean weight of female fish used in the

study (up to 22.2% over).

3. Gonadal stage was determined based on a scale that was comparable but not identical

to that provided in EPA histopathological guidelines.

These deviations do not impact the ability to interpret the results of this study.

COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements

were provided. This study was conducted in compliance with all pertinent U.S. EPA

Good Laboratory Practice regulations with the following exceptions: the most recent

water characterization performed in February 2011 was not performed in accordance

to the stated GLP standards.

A. Test Material: Piperonyl Butoxide (PBO; CAS# 51-03-6)

Description: Viscous liquid.

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa,

Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : 51-03-6

Purity: 93.7%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 7, 14, and 21

yielded recoveries of 75.5% to 110.9% of nominal concentrations. Fortification recoveries and prePage 5 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

diluter verification samples were not reported. The test material appears to be stable under the test

conditions at these treatment levels.

Storage Conditions of Test Chemicals: Stored at ambient temperature.

Page 6 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?Yes EPA recommends that fish be from a single laboratory stock.Were acclimation conditions same asdefinitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period:2 weeks EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Page 7 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities were <5% prior to preexposure.Fish did not receive any treatmentfor disease during acclimationperiod.Behavioral abnormalities or clinicalsigns were not reported. EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the

fish not be used.Type of food:Brine shrimp nauplii( Artemia salina) EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body

condition.Source of food:Not reportedFrequency of feeding:2 times/dayDetails on feeding:Food was withheld 12hours prior to tissuesampling.Page 8 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:4.5 to 6.0 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):2.53±20%=2.02-3.04A sub-sample of fish (not defined) used tostock aquaria for pre-exposure phase wereweighted. Mean and range calculated fromPre-test Weights in report appendix, p. 101. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each

sex.Range of individual weights (males)at test initiation (if determined):Range: 2.08-3.01 gIndividual weights within ± 20% of theestimated meanMean weight of females at testinitiation (if determined): 1.89±20%=1.51-2.27A sub-sample of fish (not defined) used tostock aquaria for pre-exposure phase wereweighted. Mean and range calculated fromPre-test Weights in report appendix, p. 101.Range of individual weights (females)at test initiation (if determined):Range: 1.54-2.31 gFive females over 20% of mean – up to22.2% overMean length of males at test initiation(if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:14 days EPA recommends a minimum of 14 days.Page 9 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:Not reported EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:At least 15eggs/female/day EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:> two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:NonePage 10 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Continuous flow-through. EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilution system: Flow proportional diluters Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:2.7 L/hr or at least 6.5volume additions/testvessel/day. Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Page 11 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing for flowthrough systems:Flow-through diluter system wasused and the 4 replicate testvessels.Treatment recoveries prior to testinitiation were not reported.Flow splitting accuracy was notreported. Recommended toxicant mixing for flow-through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.Aeration? No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Source of dilution water:Dechlorinated tap water Multimedia, pre-treatment, carbonand polishing filtered EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated

in the test guideline.Was dilution water analyzed forpesticides, heavy metals, and othercontaminants?YesPage 12 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:Glass aquaria EPA and OECD recommend that water-contact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are
acceptable examples).Test vessel size:40 cm x 20 cm x 20 cm EPA recommends the use of 18 L test chambers (e.g., 40

x 20 x 20 cm).Fill volume:10L; 12-15 cm depth EPA recommends 10 L solution per tank.Spawning substrate material:Two 4-inch breeding tiles of4-in diameter PVC pipe pertank EPA recommends that each tank contain three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in
length, split lengthwise.Spawning substrate size:4-inchesAdditional details on exposure system:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsTemperature (°C)24.425.525Daily EPA recommends temperature

25±1 oC; inter-replicate and intertreatment differentials should not

exceed 1 o C.pH 7.38.17.7 3X/week EPA recommends pH 6.5 to 9.0.Page 13 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsDissolved oxygen (mg/L)6.79.27.53X/week EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air

saturation)Total alkalinity (mg/L asCaCO3)32 44 38 Weekly EPA recommends total alkalinity >20 mg/L as CaCO 3.Hardness (mg/L as CaCO3) 136 152 142WeeklyTotal organic carbon (mgC/L)Not reported Not reported Not reported None EPA recommends that total organic carbon in dilution water be <2

mg/L.Unionized ammonia (mg/L) <0.06<0.06 <0.06Bi-monthly EPA recommends that unionized ammonia in the dilution water be <1

P g/L.Residual chlorine (mg/L)<0.01<0.01 <0.01Bi-monthly EPA recommends that residual chlorine in dilution water be <10

P g/L.Page 14 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsConductivity (PS/cm) Not reported Not reported Not reportedStudy authors state: “Water qualitycharacteristics of the dilution water weremonitored bimonthly for pH, dissolvedoxygen (DO), conductivity, hardness,alkalinity, ammonia, residual chlorine(oxidants); and at least annually for iodide(I-), polyaromatic hydrocarbons (PAHs),pesticides, and metals. The dilution waterwas analyzed for pesticides, PAHs, andmetals on January 28, 2011. All waterquality measurements cited above met theU.S. EPA criteria for aquatic toxicity testculture/dilution water.” General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels. Iodide was also reported at2 to 5.1Pg/L (mean 3.6Pg/L, measured semi-annually).Page 15 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesFour-day range finding studieswere conducted with adultfathead minnow, studyingmortality and morbidity (definedas abnormal development orunusual behavioral reaction toexposure). Study concentrationswere selected based on thesefour studies. EPA recommends conducting a range-finder if 96-hour LC

50 data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?Experimentally-derived MTCof 0.1 mg/L based onmorbidity and mortality4-day LC50was 12.9 mg/L, EC50was 1.6 mg/LEC50based on moderate gillhemorrhage and abnormalswimming behavior EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is appropriate.Species:Fathead Minnow( Pimephales promelas)Page 16 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsLife stage:Adult EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in

the test.Test duration:4 days EPA recommends a 96-hour exposure.Additional details:0 (control dilution water),0.01, 0.1, 1.0, 10.0 and25.0 mg a.i./L EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21-day

test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive

test be 21 days.Page 17 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:Maximum toleratedconcentration based onmorbidity and mortalityobservations in the rangefinder to ensure no overttoxic responseAlso considered using 0.33x the lower confidenceinterval of the 4-day EC50of 1.6, (0.6-4.4) mga.i./L, which is 0.2 mg a.i./L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reportedSeparation of test concentrations:0 (control), 0.011,0.033, and 0.1 mga.i./L EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or ten-fold) is scientifically acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean time-weightedaverageLimit of quantification (LOQ): 0.001 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Page 18 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsLevel of detection (LOD):Not reported.Frequency of measurement: 0, 7, 14, and 21 days It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per

treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to pre-exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Page 19 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration (ifapplicable):NA EPA recommends that the solvent not exceed 0.02

ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than

0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s) (ifapplicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Page 20 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’s surface: 652-990 lux Cool white fluorescents EPA recommends light intensity 540 – 1080 lux (at

water’s surface).Additional details:Biological Endpoint Methods:Fecundity was expressed on the basis of surviving females per reproductive(test) day per replicate. The cumulative eggs laid over the in-life exposureduration were also determined. EPA recommendations for biological endpoint measurement are specified in the method. The DER should identify the methods used, especially any alternative method(s) used.Study authors state that the recommended method was employed for fertilizationsuccess measurement but do not give details on the magnification or stage ofdevelopment when fertilization was determined.Study authors reference the EPA guideline as the method used to map nuptialtubercles.Immediately after anesthetization of an individual fish, length and weightmeasurements were taken, and fresh tissues (e.g., gonads) collected. Duringnecropsy, rapid gonad fixation in modified Davidson’s fixative was performed toprevent autolysis and cellular deterioration. Using a syringe, approximately 0.5mL of modified Davidson’s fixative was gently applied to the gonads in situ.Approximately 90 seconds following the application of fixative, the liquid fixativewithin the abdomen was removed with a gauze sponge, and the gonadsexcised. The fixed gonads were removed and weighed (fixed weight to thenearest 0.1 mg) to determine the GSI (GSI=100% x gonad wt/body wt).Page 21 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701ParameterValue(s)Details or RemarksGuideline RecommendationsGonadal stage was determined based on a scale that was comparable to thatprovided in EPA histopathological guidelines, but was somewhat more definitive.The stage scale used by EPL (Sterling, VA) was: 0=entirely immature,1=immature phases predominate, 1.5=more spermatozoa than Stage 1.0,2=Spermatocytes, spermatids, and spermatozoa present in roughly equalproportions, 2.5=mature sperm predominate, 3.0=germinal epithelium thinnerthan Stage 2.5, 3.5=germinal epithelium barely apparent and large coalescingtubule lumina are filled with spermatozoa, 4.0=not used. Findings were recordedin an electronic data recording system. The pathology report was provided.Blood was collected from the caudal artery/vein with a heparinizedmicrohematocrit capillary tubule. Plasma was separated from the blood viacentrifugation (approximately 3 minutes at 15,000 x g) and stored with proteaseinhibitors at -75oC to -85oC until analyzed for VTG. Sex steroids were notanalyzed in the present study. Non-detected (ND) samples were reported indata sets as < detection limit (DL). The measurement of VTG in plasmasamples was performed using a commercial ELISA kit (Biosense, Bergen,Norway).The stock solutions were clear and colorless with no precipitate.Page 22 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 8: Summary of Treatment Concentrations in Fish Short-Term Reproduction Assay with PBO.Treatment IDNominal Concentration(mg a.i./L)Mean Time-weightedConcentration(mg a.i./L)Mean CV(%)Details or Remarks –Mean MeasuredConcentrations (mga.i./L)Guideline RecommendationsControl (dilutionwater only)0 <LOQ NA NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.0110.010112.80.0103Treatment 20.0330.03483.00.0351Treatment 30.100.09443.70.0952Abbreviations:CVCoefficient of variation.LOQ=0.0010 mg a.i./L.E. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sexcharacterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology, and plasma vitellogeninwere evaluated.Were raw (individual) data provided?YesPage 23 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701 EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma

vitellogenin, and plasma sex steroids (testosterone and 17ȕ -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.II. RESULTS AND DISCUSSIONA. ResultsMean male and female survival was 100% at all treatment levels (Table 9).Page 24 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 9: Adult Fish Survival in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]MalesFemalesn1# Surviving% Survivaln1# Surviving% SurvivalControl (<LOQ)8810016161000.0101 8 8 10016 16 1000.0348 8 8 10016 16 1000.0944 8 8 10016 16 1001Total number of fish at test initiation.LOQ=0.0010 mg a.i./L.Mean male body weight values were 2.36, 2.29, 2.17 and 1.96 g and mean female body weight values were 1.24, 1.37, 1.26 and 1.20g in the controland TWA-measured 0.0101, 0.0348 and 0.0944 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 62.1, 64.9, 61.6and 58.0 mm and mean female body length values were 52.6, 53.1, 50.9 and 51.5 mm in the control and TWA-measured 0.0101, 0.0348and 0.0944mg a.i./L treatment levels, respectively.Page 25 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 10: Size at Test Termination in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ)4 2.36 0.14 4 1.24 0.10 NA 62.1 3.0 NA 52.6 1.70.0101 4 2.29 0.25 4 1.37 0.20 NA 64.9 1.8 NA 53.1 2.30.0348 4 2.17 0.38 4 1.26 0.01 NA 61.6 2.3 NA 50.9 1.10.0944 4 1.96 0.19 4 1.20 0.05 NA 58.0 2.0 NA 51.5 1.3Abbreviations:NANot applicable.SDStandard deviation.LOQ=0.0010 mg a.i./L.Page 26 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

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Mean fecundity values were 33.5, 35.8, 38.3 and 39.8 eggs/female/day and fertilization success was

99.0, 99.1, 98.9 and 98.95 in the control and TWA-measured 0.0101, 0.0348 and 0.0944 mg a.i./L

treatment levels, respectively (Table 11).

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( P imephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Fecundity 1 Fertilization Success 2

Mean ±SD Mean (%) ±SD

Control (<LOQ) 33.5 0.56 99.0 0.13

0.0101 35.8 2.9 99.1 0.059

0.0348 38.3 1.1 98.9 0.24

0.0944 39.8 2.2 98.9 0.17

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.

LOQ=0.0010 mg a.i./L.



### Median male tubercle scores were 54, 58, 55 and 56 in the control and TWA-measured 0.0101, 0.0348

and 0.0944 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to

have tubercles.

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Table 12: Nuptial Tubercle Score in Fathead Minnow ( P imephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

n Median Tubercle

Score 1 n Median Tubercle

Score

Control (<LOQ) 4 54 4 0

0.0101 4 58 4 0

0.0348 4 55 4 0

0.0944 4 56 4 0

1 Mean values were 54, 58, 55, and 56 (same as median values) for the negative control and low, mid, and high

treatment groups, respectively.

LOQ=0.0010 mg a.i./L.



### Mean male GSI was 1.25, 0.99, 1.20 and 1.35% and mean female GSI was 10.1, 11.8, 10.6 and 10.9% in

the control and TWA-measured 0.0101, 0.0348 and 0.0944 mg a.i./L treatment levels, respectively (Table

13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

n Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Control (<LOQ) 4 1.25 0.266 4 10.1 2.34

0.0101 4 0.99 0.149 4 11.8 2.64

0.0348 4 1.20 0.400 4 10.6 3.23

0.0944 4 1.35 0.567 4 10.9 1.90

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

LOQ=0.0010 mg a.i./L.

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Median male gonadal stages were 2, 2, 2.5 and 2 and female gonadal stages were 3.5, 3.5, 3 and 3 in

the control and TWA-measured 0.0866, 0.305 and 0.989 mg a.i./L treatment levels, respectively (Table

14). One male in the control group and two males in the 0.0101 mg a.i./L group were unable to be staged.

The testes of these fish contained primarily spermatogonia, but the presence of residual spermatozoa and

tubule formation suggested that these testes had regressed to a juvenile-like conformation, as opposed to

true Stage Juvenile testes in which germ cell maturation has not yet progressed beyond the spermatogonial

phase. One additional male in the control group was determined to be a juvenile.

Table 14: Gonadal Staging in Fathead Minnow ( P imephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

N Median Stage 1 n Median Stage 2

Control (<LOQ) 6 3 16 3

0.0101 6 2.5 16 3.5

0.0348 8 3 16 3

0.0944 8 3 16 3

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

LOQ=0.0010 mg a.i./L.



### The sole finding in males that appeared related to PBO administration was increased testicular degeneration

(minimal to mild) (Table 15, Table 16). The prevalence of this finding was higher than controls in the 0.944

mg a.i./L concentration group, but only marginally higher in the 0.0348 mg a.i./L concentration group.

According to the histopathology report, at present, little evidence exists to support the contention that

testicular degeneration is an endocrine-specific finding; for example, it is reasonable to believe that this type

of degenerative change could occur secondarily to non-endocrine testicular toxicity, systemic toxicity, or

stress. There were no findings in PBO-treated females that were substantially different from those of controls

(Table 17, Table 18). Other findings in this study were not unusual and/or were only present in single fish

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per group ( e.g. , histiocytic intraluminal cells in the testis, egg debris in the ovary), and were thus considered

incidental to the study outcome.

The reviewer notes that minimal to moderate oocyte atresia in female fish was observed in the controls and

all treatment groups. Also, one case of microsporidia infection was found in the control and 0.0348 mg

a.i./L treatment groups.

Page 30 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 15: Gonadal Histopathology in Male Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]Diagnostic Observations1SeverityIncreased Proportion ofSpermatogoniaPresence of Testis-OvaIncreased TesticularDegenerationDuct MineralizationInterstitial cell (Leydig)hypertrophy/hyperplasianIncidencenIncidencenIncidencenIncidencenIncidenceControl(<LOQ) 0 8 5 NA NA 8 7 8 7 8 81 8 1 NA NA 8 1 8 1 8 02 8 0 NA NA 8 0 8 0 8 03 8 0 NA NA 8 0 8 0 8 04 8 2 NA NA 8 0 8 0 8 00.0101 0 8 1 NA NA 8 7 8 6 8 71 8 4 NA NA 8 0 8 0 8 12 8 1 NA NA 8 1 8 2 8 03 8 0 NA NA 8 0 8 0 8 04 8 2 NA NA 8 0 8 0 8 00.0348 0 8 5 NA NA 8 5 8 7 8 81 8 1 NA NA 8 3 8 1 8 02 8 1 NA NA 8 0 8 0 8 03 8 1 NA NA 8 0 8 0 8 04 8 0 NA NA 8 0 8 0 8 0Page 31 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Treatment(mg a.i./L)[TWA measured]Diagnostic Observations1SeverityIncreased Proportion ofSpermatogoniaPresence of Testis-OvaIncreased TesticularDegenerationDuct MineralizationInterstitial cell (Leydig)hypertrophy/hyperplasianIncidencenIncidencenIncidencenIncidencenIncidence0.0944 0 8 4 NA NA 8 3 8 7 8 81 8 2 NA NA 8 4 8 1 8 02 8 0 NA NA 8 1 8 0 8 03 8 2 NA NA 8 0 8 0 8 04 8 0 NA NA 8 0 8 0 8 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.0010 mg a.i./L.Page 32 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityDecreased Proportion ofSpermatogoniaIncreased Vascular orInterstitial ProteinaceousFluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ) 0 NA NA NANA NA NA NA NA 8 71 NA NA NA NA NA NA NA NA 8 12 NA NA NA NA NANA NA NA 8 03 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 00.0101 0 NA NA NA NANA NA NA NA 8 41 NA NA NA NA NA NA NA NA 8 22 NA NA NA NA NA NA NA NA 8 23 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 00.0348 0 NA NA NA NANA NA NA NA 8 61 NA NA NA NA NA NA NA NA 8 22 NA NA NA NA NANA NA NA 8 03 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 0Page 33 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityDecreased Proportion ofSpermatogoniaIncreased Vascular orInterstitial ProteinaceousFluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.09440 NA NA NA NA NANA NA NA 8 51 NA NA NA NA NA NA NA NA 8 22 NA NA NA NA NA NA NA NA 8 13 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.0010 mg a.i./L.Page 34 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 17: Gonadal Histopathology in Female Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityIncreased Oocyte AtresiaPerifollicular CellHyperplasia/ HypertrophyDecreased Yolk FormationAggregates of macrophages,multifocalnIncidencenIncidencenIncidencenIncidenceControl(<LOQ) 0 16 6 NA NA NA NA NA NA1 16 2 NA NA NA NA NA NA2 16 2 NA NA NA NA NA NA3 16 6 NA NA NA NA NA NA4 16 0 NA NA NA NA NA NA0.0101 0 16 5 NANA NA NA NA NA1 16 3 NA NA NA NA NA NA2 16 0 NA NA NA NA NA NA3 16 8 NA NA NA NA NA NA4 16 0 NA NA NA NA NA NA0.0348 0 16 8 NA NA NA NA NA NA1 16 3 NA NA NA NA NA NA2 16 2 NA NA NA NA NA NA3 16 3 NA NA NA NA NA NA4 16 0 NA NA NA NA NA NAPage 35 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityIncreased Oocyte AtresiaPerifollicular CellHyperplasia/ HypertrophyDecreased Yolk FormationAggregates of macrophages,multifocalnIncidencenIncidencenIncidencenIncidence0.09440 16 5 NA NA NA NA NA NA1 16 2 NA NA NA NA NA NA2 16 2 NA NA NA NA NA NA3 16 7 NA NA NA NA NA NA4 16 0 NA NA NA NA NA NA1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.0010 mg a.i./L.Page 36 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityInterstitium/multifocal inflammationEgg Debris in OviductGranulomatousInflammationDecreased Post-OvulatoryFolliclesnIncidencenIncidencenIncidencenIncidenceControl(<LOQ) 0 NA NA 16 15 16 14 16 111 NA NA 16 0 16 1 16 22 NA NA 16 1 16 0 16 13 NA NA 16 0 16 1 16 24 NA NA 16 0 16 0 16 00.0101 0 NA NA 16 16 16 14 16 131 NA NA 16 0 16 2 16 12 NA NA 16 0 16 0 16 03 NA NA 16 0 16 0 16 24 NA NA 16 0 16 0 16 00.0348 0 NA NA 16 16 16 15 16 111 NA NA 16 0 16 1 16 32 NA NA 16 0 16 0 16 03 NA NA 16 0 16 0 16 24 NA NA 16 0 16 0 16 0Page 37 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityInterstitium/multifocal inflammationEgg Debris in OviductGranulomatousInflammationDecreased Post-OvulatoryFolliclesnIncidencenIncidencenIncidencenIncidence0.09440 NA NA 16 16 16 16 16 131 NA NA 16 0 16 0 16 12 NA NA 16 0 16 0 16 13 NA NA 16 0 16 0 16 14 NA NA 16 0 16 0 16 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.0010 mg a.i./L.Page 38 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

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Mean male VTG was <0.1 x 10 6 

### ng/mL at all treatment levels and mean female VTG was 25.9x10 6



### ,

25.8x10 6 

### , 26.0x10 6 

### and 26.1x10 6 

### ng/mL in the control and TWA-measured 0.0101, 0.0348 and 0.0944 mg

a.i./L treatment levels, respectively (Table 19).

Table 19: Plasma Vitellogenin in Fathead Minnow ( P imephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Plasma Vitellogenin (VTG)

Males Females

n Mean

(ng/mL plasma) ±SD n Mean

(ng/mL plasma) ±SD

Control (<LOQ) 4 <0.1 x 10 6 NA 4 25.9x10 6 0.263x10 6

0.0101 4 <0.1 x 10 6 NA 4 25.8x10 6 0.573x10 6

0.0348 4 <0.1 x 10 6 NA 4 26.0x10 6 0.364x10 6

0.0944 4 <0.1 x 10 6 NA 4 26.1x10 6 0.313x10 6

Abbreviations: NA Not applicable. SD Standard deviation.

LOQ=0.0010 mg a.i./L.



### Mean plasma testosterone and plasma 17 ȕ -estradiol in male and females were not measured (Table 20).

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Table 20: Plasma Sex Steroids in Fathead Minnow ( P imephales promelas ). Not measured.

Treatment

(mg a.i./L)

[TWA

measured] Plasma Testosterone (T) Plasma 17 ȕ -estradiol (E2)

Males Females Males Females

n Mean

(ng/mL

plasma) ±SD n Mean

(ng/mL

plasma) ±SD n Mean

(ng/mL

plasma) ±SD n Mean

(ng/mL

plasma) ±S

D

Control

(<LOQ) ND ND ND ND ND ND ND ND ND ND ND ND

0.0101 ND ND ND ND ND ND ND ND ND ND ND ND

0.0348 ND ND ND ND ND ND ND ND ND ND ND ND

0.0944 ND ND ND ND ND ND ND ND ND ND ND ND

Abbreviations: ND Not determined. SD Standard deviation.

LOQ=0.0010 mg a.i./L.



### 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 (Table 21).

All fish in the negative controls and treatment groups appeared normal and no overt signs of toxicity were

observed during the test period.

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Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( P imephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Secondary Sex Characteristics and Clinical Signs

Males Females

Type n Incidence Type n Incidence

Control (<LOQ) None 8 0 None 16 0

0.0101 None 8 0 None 16 0

0.0348 None 8 0 None 16 0

0.0944 None 8 0 None 16 0

LOQ=0.0010 mg a.i./L.



### B. Study Author’s Analysis and Conclusions

The study author analyzed survival, wet weight, tubercle score, gonadal development, GSI, fertility, fecundity,

VTG, and incidence and severity of gonad abnormalities. Data were gender specific and analyzed in

comparison to the negative controls. The study author reported using statistical procedures described in the

OECD (2006) guideline, which follow the flow chart and recommendations in the Guideline. However, results

from these statistical analyses were not presented in the study report.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant effects

were detected at p d 0.05 (software not reported). ANOVA was used to analyze male and female body

weight, female length, fertility, male and female GSI, male nuptial tubercle score, and gonadal staging.

Survival and VTG data were analyzed using a ranked ANOVA. Male length and fecundity were analyzed

using an ANOVA with a Bonferroni t-test.

There were no significant differences in survival at any treatment level compared to the negative controls

(p>0.05; Ranked ANOVA). There was a statistically significant increase in mean fecundity in the 0.0348

and 0.0944 mg a.i./L treatment levels compared to the control (p<0.05; ANOVA, Bonferroni t-test). There

were no other statistically significant differences in treated groups compared to negative controls for any

other endpoint. There were no apparent significant differences between histopathology severity scores or

gonadal stages in treated groups compared to the negative controls. Increased testicular degeneration was

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noted in the treatment groups, but the study authors concluded that “it is unlikely that this minimal to mild

testicular degeneration would have had a measurable impact on testicular function. At present, little evidence

exists to support the contention that testicular degeneration is an endocrine-specific finding; for example, it is

reasonable to believe that this type of degenerative change could occur secondarily to non-endocrine

testicular toxicity, systemic toxicity, or stress.”

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer assessed survival (mortality) data based on visual observation. Fecundity

and male weight data both exhibited monotonic trends and therefore were analyzed using the JonckheereTerpstra test. Fecundity followed an increasing trend while male body weight followed a decreasing trend. All

data were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test.

Data which met the assumptions of normality and homogeneity of variance were then analyzed using the

parametric Dunnett’s test. Female body weight data met assumptions of normality but not homogeneity of

variance and was therefore analyzed using the Dunnett T3 test. None of the surviving females were found to

have tubercles. Unless otherwise indicated, effects were considered statistically significant at p<0.05. These

analyses were conducted using CETIS (version 1.8.7.7) and backend database settings approved by EFED

on 1/30/13. Both the study author’s and reviewer relied on the time-weighted mean-measured

concentrations to discuss effects in this study.

Conclusions: Adult male and female survival was 100% in this study. There was a significant increase in

fecundity at the 0.0348 and 0.0944 mg a.i./L treatment levels of 14 and 19%, respectively, compared to

the negative control (p<0.05; Jonckheere-Terpstra test). Median male tubercle score was significantly

(p<0.05; Dunnett’s test) greater than the negative control at the low, 0.0101 mg a.i./L level, but not

different from control (p>0.05) at the middle and high concentrations. For growth parameters, there was a

significant reduction in male body weight of 17% at the 0.0944 treatment level (p<0.05; JonckheereTerpstra test). There was no other significant difference between the treatment levels and negative control for

any other endpoint. There were no apparent differences between histopathology severity scores or gonadal

stages in treated groups compared to the negative controls, with the exception of increased testicular

degeneration (minimal to mild) in males in the mid and high treatment groups compared to the control.

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.

Page 42 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( P imephales promelas)in the FSTRA with PBO.Treatment(mg a.i./L)[TWA measured]Tubercle Score4GSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative control (<LOQ) 54 NA 0NANo0 NA NA NA NA NA0.0101 58 0.010 -20.9 0.653 No NA NA NA NA NA NA0.0348 55 0.906 -4.0 0.995 Yes3NA NA NA NA NA NA0.0944 56 0.174 7.8 0.968 Yes3NA NA NA NA NA NAStatistical TestDunnett’sDunnett’sNANANANAAbbreviations:Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Increased testes degeneration at mid (marginally significant) and high concentrations.4Mean values were 54, 58, 55, and 56 (same as median values) for the negative control and low, mid, and high treatment groups, respectively.LOQ=0.0010 mg a.i./L.Page 43 of 47Data Evaluation Record on the Fish Short-TermReproduction Assay with Piperonyl ButoxideEPA MRID Number 48687701Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( P imephales promelas)in the FSTRA with PBO.Treatment(mg a.i./L)[TWA measured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p%DiffPMedianp%Diff.pEffect?(Yes/No)%Diff.p%Diff.p%Diff.pNegative control(<LOQ)0 NA 0 NA 0 NA 0 NA No0 NA NA NA NA NA0.0101 6.8 0.054 0.2 0.243 0 NA 17.10.673 No -0.7 0.843 NA NA NA NA0.0348 14.0 0.005 0 1.00 0 NA 4.70.988 No 0.3 0.983 NA NA NA NA0.0944 18.7 0.0005 -0.1 0.852 0 NA7.8 0.948 No 0.6 0.892 NA NA NA NAStatistical TestJonckheereTerpstraDunnett’s NA Dunnett’s NA Dunnett’s NA NAAbbreviations:Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.LOQ=0.0010 mg a.i./L.Page 44 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

Table 24: Growth Endpoints 1,2 

### in the Fish Short-Term Reprod uction Assay (FSTRA) with PBO.

Treatment

(mg a.i./L)

[TWA measured] Body Weight Length

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA

0.0101 -2.65 0.231 10.9 0.576 4.4 0.267 0.8 0.966

0.0348 -7.74 0.134 1.4 0.97 8 -0.8 0.980 -3.2 0.370

0.0944 -16.6 0.011 -2.8 0. 898 -6.6 0.067 -2.2 0.641

Statistical Test JonckheereTerpstra Dunnett T3 Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

2 Unless otherwise specified, effects are considered statistically significant at p<0.05.

LOQ=0.0010 mg a.i./L.



### E. Study Deficiencies

There were some deviations described in Section I. Materials and Methods of this DER regarding failure to

detect male VTG, as well as some deviations from recommendations on weight variability and gonad stage

identification.

This assay fully met the validity criteria identified in the OCSPP 890.1350 test guideline, based on the data

and other information provided in the study report. Performance criteria were met with the exception of

missing total organic carbon data. The deviations noted do not have an impact on the ability to interpret the

results of this study.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in general agreement. Both the study author’s and

reviewer’s analysis detected statistically significant increases (p<0.05) in fecundity at the 0.0348 and

Page 45 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

0.0944 mg a.i./L treatment levels compared to the control. Additionally, the reviewer’s analysis detected a

significant (p<0.05) increase in median male tubercle score (the study authors analyzed mean values) at

the 0.0101 mg a.i./L level as well as a significant decrease (p<0.05) in male body weight at the 0.0944

mg a.i./L treatment level. No other statistically significant differences were noted in the other endpoints.

The study author reported time-weighted concentration averages of 10.1, 34.8, and 94.4 P g a.i./L and noted

that variability in each set of samples primarily due to diluter flow adjustments required to fine-tune the test

concentrations. Variability between replicates during each sampling event was low.

The analysis of male VTG resulted in <0.1 x 10 6 

### ng/mL for all treatment groups, indicating the methodology

may not have been sufficiently sensitive to accurately detect male VTG.

The reviewer noted that the study author-reported replicate means in Table 9 (p. 42 of 286) for mean male

and female body weights differed from those calculated using the provided raw data which accompanied this

study (both in the Appendix and electronically). The reviewer relied on the raw data for analysis.

III. REFERENCES

USEPA, Federal Insecticide, Fungicide, Rodenticide Act (FIFRA). Good Laboratory Practice

Standards, Title 40 Code of Federal Regulations Part 160 (effective October 16, 1989).

FEL Quality Assurance Management Plan (QAMP), Fort Environmental Laboratories, Stillwater, OK,

July 23, 2010 (current version).

OPPTS. 890.1350 Fish Short-Term Reproduction Assay, United State Environmental Protection

Agency, Washington DC, EPA 740-C-09-007, October 2009.

USDA, Volume 1, Subchapter A – Animal Welfare, 9 CFR – Animals and Animal Products, 2009.

USEPA. A Short-term Method for Assessing the Reproductive Toxicity of Endocrine Disrupting

Chemicals Using the Fathead Minnow (Pimephales promelas). EPA/600/R-01/067, 2002.

Page 46 of 47

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Piperonyl Butoxide

EPA MRID Number 48687701

USEPA. Histopathology Guidelines for the Fathead Minnow (Pimephales promelas) 21-day

Reproduction Assay, 2006.

OECD. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application.

Environmental Health and Safety Publications. Series on Testing and Assessment, No. 54, Paris,

France, 2006.

Ankley G.T., Jensen K.J., Kahl M.D., Korte J.J., and Makynen E.A. Description and evaluation of a

short-term reproduction test with the fathead minnow (Pimephales promelas).” Environ. Toxicol.

Chem. 20:1276-1290, 2001.

Ankley, G.T., Jensen, K.M., Makynen, E.A., Kahl, M.D., Korte, J.J., Hornung, M.W., Henry, T.R.,

Denny, J.S., Leino, R.L., Wilson, V.S., Cardon, M.C., Hartig, P.C., and Gray, L.E. Effects of the

androgenic growth promoter 17-B-trenbolone on fecundity and reproductive endocrinology of the

fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 22:1350-1360, 2003.

SOP 4.10, Study File Document Collection and Organization, Fort Environmental Laboratories, 2001.

SOP 4.11, Maintenance of Study Archives, Fort Environmental Laboratories, 2001.

SOP 12.1, Archiving of Test Specimens, Fort Environmental Laboratories, 2003.

Page 47 of 47

### CETIS Summary Report Report Date: 29 Apr-13 15:12 (p 1 of 5)

Test Code: 067501 48687701 | 03-1763-0053

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Fort Evironmental Laboratories, Inc.

Batch ID: 12-3195-1361

Start Date: 08 Nov-12

Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Dechlorinated Tap Water

Brine: Not Applicable Species: Pimephales promelas

Source: Lab In-House Culture Analyst:

Age: 4-6m

Sample ID: 16-0806-5102

Sample Date: 08 Nov-12

Receive Date: Code: 48687701

Sample Age: NA Source: Piperonyl Butoxide Task Force II

Station: Client: CDM Smith

Project: Insecticide Material: Piperonyl Butoxide

in-life dates not reported in study; 067501 48687701

067501 48687701 Batch Note:

Sample Note:

Comparison Summary

NOEL LOEL TOEL Endpoint Analysis ID Method PMSD TU

10.1 34.8 18.75 13-4760-3726 Jonckheere-Terpstra Step-Down Test NA Fecundity

10.1 34.8 18.75 11-6044-2142 Dunnett Multiple Comparison Test 9.19% Fecundity

94.4 >94.4 NA 07-1481-5287 Dunnett T3 Multiple Comparison Test 16.5% FemaleBodyWt

94.4 >94.4 NA 18-7888-2608 Dunnett Multiple Comparison Test 48.4% FemaleGSI

94.4 >94.4 NA 13-5904-8490 Dunnett Multiple Comparison Test 5.89% FemaleLength

94.4 >94.4 NA 20-0951-1690 Dunnett Multiple Comparison Test 2.71% FemaleVTG

94.4 >94.4 NA 03-7035-4533 Dunnett Multiple Comparison Test 0.31% Fertility

34.8 94.4 57.32 16-6147-4066 Jonckheere-Terpstra Step-Down Test NA MaleBodyWt

94.4 >94.4 NA 03-5486-3646 Dunnett Multiple Comparison Test 20.6% MaleBodyWt

94.4 >94.4 NA 10-1798-4877 Dunnett Multiple Comparison Test 57.4% MaleGSI

94.4 >94.4 NA 02-2355-7914 Dunnett Multiple Comparison Test 7.08% MaleLength

<10.1 10.1 NA 02-4393-0165 Dunnett Multiple Comparison Test 5.74% MaleMedianTubercleScore

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 29 Apr-13 15:12 (p 2 of 5)

Test Code: 067501 48687701 | 03-1763-0053

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Fort Evironmental Laboratories, Inc.

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L Fecundity Summary

95% LCL 95% UCL %Effect

0 Negative Control 33.5 33 34.3 0.556 4 1.66% 0.278 32.6 34.4 0.0%

10.1 35.8 33.4 39.9 2.88 4 8.04% 1.44 31.2 40.4 -6.79%

34.8 38.2 37 39.7 1.14 4 2.98% 0.569 36.4 40 -14.0%

94.4 39.8 37.2 42.4 2.15 4 5.4% 1.07 36.4 43.2 -18.7%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L FemaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.24 1.1 1.33 0.102 4 8.25% 0.0512 1.08 1.4 0.0%

10.1 1.38 1.22 1.66 0.195 4 14.2% 0.0977 1.06 1.69 -10.9%

34.8 1.26 1.25 1.26 0.005 4 0.4% 0.0025 1.25 1.27 -1.41%

94.4 1.21 1.14 1.26 0.05 4 4.15% 0.025 1.13 1.28 2.82%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L FemaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 10.1 6.61 11.7 2.35 4 23.3% 1.18 6.36 13.8 0.0%

10.1 11.8 8.8 14.2 2.65 4 22.4% 1.33 7.6 16 -17.1%

34.8 10.6 7.79 14.9 3.21 4 30.4% 1.61 5.46 15.7 -4.68%

94.4 10.9 9 13.4 1.9 4 17.4% 0.95 7.87 13.9 -7.8%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L FemaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 52.6 50.3 54.3 1.68 4 3.2% 0.841 50 55.3 0.0%

10.1 53.1 50.8 56 2.24 4 4.22% 1.12 49.5 56.6 -0.81%

34.8 51 49.8 52.3 1.08 4 2.11% 0.538 49.3 52.7 3.18%

94.4 51.5 50 53 1.29 4 2.51% 0.645 49.4 53.6 2.18%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L FemaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 2.59E+7 2.57E+7 2.63E+7 2.63E+5 4 1.01% 1.31E+5 2.55E+7 2.63E+7 0.0%

10.1 2.58E+7 2.54E+7 2.66E+7 5.74E+5 4 2.23% 2.87E+5 2.48E+7 2.67E+7 0.68%

34.8 2.60E+7 2.57E+7 2.63E+7 2.94E+5 4 1.13% 1.47E+5 2.55E+7 2.65E+7 -0.29%

94.4 2.61E+7 2.58E+7 2.64E+7 2.50E+5 4 0.96% 1.25E+5 2.57E+7 2.65E+7 -0.58%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L Fertility Summary

95% LCL 95% UCL %Effect

0 Negative Control 99 98.8 99.1 0.128 4 0.13% 0.0641 98.7 99.2 0.0%

10.1 99.1 99.1 99.2 0.0585 4 0.06% 0.0292 99.1 99.2 -0.2%

34.8 98.9 98.6 99.1 0.238 4 0.24% 0.119 98.6 99.3 0.0%

94.4 98.9 98.7 99.1 0.17 4 0.17% 0.0851 98.6 99.1 0.08%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L MaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 2.36 2.24 2.55 0.14 4 5.93% 0.0699 2.13 2.58 0.0%

10.1 2.3 2.13 2.65 0.246 4 10.7% 0.123 1.9 2.69 2.65%

34.8 2.18 1.69 2.63 0.384 4 17.7% 0.192 1.56 2.79 7.74%

94.4 1.97 1.77 2.19 0.187 4 9.53% 0.0937 1.67 2.26 16.6%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L MaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.26 1.01 1.6 0.268 4 21.3% 0.134 0.829 1.68 0.0%

10.1 0.993 0.88 1.2 0.151 4 15.2% 0.0756 0.752 1.23 20.9%

34.8 1.2 0.78 1.62 0.399 4 33.1% 0.2 0.57 1.84 3.98%

94.4 1.35 0.86 1.85 0.569 4 42.0% 0.284 0.448 2.26 -7.77%

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 29 Apr-13 15:12 (p 3 of 5)

Test Code: 067501 48687701 | 03-1763-0053

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Fort Evironmental Laboratories, Inc.

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L MaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 62.1 58 65 2.95 4 4.76% 1.48 57.4 66.8 0.0%

10.1 64.9 62.5 67 1.84 4 2.84% 0.921 61.9 67.8 -4.43%

34.8 61.6 59.5 64.5 2.32 4 3.77% 1.16 57.9 65.3 0.81%

94.4 58 57 61 2 4 3.45% 1 54.8 61.2 6.64%

Control Type Mean Min Max Std Dev Count CV% Std Err C- μ g ai/L MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 53.9 53 54.5 0.75 4 1.39% 0.375 52.7 55.1 0.0%

10.1 58 56.5 59 1.08 4 1.86% 0.54 56.3 59.7 -7.66%

34.8 54.5 51.5 57.5 2.94 4 5.4% 1.47 49.8 59.2 -1.16%

94.4 56.1 55.5 56.5 0.479 4 0.85% 0.239 55.4 56.9 -4.18%

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 29 Apr-13 15:12 (p 4 of 5)

Test Code: 067501 48687701 | 03-1763-0053

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Fort Evironmental Laboratories, Inc.

Control Type C- μ g ai/L Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 33.5 33 34.3 33.3

10.1 39.9 35.6 34.3 33.4

34.8 39.7 37.8 38.4 37

94.4 40.2 37.2 39.4 42.4

Control Type C- μ g ai/L FemaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.3 1.23 1.1 1.33

10.1 1.29 1.33 1.66 1.22

34.8 1.25 1.26 1.26 1.26

94.4 1.22 1.26 1.14 1.2

Control Type C- μ g ai/L FemaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 11.7 11.2 6.61 10.9

10.1 10.4 13.9 14.2 8.8

34.8 7.79 11.1 14.9 8.51

94.4 9 11.2 9.96 13.4

Control Type C- μ g ai/L FemaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 53 53 50.3 54.3

10.1 50.8 52 56 53.5

34.8 50.5 49.8 51.3 52.3

94.4 52 53 50 51

Control Type C- μ g ai/L FemaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 2.59E+7 2.57E+7 2.63E+7 2.58E+7

10.1 2.66E+7 2.54E+7 2.56E+7 2.54E+7

34.8 2.63E+7 2.62E+7 2.58E+7 2.57E+7

94.4 2.58E+7 2.61E+7 2.60E+7 2.64E+7

Control Type C- μ g ai/L Fertility Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 98.9 99 99.1 98.8

10.1 99.1 99.1 99.2 99.2

34.8 99.1 99.1 98.6 99

94.4 98.7 98.8 99.1 98.9

Control Type C- μ g ai/L MaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 2.55 2.37 2.24 2.27

10.1 2.65 2.27 2.13 2.13

34.8 2.19 2.19 1.69 2.63

94.4 2.04 1.86 2.19 1.77

Control Type C- μ g ai/L MaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.6 1.08 1.33 1.01

10.1 1.01 0.88 1.2 0.88

34.8 0.78 0.96 1.62 1.46

94.4 0.86 0.86 1.84 1.85

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

### CETIS Summary Report Report Date: 29 Apr-13 15:12 (p 5 of 5)

Test Code: 067501 48687701 | 03-1763-0053

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Fort Evironmental Laboratories, Inc.

Control Type C- μ g ai/L MaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 58 65 62.5 63

10.1 67 65 62.5 65

34.8 62.5 59.5 60 64.5

94.4 61 57 57 57

Control Type C- μ g ai/L MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 54.5 54.5 53.5 53

10.1 56.5 59 58 58.5

34.8 52.5 51.5 57.5 56.5

94.4 55.5 56.5 56.5 56

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: O CSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618805)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Rebecca L. Bryan, B.S. Date: 4 / 5 /2012

Secondary Reviewer Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 4/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/ 15 /2012

Quality Assurance: Signature:

Steven Brecher

, Ph.D., D.A.B.T. Date: 4/17 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS -Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Ti er 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay shoul d not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIP E RO N YL B TOXID E/06750 1



## P rim ary Reviewer: Stephen C

. Dap son. Ph.D.



## Se nior · Pharmaco l og i s t

, RAB6

, RED (7509 P )

S econdary R ev i ewer : Orel! Akerm an. Ph

. D.



## H e alth Effects Div i s ion /11 v i vo H ers hb e r ger Assay (20 11 )/ Pa ge I o f 1 3



### OCS P P 890. 1-' 00 / 

### OE C D -'-' 1



## S i gnat ure:

# ~<2o& 

# J...,- SJ:>



## Date:

# ~ S i g n a ture: 

# ,/S

Dat e:**~** 

# l \ 9' ( 1, .; Te m p l ate ve r s i o n 1 0120 11



## DATA E VAL UAT IO N R ECO RD

STU DY TYPE: In Vivo H ers hberger Assay (Rat); OCS PP 890.1400; O EC D 44 1

P C C OD E : 067501 

## DP BARCODE: D3985 58



## TXR#: 0056259 

## CAS# : 51-03-6



## TEST MATERIAL (P U RITY): Pi peronyl butoxide (93

. 8% a .i. )

SYN O NYMS : 5-[[2-(2-butoxyetho.>ry)ethoxy]methylj-6-propyl

- l .3-ben zod i oxo l e

C I TAT IO N: Zorilla, 

## L. (20 11 ). The Hershberger Bio assay fo r Pi pe r ony l Butox id e

. Int egra t ed La b orato r y Systems

, I n c

.. Dur h a m, NC. L a b o r ato r y Proj ect Study

lD : C 192-200. Nove mb er 10

, 20 11. U npublished. EPA MR ID N um ber

48618805.



## SP O NS OR: Consumer pecialty Product s Assoc iation Inc. for the Piperonyl But oxide Task

Force IL 1667 K Street NW, Suite 300, Washingto n. D C 20006

TEST 

## ORD E R # : C O N -067501-9



## EXEC UTIVE SUMMARY: In a H e r s hb e r ge r assay ( MRJD 48618805) sc r ee nin g for androge ni c act i v it y, piperonyl butoxide (93 .8 % a. i

.. Lot # E 104-IO ) in co rn o i l was a dmini s ter ed daily via oral gavage to gro up s of 58

- t o 59-day old, castrated male Sprag u e Dawley rats (8 / group) at dose level s of 0 (veh i cle)

. 320. o r I 000 ( limit dose) m g/kg/day

. An 

### a ndro gen i c positive control group cons i st ing of eight castrated rats exposed to 0.4 m g/ kg / day testostero n e propionate (T P ) in corn oil by s ubcutaneous (s .c. ) injection was a l so included. To sc re en for potential anti

- androgenic activ ity. piperonyl butoxide in co rn o il was adm ini stered daily via oral

gavage to 58

- to 59-day old, castrated male Sprague Dawley ra ts (8 / g r o up ) a t dose l eve l s of I 00.

320. or 1000 mg / kg / day in conju nction wi th a daily dose of r efere n ce a ndro ge n TP at 0.4

mg / kg / day by s

. c

. in jection. T h e n eg ativ e co ntr o l g r ou p cons i sted of e i g ht cas trated m a l e rat s
dosed dai l y with TP (0.4 mg / kg / day) b y s

. c. inj ect i o n , a nd th e a nti- androge ni c posit i ve control

g roup consisted or eight cas t rated m a le rats dosed o r a ll y w ith flutan1id e (FT) in co rn o il at 3

m g/ kg/day in conjugation with a 

## daily s

. c. dose of TP (0.4 mg/kg/da y).In vivo Hershberger Assay (2011 ) / Page 2 of 13

PIPERONYL BUTOXIDE/067501 O CS PP 8 9 0. 1400/ OECD 441



## 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 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 1 10%). 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 a t 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 wa s 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 weight s. Piperonyl butoxide was negative for androgenicity and anti-androgenicity in the Hershberger assay

.

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 890.1400).In vivo Hershberger Assay (2011 ) / Page 3 of 13

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## COMP LIANCE : Signed and dated GLP Compliance and Quality Assurance statements were provided.

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratory Systems, Inc.

Location: Durham, NC Study Director: L. Zorrilla

Other Personnel: S. Borghoff (Study Toxicologist ) Study Period: March 2 3 , 2011 to November 10 , 2011



## 2. Test Substance : Piperonyl butoxide

Description: Pale, oily liquid

Source: Endura Fine Chemicals

Lot/Batch #: E104

- 10 , expires July 2012 Purity: 93.8 % a.i. Stability: Stable in corn oil for up to 2 0 days at 1

- 10 ° C

CAS #: 51

- 03

- 6

Structure:

O O

O O CH 3

O CH 3



## 3

. Reference Androgen : Testosterone propionate (TP)

Supplier : Sigma

- Aldrich Company (St. Louis, MO)

Batch #: 048K1328 Purity: 100.2%

CAS # : 57

- 85

- 2



## 4

. Reference Anti

- androgen : Flutamide ( FT )

Supplier : Sigma

- Aldrich Company (St. Louis, MO)

Lot #: 107K1293 Purity: >99%

CAS # : 13311

- 84

- 7



## 5

. Solvent/Vehicle Control : Corn Oil

Supplier : MP Biomedicals, LLC (Solon, OH)

Lot/Batch #: 7862K Rationale (if other than water) : Not provided , report stated that the same vehicle was used as in the Dose Range Finding study

Final concentration : Not applicableIn vivo Hershberger Assay (2011 ) / Page 4 of 13

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## 6. Test Animals : Species: Rat (castrated males)

Strain: Sprague Da w ley (Crl:CD ® [SD] IGS ) Age/weight at dose initiation: Post

- natal day (PND) 58/59 ; 254.9

- 336.1 g Source : Charles River Laboratories International, Inc., Raleigh, NC Housing: Rats were housed up to 2 /cage in polycarbonate cages with absorbent heat

treated hardwood bedding

. Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI),

ad libitum ; total genistein (genistein plus daidzein) equivalents = 6.2 μg/g diet.

Water: Reverse osmosis

- treated t ap water , ad libitum

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22

- 24 oC

33

- 57 % Not reported 12 hrs light/12 hrs dark Acclimation period: 8 days



## B. STUDY DESIGN

1. In

- life dates : Start: 4/2/11 End: 4/13/11

2. Study Design : This Hershberger Assay was conducted to screen for both potential androgenic and anti-androgenic activity. In the test for androgenic activity, piperonyl butoxide was administered daily via oral gavage to groups of castrated male rats. Positive androgenic act ivity is defined as a significant increase in two or more target organ weights compared to the vehicle control group. The positive control group for androgenic activity consisted of castrated male rats dosed by s.c. injection with TP at 0.4 mg/kg/day. In the test

for anti -androgenic activity, piperonyl butoxide was administered daily via oral gavage to groups of castrated male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection.

Anti

- androgenic activity is indicated by a statistically signific ant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP-only control group. The positive control group for anti- androgenic activity consisted of castrated male rats dosed by s.c. injection with TP (0 .4 mg/kg/day) in conjunction with an oral dose of FT (3.0 mg/kg/day). For both assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration for measurement of target organ weights.

3. Study Schedule : Following the initial 8

- day acclimation period, castrated male rats (surgical manipulation performed by Supplier at PND 42) were administered the test substance from PND 58-59 to PND 67-68. Rats were euthanized on PND 68-69, approximately 24 hours after the last dose and necropsied for organ weight measurements.

4. Animal Assignment : Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significa nt differences in group means at study initiation.In vivo Hershberger Assay (2011 ) / Page 5 of 13

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### Table 1. Study design a


### Test group Dose (mg/kg/day) # of Males
Androgen Agonist Assay
Vehicle control (corn oil) 0 8
Low ( Piperonyl butoxide ) 320 8 High ( Piperonyl butoxide ) 1000 8
Positive control (corn oil) +TP b 

### 0.4 8

Anti

- Androgen Assay
Vehicle control (corn oil) +TP b 

### , negative control 0 + 0.4 8
Low ( Piperonyl butoxide +TP) 100 + 0.4 8
Mid ( Piperonyl butoxide +TP) 320 + 0.4 8 High ( Piperonyl butoxide +TP) 100 0 + 0.4 8 Flutamide (oral gavage) + TP (s.c.), positive control 3 + 0.4 8

a Data were obtained from Tables 1 and 2 on page 16 of the study report.

b Same animals dosed for both androgen agonist (serves as positive control) and anti

- androgen (served as negative control) assays. This did not affect the outcome or conclusions of this study.



## 5. Dose Selection Rationale : The piperonyl butoxide dose levels were selected based on the results of a range -finding study (ILS Inc., Study No. C192- 500). Peripubertal male rats were administered piperonyl butoxide at doses of 0, 200, 400, 600, 800, or 1000 mg/kg (3/group) for 14 days. No effects on body weights or clinical signs were observed at any dose group. Based on these results, doses of 1000 mg/kg/day (limit dose) and 320 mg/kg/day (approximately half-log of high dose) were used for both the androgen agonist and antiandrog en assays

.

6. (a) Dose Preparation : Dose formulations were prepared at ILS by adding the appropriate amount of piperonyl butoxide and/or positive control into corn oil. Dose formulations were prepared at least two (20 mg/mL) or three (64 and 200 mg/mL) times during the study.

( b) Dose Analysis : Samples of dose formulations were collected from the top, middle, and bottom of the formulation and sent for analysis at Smithers Viscient, LLC (Wareham, MA). Smithers Viscient analyzed, in duplicate, samples r eceived for concentration and homogeneity. The report stated that dose formulations of piperonyl butoxide in corn oil held from 1 to 10°C for up to 20 days were considered stable (Smith, 2011 1 

## ); however, no data were submitted to allow independent verification.

Results

Homogeneity (%CV) : 0.57-6.74%

Stability (% of Day 0): Data not provided

Concentration (% of nominal): 88.5-110.3%

1 

### Smith, R. (2011). Storage Stability of Piperonyl Butoxide in Corn Oil. Unpublished study report prepared by

Smithers Viscient Inc. Study No.13513

- 6132.In vivo Hershberger Assay (2011 ) / Page 6 of 13

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## The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable. The study referenced above should be submitted for verification of the stability findings.

7. Dos age administration : Test formulations of piperonyl butoxide and FT were administered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw. Dose volumes were adjusted daily based on the concurrent individual body weight measurement.

The dose formulations were administered on a staggered start for 10 consecutive days (PND 58/59 through PND 67/68). The first four animals from each group were dosed beginning on PND 58 and the second four from each group on PND 59. Dosing occurred 24 hours (± 2 hours) from the previous dose. Dose volume was determined on individual animal daily body weight. The dosing sequence was stratified across dose groups; one animal from each group and then repeated until all animals were dosed.

A control study (ILS study number C193-999) was conducted to compare TP alone to TP and corn oil co-administered by gavage. This study is summarized in the Appendix at the

end of this DER.

8. Statistics : Final body weights, body wei ght gains, and accessory sex organ weights were subjected to the following statistical analyses: Studentized residual plots for outliers, Levene’s test for homogeneity of variance, and one- way ANOVA followed by Dunnett’s t

tests. Since one tissue was sig nificantly increased in the antagonist assay, multivariate analysis was performed on the data sets, as suggested by the guidelines (U.S. EPA, 2009). The positive controls were compared to the negative controls using the t- test. Significance

was denoted a t p < 0.05. Statistical analyses were performed using MS EXCEL (descriptive statistics only) and SAS Version 9.1. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations : Animals were checked for mortality or moribundity twice daily on weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity at randomization, daily prior to dosing throughout the dosing period, and at termination.

2. Body Weight : Animals were weighed at randomization, daily prior to dosing throughout the dosing period, and at termination.

3. Food Consumption (Optional) : Food consumption was not determined.

4. Serum Hormone Measurements (Optional) : Serum hormone measurements were not

conducted.

5. Dissection and Measurement of Tissue and Organ Weights : On PND 68 -69 (approximately 24 hours after the final administration of the test substance) , all rats wereIn vivo Hershberger Assay (2011 ) / Page 7 of 13

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## weighed, euthanized by CO 2 

## asphyxiation followed by cervical dislocation, and subjected to

a gross necropsy. The five mandatory androgen -dependent organs (ventral prostate, seminal vesicles with coagulating glands and fluid, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Preputial separation (PPS) was also evaluated at necropsy (results not reported).

II. RESULTS

A. OBSERVATIONS

1. Mortality : One 1000 mg/kg/day animal was found moribund and euthanized on Day 3 in the androgen agonist assay, and one positive control animal was found dead on Day 5 in the androgen antagonist assay. No evidence of dosing error was observed in either animal. All

other animals survived until scheduled termination.

2. Clinical signs o f toxicity : In the androgen agonist assay, no clinical signs of toxicity were observed in the 320 mg/kg/day dose group. At 1000 mg/kg/day, clinical signs included one rat with piloerection and uncoordinated movements on Day 5 and one rat with fur discoloration, dried blood (around eyes, nose, and paws), hunched, piloerection, uncoordinated movements, and/or oily around anus area on Days 4-6.

In the androgen antagonist assay, no clinical signs of toxicity were observed in the 100 and 320 mg/kg/day dose groups. One 1000 mg/kg/day (+TP) rat exhibited fur discoloration, dried blood, and/or lethargy on Days 4-6.

B. BODY WEIGHT AND WEIGHT GAIN : Selected body weight and body weight gains for the androgen agonist assay are presented in Table 2. 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 [NS]) by only 7%, but overall (Days 1-11) body weight gains were decreased (p<0.05) by 96%. Animals dosed with TP had increased (p<0.05) body weights on Day 11( ↑ 9%) and increased (p<0.05) overall body weight gains ( ↑ 110%).In vivo Hershberger Assay (2011 ) / Page 8 of 13

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### TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative B ody Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control

Vehicle + TP (0.4) b Piperonyl butoxide ( 320 ) Piperonyl butoxide ( 100 0)

N Mean SD N Mean SD N Mean SD N Mean SD

1 8 301.7 17.6 8 299.9 16.7 8 297.8 21.0 8 302.9 19.4

4 8 307.5 20.0 8 317.1 20.6 8 306.5 23.2 7 c 289.9 19.4

7 8 318.0 24.0 8 335.0 23.4 8 329.1 24.7 7 297.3 20.6

11 8 328.8 24.8 8 357.0 * (↑ 9 ) 27.5 8 327.8 28.8 7 305.9 (↓7) 24.6

BWG (Days 1

- 11) 8 27.2 8.7 8 5 7.1 * (↑ 110 ) 11.4 8 30.0 10.5 7 1.1 * (↓ 96 ) 9.9

a D ata were obt ained from Table 6 on page 2 3 and Appendix V on page 1 14 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the negative control for the anti

- androgen. This did not affect the outcome or conclusions of this study.

c One 100 0 mg/kg/day animal was found moribund and euthanized on day three.

N Number of animals in the group ; SD Standard Deviation

* Significantly different from controls at p ≤ 0.05



## Body weight and body weight gain data for the anti- androgen assay are presented in Table 3. Body weights and overall body weight gains for the 100 mg/kg/day treatment group were comparable to controls throughout the duration of the assay. Terminal body weight was decreased (NS) by 4% at 320 mg/kg/day and 9% at 1000 mg/kg/day. T he 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 1 00 0 mg/kg/day. Animals dosed with TP +FT had decreased (p<0.05) overall body weight gains ( ↓40 %).

TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti

Andr ogen Assay a

Study Day # Dose (mg/kg/day)
Negative Control

Vehicle + TP (0.4) b Positive Control

TP + FT (0.4 + 3.0) Piperonyl

butoxide +TP

( 100 + 0.4) Piperonyl

butoxide +TP

( 320 + 0.4) Piperonyl

butoxide +TP

(100 0 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 8 299.9 16.7 8 303.5 13.7 8 301.9 16.8 8 300.7 16.9 8 303.0 19.3

4 8 317.1 20.6 8 311.1 15.8 8 312.8 19.6 8 308.5 17.7 8 290.7 19.1

7 8 335. 0 23.4 7 c 326.0 19.7 8 334.9 18.3 8 326.8 22.3 8 309.1 21.9

11 8 357 .0 27.5 7 344.1 19.6 8 358.2 19.2 8 343.6 (↓4) 25.1 8 328.1 (↓9) 20.7

BWG (days 1

- 11) 8 67.1 11.4 7 40.3 * (↓ 40 ) 6.2 8 56.3 10.1 8 42.9 * (↓ 36 ) 10.1 8 25.1 * (↓ 62 ) 10.9

a Data were obtained from Table 7 on pages 24 and Appendix V on pages 1 15 of the study report. Percent differences from

vehicle controls (calculated by the reviewers) are included in parentheses.

b The negative control for the anti

- androgen is same as the positive control group for the androgen agonist. This did not affect the outcome or conclusions of this study.

c One positive control animal was found dead on day 5.

N Number of animals in the group

SD Standar d Deviation

* Significantly different from vehicle controls at p<0.05.



## C. FOOD CONSUMPTION (Optional) : Food consumption was not determined.In vivo Hershberger Assay (2011 ) / Page 9 of 13

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## D. SERUM HORMONE CONCENTRATIONS (Optional) : Serum hormone concentrations

were not determined.

E. ORGAN WEIGHTS : Accessory sex organ weights for the androgen agonist assay are presented in Table 4. There were no treatment

- related effects in any of the accessory sex organs of animals dosed with piperonyl butoxide. 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 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 guidelinespecified %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 recommen ded values.

Table 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague

- Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control Piperonyl butoxide (320 ) Piperonyl butoxide (100 0) Positive Control

Vehicle + TP (0.4) b

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal

vesicles 8 39.9 10.2 26 8 43.2 7.9 18 7 42.4 8.1 19 8 757.9 * (↑ 1799 ) 216.3 9

Ventral

prostate 8 15.4 8.9 58 c 8 13.9 4.2 30 7 13.5 4.0 30 8 169.0 * (↑ 997 ) 6.3 37

LABC 8 168.3 29.7 18 8 177.3 24.9 14 7 149.4 16.5 11 8 574.9 * (↑ 242 ) 64.7 11

Cowper’s glands 8 6.6 3.6 54 8 6.3 2.1 34 7 6.2 3.6 58 c 8 44.3 * (↑ 571 ) 11.8 27

Glans penis 8 57.6 14.9 26 c 8 61.0 9.4 15 7 52.6 8.4 16 8 87.9 * (↑ 53 ) 18.1 21

a Data were obtained from Table 8 on page 26 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the negative control for the anti

- androgen. This did not affect the outcome or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group ; SD Standard Deviation ; CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.



## Accessory sex organ weights for the anti -androge n assay are presented in Table 5

. There

were no treatment

- related effects in any of the accessory sex organs of animals dosed with 100 and 320 mg/kg/day piperonyl butoxide with TP. At 1 000 mg/kg/day

, the LABC weight

was decrease d (p≤0 .05) by 21 %. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 90 % in seminal vesicles;

85 % in ventral prostate; 5 6 % in LABC; and 66% in Cowper’s gland . T he maximum guideline- specified %CV was exceeded for the glans penis in the negative control (20% vs. 17%) and for the ventral prostate in the TP + FT positive control (46% vs. 40%). All other

%CV values were less than the maximum recommended values.In vivo Hershberger Assay (2011 ) / Page 10 of 13

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### Ta ble 5. Accessory Sex Organ Weights (mg) from Anti

- Androgen Agonist Assay in Sprague

- Dawley Rats a

Organ Dose (mg/kg/day)

Negative Control

Vehicle +TP (0.4) b Piperonyl butoxide

+TP (100 + 0.4) Piperonyl butoxide

+TP (320 + 0.4) Piperonyl butoxide

+TP (100 0 + 0.4) Positive Control

TP + FT

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal

vesicles 8 757.9 216.3 29 8 640.6 104.0 16 8 696.2 144.6 21 8 621.1 87.2 14 7 74.0 * (↓ 90 ) 24.1 33

Ventral

prostate 8 169.0 6.3 37 8 190.6 34.2 18 8 178.2 41.8 23 8 163.4 38.0 23 7 25.5 * (↓ 85 ) 11.8 46 c

LABC 8 574.9 64.7 11 8 544.4 56.1 10 8 534.4 55.9 10 8 454.2 * (↓ 21 ) 70.6 16 7 253.4 * (↓ 56 ) 26.7 11

Cowper’s glands 8 44.3 11.8 27 8 46.1 10.7 23 8 41.3 10.3 25 8 41.7 11.5 28 7 14.9 * (↓ 66 ) 4.8 32

Glans penis 8 87.9 18.1 20 c 8 91.0 14.2 16 8 98.9 16.9 17 8 92.6 14.6 16 7 78.0 10.3 13

a Data were obtained from Tables 9 on page 27 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The negative control for the anti

- androgen is same as the positive control group for the androgen agonist. This did not affect the

out come or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group ; SD Standard Deviation ; CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS ’ CONCLUSIONS (from the study report) : Sixty

- four castrated male SD rats were allocated to one of eight designated dose groups. To evaluate the test substance for agonist properties, animals were administered one of two dose levels (320 and 1000 mg/kg) of PBO, the vehicle control, or an agonist reference substance, TP (0.4 mg/kg). To evaluate PBO for antagonist properties, animals were co-administered one of three dose levels (100, 320, and 1000 mg/kg) of PBO, or FT (antagonist positive control, 3 mg/kg) with TP (0.4 mg/kg). Animals were orally administered for 10 consecutive days with PBO, FT or

co -administered subcutaneously with TP. Approximately twenty-four hours following the final dose administration, the animals were humanely euthanized; the glans penis, ventral prostate, LABC, Cowper’s glands, and seminal vesicle with coagulating gland with fluid were excised and weighed.

The positive control in the agonist assay, TP, induced a statistically significant increase in glan s penis, Cowper’s glands, seminal vesicle, ventral prostate and LABC weightscompared to the vehicle control group. In the antagonist assay, the positive control FT (coadministered with TP) induced a statistically significant decrease in Cowper’s glands, seminal vesicle, ventral prostate, and LABC weights compared to the control group (TP only).

In the agonist assay, mean body weight gain was significantly decreased in animals administered 1000 mg/kg PBO. Dose levels of 320 and 1000 mg/kg PBO did not inc rease androgen dependent tissue weights compared to the weights of these tissues in the vehicle

control animals.In vivo Hershberger Assay (2011 ) / Page 11 of 13

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## In the antagonist assay, final body weights of animals administered 100, 320, or 1000 mg/kg of PBO were not statistically different as compare d to TP control. Mean body weight gain was significantly decreased in animals co -administered 320 and 1000 mg/kg PBO and TP compared to TP alone.

When PBO at 1000 mg/kg was co-administered with TP, only the LABC showed a statistically significant reductio n in tissue weight, with a suggestion of a weight reduction in the seminal vesicle, although this apparent reduction was not statistically significant. No other tissues showed any degree of reduced growth.

Although the multivariate analysis of the five androgen dependent tissues showed statistical significance (p=0.0420), because of the observation of statistically significant dose- related changes in only one of five of the androgen dependent tissues, and the lack of reduced growth in the other tissues, PBO is not considered to be an androgen antagonist.

In conclusion, based on the results of this study using the castrated rat model Hershberger bioassay (OPPTS 890.1400), the oral administration of PBO, up to and including the limit dose level of 1000 mg/kg, indicates that PBO is neither an androgen agonist nor antagonist.

A. AGENCY ’S COMMENTS : In the androgen agonist assay, no adverse, treatment

- related findings were observed on mortality, clinical signs, or accessory sex organ weight. At 1000 mg/kg/day, clinical signs included one rat with piloerection and uncoordinated movements on Day 5 and one rat with fur discoloration, dried blood (around eyes, nose, and paws), hunched, piloerection, uncoordinated movements, and/or oily around anus area o n Days 4 -6. As similar findings were observed in 2/16 animals (one in the androgen agonist assay and

one in the anti- androgen assay) , these findings may have been treatment- related, but were transient. Similar signs were not reported in the dose range finding study. 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, overall (Days 1-11) body weight gains were decreased (p<0.05) by 96%.

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

on Day 11( ↑ 9%) and increased (p<0.05) overall body weight gains ( ↑ 110%). Animals in the TP group had increases (p≤0.05) in accessory sex organ weights as follows: se minal

vesicles

, ventral prostate

, LABC

, Cowper’s gland , and glans penis. For the vehicle control, the maximum guideline

- specified %CV values were exceeded for the ventral prostate and glans penis. The maximum guideline

- specified %CV was slightly exceeded for the Cowper’s gland in the 1000 mg/kg/day piperonyl butoxide group. 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. T he 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. At 1000 mg/kg/day, the LABC weight was decreased (p≤0.05) by 21%. This finding was slight in magnitude and observed in only one accessoryIn vivo Hershberger Assay (2011 ) / Page 12 of 13

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## sex organ; therefore, the finding was considered incident al. Animals dosed with TP +FT had decreased (p<0.05) overall body weight gains ( decreased 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: seminal ve sicles

, ventral

prostate, LABC , and 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 weight s. Piperonyl butoxide was negative for androgenicity and anti-androgenicity in the Hershberger assay

.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• In the androgen agonist assay, t he maximum guideline

- specified %CV values were exceeded for the ventral prostate (58% vs. 45%) and glans penis (26% vs. 22%) in the vehicle control, and the Cowper’s gland (58% vs. 55%) in the 1000 mg/kg/day piperonyl butoxide group. However, the higher CVs did not alter the conclusions of the androgen agonist assay.

• In the anti-androgen assay, the maximum guideline

- specified %CV was exceeded for the glans penis (20% vs. 17%) in the negative control and the ventral prostate in the positive control (46% vs. 40%). All other %CV values were less than the maximum

recommended values. Th is higher CV did not alter the conclusions of the anti -androgen

assay.In vivo Hershberger Assay (2011 ) / Page 13 of 13

PIPERONYL BUTOXIDE/067501 O CS PP 8 9 0. 1400/ OECD 441



## APPENDIX

Comparison of Negative Controls Study

In a control group study (ILS Project Study Number C193-999), two groups of eight castrated male Sprague

- Dawley (Crl:CD ® 

## [SD]IGS) rats were administered 0.4 mg/kg testosterone propionate (TP) in corn oil by subcutaneous injection (dose volume 0.5 mL/kg) daily for 10 consecutive days. One group was also concurrently dosed with corn oil by oral gavage (dose volume 5 mL/kg) just prior to receiving the TP injection. The stated purpose of this study was to evaluate if there was a difference in androgen

- dependent tissue weights in animals administered

TP alone or co -administered with corn oil (vehicle control). The rats were routinely observed for mortality, morbidity, and clinical signs of toxicity, and body weights were recorded daily. Twenty -four hours after the final dosing, the rats were euthanized, and the androgen- dependent tissues (ventral prostate, seminal vesicles, LAB C, Cowper’s glands, and glans penis) were excised and weighed.

All rats survived to scheduled euthanasia; no clinical observations were noted. There were no differences in initial or final body weights, or in overall (Days 1- 11) body weight gains.

In th e androgen

- dependent tissues, ventral prostate was decreased (p≤0.05) by 22% in the TP + corn oil group compared to the TP only group. All other tissue weights were similar between groups. All tissue weights were within the acceptable criteria as stated in the Guidelines (OCSPP 890.1400). It was stated that co-administration of corn oil appeared to increase the variability of the tissue weights, although all tissue CVs were less than the maximums specified

in the Guidelines. The observed difference in t he ventral prostate was ascribed to the differing variability between the two groups: 9.0% CV for the TP only group vs. 19.6% CV for the TP + corn oil group. It was considered that if the variability between the groups had been similar, statistical signi ficance would not have been attained. In the absence of effects on the other four tissues, the slight weight change in the ventral prostate was not considered to the biologicallysignificant. Therefore, co-administration of corn oil with TP had no effect on the androgendependent tissue weights.

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: O CSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618806)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Rebecca L. Bryan, B.S. Date: 4/ 11 /2012

Secondary Reviewer Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 4/18 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/ 23 /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/ 23 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIP E RO N YL B TOXIDE / 0 6750 1



## Primary Re v iew er: Yung 

## G. Ya n g

. 

## P h. D.

Risk As s ess m e nt Branch VI, H ea lth E ff ec t s Divi s i o n S eco ndary Reviewer : E li za b e th M e nd ez. Ph.D

. 

## Health E ffect s Division femal e Pub e rtal A ss a y ( 20 11 ) I Page I o f 1 6

O C PP 890.1 .t SO / OECD No n e



## Sign a tu 

## r e :**--H- ----..** **~-=---.R"--.9-** 

## D a t e:

## -:::::::="'**r~S::""'D'7"(** S i g na tu 

## r e

. .... ·**-~ll"'l'OQllll~r;tri'o W!f"**  

## D ate :**---- IH-'+-'-** **~-**



## DATA EVAL U AT IO N RECO RD

STUDY TYPE: Female Pubertal Assay; OCSP P 890

. 1450; OEC O None.



## P C CO D E : 0 67501 

## DP BAR C ODE: D 3 98 558



## TX R #: 0 0 562 59 

## CAS#: 51

- 0 3

- 6



## TEST MAT E RIAL (PURJTy): P iperonyl butox i dc (93.8% a.i.)



## S YNONYMS: 5

- l[2-(2-b u toxyet h oxy )e t hoxy] me th y l ]

- 6 -p ropyl

- 1.3

- benzo d i oxo l e



## C I TATION : Zor i lla, 

## L. (20 11 )

. Pub e rt a l deve l op m e nt and th y r o i d fu n ctio n in int ac t ju vc nil e / p e ripub e rtal fe m a l e a n d m a l e r a t s w ith pip ero n y l b ut o xid e . I nte g rat e d

L abo r a t o r y Sys tems

, In c

. , Durh a m
, NC. Laborat o r y P ro j ec t S tud y ID:

C J 92-300, Nove mb e r 10

, 20 l 

### l. MRID 48 6 188 06

. U n p ubli s h e d.



## PONSOR: Pi pero n y l B moxide Task Fo r ce 

### 11 c / o Cons wn e r Specia lt y Prod u cts Assoc i a ti o n

. 1667 

## K S t reet NW, S ui te 300

. Was hi ng t o n. D C



## T EST ORDER#: CON

- 067501

- 9



## EXECUT IV E SUMMARY: In a fe rnaJ e pub er t a l assay ( MRlD 48 6 1880 6), 

### 1 6 female Spr ag u e

Daw l ey rat s/ d ose g r o up we r e tr e ated dail y v i a o ral gavage with pip e ron y l but ox i d e ( 9 3 .8 % a.i.

, Batc h / l o t # E l0 4 -I 0) in co rn o il a t do ses of 0 (ve hi c l e). 5 0 0

, o r 100 0 (l i m i t d ose) m g/ k g / da y fr o m

pos t-n a t a l da y ( P ND) 22 t o 42 / 43

. Anim a l s we r e exa min ed fo r vag in a l o p e nin g 

## (VO) dail y beg i n ni ng on P ND 22. and weig h t at day of a t ta in me n t was r eco rd ed. Fo ll ow i ng sac rifi ce o n

PND 42 or 43. tota l serum leve l s of t h yrox in e 

### (T.1) and th yroid st imul ati n g h o rm o n e (TS H ) levels

were determined using a radioimmu n oassay: serum was a l so analyzed fo r l eve l s of se l ec t ed

clinical c h e mi st r y parame t ers. A dr ena l. kidneys. l i ve r
, pi tuit ary

. t h yro i d. and ur oge nit a l o r gans

were we i g h ed

. a n d m icroscop i c exa min at i ons were pe r fo rm e d o n th e ova ri es. ut e ru s, t h yr oid

, a n d

k i d n eys

.

No t r eat m e n t -r e l a t e d cffocts we r e o b served o n m o rt a lit y o r cl inic a l s i g n s of t ox i c it y

. Ther e we r e
also no trea t men t-r e l ated effects o n th e we i g ht s of p ituit a r y . u te ru s. or thyro id. Wi t h r ega rd s t o reproductive developmen t

, no t r ea tm ent

- related effec t s we r e observed o n t h e age a t Y O

, we i g ht

at YO

. and number of animals ana inin g VO. mean age at first vagina l estrus, m ea n cy cl e l e n gth

. percem cycling, and percent r egu l arly cycling. number of ovary fo ll icles

. fo lli cular cys t s o r

corpora l u tea

.

A t 500 an d 1 000 m g / k g / day, th e fin al a dju s t e d b ody we i g ht was d ec r eased ( p < 0 .0 5) b y 5 a nd

7%, r es p e cti v el y

. a nd th e a dju s t e d bod y w e i g ht ga in was d e cr e a s ed ( p < 0

. 05) by 9 and I 0 %,Female Pubertal Assay (2011) / Page 2 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## re spectively. 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 s ignificant 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 T 4 

## 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 G rades 1 –3 at 500 mg/kg/day and G rades 3 –5 at 1000 mg/kg/day. Follicular colloid area was G rades 4 –5 in the controls compared to G rades 3

– 4 at 500 mg/kg/day and G rades 1 –3 at 1000 mg/kg /day.

The doses tested were adequate since the high dose was the Limit Dose ( 1000 mg/kg/day ).

The assay satisfies the EDSP Tier 1 Test Order requirement s for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE : Signed and dated D ata C onfidentiality, GLP Compliance, and Quality

Assurance statements were provided.Female Pubertal Assay (2011) / Page 3 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratory Systems, Inc.

Location: Durham, NC Study Director: L. Zorilla

Other Personnel: S. Borghoff (Study Toxicologist ) Study Period: March 1 6

– November 10 , 2011



## 2

. Test S ubstance : Piperonyl butoxide

Description: Pale, oily liquid

Source: Endura Fine Chemicals

Lot/Batch #: E104

- 10 (expiration date July 2012) Purity : 93.8 % Stability: Stable in corn oil for up to 2 0 days at 1

- 10 ° C

CAS #: 51

- 03

- 6

Structure:

O O

O O CH 3

O CH 3



## 3

. Vehicle : Corn oil

4

. Test A nimals : Species: Rat (females only)

Strain: Sprague Dawley [Crl:CD®(SD) IGS] Age/Weight at Study

Initiation: PND 22 / 47.4

- 70.6 g
Source: Charles River Laboratories International, Inc. ( Raleigh , NC) Housing: Dams were housed individually in polycarbonate cages with micro

- isolator top and

absorbent heat

- treated hardwood bedding; after weaning, pups were housed two per

cage
Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI)

, ad libitum Phytoestrogen content of diet = 6.2 μg /g genistein equivalents

Water: Reverse

- osmosis treated tap water, ad libitum

Environmental

Conditions: Temperature: Humidity: Air changes: Photoperiod: 21

- 23 °C

32

- 63 % Not reported

14 hrs light/ 10 hrs dark



## B. ST UDY DESIGN

1. In -L ife D ates : Start: April 22, 2011 End: August 9, 2011

2. Mating : Time

- mated pregnant dams were received from the supplier on gestation D ay 8.

Litters were standardized to 8 pups, with equal numbers of males and females, between

PND 3 and 5.Female Pubertal Assay (2011) / Page 4 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## 3. Animal A ssignment : Animals were assigned to the test groups noted in Table 1 using a procedure that stratifie d animals across groups by body weight such that the mean body weight of each group was not statistically different from any other group. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a


### Test group Dose (mg/kg/day) # of Fem ales

Control 0 16

Low 500 16 High 1 000 16

a Data were obtained from Table 1 on page 14 of the study report.



## 4. Dose S election Rationale : The dose levels were selected based on the results from a dose

range -finding study conducted by the performing laboratory prior to initiation of dosing. 1 

## In the study, female rats were administered (route not specified, but oral gavage assumed) the test substance at 0, 200, 400, 600, 800, and 1000 mg/kg/d a y for 1 4 days. No significant effects on body weights or overall body weight gains were observed. At 1000 mg/kg/day, absolute liver weights were significantly increased. At 600 mg/kg/day and above , relative liver weights were increased . At 600 mg/kg/day only, absolute kidney weights were significantly decreased. A high dose of 1000 mg/kg/day f or fe males was selected since 1000 mg/kg/day is the limit dose

.

5. Dose P reparation and Analysis : Dose formulations were prepared t wo times during the study by mixing appropriate amounts of test substance with corn oil. Samples of dose

formulations from all preparations were analyzed for h omogeneity by collection of samples from the top, middle, and bottom of the formulation ; the mean results of the homogeneity analyses were used to demonstrate achieved concentrations. Stability was tested in a separate study; 2 

## the test substance was found to be stable in corn oil for up to 20 days at

1-10 °C ; however, no data were submitted to allow independent verification.

Results of D ose Analysis

Homogeneity (% CV) : 2.26-6.65%

Stability: Provided in Study No. 13513-6132

Concentration (% of nominal): 95.5-109.0%

The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable.

1 . Zorrilla, L. (2011). Range Finder Study for Piperonyl Butoxide (PBO) Tier 1 Endocrine Disrupting Screening Program in vivo Mammalian Assays. Unpublished Study Report Prepared by ILS, Inc. Study No. C182

- 500.

2 . Smith, R. (2011). Storage Stability of Piperonyl Butoxide in Corn Oil. Unpublished study report prepared by Smithers Viscient Inc. Study No. 13513

- 6132.Female Pubertal Assay (2011) / Page 5 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## 6. Dosage A dministration : All doses were administered once daily by gavage in a volume of 5 mL/kg of body weight. The dose formulations were administered for 2 1 or 22 consecutive days on PND 22–42/43. Dosing occurred 24-hours (±2 hours) from the previous dose. Dose volume was determined daily on individual animal body weight. The dosing sequence was stratified across all dose groups; one animal from each group and th en repeated until all

animals were dosed.

7. Statistics : Statistics were analyzed using Microsoft Excel and SAS (version 9.2). Studentized residual plots were used to detect possible outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, multiplicative inverse, and square root) and if still heterogeneous

, analyzed using the non- parametric Kruskal -Wallis and Dunn’s test. If data sets were not homogenous, ANCOVA analyses were not performed. Trend tests were performed on body weight and tissue weight data sets and reported when significant (p<0.05) for endp oints that did not show any significant pair- wise comparisons.

Classification Procedure

Initial body weights
Final body weight (use last day all body weights collected)
Final body weight gain (use last day all body weights collected)
Age and body weight at VO / PPS
Age at first estrus
Cycle length
Ovarian counts (follicles, cysts, corpora lutea) Homogeneous data sets:

O ne

- way analysis of variance (ANOVA) followed by pair

wise comparisons performed using Dunnett’s two

- tailed

t- tests

Tissue weights
Relative tissue weights (liver, kidneys, pituitary and adrenals)

Hormone levels
Clinical chemistry levels T wo

- way ANOVA with treatment and necropsy day (if >1 day) as main effects.

Pair

- wise comparisons were performed using Dunnett’s

two

- tailed t

- tests.

Initial body weights
Final body weight (use last day all body weights collected)
Final body weight gain (use last day all body weights collected)
Age and body weight at VO / PPS
Tissue weights T wo

- way analysis of covariance (ANCOVA) with PND 22/23 body weight (weight on first day of administration) as

the covariable.

Pair

- wise comparisons were performed using Dunnett’s

two

- tailed t

- tests.

C ycling status

P ercent of animals cycling regularly Chi- square analysis

T hyroid scoring (colloid area and follicul ar cell height) Fisher’s Exact test, and when statistically significant,

f ollowed by Kruskal

- Wallis and Dunn’s test.



## Significance was reported as exact p -values, except no p-value was reported for Dunn's Test (p<0.05). The statistical analyses were considered appropriate.Female Pubertal Assay (2011) / Page 6 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## C. METHODS

1. Mortality and C linical Examinations : All animals were examined for mortality and moribundity twice daily on weekdays and once daily on weekends or holidays. Clinical

examinations were conducted at allocation

, daily prior to dosing, and prior to euthanasia.

2. Body W eight : Animals were weighed at randomization , daily prior to dosing, and at

termination

.

3. Vaginal O pening : Beginning on PND 22 , all animals were examined daily for onset of

VO . Age and weight on the day of completion of VO were recorded.

4. Estr o us C yclicity : Beginning on the day of VO , up to and including the day of necropsy, daily vaginal smears were obtained and classified as diestrus, proestrus, or estrus. Metestrus was classified as diestrus. The overall cycling pattern was classified as regular, irregular, or not cycling. The age at first vaginal estrus was recorded. For each dose group, summary information was provided which included the me an age at fi rst vaginal estrus, the mean cycle length for each group, the percent of each group cycling, the percent of each group cycling regularly, and the stage of the cycle at the time of necropsy.

5. Sacrifice and Pathology : Beginning at the initiation of dosing, any rats found moribund or dead were necropsied and the cause of death determined, if possible. Moribund rats were euthanized by carbon dioxide asphyxiation followed by cervical dislocation. On the day of termination, rats were removed to a holding room at least 2 hours before termination. All surviving animals were sacrificed

, in the same order as they were dosed

, by decapitation on PND 42 or 43, approximately 2 hours post-dosing. Blood from the trunk of the animals was collected imme diately into serum separation tubes, processed by centrifugation, and the

serum was stored at ≤−70 °C for subsequent hormone and clinical chemistry evaluations

. Serum samples were shipped on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis.

a. Hormone Analysis : Total serum T 4 

## and TSH levels were determined using radioimmunoassays. Samples were assayed in duplicate and in conjunction with multiple quality control samples.

b. Clinical C hemistry : The study author noted that the performing laboratory did not have a database of historical hormone and clinical chemistry values for female Sprague Dawley rats at the age of rats in this study. R eference ranges were provided from published

literature ( Table 1 0 on page 30 of th e MRID ). The following CHECKED (X) parameters

were examined

.Female Pubertal Assay (2011) / Page 7 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALK) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH)

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in female rats based on G uideline 890.1 45 0.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were

collected

, weighed , and grossly examined. In addition, the (XX) organs were subjected to histological examination

.

UROGENITAL OTHER

XX Ovaries ( paired, without oviducts) * + XX Thyroid * +

XX Uterus* + X Liver *

XX Kidneys (paired)*+ X Adrenals (paired) *

X Pituitary *

* Weights required based on guideline 890.1 4 50

+ Histopathological examination required based on guideline 890.1 4 50



## All organs collected, except the thyroid/trachea , were weighed prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed again following removal of the fluid in the lumen (blotted weight).

The ovaries ( left ), kidney (left), uterus, and thyroid were fixed in 10% neutral buffered

formalin for at least 24 hours and stored in 70% histology grade alcohol prior to embedding ; f

ollowing fixation in 10% neutral buffered formalin, the thyroid w as dissected from the

trachea before transfer to alcohol storage

. All collected tissues were routinely processed, sectioned, stained with hematoxylin and eosin, and examined microscopically.

T wo serial sections from each of the two lobes of the thyroid were evaluated for follicular cell height and colloid area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest) . Five random sections of the left ovary were evaluated for any abnormalities/lesions (such as ovarian atrophy) and follicular development, including presence/absence of tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum development, and changes in number of both primary and atretic follicles.Female Pubertal Assay (2011) / Page 8 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## II. RESULTS

A. MORTALITY : One 500 mg/kg/day animal died immediately following dosing on Day 17 (PND 38) ; this death was attributed to gavage error . In the 1000 mg/kg/day group, one animal was moribund (decreased movement, lethargy, and thin) and euthanized on Day 3 (PND 24). Gross examination of this animal did not reveal any evidence of dosing error.
However, there were no clinical signs of toxicity, decreases in body weights, or other toxicity data that implicated the test compound as the cause of death. In addition, m ortality was not observed in the range-finding study at this dose. Therefore, the single death (1/16)

was considered incidental. All other rats survived until scheduled sacrifice.

B. CLINICAL SIGNS OF TOXICITY : No treatment

- related clinical signs of toxicity were observed in animals from any dose group. Three rats in the 1 000 mg/kg/ day dose group exhibited an ungroomed appearance on at least one day, and one of these rats was also noted as thin on one day. These findings were of low incidence and transient. All other rats were observed as normal throughout the study.

C. GENERAL GROWTH AND VAGINAL OPENING : Body weights, body weight gains, age of attainment of VO , and weight at day of attainment are presented in Table 2. Unadjusted mean final body weights and body weight gains in the 500 and 1000 mg/kg/day

group s were similar to control. However, when adjusted for initial body weight on PND 21,

the f inal body weight s w ere decreased ( p<0.05) by 5% and 7% in the 500 and 1000 mg/kg/day group s, respectively. In addition, the adjusted body weight gains were decreased (p<0.05) by 9% and 10% in the 500 and 1000 mg/kg/day group s, respectively . A ge at VO, weight at VO , and number of animals that attained VO were comparable in the treated and control groups.

With regards to the performance criteria, the mean age at VO, body weight at V O, and final body weight in the control group were within the acceptable range provided in the Guideline (890.1450), however, the following CV s w ere outside the acceptable range: 7.4 % CV for age at VO (maximum 6.52 ) and 13.0 % CV for final body weight (maximum 8.93%).Female Pubertal Assay (2011) / Page 9 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



### TABLE 2

. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control 500 mg/ kg/day 1000 mg/kg/day

N Mean SD CV (%) N b Mean SD CV (%) N b Mean SD CV (%)

Initial body weight (PND 22; g) U 16 58.1 5.3 9.2 16 57.9 5.7 9.9 16 58.5 6.0 10.2

A 16 NA NA NA 16 NA NA NA 16 NA NA NA

Body weight at VO (g) U 16 129.5 18.0 13.9 16 126.7 18.2 14.4 14 123.5 16.2 13.1

A 16 129.9 NA NA 16 127.3 NA NA 14 122.3 NA NA

Final body weight c (g) U 16 165.7 21.6 13.0 15 155.8 16.8 10.8 15 156.9 20.1 12.8

A 16 166.2 NA NA 15 157.2 * (↓5) NA NA 15 155.0 * (↓7) NA NA

Final body weight (% of control) U NA NA NA NA 15 94.0 NA NA 15 94.7 NA NA

A NA NA NA NA 15 94.6 NA NA 15 93.3 NA NA

Body weight gain (final

– initial; g) U 16 107.7 18.3 17.0 15 98.1 13.8 14.1 15 97.8 16.2 16.6

A 16 108.0 NA NA 15 98.7 * (↓9) NA NA 15 96.8 * (↓10) NA NA

Age at VO (PND) U 16 35.4 2.6 7.4 16 36.3 2.7 7.5 14 36.4 2.9 7.9

A 16 35.3 NA NA 16 36.2 NA NA 14 36.6 NA NA
Proportion unopened (#/N) 0 /16 0/16 1/15

a Data were obtained from Table 5 on page 24 of the study report . Percent differences from controls, calculated by the reviewers, are included in parentheses.

b Number of animals examined varied as one 500 mg/kg/day animal died on PND 38 (gavage error), one 1000 mg/kg/day animals died on Day 3, and one 1000 mg/kg/day animal did not attain VO.

c Body weight on PND 4 3 , the last day all body weights were collected.

U Unadjusted for body weight on PND 2 1

A Adjusted for body weight on PND 2 1

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

NA Not applicable

* Significantly different from controls at p<0.05.



## D. ORGAN WEIGHTS : Or gan weights at necropsy are presented in Table 3. There were no

dose- related effects on the weights of pituitary, uterus, or thyroid. At 500 and 1000mg/kg/day, the adjusted and relative liver weights were increased (p<0.05) dosedependently by 15 -35% and 22-44 %, respectively , and the unadjusted liver was increased (p<0.05) by 36% at 1000 mg/kg/day. Incre ases (p<0.05) in relative kidney weight (↑6%) and unadjusted, adjusted, and relative adrenal weights (↑20 -29%) were also noted a t 1000 mg/kg/day compared to the controls, and t he adjusted weights for ovaries were decreased (p<0.05) by 16%.

The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1450), with the exception

of mean adrenal gland s weight ( 34.6 mg; acceptable range 38.34-48.84 mg). T he % CV s for liver ( 15.7 %; maximum 13.13%) and ovaries (24.0 %; maximum 23.20 %) were also outside the acceptable ranges

.Female Pubertal Assay (2011) / Page 10 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



### TABLE 3

. Organ Weights at Necropsy a

Organ Vehicle Control Piperonyl butoxide

500 m g/kg/day Piperonyl butoxide

1 000 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 16 7.73 1.22 15.7 15 8.86 0.90 10.1 15 10.54 * (↑36) 1.55 14.7

A 16 7.76 NA NA 15 8.93* (↑15) NA NA 15 10.44* (↑35) NA NA

R 16 4.66 0.32 6.9 15 5.70* (↑22) 0.34 6.0 15 6.71* (↑44) 0.38 5.7

Kidneys (g) U 16 1.26 0.12 9.8 15 1.22 0.13 10.6 15 1.29 0.17 13.5

A 16 1.26 NA NA 15 1.23 NA NA 15 1.28 NA NA

R 16 0.77 0.06 7.4 15 0.78 0.05 6.8 15 0.82* (↑6) 0.05 6.0

Pituitary (mg) U 16 7.5 1.2 16.6 15 6.9 1.1 16.0 15 6.7 1.5 22.0

A 16 7.5 NA NA 15 6.9 NA NA 15 6.7 NA NA

R 16 4.6 0.9 21.5 15 4.5 0.9 19.8 15 4.3 0.9 19.5

Adrenals (mg) U 16 34.6 5.7 16.6 15 35.6 4.5 12.8 15 41.7* (↑21) 6.0 14.4

A 16 34.7 NA NA 15 35.8 NA NA 15 41.5* (↑20) NA NA

R 16 20.7 11.4 2.4 15 22.8 2.9 12.5 15 26.7* (↑29) 4.3 16.2

Ovaries (mg) U 16 68.1 16.3 24.0 15 64.0 7.4 11.6 15 58.2 16.2 27.9

A 16 68.3 NA NA 15 64.6 NA NA 15 57.4* (↓16) NA NA

Uterus, wet (mg) U 16 291.1 115.3 39.6 15 281.2 136.7 48.6 15 240.9 142.0 59.0

A 16 291.3 NA NA 15 281.9 NA NA 15 239.9 NA NA

Uterus, blotted (mg) U 16 252.2 67.2 26.7 15 236.5 76.8 32.5 15 206.1 78.0 37.9

A 16 252.4 NA NA 15 237.2 NA NA 15 205.0 NA NA
Thyroid, fixed (mg) U 16 12.2 1.7 13.6 15 13.1 2.8 21.5 15 13.2 1.8 13.9

A 16 NR b NA NA 15 NR b NA NA 15 NR b NA NA

a Data were obtained from Table 6 on page 26 of the study report

. Percent differences from controls, calculated by the reviewers, are included in parentheses.

b No value given for adjusted ANCOVA mean.

U Unadjusted for body weight on PND 2 1

A Adjusted for body weight on PND 2 1

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

R Organ

- to

- body weight ratio (relative to body weight)

NA Not applicable

NR Not reported

* Significantly different from controls at p<0.05.



## E. ESTROUS CYCLICITY : Estr ous cycle data are provided in Table 4. There were no effects of treatment on mean age at first vaginal estrus, mean cycle length, percent cycling, and percent regularly cycling.Female Pubertal Assay (2011) / Page 11 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



### TABLE 4

. Estrous Cyclicity a

Dose Level (mg/kg/day) N Mean A ge at

F irst V aginal

E strus (PND) Mean C ycle

L ength (days) Cycling (%) Regularly

C ycling

(%) Cycle S tatus at N ecropsy (# F emales) b

Diestrus Proestrus Estrus Not

C ycling

Vehicle 16 36.6 4.1 100 81.25 6 4 5 0

500 16 37.4 4.2 100 81.25 6 0 8 0

1 000 14 37.2 3.8 100 71.43 6 1 6 0

a Data were obtained from Tables 6 and 7 on page s 26 and 27 of the study report.

b Day of cycle at necropsy. No cells present at necropsy for one vehicle control animal and one 1000 mg/kg/day animal . One

500 mg/kg/day animal not included due to mucus present at necropsy.

N Number of animals



## F. CLINICAL CHEMISTRY AND HORMONE LEVELS : Mean hormone and clinical chemistry levels are presented in Table 5 . Levels of alkaline phosphatase were decreased (p<0.05) in rats by 24 % and 30% at 500 and 1000 mg/kg/day

, respectively

. Levels of total protein were increased (p<0.05) by 9% in rats dosed at 500 and 1000 mg/kg/day. At 1000 mg/kg/day the following increases (p<0.05) were observed compared to controls: sodium (↑ 3% ), phosphorous (↑ 17%), and blood urea nitrogen (↑ 18%).

There were no dose- related effects on serum T 4 

## or TSH, or on the other evaluated clinical chemistry parameters. The results for serum T 4 

## in the control group were within the acceptable range of th e performance criteria provided in the Guideline (890.1450).

TABLE 5

. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 16 3.49 0.76 21.9 15 3.22 0.63 19.5 15 3.65 0.71 19.5

Serum TSH (ng/mL) 16 1.26 0.53 42.0 15 1.92 1.53 80.0 15 1.97 1.21 61.5

Clinical Chemistry Sodium (meq/L) 15 135 4 3 15 135 5 4 15 139 * (↑3) 4 3

Potassium (meq/L) 1 5 9.3 1.5 16.6 15 9.1 1.1 12.0 15 9.0 1.5 16.6
Chloride (meq/L) 15 100 4 4 15 99 4 4 15 101 3 3
Calcium (mg/dL) 1 5 10.3 0.5 4.7 15 10.3 0.5 4.9 15 10.4 0.4 3.4
Phosphorus (mg/dL) 15 9.6 1.3 13.7 15 10.4 1.1 10.7 15 11.2 * (↑17) 0.9 7.7

Aspartate transferase (U/L) 15 292 171 58 15 286 102 36 15 280 158 56

Alanine transferase (U/L) 15 101 37 37 15 104 26 25 15 107 36 33

Gamma glutamyl transferase (U/L) 15 0 1 245 15 0 1 387 15 1 1 172

Alkaline phosphatase (U/L) 15 400 107 27 15 304 * (↓ 2 4) 77 25 15 279 * (↓30) 83 30

Blood urea nitrogen (mmol/L) 15 11 2 18 15 12 2 19 15 13 * (↑18) 3 20

Creatinine (μmol/L) 15 0.4 0.1 18.0 15 0.4 0.1 14.6 15 0.4 0.0 6.6

Total bilirubin (mg/dL) 15 0.1 0.1 112.2 15 0.1 0.1 146.4 15 0.1 0.1 185.2Female Pubertal Assay (2011) / Page 12 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



### TABLE 5

. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Sorbital dehydrogenase (U/L) 15 28 5 19 15 30 6 20 15 28 7 26

Total protein (g/dL) 15 5.5 0.4 6.7 15 6.0 * (↑9) 0.6 10 15 6.0 * (↑9) 0.4 6.0

Albumin (g/dL) 1 5 4.6 0.3 7.0 15 4.8 0.5 10.3 15 4.8 0.3 7.3

a Data were obtained from Tables 8 and 9 on pages 27 and 29 of the study report

. Percent differences from controls, calculated by the reviewers, are included in parentheses.

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05.



## G. HISTOPATHOLOGY : The thyroid gland follicular cell height and colloid area data are presented in Table 6 ; follicular cell shape was not reported. There was a d ose -dependent increase in the thyroid gland follicular epithelial cell height and concomitant wi th a decrease in the colloid area. Follicular cell height was Grade 1 in controls compared to G rades 1

– 3 at 500 mg/kg/day and G rades 3 –5 at 1000 mg/kg/day. Follicular colloid area was G rades 4 –5 in the controls compared to G rades 3 –4 at 500 mg/kg/day and G rades 1 –3 at 1000 mg/kg/day.

TABLE 6

. Thyroid Gland Follicular Cell He i ght and Colloid Area a



### Grades Vehicle Control Piperonyl butoxide

500 m g / k g /d ay Piperonyl butoxide

1 000 m g / k g /d ay *

# of animals

examined 16 14 c 

### 15

Follicular cell height b



### 1 1 6 ( 100 %) 8 (57 %) 0

2 0 3 (21 %) 0

3 0 3 (21 %) 9 ( 60 %)

4 0 0 5 ( 33 %)

5 0 0 1 ( 7 %)

Follicular colloid area b



### 1 0 0 1 (7 %)

2 0 0 1 ( 7%)

3 0 1 ( 7 %) 13 (8 7%)

4 8 ( 5 0 %) 13 (93 %) 0

5 8 (50 %) 0 0

a Data were obtained from Table 1 2 on page 3 2 of the study report.

b A five

- point grading scale (1 = shortest / smallest; 5 = tallest / largest) was used.

c The thyroid tissues from one 500 mg/kg/day animal was lost.

* Significantly different from controls at p<0.05.Female Pubertal Assay (2011) / Page 13 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## The incidence of histopathological findings of the uterus and kidneys are presented in

Table 7. No treatment -related histopathological effects were observed in the kidney, and no

treatment

- related e ffect s were observed in the uterus at 500 mg/kg/day. However, at 1000 mg/kg/day, 2 of 14 rats had m oderate atrophy of the myometrium, uterine stroma, and endometrial glands , which was not found in control rats. Additionally, mild atrophy of the

endometrial gland was observed in two other 1000 mg/kg/day rats. Mild luminal dilation

was observed in two 1000 mg/kg/day rats compared to 0 controls.

TABLE 7

. Incidence of Histopathological Lesions of the Uterus and Kidney a

Findings Vehicle Control Piperonyl butoxide

500 m g / k g /d ay Piperonyl butoxide

1 000 m g / k g /d ay
# Observed # Examined # Observed # Examined # Observed # Examined b

Uterus Lumen: Eosinphilic material

Minimal 1 16 3 15 3 14

Mild 1 16 1 15 1 14

Moderate 1 16 0 15 0 14
Glandular Lumens: Inflammatory cell/debris

Minimal 0 16 0 15 1 14

Lumen: Dilatation

Mild 0 16 1 15 2 14
Endometrial folding

Mild 0 16 0 15 1 14
Myometrium: Atrophy

Moderate 0 16 0 15 2 14
Endometrial glands: Atrophy

Mild 0 16 0 15 2 14

Moderate 0 16 0 15 2 14
Stroma: Atrophy

Moderate 0 16 0 15 2 14
Kidney
Cortex: Tubular Epithelium

- Basophilic

Minimal 6 16 5 15 4 15

Cortex/Cortico

- Medullary Junction: Mineral Deposit(s)

Minimal 4 16 1 15 0 15

Cortex/Cortico

- Medullary Junction: Cyst(s)

Minimal 1 16 0 15 1 15
Subacute/Chronic Interstitial Inflammation/Chronic Nephropathy

Minimal 1 16 4 15 0 15

Mild 2 16 0 15 0 15

a Data were obtained from Appendix X ( Table 3 and Appendix 7 ) on page s 146 and 154 of the study report.
b At the 1000 mg/kg/day dose, uterus was not processed for one animal.



## The number of ovary follicles, follicular cysts, and corpora lutea are presented in Table 8.

No dose- related effects were observed for number of ovary follicles, follicular cysts or corpora lutea.Female Pubertal Assay (2011) / Page 14 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



### TABLE 8. Number of Ovary Follicles, Follicular Cysts, and Corpora Lutea a

Parameter Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Small Follicles 16 15.0 6.5 43.3 15 15.9 7.5 47.2 15 16.0 8.3 51.9

Medium Follicles 16 10.8 3.9 36.1 15 9.2 2.4 26.1 15 11.3 4.4 38.9

Antral Follicles 16 7.2 4.0 100.6 15 7.3 3.5 47.9 15 7.2 2.7 37.5

A ntretic Follicles 16 10.1 3.3 9.9 15 10.4 3.7 35.6 15 12.9 5.2 40.3

F ollicular Cysts 16 0.03 0.11 366.7 15 0.03 0.11 366.7 15 0.02 0.06 300.0

C orpora Lutea 16 5.5 1.6 29.1 15 4.4 2.2 50.0 15 4.3 3.3 76.7

a Data were obtained from Table 1 1 on page 31 of the study report

. N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS : Female rats administered 500 and 1000 mg/kg piperonyl butoxide showed no difference in terminal body weights or body weight gain.

The liver, re lative kidney and adrenal weights were significantly increased compared to

control animals . T here were no consistent changes in clinical chemistry analytes that reflect overt toxicity due to piperonyl butoxide administration. No histopathological changes due to piperonyl butoxide administration were observed in the kidney. The terminal body weight in the high dose rats was 94.7% of control animals, suggesting the limit dose of 1000 mg/kg approached, but did not exceed the maximum tolerated dose

.

No differences were observed in the mean age at VO, mean body weight at VO, the time of first estrus, estrous cycle length, a nd cyclicity with piperonyl butoxide administration at either dose level. No effects were observed in uterine or ovarian weights, however uterine gland atrophy was observed in 4 of 15 animals administered 1000 mg/kg piperonyl butoxide. No changes in anima ls administered piperonyl butoxide at either dose level were observed in the number of follicles, corpora lutea, or ovarian cysts as compared to controls. There were also no significant changes in the absolute and relative pituitary gland weights following administration of piperonyl butoxide (at either dose level) compared to controls.

After 21/22 days of piperonyl butoxide administration there were no changes in thyroid gland weight or circulating T 4 

## or TSH concentrations with administration of piperonyl butoxide. However, administration of piperonyl butoxide did result in statistically significant histopathological changes in the thyroid gland, including an increase in follicular cell height (1000 mg/kg) and a decrease in colloid area (1000 mg/kg) compared to the control group.

Increases in relative liver weight, which was significantly increased in animals administration 1000 mg/kg piperonyl butoxide compared to the vehicle control, is typically observed with hepatic-enzyme inducers. Histopat hological changes that occurred in the thyroid gland in the high (1000 mg/kg) dose group are a sensitive indicator of thyroid gland

modulation.Female Pubertal Assay (2011) / Page 15 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## Overall, administration of piperonyl butoxide to intact juvenile/peripubertal female rats does not appear to show an effect on pubertal development; however, the effects observed in thyroid (i.e., histopathological changes) following administration of 1000 mg/kg piperonyl

butoxide may indicate potent ial modulation of thyroid function at the limit dose level.

B. AGENCY COMMENTS : No treatment

- related effects were observed on mortality, clinical signs of toxicity , and the weights of pituitary, uterus, or thyroid. Tr eatment

- related

effect s w ere also not observed on 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. There were

also no treatment

- related effects on serum T 4 

## or TSH.

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) dose-dependently by 15 -35% and 22-44% at 500 and 1000 mg/kg/day, respectivel y, and the unadjusted liver weight was increased (p<0.05) by 36% at 1000 mg/kg/day . Compared to the contro ls , increases (p<0.05) in the relative kidney weight (↑6%) and unadjusted, adjusted, and relative adrenal weights (↑20 -29%) were also noted 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

.

At 500 and 1000 mg/kg/day, serum levels of alkaline phosphatase were decreased (p<0.05) by 24 and 30%, respectively, and total protein levels were increased (p<0.05) by 9%. No other changes in clinical chemistry were noted at 500 mg/ kg/day. However, at 1000 mg/kg/day , increases (p<0.05) were observed in the levels of sodium (↑3%), phosphorous (↑17%), and blood urea nitrogen (↑18%).

Although no changes were noted in uterine weights, selected histological findings were

noted in uteri of rats in the high dose group. At 1000 mg/kg/day, m oderate 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 for these findings.

In the thyroid, there were no dose- related effects on serum T 4 

## or TSH levels; however, ther e was a d ose- dependent increase in the thyroid gland follicular epithelial cell height with a

concomitant decrease in the colloid area.

C. STUDY DEFICIENCIES : The following minor deficiencies were noted that are not
considered to have had an adverse impact on the results , interpretation or conclusions of this study:

• Control CV for age at VO (7.4% ) was higher than the performance criteria maximum of

6.52%.

• Control CV for final body weight (13.0% ) was higher than the performance criteria

maximum of 8.93%.Female Pubertal Assay (2011) / Page 16 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None



## • Control CV for liver weight (15.7% ) was higher than the performance criteria

maximum of 13.13%.

• Control CV for weight of ovaries (24.0% ) was higher than the performance criteria

maximum of 23.20 %.

• Control m ean adrenal weight (34.6 mg ) was below the performance criteria acceptable

range (38.34-48.84 mg).

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: O CSPP 890.1500, M ale Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618806)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 4/5 /2012

Secondary Reviewer : Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 4/20/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5 / 08 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5 / 08 / 2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS -Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be con sidered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PIPERO N YL llUTOXIDE I 06 7 50 1



### Prim a r y Reviewer: 

## Pati e n ce Browne. Ph.D

. Ri s k Assessme nt Branch V I

, Health Effects D i vision Secondary Rev i ewe r: 

## John Li cc ion e. Ph.D.

Health Effects Di v i s i on Male Pubertal A ss a y (20 1 1 ) I P age I o f 1 6


### O CS PP 890. 1 500 / OECO No ne

# natur**e: ~** **Date: ~**  Signat ur e: 

# =

...

D a t e: :;;,:(.

,. os 1 2011



### DATA EVALUAT I ON R ECO RD

STUDY TYPE: 

## Male Pubertal Assay: OCS PP 890.1500 ; OEC D No n e.

PC CODE: 

## 06750 1 OP BARCODE: 

## 0 398558

TXR #: 

## 0056259 CAS 

## No

. : 51-03-6

TEST MATERIAL (PUR I TY): 

## Pip ero n yl butoxide (93.8 % a.i. )



## SYNONYMS : 5-[[ 2-(2-bu toxyethox y)e th oxy ]meth y l]-6-prop y l-1 ,3 -b e n zod ioxole



## CITAT I ON: Zo rilla. 

## L. (20 11 ) Pub e rt a l developmental and th y roid function in int ac t j u ven il e / per ipub e rt al fema l e and m a l e rats w ith piperonyl b ut oxide.

Int egrated Labora t orie s, In c.
, Durham

, NC. Labora t ory S tud y No.: C l 92-300.

Novembe r 1 0, 20 11. MRID 48618806. U npubli s hed.

SPONSOR: 

## Pip ero n y l 13utoxide Ta sk Fo r ce 11

, 

## c / o Co n su mer Specialty P roducts
Association, 1 667 K Street NW

. Suit e 300. Washington D C

TEST ORDER#: 

## CON-06750 1- 9

EXECUT IV E SUMMARY: 

## In a Mal e Pubert a l Assay (MRID 48618806)

, 1 6 mal e SpragueDawl ey (C rl: C D ® (SD) I GS) r a t s / dose group were treated dail y via ora l gavage wi th piperon y l

butox id e (93.8 % a. i

., Lot # : E 104-IO ) in corn o il a t do ses o l' O, 500

. or 1000 ( li mit d ose)

m g/ k g/ da y from post-natal day ( P D) 23 to 53 / 54

. The an im a l s were exami n ed fo r preputial

s epara t ion ( PP S) da il y b e ginning o n P D 30

, and body weight at day of a tt a inm e nt was
recorded. Fo ll ow in g sac rifi ce o n P ND 53 / 54. t ota l serum t es t os t ero n e, th yrox in e (T4)

, and thyroid s timul a ting horm o n e (TS H) level s were d e termined usin g radioimmun oassays. Adrenal

, liv e r. th yro id

. pituitar y
, and uro ge nital organ weights we r e r eco rded

, and micr osco pic

exam in at i ons of the testes

, epid id y mid es
, thyroid

, a nd kidn eys were performed.

Although four rats died during th e st ud y ( 1 co ntr ol

, 2 l ow-dose. and I high-dose). t here were no

tr eatmen t- re l ated effec t s on mortality as th e dea th s were a ttribut ed to gavage e rr o r s. There we r e

no clinical s i g n s of toxi c it y in a n y grnup
, 

## and no treatm e nt-r e l a t e d e ffect s w e r e observed on age

a t PPS

, weight al PPS

, and sc rum l ev el s of 

## T.

. 1

a nd TS I-1. T h e r e we r e a l so no tr ea tm e nt-relat e d histopathological findin gs noted in th e t este s
, cp idid y mid es, o r kidneys at either dose

.

General g r o wth was decrea s ed in a dose dependent m a nn er. T h e final body weig ht (adj u s t ed for body wei g ht on PND 23) wa s decreased by 5 % ( n ot s tati st i ca ll y sig nifi ca nt [NS]) and 9%Male Pubertal Assay (2011) / Page 2 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None



## (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

follicul ar 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 a t 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 (T 4 

## 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% (p<0.05). At 1000 mg/kg/day, adjusted and unadjusted LABC weights were decreased b y 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 b ased on the 11% decrease in

ad justed body weight grain observed at 1000 mg/kg/day, which also is the dose limit for the

assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for a M ale Pubertal Assay (OCSPP

890.150 0).

COMPLIANCE : Signed and dated GLP, Data Confidentiality, and Quality Assurance

statements were provided.Male Pubertal Assay (2011) / Page 3 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratories, Inc.

Location: Durham, NC Study Director s : Zorilla, L.

Other Personnel: Not reported Study Period: March 16 , 2011 through November 10

, 2011



## 2

. Test S ubstance : Piperonyl butoxide

Description: Pale, oily liquid

Source: Endura Fine Chemicals , Bologna, Italy

Lot #: E104

- 10 ( retest date of July 2012 ) Purity : 9 3.8 % Stability: Report stated that the compound is stable in the vehicle at 1

- 10°C for up to 20 days

CAS #: 51

- 03

- 6

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Vehicle : Corn oil

4

. Test A nimals : Species: Rat (males only)

Strain: Sprague

- Dawley ( Crl:CD ® (SD ) IGS ) Age/ Mean W eight

at S tudy I nitiation: PND 23 / 4 6.0

- 75.9 g

Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: P arental dams were individually housed in polycarbonate cages with micro

- isolator tops and

hardwood be dding; each dam was housed with her litter until weaning on PND 21. After weaning,

up to 2 males were housed per cage.
Diet: Teklad Global 1 6 % Protein Rodent Diet (T e klad Diets , Madison, WI), ad libitum . It was stated

that t he total genistein equivalents of genistein plus daidzein was determined to be 6.2 μg/g of

feed.

Water: Reverse osmosis

- purified tap water

, ad libitum

Environmental

C onditions: Temperature: Humidity: Air changes: Photoperiod : 2 1

- 2 3 °C

32-63% Not reported

14 h light / 10 h dark



## B. ST UDY DESIGN

1. In -L ife D ates : Start: April 2 3, 2011 End: May 24 , 2011 (Dates estimated by the reviewers)

2. Mating : Time

- mated

, pregnant nulliparous dams (age not reported ) were received from the supplier on gestation day ( GD ) 8 and were allowed to deliver naturally and rear their pups to

PND 21. Between PND 3 and 5, litters with the same date of birth were standardized to 8 pups with equal numbers of males and females.Male Pubertal Assay (2011) / Page 4 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None



## 3. Animal A ssignment : Following weaning on PND 21, animal s were assigned using a procedure that stratifies animals across groups by body weight such that the mean body weight of each group was not statistically different from any other group (male and females performed as separate analyses) using analysis of variance. Care was taken to avoid placing

littermates in the same dose group. The test groups a re presented in Table 1.

TABLE 1. Study design a

Test group Dose (mg/kg/day) # of M ales

Control

– vehicle control 0 16

Low

- Piperonyl butoxide 500 16

High

- Piperonyl butoxide 1000 16

a Data were obtained from page 14 of the study report.



## 4. Dose S election Rationale : The doses were selected based on the results of a dose range finding study. 1 

## Three animals per sex were administered piperonyl butoxide at 0, 200, 400,
600, 800, or 1000 mg/kg/day for 14 days. Male rats showed no changes in body weight or body weight gain in any dose group. Absolute liver, kidney , and adrenal weights were also not significantly different than controls. Relative adrenal weights were significantly increased at 400 and 1000 mg/kg/day. Relative liver weights were significantly increased at 800 and 1000 mg/kg/day. T he high dose selected for males was 1000 mg/kg/day, which is

the limit dose.

5. Dose P reparation and Analysis : Dose formulations were prepared at ILS twice by adding the appropriate amount (adjusted for purity) of piperonyl butoxide to corn oil. Samples of dose formulations prepared on April 19 and 26, 2011 were collected from the top, middle,

and bottom of the formulation and sent to Smithers Viscient, LLC (Wareham, MA) for analysis. Smithers Viscient analyzed, in duplicate, samples received for concentration and homogeneity. T he mean results of the homogeneity analyses were used to demonstrate

achieved concentrations. The report stated that d ose formulations of piperonyl butoxide in corn oil held from 1 to 10°C for up to 20 days were considered stable based on a separate study; 2 

## however, no data were submitted to allow independent v erification.

Results

Stability (% of time zero): Not reported

Concentration (% of nominal): 95.6–108.2%

Homo geneity (% CV ): 2.3–6.6

1 

### Zorrilla, L. (2011) Range Finder Study for Piperonyl Butoxide (PBO) Tier 1 Endocrine Disrupting Screening

Program in vivo Mammalian Assays. Unpublished Study Report Prepared by ILS, Inc. Study No. C182

- 500.

2 

### Smith, R. (2011) Storage Stability of Piperonyl Butoxide in Corn Oil. Unpublished study report prepared by

Smithers Viscient Inc. Study No.13513

- 6132.Male Pubertal Assay (2011) / Page 5 of 16

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## The analytical data indicated that the mixing procedure was adequate and the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage A dministration : D ose formulations were administered by oral gavage at a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31 or 32 consecutive days on PND 23 through 53/54. Dosing occurred 24 hours (±2 hours) from the previous dose. Dose volume was adjusted daily based on individual animal body weight. The dosing sequence was stratified across all dose groups; one animal from each group and then repeated until all animals were dosed.

7. Statistics : Descriptive statistics (mean, standard deviation, coefficient of variance, and sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets

listed below were analyzed using Statistical Analysis System

, version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possible outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were

transfor med (logarithm, square root, and multiplicative inverse) and if still heterogeneous analyzed using the non- parametric Kruskal -Wallis and Dunn’s test. If data sets were not homogenous, ANCOVA analyses were not performed. Trend tests were performed on body weight and tissue weight data sets and reported when significant (p<0.05) for endpoints that did not show any significant pair wise comparisons.

Classification Procedure

Initial body weights
Final body weight (use last day all body weights collected)
Final body weight gain (use last day all body weights collected)
Age and body weight at PPS Homogeneous data sets:

O ne

- way analysis of variance (ANOVA) followed by pair

wise comparisons performed using Dunnett’s two

- tailed

t- tests

Tissue weights

Relative tissue weights (liver, kidneys, pituitary and adrenals)

Hormone levels
Clinical chemistry levels T wo

- way ANOVA with treatment and necropsy day (if >1 day) as main effects.

Pair

- wise comparisons were performed using Dunnett’s

two

- tailed t

- tests.

Initial body weights
Final body weight (use last day all body weights collected)
Final body weight gain (use last day all body weights collected)
Age and body weight at PPS
Tissue weights T wo

- way analysis of covariance (ANCOVA) with PND 23 body weight (weight on f irst day of administration) as the

covariable.

Pair

- wise comparisons were performed using Dunnett’s

two

- tailed t

- tests.

T hyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant,

f ollowed by Kruskal

- Wallis and Dunn’s test.



## Significance was reported as exact p -values, except no p-value was reported for Dunn's Test (p<0.05) . The statistical analyses were considered appropriate.Male Pubertal Assay (2011) / Page 6 of 16

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## C. METHODS

1. Mortality and C linical Examinations : Animals were observed twice daily for mortality and moribundity on weekdays and once daily on weekends and holidays. Clinical

observations were performed daily prior to dose administration and prior to euthanasia.

2. Body W eight : Body weights were recorded prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia.

3. Food Consumption : Food consumption was not measured.

4. Preputial Separation (PPS) : Beginning on PND 30, males were examined daily following dosing for PPS

. PPS was recorded as: progression not initiated, partial separation, a persistent thread of tissue between the glans and prepuce, or complete PPS

.

5. Sacrifice and Pathology : Beginning on the first day of dose administration, any animals found moribund or dead were necropsied and cause of death was determined and recorded, if possible. Moribund animals were euthanized by CO 2 

## inhalation and death confirmed by cervical dislocation. For animals surviving to scheduled termination, animals were humanely euthanized by decapitation a t least 2 hours after the final dose administration in the same order as they were dosed. The method for euthanasia of culled animals was not provided.

After euthanasia and prior to tissue collection, trunk blood was collected, and blood serum was obtained. All serum samples were stored at or below

− 70°C. Serum samples were shipped on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis.

a. Hormone Analysis : T 4 

## , TSH , and testosterone were measured in serum using radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with multiple quality control samples.

b. Clinical C hemistry : The following CHECKED (X) parameters were examined.Male Pubertal Assay (2011) / Page 7 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkali ne phosphatase (ALP ) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Albumin/globulin ratio (calculated)

Lactic acid dehydrogenase (LDH) Bile acids

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in male rats based on guideline 890.1 500.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were collected and weighed and grossly observed from all study animals . The (XX) organs, in addition, were subjected to histological examination.

UROGENITAL OTHER

X X Testes (left and right separately) * + X X Thyroid a * +

X X Epididymides (left and right separately) * + X Liver *

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)*

X Ventral prostate* X Pituitary*

X Dorsolateral prostate*

X Levator ani/bulbocavernosus (LABC) muscle complex*

X X Kidneys (paired)* +

Gross lesions and masses

a Thyroid was weighed post

- fixation.

* Weights required based on guideline 890.1 50 0.

+ Histopathological examination required based on guideline 890.1 50 0.



## Left kidney and thyroid were fixed in 10% neutral buffered formalin for at least 24 -hours. Left testis and left epididymis were each placed in Bouin’s fixative for 18 - to 24-hours and then washed in 70% histology grade alcohol. After fixation, tissues were transferred to 70%

hi stology grade alcohol, histologically processed, embedded, sectioned, and stained with hematoxylin and eosin

. Histologically

- processed tissues (thyroid, kidneys, epididymis, and testis) from all animals were microscopically evaluated. Two sections of each of the 2 lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used , and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA.Male Pubertal Assay (2011) / Page 8 of 16

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## II. RESULTS

A. Mortality : One control rat a nd one 1000 mg/kg/day rat were found dead on D ay s 39 and 25, respectively. Two rats administered 500 mg/kg died immediately following dose administration on Days 38 and 39. All four animals showed signs of gavage error. No other animals showed signs of moribundity.

B. Clinical Signs of Toxicity : No treatment

- related clinical signs of toxicity were observed in

animals from 500 mg/kg/day dose group. The clinical signs obse rved at 1000 mg/kg/day

included: (i) o ne rat was thin on D ay 3; (ii) one rat had an ungroomed appearance on D ay 4 ; and (iii) s ix rats were salivating prior to dose administration on Day 12. These findings

were of low incidence and transient. All other rats were observed as normal throughout the study.

C. General Growth and Preputial Separation : Body weights, body weight gains, age of

attainment of PPS

, weight at PPS , and proportion/incidence of unseparated are presented in

Table 2.

G eneral growth was slightly attenuated in a dose dependent manner. The adjusted (for PND 23 body weight) final body weight was decreased by 5% ( NS ) and 9% (p<0.05) at 500 and 1000 mg/kg/day , respectively . The adjusted overall body weight gain was decreased by 6% (NS) and 11% (p<0.05) at 500 and 1000 mg/kg/day , respectively

.

Additionally at 1000 mg/kg/day, an increase (p< 0.001 ) in the age at partial PPS was noted (PND 42.5 treated vs. PND 40.3 controls), but was considered unimportant as no difference s

were noted in the age at complete PPS in the treated groups compared to the control. Also, body weights at complete PPS were decreased (NS) by 10% at this dose, but body weights were also decreased by 10% at PND 23.

The following Guideline criteria were no t met: (i) %CV=7.1 for age at PPS (top of acceptable range: 5.67); (ii) %CV=18.7 for body weight at PPS ( top of acceptable range: 7.57); and (iii) %CV=11.3 for final body weight ( top of acceptable range: 7.47). Additionally, weaning weight (PND 21) was not reported.Male Pubertal Assay (2011) / Page 9 of 16

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### TABLE 2 . General Growth and Preputial Separation (PPS) a

Parameter

Evaluated Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

# of

Males Mean SD CV (%) # of

Males Mean SD CV

(%) # of

Males Mean SD CV

(%)

Age at PPS (PND) U 14 44.7 3.2 7.1 13 44.4 2.4 5.3 14 45.6 2.5 5.5

A 14 44.6

--- --- 13 44.3

--- --- 14 45.9

--- ---
Body weight at PPS (g) U 14 230.0 43.1 18.7 13 215.8 29.0 13.5 14 206.0 (↓10) 21.0 10.2

A 14

--- --- --- 13

--- --- --- 14

--- ---- ---

Age at partial PPS (PND) U 15 40.3 1.0 2.4 14 41.4 1.4 3.4 15 42.5* ** (↑5) 2.0 4.7

A 15

--- --- --- 14

--- --- --- 15 X* **

--- ---

Initial body weight
(PND 23; g) U 16 67.4 5.8 8.5 16 67.6 5.9 8.7 16 60.7 (↓10) 9.8 16.2

A 16

--- --- --- 16

--- --- --- 16

--- --- ---

Final body weight (g) U 15 298.4 33.3 11.3 14 284.9 (↓5) 34.5 12.1 15 258.1 ** (↓14) 35.3 13.8

A 15 293.9

--- --- 14 279.5 (↓5) --- --- 15 267.7 * (↓9) --- ---

Final body weight

(% of control) U 15

--- --- --- 14 95.5

--- --- 15 86.5

--- ---

A 15

--- --- --- 14 95.1

--- --- 15 91.0

--- ---

Body weight gain

(final

– initial; g) U 15 235.2 32.0 13.6 14 220.4 30.5 13.8 15 200.0 ** (↓15) 32.0 16.0

A 15 228.7

--- --- 14 214.2 (↓6) --- --- 15 202.4 * (↓11) --- ---

Proportion unseparated (#/N) 15 1/15 14 1/14 15 1/15

a Data were obtained from Table 16 on page 39 of the study report

. Percent difference from controls (calculated by reviewers) is presented parenthetically.

U Unadjusted for body weight on PND 23

A Adjusted for body weight on PND 23

X No mean given for transformed ANCOVA data

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05.

** Significantly different from controls at p<0.0 1.

* ** Significantly different from cont rols at p<0.0 01

.



## D. Organ Weights : Organ weights at necropsy are presented in Table 3. At 500 mg/kg/day, adjusted LABC weight was decreased (p<0.05) by 14% ; a ll other androgen-dependent tissue weights were similar to controls at this dose. At 1000 mg/kg/day , the following dec reases (p<0.05) were observed in the androgen- dependent tissues : (i) unadjusted and adjusted weights of seminal vesicles with coagulating gland and fluid (↓ 28– 35%); (ii) unadjusted and adjusted weights of seminal vesicles with coagulating gland and without fluid ( ↓21 –26%);

and (iii) unadjusted and adjusted weights of LABC muscle weights ( ↓20

– 26%); and (iv) unadjusted weight of left epi didymis (↓10%).Male Pubertal Assay (2011) / Page 10 of 16

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## There were differences (p<0.05, except as noted ) in the weights of other organs in the 500 and 1000 mg/kg/day groups as follows: (i) relative to body liver weights (↑ 15-34 %); (ii) relative to body kidney weights (↑8

– 11%); and (iii) unadjusted and adjusted pituitary weight (↓16

– 17%; NS for the adjusted weight in the 1000 mg/kg/day group). Additionally at 1000 mg/kg/day, unadjusted and adjusted liver weights were increased (p<0.05) by 16–20%, and adjusted and relative to body adrenal weights were increased by 15–31%.

The following Guideline cri teria were not met: (i) mean kidney weight of 2.00 g (acceptable range : 2.242–3.050); (ii) %CV=24.3 for seminal vesicles with coagulating gland and fluid (top acceptable range: 21.06); and (iii) %CV=26.7 for ventral prostate (top acceptable range: 2 2.32).

TABLE 3

. Organ Weights at Necropsy a

Organ Vehicle Control Piperonyl butoxide

50 0 m g/kg/day Piperonyl butoxide

100 0 mg/kg/day

# of

Males Mean SD CV # of

Males Mean SD CV # of

Males Mean SD CV

Liver
(g) U 15 13.97 2.08 14.9 14 15.22 1.88 12.4 15 16.17* (↑16) 2.17 13.4

A 15 13.8

--- --- 14 15.0

--- --- 15 16.6* ** (↑ 20 ) --- ---

R 15 4.61 0.31 6.8 14 5.29*** (↑ 1 5) 0.18 3.4 15 6.20*** (↑ 34 ) 0.37 5.9

Kidneys
(g) U 15 2.00 0.19 9.3 14 2.03 0.28 13.9 15 1.92 0.29 15.0

A 15 1.97

--- --- 14 2.00

--- --- 15 1.97

--- ---

R 15 0.66 0.05 7.2 14 0.71* (↑8) 0.06 7.9 15 0.73*** (↑11) 0.06 7.8

Pituitary
(mg) U 15 8.6 1.2 14.1 14 7.2* (↓ 16) 1.7 24.3 15 7.1* (↓ 17) 1.8 25.3

A 15 8.6

--- --- 14 7.1* (↓ 17) --- --- 15 7.2
(↓16) --- ---

R 15 2.9 0.4 15.0 14 2.5 0.6 22.7 15 2.7 0.8 28.1

Adrenals
(mg) U 15 39.6 5.7 14.4 14 40.9 6.7 16.3 15 44.1 5.9 13.3

A 15 39.2

--- --- 14 40.4

--- --- 15 44.9* (↑15) --- ---

R 15 13.1 1.6 12.4 14 14.2 1.6 11.4 15 17.1* ** (↑31) 3.5 20.5

Seminal vesicle + coagulating gland, with fluid (mg) U 15 505.4 122.8 24.3 14 460.6 157.1 34.1 15 326.4* * (↓35) 124.6 38.2

A 15 493.0

--- --- 14 445.4

--- --- 15 352.9* (↓28) --- ---

Seminal vesicle + coagulating gland, without fluid (mg) U 15 298.1 66.6 22.3 14 258.8 54.4 21.0 15 219.4* * (↓26) 69.5 31.7

A 15 292.2

--- --- 14 251.7

--- --- 15 231.9* (↓21) --- ---

Ventral prostate (mg) U 15 197.4 52.7 26.7 14 183.8 41.1 22.4 15 166.7 45.7 27.4

A 15 193.6

--- --- 14 179.2

--- --- 15 174.6

--- ---Male Pubertal Assay (2011) / Page 11 of 16

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### TABLE 3

. Organ Weights at Necropsy a

Organ Vehicle Control Piperonyl butoxide

50 0 m g/kg/day Piperonyl butoxide

100 0 mg/kg/day

# of

Males Mean SD CV # of

Males Mean SD CV # of

Males Mean SD CV

Dorsolateral prostate (mg) U 15 142.2 34.5 24.2 14 139.9 40.8 29.2 15 110.0 39.4 35.8

A 15 138.9

--- --- 14 135.8

--- --- 15 117.1

--- ---

LABC
(mg) U 15 493.5 69.8 14.1 14 429.7 79.1 18.4 15 366.5 * ** (↓26) 99.7 27.2

A 15 484.0

--- --- 14 418.2* (↓14) --- --- 15 386.6 ** (↓20) --- ---

Epididymis, left (mg) U 15 205.7 18.8 9.1 14 198.6 23.0 11.6 15 185.2 * (↓10) 29.7 16.1

A 15 202.6

--- --- 14 194.9

--- --- 15 191.7

--- --- Epididymis, right (mg) U 15 203.7 20.1 9.9 14 204.5 21.6 10.6 15 187.5 28.7 15.3

A 15 200.4

--- --- 14 200.4

--- --- 15 194.6

--- --- Testis, left (mg) U 15 1409.9 76.7 5.4 14 1443.8 123.5 8.6 15 1389.8 156.9 11.3

A 15 1398.1

--- --- 14 1429.4

--- --- 15 1415.0

--- --- Testis, right (mg) U 15 1445.6 79.7 5.5 14 1460.7 120.9 8.3 15 1385.4 154.4 11.1

A 15 1431.8

--- --- 14 1443.8

--- --- 15 1414.9

--- --- Thyroid, fixed
(mg) U 15 18.8 2.2 11.9 14 19.0 3.7 19.5 15 20.1 3.3 16.6

A 15 18.5

--- --- 14 18.7

--- --- 15 20.7

--- ---

a Data were obtained from Table 17 on page s 41

– 42 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically.

U Unadjusted for body weight on PND 23

A Adjusted for body weight on PND 23

R Organ

- to

- body weight ratio (relative to body weight)

SD Standard Deviation

CV Coefficien t of Variation

* Significantly different from controls at p<0.05.

** Significantly different from controls at p<0.01.

* ** Significantly different from controls at p<0.001.



## E. Clinical Chemistry and Hormone Levels : Clinical chemistry and h ormone levels are presented in Table 4. Serum testosterone levels were decreased by 36% ( NS) and 67% (p<0.05) at 500 and 1000 mg/kg/day , respectively; however, no changes were observed in

s erum T 4 

## and TSH levels in the treated groups compared to the controls.

The following clinical chemistry parameters differed (p<0.05) in the treated groups compared to controls: (i) sodium was increased by 2% at 1000 mg/kg/day; (ii) alkaline phosphatase was decreased by 32 and 42% respectively at 500 and 1000 mg/kg/day ; (iii) blood urea nitrogen was increased by 20% at 1000 mg/kg/day; (iv) total protein was increased by 7 and 16% respectively at 500 and 1000 mg/kg/day; and (v) albumin was increased by 9% at 1000 mg/kg/day.

There were no significant differences in ser um thyroid hormone (T4 and TSH) levels among treated and control groups. However, t he following Guideline criteria for control levels wereMale Pubertal Assay (2011) / Page 12 of 16

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## not met: (i) mean serum T4 level of 3.65 μg/dL (acceptable range : 4.056–7.376 ); and mean serum T SH level of 2.25 ng/ m L (acceptable range: 4. 212–24.112 ).

TABLE 4 . Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

# of

Males Mean SD CV # of

Males Mean SD CV # of

Males Mean SD CV

Hormones

Serum T 4 , Total (μg/dL) 15 3.65 0.47 13.0 14 4.00 0.74 18.5 15 4.11 0.90 21.9

Serum TSH (ng/mL) 14 2.25 0.84 37.3 14 2.67 2.17 81.3 15 2.94 1.61 54.8

Serum

testosterone (ng/ m L) 15 1.29 0.68 52.9 14 0.83 (↓36) 0.76 91.6 15 0.43* * (↓ 67 ) 0.52 121

Clinical Chemistry

Sodium (mEq/L) 15 142 2 2 14 142 3 2 15 145* (↑ 2) 4 3

Potassium (mEq/L) 15 9.5 1.2 12.6 14 9.3 1.5 16.3 15 8.9 1.7 18.6

Chloride (mEq/L) 15 105 2 2 14 103.1 2.2 2.2 15 104 3 3

Calcium (mg/dL) 15 10.3 0.5 5.3 14 10.2 0.8 8.2 15 10.4 0.8 7.6

Phosphorus (mg/dL) 15 10.6 1.3 12.6 14 10.6 1.0 9.5 15 11.1 1.4 12.5

AST (U/L) 15 283 121 43 14 317 157 50 15 277 158 57

ALT (U/L) 15 113 29 25 14 118 41 35 15 106 48 45

GGT (U/L) 15 0 1 387 14 0 0

--- 15 0 0 387

ALP (U/L) 15 552 86 16 14 377* ** (↓32) 92 24 15 318* ** (↓42) 62 20

BUN (mg/dL) 15 10 2 17 14 11 2 21 15 12* (↑20) 2 16

Creatinine (mg/dL) 15 0.4 0.0 11.3 14 0.4 0.0 11.4 15 0.5 0.1 20.2

Total

Bilirubin (mg/dL) 15 0.1 0.1 109 14 0.1 0.1 167 15 0.0 0.1 178.7

SDH (U/L) 15 30 5 17 14 31 6 20 15 30 7 24

Total Protein (g/dL) 15 5.6 0.3 5.2 14 6.0* * (↑7) 0.5 7.8 15 6.5* ** (↑16) 0.4 5.6Male Pubertal Assay (2011) / Page 13 of 16

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### TABLE 4 . Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control Piperonyl butoxide

500 mg/kg/day Piperonyl butoxide

1000 mg/kg/day

# of

Males Mean SD CV # of

Males Mean SD CV # of

Males Mean SD CV

Albumin (g/dL) 15 4.6 0.3 7.4 14 4.7 0.4 7.8 15 5.0* * (↑9) 0.4 7.4

a Data were obtained from Table 18 on page 44 and Table 19 on page 45 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically.

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05.

** Significantly different from controls at p< 0.01.

* ** Significantly different from controls at p<0.001.



## F. Gross pathology : There were no effects of treatment observed at necropsy.

G. Histopathology : The incidences of histopathological findings of the thyroid gland are presented in Table 5 ; follicular cell shape was not reported

. There was a dose -dependent

increase in the height of thyroid gland follicular epithelial cell s and concomitant with a

decrease in the colloid area. In the controls, 15/15 rats had colloid areas graded at 4 or 5 (largest) , compared to 4/14 rats at 500 mg/kg/day, and 3/15 rats at 1000 mg/kg/day. Conversely, 0/15 controls had follicular cell height graded at 4/5 (tallest) , compared to 3/14 rats at 500 mg/kg/day and 10/15 rats at 1000 mg/kg/day.

TABLE 5 . Inciden ce of Histopathological Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade Incidence

O E O E

Vehicle Control 1 0 15 1 9 15

2 0 15 2 6 15

3 0 15 3 0 15

4 7 15 4 0 15

5 8 15 5 0 15

Low Dose ( 50 0 mg/kg/day) 1 0 14 1 1 14

2 0 14 2 4 14

3 10 14 3 6 14

4 4 14 4 3 14

5 0 14 5 0 14

High Dose ( 100 0 mg/kg/day) 1 1 15 1 0 15

2 3 15 2 1 15

3 8 15 3 4 15

4 3 15 4 6 15

5 0 15 5 4 15

a Data were obtained from Table 21 on page 47 of the study report.

b A 5

- point grading scale (1=shortest/smallest; 5=tallest/largest) was used.

O No. Observed

E No. ExaminedMale Pubertal Assay (2011) / Page 14 of 16

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## The incidence of histopathological findings of the testes, epididymides, liver, thyroid, and kidneys are presented in Table 6. There were no treatment -related histopathological findings noted in the testes, epididymides, or kidneys at any dose.

TABLE 6 . Incidence of Histopathological Lesions of the Epididymides , Thyroid, and Kidney a

Findings Dose L evel (mg/kg bw/day)

Vehicle Control Piperonyl butoxide ( 50 0 ) Piperonyl butoxide ( 100 0 )

# Observed # Examined # Observed # Examined # Observed # Examined
Epididymides

Minimal inflammation 9 15 7 1 4 7 15
Thyroid Ectopic t hymus 1 1 4 1 1 3 0 15

Minimal C

- cell hyperplasia 1 1 4 1 1 3 0 15
Kidney s Minimal basophilic tubules 5 15 8 1 4 6 15
Minimal mineral deposits at the cortex/corticomedullary junction 0 15 0 14 0 15

Minimal cysts at the cortex/ corticomedullary junction 0 15 3 14 0 15

Minimal interstitial

inflammation/ chronic nephropathy 5 15 6 14 5 15

Mild interstitial inflammation/ chronic nephropathy 1 15 0 14 0 15

a Data were obtained from on page s 145

– 173 of the study report

. No abnormal effects were noted in the testis.



## III

. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : A dministration of piperonyl butoxide to intact juvenile/peripubertal male rats shows changes in specific endpoints that suggest an effect on pubertal development, and the effects observed in thyroid (i.e., histopathological changes) following administration of 1000 mg/kg /day piperonyl butoxide may indicate potential modulation of thyroid function at the limit dose level.

B. AGENCY COMMENTS : No treatment

- related effects were observed on mortality, clinical signs of toxicity, age at PPS, weight at PPS, and s erum levels of T 4 

## and TSH. There

were also no treatment -related histopathological findings noted in the testes, epididymides, or kidneys at either dose.

General growth was slightly attenuated in a dose dependent manner. Decreases in body weights and body weight gains were noted; however, t he exact magnitude s of th ese effect s

w ere confounded by an experimental error. Between allocation of animals to groups and the first day of dosing (PND 21 -23) and prior to the administration of the first dose on PND 23, 8 of the 16 rats in the 1000 mg/kg/day group lost weight. The report stated that the cause of the weight loss appeared to be a problem with the water bottles, because when the bottles

were changed, the animals began to gain weight. As a result of this issue, the 1000 mg/kg/day animals weighed approximately 10% less than the control group on PND 23

.Male Pubertal Assay (2011) / Page 15 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None



## The adjusted (for body weight on PND 23) final body weight was decreased by 5% ( NS) and 9% (p<0.05) at 500 and 1000 mg/kg/day , respectively, and t he adjusted overall body weight gain was decreased by 6% (NS) and 11% (p<0.05) at 500 and 1000 mg/kg/day

, respectively

. The decreases in body weight and body weight gain at 1000 mg/kg/day indicate that high dose was adequate

.

Serum testosterone levels were decreased by 36% (NS) and 67% (p<0.05) at 500 and 1000 mg/kg/day , respectively

. Decreases in the androgen

- dependent tissues were also noted; however, b ecause of the discrepancy in initial mean weights, adjusted tissue weights we re required for meaningful comparisons. At 500 mg/kg/day, a djusted LABC weight was decreased (p<0.05) by 14 %. At 1000 mg/kg/day, adjusted tissue weights were decreased (p<0.05) as follows : (i) seminal vesicles with coagulating gland and fluid by 28%; (ii) seminal vesicles with coagulating gland and without fluid by 21%; and (iii) LABC by 20%.

There were diff erences (p<0.05, except as noted) in the weights of other organs in the 500 and 1000 mg/kg/day groups as follows: (i) relative to body liver weights (↑15

- 34%); (ii) relative to body kidney weights (↑8 –11%); (iii) unadjusted and adjusted pituitary weight ( ↓16

– 17%; NS for the adjusted weight in the 1000 mg/kg/day group). Additionally at 1000 mg/kg/day, unadjusted and adjusted liver weights were increased (p<0.05) by 16–20%, and adjusted and relative to body adrenal weights were increased (p<0.05) by 15 –31%.

Although there on no treatment related effect on thyroid weight, there was a dose-dependent

decrease in the colloid area of thyroid with a concomitant increase in the thyroid gland follicular epithelial cell height. In the controls, 15/15 rats had colloid areas graded at 4 or 5, compared to 4/14 rats at 500 mg/kg/day, and 3/15 rats at 1000 mg/kg/day. Conversely, 0/15 controls had follicular cell height graded at 4/5, compared to 3/14 rats at 500 mg/kg/day and 10/15 rats at 1000 mg/kg/day. There were no significant differences in serum thyroid hormone (T4 and TSH) levels among treated and control groups. However, t he following

Guideline criteria for control levels were not met: (i) mean serum T4 level of 3.65 μg/dL (acceptable range : 4.056–7.376 ); and mean serum T SH level of 2.25 ng/m L (acceptable range: 4. 212–24.112 ).

The following clinical chemistry parameters differed (p<0.05) in the treated groups compared to controls: (i) 2% increase in sodium at 1000 mg/kg/day; (ii) 32 and 42%

decreases in alkaline phosphatase at 500 and 1000 mg/kg/day , respectively ; (iii) 20%

increase in blood urea nitrogen at 1000 mg/kg/day; (iv) 7 and 16% increases in total protein at 500 and 1000 mg/kg/day , respectiv e ly ; and (v) 9% increase in albumin at 1000 mg/kg/day (↑9%). These findings were considered equivocal or not adverse due to lack of corroborating evidence of toxicity and/or slight magnitude of difference.

In summary, dosing with piperonyl butoxide at ≥ 500 mg/kg/day resulted in decreases in LABC weight, serum testosterone levels, and thyroid follicular cell height. In addition, at a dose of 1000 mg/kg/day, decreases were also observed in final body weight, body weight gain, weight of the seminal vesicle s with coagulating gland (with/without fluid), and thyroid

colloid area.Male Pubertal Assay (2011) / Page 16 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None



## C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• %C V=7.1 for age at PPS (top o f acceptable range: 5.67)

• %CV=18.7 for body weight at PPS (top of acceptable range: 7.57)

• %CV=11.3 for final body weight (top of acceptable range: 7.47)

• weaning weight (PND 21) was not reported

• mean serum T4 level of 3.65 μg/dL (acceptable range: 4.056–7.376)

• mean serum TSH level of 2.25 ng/mL (acceptable range: 4.212–24.112)

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: O CSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618807)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Scott D. Studenberg

, Ph.D., D.A.B.T. Date: 4/05 /2012

Secondary Reviewer : Signature: Michelle Sharpe

- Kass , M.S. Date: 4/13 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/17/ /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/17 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### PI P ERONY L B TOX ID E / 06750 1



## P r i m ary R ev iew e r: Yung 

## G. Yang

. Ph.D.



## Ri s k A ss e ss m e n t Br a n c h VI

, He a lt h E f fec t s D i v i s i on
Se condary Revi e wer : John Liccione. Ph

. D.



## H e a lth E ffect s D i v i s ion Steroi d ogene s i Assay (20 11 ) I P age I of 1 9

O CS PP 890. 1 550 / OEC D None



## D A T A E V ALUAT I O N R EC ORD

STU D Y TYP E : Steroidogenesis Assay ( H 295R Cells); OCSPP 890.1550



## PC C OD E: 06750 I 

## DP B A R C OD E : 0398558



## T X R #: 0056259 

## C A S N o.: 51

- 03-6



## TES T MATE R IAL ( P U RI TY): Piperon yl Butoxid e (93.8% a.i.)



## SYN O N Y MS : 5-[[2-(2-butoxyethoxy)ethoxy]methyl]

- 6-propyl-l .3-benzodioxole; 

## PBO

C I TAT IO N : Wagner, 

## H

. (2011). 

### Piperonyl butoxide: H295R steroidogenesis assay.
CeeTox. I nc

.. Kalamazoo

, 

## M l. Laboratory Study 

## No. 9146Y-100377STER,

November 17, 2011. MRJD 48618807

. Unp u b li shed

.



## S PO NS OR: Piperonyl Butoxide Task Force 

## I L c / o Consumer Specialty Products

Assoc iati on. Inc.

, 1900

- 

### l 7 111 

### S tr ee t NW. Suite 300. Washington, DC.



## TES T ORD E R #: CO -067501-9



## EXECUTIVE SU MM A RY : [n a steroidogenesis assay (MRID 48618807). H295R cells

cu ltured 

## in vitro in 24-well plates were incubated with piperonyl butoxide

, 

## (PBO; 93.8% purity.

Batch #E 104 / JO) at log concentrations from 0.0001to100 μM in trip li cate fo r 48 h ou r s. Dimethyl sulfoxide (D M SO) was used as the vehic l e

. at a final concentration of 0

. 05%

.

Testosterone and cstradiol concentrations we r e measured by HPLC / MS-MS with positive ion multiple reaction monitoring. Four indep endent expe riments were performed. A Quality

Contro l (QC) plate was run concurrently with each independent run of a test chemica l plate LO



### demonstrate that the assay responded properly t o positive control agents at two conce n tration
levels; positive controls included the known inhibitor ( prochloraz ) and inducer (forskolin) of

estrad iol and testo s teron e product i on.

Laboratory proficienc y t es tin g was not conducted in the curre n t st u dy

. The ame n dment sta t ed

that the laborator y proficiency data was previously dctem1ined for thi s batch or cells; however,

details of a previous proficiency determination. including estimates of EC ;;o s fo r prochloraz and

forskoli n were not included in Lhe study report. Data from the highest tested PBO concentration.

I 00 μM. were not evalua t ed due to precipitation that was observed post

- expos ure.

All guide line acceptability requirements were met

, including lack of cytotoxicity, adequate production of testosteron e and estradiol, acceptab l e r eproducibi li ty (low %CY), and approp r iate

induction and in h ib i tion w i th pos i t i ve controls

.Steroidogenesis Assay (2011) / Page 2 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



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

test runs after incubation with PBO

. However, f our 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 μ M. The remaining PBO concentrations in Run 4 (0.0001 to 1 μ M) exhibited statistically significant decreases (0.78- to 0.89- fold) in estradiol production, but these

de creases 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.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Steroidog enesis a ssay (OCSPP 890.1550).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Steroidogenesis Assay (2011) / Page 3 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: Kalamazoo, MI Study Director: H. Wagner

Other Personnel: C. Toole, Director of Project Management K. Rutherford, Director of laboratory Operations D. Blakeman, Senior Scientist B. Wallace, Lead Cell Culture Scientist S. McColley, Scientist

C. H aines, Scientist F. Wong, Scientist J. Burgam, Associate Scientist

L. Blakeman, Associate Scientist Study Period: May 23, 2011 to November 17, 2011



## 2

. Test S ubstance : Piperonyl Butoxide (PBO)

Description: T echnical Grade, pale yellow oily liquid

Batch #: (Expiration Date) E104/10 (retest date: July 2012) Purity: 93.8 % Solubility (in Solvent): Soluble in DMSO up to 200 mM Volatility: Not reported Stability: Stability in test preparations was not conducted. Storage conditions: Ambient

CAS #: 51

- 03

- 6 Molecular weight: 338.43

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Positive C ontrol (Inducer) : Forskolin

Description: White powder, 410.50

Source: Sigma Aldrich, St. Louis, MO ( catalog # not reported)

Lot #: (Expiration Date) : 097K50653V and 109K50571V (July, 2016) Purity: 99% and 98%, respectively Solubility (in Solvent): Soluble in DMSO up to 100 mM Storage conditions: Room temperature

CAS #: 66575

- 29

- 9



## 4. Positive C ontrol (Inhibitor) : Prochloraz

Description: White powder, 376.67

Source: Sigma Aldrich, St. Louis, MO (catalog # not reported)

Lot #: (Expiration Date) SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in Solvent): Soluble in DMSO up to 100 mM Storage conditions: Room temperature

CAS #: 67747

- 09

- 5Steroidogenesis Assay (2011) / Page 4 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## 5

. Solvent/ V ehicle C ontrol : Dimethyl Sulfoxide ( DMSO )

Description: Not reported

Source: Sigma Aldrich, St. Louis, MO (catalog # not reported) Lot #: (Expiration Date) RNBB7617 (expiration date not reported) Purity: Not reported Storage conditions: Not reported

CAS #: Not reported

Justification for choice of

solvent: Not reported ( PBO , forskolin, and prochloraz were soluble at test concentrations)

Final concentration: (% volume in assay) 0.05% (v/v)



## 6. Solvent/Vehicle Control : Ethanol

Description: Not reported

Source: Not reported Lot #: (Expiration Date) Not reported Purity: Not reported Storage conditions: Not reported

CAS #: Not reported

Justification for choice of

solvent: Not reported (22R

- hydroxycholesterol soluble at test concentrations)

Final concentration: (% volume in assay) 0.025% (v/v)



## 7

. Other Materials : 22R

- Hydroxycholesterol

Description (molecular weight) : White powder (402.65)

Source: Sigma

- Aldrich (St. Louis, MO) Lot # (expiration date): 089K4132, 060M4098 (not provided) Purity: 99.0% Storage conditions: Room temperature

Solvent used: Ethanol, final concentration in assay, 0.025% v/v

CAS #: 17954

- 98

- 2



## 8

. Stock Medium : Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/Ham

- F12)

Description: Included 15 mM HEPES

Source: Not reported Lot / Batch #: (Expiration Date) Not reported

Sodium bicarbonate: Not reported

Nu

- Serum: Becton Dickinson, Catalog #355500, Lot #81515; tested for background hormone concentrations by performing laboratory ITS+ premix: Lot number (not reported)



## 9. Test Cells : H295R human adrenocortical carcinoma cells (ATCC , Manassas, VA, CLR2128; L ot #7635054 ). Cells were maintained in DMEM/Ham -F12 with 15 mM HEPES, sodium bicarbonate, ITS+Premix, and 2.5% Nu-Serum in a 5% CO 2 

## incubator at 37±2 oC. Cells were initially grown for five passages, and then frozen in liquid nitrogen. After thawing, cells were cultured additionally for seven or eight passages prior to use in the steroidogenesis assay. 22 R-Hydroxycholesterol (10 μ M) was added to the culture medium at plating, dosing, and harvesting. Cells were plated into wells of 24

- well culture plates at a density of approximately 3 × 10 5 

## cells/mL. Plates were incubated in a 5% CO2 atmosphere

at 37 ±2 oC for approximately 24 hours prior to exposure to the test chemical or the positive

controls.

The following performance criteria were met (indicated by an “x”):

X Cell passage identifier . Cell Passage # : ≥ 7.5

X Cells frozen down at passage 5Steroidogenesis Assay (2011) / Page 5 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None

X Frozen cells cultured for 4 additional passages Total number of passages does not exceed 10 (not reported)



## B. METHODS

1. Pre -T est Information

a. Hormone Assay I nterference T est : Hormone cross reactivity or interference tests were not conducted because testosterone and estradiol concentrations were determined by

HPLC/MS

- MS analysis; no interference was expected.

b. Hormone Extraction : Testosterone and estradiol were extracted from H295Rsupplemented medium by liquid/liquid extraction with methyl tert -butyl ether spiked with
[ 2 

## H 5 

## ]-testosterone and [ 2 

## H 5 

## ]- estradiol as internal standards. The extracts were analyzed using HPLC /MS

- MS with positive ion multiple reaction monitoring. T he method MDL s for testosterone and estradiol were 100 and 10 pg/mL, respectively, for a 300-μL portion of

medium.

c. Laboratory P roficiency T est : Laboratory proficiency testing w as not conducted for the current study, because laboratory proficiency data was reported as previously determined for this

batch of cells.

2. Test Solutions : A 200-mM stock of PBO was prepared in DMSO, followed by serial dilutions (1:10) in DMSO. A 40 -mM stock of 22R-hydroxycholesterol was dissolved in ethanol and further diluted in supplemented medium to achieve a final concentration of 10

μ M. Mastermix solutions with PBO were prepared by 1:2000 dilutions in supplemented medium containing 10 μM 22R- hy droxycholesterol. Forskolin and prochloraz solutions were prepared in a similar manner by preparing 100 mM solutions in DMSO, followed by serial dilutions in DMSO and 1:2000 dilutions in supplemented medium containing 10 μ M 22R-hydroxycholesterol. Final concentrations after addition to the 24- well culture plates were 1 and 10 μM for forskolin, 0.1 and 1 μM for prochloraz, and 0.0001 to 100 μ M for

PBO . Final concentrations of DMSO and ethanol in the medium were 0.05% and 0.025% (v/v), respectively.

3. Cell Plating and P reincubation : H295R cells (ATCC CLR -2128) were initially grown for five passages, then frozen in liquid nitrogen. After thawing, cells were cultured additionally for seven or eight passages prior to use. 22-Hydroxycholesterol (10 μ M) w as added to the culture medium at plating, dosing, and harvesting. Cells (1 mL) were seeded into wells of

24- well culture plates at a density of approximately 3 × 10 5 

## cells/mL. Plates were incubated

in a 5% CO 2 

## atmosphere at 37±2oC for approximately 24 hours prior to exposure to the test chemical or the positive controls. The percent confluency after 24-hour incubation was not reported , but the protocol stated that 50-60% confluency was expected after plating

2.5 × 10 5 

## cells/mL followed by a 24 -hour incubation.

4. Exposure : The cells were checked microscopically for good attachment and proper morphology, and the medium was removed and replaced with 1 mL of supplemented medium containing 10 μM 22R-hydroxycholesterol. The cells were then exposed to

identical volumes of either each serial dilution of the test compound or DMSO (SC) in triplicate according to the schematic presented in Table 1.Steroidogenesis Assay (2011) / Page 6 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



### TABLE 1

. Dosing S chematic for the E xposure of H295R C ells to Piperonyl Butoxide ( F inal

C oncentrations in μM) a

1 2 3 4 5 6

A DMSO DMSO DMSO 0.1 0.1 0.1

B 100 100 100 0.01 0.01 0.01

C 10 10 10 0.001 0.001 0.001

D 1 1 1 0.0001 0.0001 0.0001

a Data were obtained from page 15 of the study report. Dosing was calculated based on a total volume of 1 mL per well.



## A concurrent quality control (QC) plate was included with each of the four independent runs of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol production). The QC plate was prepared and dosed in the same manner with either

forskolin or prochloraz according to the schematic presented in Table 2.

TABLE 2

. Dosing S chematic for the QC P late for P ositive C ontrols ( F inal C oncentrations in μM) a

1 2 3 4 5 6

A Blank b Blank Blank B ackground c Background Background

B DMSO DMSO DMSO DMSO + MeOH d DMSO + MeOH DMSO + MeOH

C Forskolin 1μM e Forskolin 1μM e Forskolin 1μM e Prochloraz 0. 1μM Prochloraz 0.1μM Prochloraz 0.1μM

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM

a Data were obtained from page 15 of the study report. Dosing was calculated based on a total volume of 1 mL per well.

b Blank wells received containing 10μM 22R

- hydroxycholesterol.

c Background wells received media without 22R

- hydroxycholesterol.

d MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal.

e In Run 1, the wells for 1μM forskolin were dosed with 3.33 μM forskolin; Runs 2

- 4 contained 1μM forskolin.



## Following dosing, the plates were incubated for 48±2 hours under the conditions described previously. After the incubation period, each well was examined microscopically, and images w ere taken of the solvent control wells and the two greatest non-cytotoxic concentrations. Precipitation, if present, was noted. The medium from each well was removed, split into two equal volumes, and frozen at −80 ±10°C until hormone

measurements were conducted using HPLC/MS

- MS

. Cell viability was determined after media removal was completed.

5. Cell V iability/ Cytotoxicity A ssay : Cell viability was determined with the MTT [3 -(4,5- dimethylthiazol -2- yl) -2,5- diphenyl tetrazolium bromide] test desc ribed by Mosman (1983) 1


## immediately after removal of the culture medium after the 48 -hour incubation. The QC plate wells designated to receive methanol (MeOH) were rinsed twice with phosphate buffered saline ( PBS ), followed by an incubation with MeOH for 30 minutes at room temperature, and three additional rinses with PBS. Next, 0.5-mL portions of a 0.5 mg/mL MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol were added to each well on the test chemical and QC plates. MTT

- treated plates were incubated

in a 5% CO 2 

## atmosphere at 37±2oC for 3 hours, followed by removal of the MTT solutions

1 Mosman T. (1983). Rapid colorimetric assay for growth and survival: application to proliferation and cytotoxicity. J. Immunol. Methods . 100:45

- 50.Steroidogenesis Assay (2011) / Page 7 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## and addition of 0.5 mL of isopropanol to each well. Plates were incubated for a further 20 minutes at room temperature with shaking. After the 2 0- mi nute incubation, the absorbance

values at 570 and 650 nm were determined with a Packard Fusion plate reader, and the

absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT

value. A reduction of ≥20% in cell viability wa s considered evidence of cytotoxicity.

6. Hormone M easurement S ystem : Concentrations of testosterone and estradiol in the supplemented medium were determined by HPLC- MS/MS at a separate analytical facility (OpAns, LLC, Durham , NC). The extraction method (with spiked, labeled internal standards) was described previously (B.1.b.). All back

- calculated calibration standard concentrations, except for one 10 pg/mL standard measurement, and QC concentrations were acceptable. The percent r ecovery across the test runs for the QC samples ranged from

91.5-105.4% for testosterone and 87.4- 106.3% for estradiol.

The following performance criteria were met (indicated by an “x”) :

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount from triplicate samples within 30% of nominal concentration)

NA Hormone cross

- reactivity (antibody

- based assays only; ≤30% of basal production of the respective hormone)

NR Solvent control within 75% range below maximum response on standard curve

NA Test compound tested for interference with measurement system



## C. DATA ANALYSIS : Mean values (pg/mL) and standard deviations (SD) for testosterone

and estradiol concentrations were calculated for each concentration of the reference chemicals and SC, as well as the blank and background wells, on the QC plates and for each

concentration of PBO and SC on the test chemical plate. Relative changes in hormone production were calculated with the following equation:

Relative change = (hormone concentration in each well) ÷ (mean SC hormone concentration)

For forskolin induction of testosterone, the background hormone production was subtracted from the forskolin-treated wells, and blank and SC wells, prior to calculation of the relative change. Background hormone production was determined from three wells on the QC plate

that received H295R cells with no 22R-hydroxycholesterol.

Concentrations that exhibit ed ≥ 20% c ytotoxicity , or where precipitation was observed, wer e omitted from further analysis

.

Normality of the data was evaluated with the Shapiro -Wilk’s test. Homogeneity of the variances between the treatment groups was evaluated by using Levene’s test. S tatistical significance between each treatment group and control was evaluated with Dunnett ’s test if p>0.05 in both tests . If p ≤ 0.05 in the normality or homogeneity tests, a log transformation was performed on the data to attempt to approximate a normal distribution. Dunnett’s test was conducted to evaluate statistical significance between th e treatment and control groups if p>0.05 in both the normality and homogeneity tests after transformation. If p ≤ 0.05 in either the normality or homogeneity tests after transformation, the non

- transformed data were analyzed with the nonparametric Kruskal -W allis test, followed by Dunn’s test, toSteroidogenesis Assay (2011) / Page 8 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## evaluate statistical significance between the tre atment and control groups. The se analyses are considered acceptable

.

II. RESULTS

A. TEST COMPOUND : The hormone concentrations after exposure to PBO , and the fold

difference change relative to SC and mean ± SD for the 4 assay runs, are presented in Table 3. The fold-changes in hormone concentration for PBO in the four runs are shown graphically in Figures 1 – 4 for testosterone and Figures 5 – 8 for estradiol. Data from the highest tested PBO concentration, 100 μM, were not evaluated for induction or inhibition due to precipi tation that was observed post-exposure.

The % CVs for absolute testosterone concentrations in the SC replicates within the test plates ranged from 0.88-5.93%, and met the ≤ 30% performance criteria recommended in the test guideline. Similarly, the % CVs for absolute estradiol concentrations in the SC replicates within the test plates ranged from 1.50 -3.34%. Between plate % CV for SC bas ed

on absolute hormone concentrations in all four test runs ( calculated by the reviewer s) was 13.7% for testosterone and 25.4% for estradiol.

No statistically significant effects on testosterone production were observed in Runs 1 through 3 after incubation with PBO . Four of the six evaluable concentrations exhibited statistically significant decreases in testosterone production in run 4, but these decreases

were not dose related and were not considered an effect of PBO exposure. Statisticallysignificant increases in estradiol production were observed in Runs 1, 3, and 4 (with a nonsignificant increase in Run 2) at the concentration of 10 μM. The remaining concentrations in Run 4 (0.0001 to 1 μ M) exhibited statistica lly significant decreases in estradiol production (similar to those observed with testosterone) ; h owever, t hese non -dose- related

effects were not considered an effect of PBO exposure since no effect was observed in previous three runs

TABLE 3

. Mean (± SEM ) Hormone Concentrations Following Treatment with Piperonyl Butoxide for

48 a 

### Hours. a

Nominal

Concentration (μM) Run 1 Run 2 Run 3 Run 4 Run 1 Run 2 Run 3 Run 4 Mean b ± S D b

Testosterone (pg/mL) Fold Difference
DMSO (SC) 2533 2655 1961 2676 1.00 1.00 1.00 1.00 1.00

-- 0.0001 2458 2647 1891 2434 0.97 1.00 0.96 0.91* 0.98 0.0 2

0.001 2507 2623 1864 2357 0.99 0.99 0.95 0.88* 0.98 0.0 2

0.01 2441 2566 1932 2331 0.96 0.97 0.99 0.87* 0.97 0.0 2

0.1 2684 2673 1952 2498 1.06 1.01 1.00 0.93 1.02 0.0 3

1 2498 2712 2015 2429 0.99 1.02 1.03 0.91* 1.01 0.0 2

10 2552 2996 2030 25 83 1.01 1.13 1.03 0.97 1.06 0.0 6

100 2019 2065 2066 2295 NA NA NA NA NA NA

Estradiol (pg/mL) Fold Difference
DMSO (SC) 164 255 160 252 1.00 1.00 1.00 1.00 1.00

-- 0.0001 160 231 152 198 0.97 0.91 0.96 0.78* 0.95 0.0 3

0.001 163 235 156 201 0.99 0.92 0.98 0.8* 0.96 0.0 4

0.01 157 237 160 199 0.96 0.93 1.00 0.79* 0.96 0.0 4Steroidogenesis Assay (2011) / Page 9 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



### TABLE 3

. Mean (± SEM ) Hormone Concentrations Following Treatment with Piperonyl Butoxide for

48 a 

### Hours. a

Nominal

Concentration (μM) Run 1 Run 2 Run 3 Run 4 Run 1 Run 2 Run 3 Run 4 Mean b ± S D b

0.1 165 239 163 224 1.01 0.94 1.02 0.89* 0.99 0.0 4

1 167 269 175 216 1.02 1.06 1.1 0.86* 1.06 0.0 4

10 214 369 207 281 1.31* 1.45 1.30* 1.11* 1.35 0.0 8

100 167 252 212 226 NA NA NA NA NA NA

a Data were obtained from page 27

- 28 and 37

- 40 of the study report.

b Mean and SD were calculated by the reviewers.

NA Not applicable ; fold change not determined due to the presence of precipitation post

- exposure. SC Solvent control

* Statisti cally significant (p ≤ 0.05)



## F IGURE 1 . Change in Testosterone P roduction Relative to PBO Concentration in

Test Run 1

.Steroidogenesis Assay (2011) / Page 10 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 2. Change in Testosterone Production Relative to PBO Concentration in

Test Run 2.

FIGURE 3. Change in Testosterone Production Relative to PBO Concentration i n Test Run 3.Steroidogenesis Assay (2011) / Page 11 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 4. Change in Testosterone Production Relative to PBO Concentration in

Test Run 4.

* Significantly different from the solvent control at p≤0.05.



## FIGURE 5. Change in Estradiol Production Relative to PBO Concentration in

Test Run 1.

* Significantly different from the solvent control at p≤0.05.Steroidogenesis Assay (2011) / Page 12 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 6. Change in Estradiol Production Relative to PBO Concentration in

Test Run 2.

FIGURE 7. Change in Estradiol Production Relative to PBO Concentration in

Test Run 3.

* Significantly different from the solvent control at p≤0.05.Steroidogenesis Assay (2011) / Page 13 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 8. Change in Estradiol Production Relative to PBO Concentration in

Test Run 4.

* Significantly different from the solvent control at p≤0.05.



## B. CYTOTOXICITY : No evidence of cytotoxicity was present from the MTT cell viability assay conducted on the QC and PBO test plates (Table 4 and Figures 9-12 ). Cell viability values ranged from 93.5-109.3% on the QC plates and 84.7-102.1% on the test chemica l plates; methanol exposure reduced cell viability to 5.1-11.4%.Steroidogenesis Assay (2011) / Page 14 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



### T ABLE 4 . Mean (±SD) MTT Cell Viabilit y Results after Treatment with F orskolin, Prochloraz, or Piperonyl Butoxide for 48 Hours. a

Compound Concen. (μ M) Cell Viability

– Run 1 Cell Viability

– Run 2 Cell Viability

– Run 3 Cell Viability

– Run 4

Mean SD Mean SD Mean SD Mean SD

Blank NA 100.4 4.97 104.3 2.69 104.3 2.81 100.7 3.98
Background NA 94.7 2.09 95.4 4.14 102.5 1.79 98.9 0.94

SC + Methanol NA 9.1 0 1.98 5.6 0 2.51 11.4 1.99 5.1 0 0.14

Forskolin b 1 101.9 1.19 107.8 1.52 109.3 1.17 105.0 1.55

Forskolin 10 97.5 4.35 107.0 2.43 108.5 4.17 102.4 2.58

Prochloraz 0.1 93.5 1.49 98.3 2.20 101.2 0.47 100.7 0.86

Prochloraz 1 94.0 3.07 99.4 1.19 101.5 0.78 99.5 2.37

PBO 0.0001 96.0 1.75 95.4 4.68 94.2 2.14 100.6 0.88

PBO 0.001 96.5 2.79 96.7 1.20 96.8 1.31 99.1 1.82

PBO 0.01 95.3 1.99 97.0 4.19 97.8 0.97 99.5 1.91

PBO 0.1 99.0 2.58 102.1 4.57 98.4 2.14 100.2 1.07

PBO 1 96.8 1.41 99.1 2.27 100.1 5.33 96.7 4.11

PBO 10 97.2 0.35 97.6 2.19 97.8 1.42 99.8 1.42

PBO 100 86.9 16.44 84.7 17.89 100.6 5.58 91.7 3.36

a Data were obtained from page 21 -22 of the study report.

b Forskolin concentration was 3.33 μM in Run 1.

NA Not applicable

SC Solvent control



## FIGURE 9 . MTT Cell Cytotoxicity Results From Test Run 1.Steroidogenesis Assay (2011) / Page 15 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 1 0 . MTT Cell Cytotoxicity Results From Test Run 2.

FIGURE 1 1 . MTT Cell Cytotoxicity Results From Test Run 3.Steroidogenesis Assay (2011) / Page 16 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## FIGURE 1 2 . MTT Cell Cytotoxicity Results From Test Run 4.

C. QC PLATE : The hormone concentrations after exposure to the reference chemical, SC, blank, and background samples, as well as the fold- difference change relative to SC (individual and mean ± SD) for the 4 assay runs, are presented in Table 5. Mi nimum basal hormone production was met in all blank and SC wells (500 pg/mL for testosterone, 40 pg/mL for estradiol) , and it met the b asal hormone production increase criteria in SC of
≥5

- fold for testosterone and ≥2.5

- fold for estrogen a bove the MDL of the assay

. The medium was supplemented with 10 μM 22R-hydroxycholesterol to ensure that estradiol production met the minimum guideline levels. Exposure to 10 μM forskolin induced testosterone production 1.3- to 1.5-fold, which is below the Guideline recommendation of
≥2 .0- fol d; however, when the background levels are subtracted 10 μM forskolin induced testosterone production by 2.1- to 2.6- fold . E stradiol production was induced by 10 μM

forskolin ≥7.5 -fold ( actual 9.3- to 13.1- fold) over SC. Exposure to 1 μM prochloraz

inhibited synthesis of testosterone and estradiol by ≥50% ( actual 51-73% and 55-71%, respectively) compared to SC.

The % CVs for absolute testosterone concentration in SC well replicates within the QC plates ranged from 0.83-5.44%, and t he % CVs for absolute estradiol concentration in SC well replicates within the QC plates ranged from 2.90 -6.16%. The between- plate variability data for the QC plates, based on the absolute hormone concentrations in the SC wells, yielded % CVs of 12.3% for testosterone and 15.8% for estradiol. These data me t the performance criteria recommended by the Guideline ( ≤ 30%).Steroidogenesis Assay (2011) / Page 17 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



### TABLE 5 . Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours. a

Compound Conc. (μM) Run 1 Run 2 Run 3 Run 4 Run 1 Run 2 Run3 Run 4 Mean b ± S D b

Testosterone (pg/mL) Fold Difference

Background NA 1056 1135 1073 1262 0.49 0.45 0.57 0.54 0.51 0.0 5

Blank NA 2174 2581 1873 2304 1.01 1.03 1.00 0.99 1.01 0.0 2

DMSO NA 2143 2511 1874 2339 1.00 1.00 1.00 1.00 1.00

-- Forskolin c 1 2930 3764 2595 3120 1.37 d 1.50 d 1.38 d 1.33 d 1.40 d 0.0 7

Forskolin 10 3349 4751 3019 3780 1.56 e 1.89 e 1.61 e 1.62 e 1.67 e 0. 15

Prochloraz 0.1 1914 1816 1149 1818 0.89 0.72 0.61 0.78 0.75 0. 12

Prochloraz 1 1009 1188 501 1153 0.47 0.47 0.27 0.49 0.43 0. 10

Estradiol (pg/mL) Fold Difference
Background NA 72 41 56 77 0.47 0.21 0.31 0.35 0.34 0. 11

Blank NA 158 208 182 228 1.04 1.04 1.01 1.02 1.03 0.0 2

DMSO NA 153 200 179 223 1.00 1.00 1.00 1.00 1.00

-- Forskolin 1 1787 1343 1254 1538 11.69 6.72 7.01 6.91 8.08 2.41

Forskolin 10 2002 2116 1661 2477 13.10 10.60 9.28 11.13 11.03 1.59

Prochloraz 0.1 148 133 104 171 0.97 0.67 0.58 0.77 0.75 0. 17

Prochloraz 1 68 57 52 83 0.45 0.29 0.29 0.37 0.35 0.0 8

a Data were obtained from page 23 and 24 of the study report.

b Calculated by the reviewers.

c Forskolin concentration was 3.33 μ M in Run 1.

d When the background is subtracted, the values are 1.73, 1.91

- , 1.90

- , and 1.73

- fold difference for Runs 1, 2, 3, and 4, respectively, with a mean of 1.8 2- fold difference.

e When the background is subtracted, the values are 2.11, 2.63

, 2.43 , and 2.34

- fold difference for Runs 1, 2, 3, and 4

, respectively, with a mean of 2.38

- fold difference.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Exposure to PBO yielded no statistically significant effects on testosterone production in three out of four test runs; non-dose- related statistically significant decreases were observed in the fourth run at PBO concentrations of 0.0001, 0.001, 0.01, and 1 μM, but not 0.1 and 10 μ M. These changes were consider ed to

be random effects unrelated to test chemical treatment. Exposure to PBO yielded statistically significant increases to estradiol production in three out of four test runs at the highest evaluable concentratio n (10 μ M). Similarly, non -dose- related statistically significant decreases were observed in the fourth run at PBO concentrations of 0.0001 to1 μM, but not at 10 μ M. These decreases were consider ed to be random effects unrelated to

test chemical treatment

.

B. AGENCY COMMENTS : Laboratory proficiency testing w as not conducted in the current study because proficiency data was reportedly generated previously for the batch of cells used in this study. Howeve r, t he details and results of this previous proficiency test

, including estimates of EC 50 

## s for prochloraz and forskolin, were not included in the current study report.

Four independent test runs of the steroidogenesis assay with PBO were conducted. Although samples from only thre e of the four runs were initially analyzed, a difference in the data from one of the runs resulted in analysis of the fourth run and presentation of all of

the test run data. Due to precipitation that was observed after the 48-hour incubation in

wells con taining 100 μ M PBO , the highest concentration of PBO in the assay deemedSteroidogenesis Assay (2011) / Page 18 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## suitable for evaluation was 10 μM. No cytotoxicity was evident at any of the test chemical

concentrations us ed in the current assay

.

The QC plate results met the m inimum basal hormone production in all blank and SC wells ( ≥ 500 pg/mL for testosterone, ≥ 40 pg/mL for estradiol), and met the basal hormone production criteria in SC of ≥5 -fold increases for testosterone and ≥2.5

- fold increases for estrogen above the MDL of the assay. The cell medium was supplemented with 10 μ M 22R-hydroxycholesterol. Exposure to 10 μM forskolin induced testosterone production 1.3-

to 1.5- fold, which is below the Guideline recommendation of ≥2.0 -fold; however, when the background levels ar e subtracted 10 μM forskolin induced testosterone production by 2.1- to 2.6-fold. Estradiol production was induced by 10 μM forskolin ≥7.5 -fold ( actual 9.3- to 13.1-fold) over SC. Exposure to 1 μM prochloraz inhibited synthesis of testosterone and

estrad iol by ≥50% ( actuals 51-73% and 55-71%, respectively). The % CVs for absolute

testosterone concentration in SC well replicates within the QC plates ranged from 0.83- 5.44%, and the % CVs for absolute estradiol concentration in SC well replicates within the QC plates ranged from 2.90-6.16 %. The between

- plate variabilit y data for the QC plates

, based on the between plate % CVs for the absolute hormone concentrations in the SC wells

, yie lded % CVs of 12.3% for testosterone and 15.8 % for estradiol (calculated by the reviewers). These data meet the performance criteria recommended by the Guideline.

The % CVs for absolute testosterone concentration in SC replicates within the test plates ranged from 0.88-5.93%, and the %CVs for absolute estradiol c oncentration in SC replicates within the test plates ranged from 1.50-3.34%. Between- plate % CVs for SC based on the

absolute hormone concentrations in the SC, were 13.7% for testosterone and 25.4% for estradiol (calculated by the reviewers). These data meet the recommended performance

criteria of ≤ 30%.

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

four test runs after incubation with PBO . In the fourth run, four of the six suitable

concentrat ions exhibited statistically significant decreases in testosterone production, but

these decreases (0.87- to 0.91

- fold) were not dose related and are not considered an effect of

PBO exposure. Statistically significant increases in estradiol production wer e observed at the highest evaluable PBO concentration, 10 μM, in three test runs (1.11- to 1.31-fold), with

a non- significant increase of 1.45- fold the fourth test run. The remaining concentrations in Run 4 (0.0001 to 1 μ M) exhibited statistically signifi cant decreases (0.78- to 0.89-fold) in estradiol production, but are not considered an effect of PBO exposure.

T he results of the steroidogenesis assay with PBO indicate that PBO is neither an inducer nor an inhibitor of testosterone synthesis in this assay. The results of this assay also indicate

that , although PBO is not an inhibitor of estradiol synthesis, it may be an inducer of estradiol production at concentrations at ≥ 10 μ M. This was not confirmed at the next highest concentration (100 μ M) due to precipitation in the test wells after incubation.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• Laboratory proficiency testing prior to the initiation of the current study was not conducted, and the details of a previous proficiency determination with the currentSteroidogenesis Assay (2011) / Page 19 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None



## batch of cells, including estimates of EC 50 

## s for prochloraz and forskolin, were not reported in the study report.

• Although it was stated that cells were thawed after being frozen down at passage 5, and cultured for an additional 7 or 8 passages, the specific cell passage identification

for each test run was not provided in the study report.

# DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: O CSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-32-2012 (MRID 48618808)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Sandra Hastings Date: 4 / 4 /2012

Secondary Reviewer : Signature: Michelle Sharpe

- Kass , M.S. Date: 4/13 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/13 /2012

Quality Assurance: Signature:
Steven Brecher, Ph.D., D.A.B.T. Date: 4 / 1 3 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).P IP ERONY L B TOXIOE / 06750 I In vfro U ter o tropbi c A ssay ( 20 11 ) I P age I or9

O CS PP 890. 1 60 0 / OE C D 440



## Primary Revi e wer : Yung G. Yang, Ph

. D. 

## S ign at ur e :

# V'~ G 

# <r/-a

Risk Assess m e nt Bra n c h VI

, Health E ff ects Divi s ion Date: 

## r/,,1/go1'.<'

Seco ndary Review e r:

_ .....; J :;...;:e=s=-s-=- R

=o'""'"w .:....:cl =a n ;.:..;

d""--

____ _ _ _ __ 

## S i gnat ur e:**~a..;.~** 

## Healt h E ff ec t s Divi sio n Date: 5:1J..7 I1 )
Template ve r s i on 09 / 20 11



## DAT A EVA L UA TION RE C ORD

STU DY TYPE: Uterot rophic Assay ( Rat): OCSPP 890.1600; OECO 440



## P C C OD E : 06750 I 

## DP BARCODE: 0 398558



## TXR# : 0056259 

## CAS# : 5 1-0 3

- 6



## TEST MATERIAL (PURITY): Pip e ronyl butoxide (93 .8 % a.i. )



## SYN O NYMS: 5

- [2- ( 2

- butoxyethoxy)ethoxymethyl] -6-pr opyl

- J 

## ,3 -b enzodioxo le



## C ITATIO N: Zo rrill a

. L. 

## (20 11 ). Th e U terotrophi c Assay for Piperon y l Bu t oxide ( PBO ). Integrated Laboratory Syste m s

. Inc.

, Durham

, NC. Laboratory Project Study No.:

C l 92 -IOO. Nove mber 18

, 201 J 

## ( re v i se d r eport dat e)

. MRfD 48618808. Unpublished

.



## SP O NSO R : Consumer S pecialty P roducts Assoc i a tion

, Inc

. For Pip e ronyl Butoxid e Ta s k

Force II. 1 667 K S treet NW

. S uite 300. W as hingt o n
, D. C.



## TE S T O RD E R #: CON-06750 1- 9



## EXECUTIVE SUMMARY: In a Uterotroph i c Assay ( MRlD 486188 0 8) conduc t e d to sc reen for pot ent ial estrogenic acti v i ty, piperon y l butoxide (9 3.8 % a .i.

, Batch # E l 04 / 10 ) in corn oil was ad mini s t e red da il y via oral gavage t o gro up s of e i ght ovar i e c t omized fe mal e Sp ragu e Dawl ey

rat s at dose l eve ls of 0 (veh icle )

. 320 or I 000 (limit dose) mg/k g/ da y o n post-natal days ( P N D )

58-60

. A po s itive control g roup was treated w ith a da il y dose of I 7a- et h yny l es tradiol (EE) at
0 .1 m g/ kg / da y by oral gavage. B o d y we ight s were determined daily

. All animals were terminated and necrop s ied o n P N D 61 a pproximat e l y 24 h o urs after the final dose admini s tration

to determine wet and blott e d uterine w e i g ht s.

A ll animals s urvived until sc h edu l ed termination. Two animals adm i ni s tered piperonyl butoxicl e

a t 320 mg/kg ex hibit ed abnonnal clinical s i gns ( unco o rdin a ted m ove m e nt s or l e thargy) during

d os ing

, but all anima l s wer e noted as normal p ri or t o te rm ination. A ll a nim a l s dosed with IOOO

m g/ kg piperon yl but ox id e gro up e xhibit ed abnormal clin i ca l s ign s ( de creased o r un coo rd inated

movements, hunch ed posture, lethargy

, a n d/o r nas a l d i sc h arge) by D ay 3 of dosing. At

t e rmination

, all high close animals were not ed as normal

, exce pt for o n e rat which ex hibit ed multipl e s ign s of toxicity.

Bod y weig ht s we r e decrea s ed (p < 0.05) on Da y 4 (l 

## 10 %) in the hi g h do se group com p ared to th e vehicle co ntrol s

. re s ulting in a dec r ease (p<0.05) in ove rall bod y we i ght ga in (BWG: 12 40 %)

. B o dy weights in th e low-dose gro up were comparable to th e ve hi c l e controls throu g hout th e du r a tion of th e assay. S tati s tical s ignificance was not determ in ed for th e bod y we ight s and BWGIn vivo Uterotrophic Assay ( 2011 ) / Page 2 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## of animals in the positive control (EE) group. However, body weights were decreased by Day 4 (↓7%) in the EE group compared to th e 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 chan ges were seen in uterine weight in this assay. Piperonyl butoxide is negative in the uterotrophic assay.

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE : Signed and dated GLP Compliance and Quality Assurance statements were provided.In vivo Uterotrophic Assay ( 2011 ) / Page 3 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratory Systems, Inc. (ILS)

Location: Durham, NC Study Director: L. Zorrilla

Other Personnel: S. Borghoff (Study Toxicologist); P. Sproul (Toxicology Study Manager); A. Glasscock (Animal Facility Operations Manager); T. Hackett (Necropsy Manager); D. Kurtz (Facility Veterinarian); C. Cachafeiro (Health and Safety Officer) Study Period: March 23, 2011 to November 1 8 , 2011



## 2. Test Substance : Piperonyl Butoxide

Description: Technical; oily yellow liquid

Source: Endura Fine Chemicals, Italy

Lot/Batch #: E104/10 (expiry date: July 2012) Purity: 93.8 % a.i. Stability: Stable in corn oil for up to 20 days at 1 to 10 ° C

CAS #: 51

- 03

- 6

Structure:



### O O

O O CH 3



### OC 4 

### H 9



## 3

. Reference E strogen : 17α

- ethynyl estradiol (EE)

Supplier : Sigma

- Aldrich ( St. Louis, MO )

Lot/ Batch #: 090M1241V ( expir y date : February 2012) Purity: > 98 %

CAS # : 57

- 63

- 6



## 4. Solvent/Vehicle Control : Corn oil

Supplier : MP Biomedicals, LLC (Solon, OH)

Lot/Batch #: 7862K Rationale (if other than water) : Corn oil was the vehicle used in oral gavage studies conducted by the Sponsor, including a range finding study conducted by ILS

Final concentration : Not applicable



## 5

. Test A nimals : Species : Rat (ovariectomized)

Strain: Sprague Dawley , Crl:CD ® (SD) IGS Age/weight at dose initiation : 58 days old/ 223.2

– 270.8 g fe males

Source: Charles River Laboratories International, Inc. (Raleigh, NC).

Housin g: Upon receipt animals were maintained 1 animal / cage and after group allocation maintained 2 animals/ cage in polycarbonate cages with micro

- isolator tops and

absorbent heat

- treated hardwood bedding.

Diet : Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison WI) ad libitum Total genistein equivalents (genistein plus daidzein) of 6.2 μg/g of feed.

Water: Reverse

- osmosis filtered tap water , ad libitum

Environmental conditions: Temperature:

Humid ity: Air changes: Photoperiod: 22

- 24 oC

33

- 57 % Not reported

12 hrs light/ 12 hrs dark Acclimation period: 8 daysIn vivo Uterotrophic Assay ( 2011 ) / Page 4 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## B. STUDY DESIGN

1. In L ife D ates : Start: April 8, 2011 End: April 11, 2011

2. Study Design : Sexually mature ovariectomized female rats (ovariectom ized at PND 42 )

were received from Charles River Laboratories (PND 49) . Animals were acclimated for 8 days prior to initiation of dosing. The dose administration period was from PND 58 through

60 . Rats were euthanized approximately 24 hours later on PND 61 and necropsied for uterine weight measurements.

Vaginal smears were conducted for 5 consecutive days immediately preceding dose administration and evaluated for stage of estrous cycle. If an animal indicated evidence of cyclicity, the animal w ould not be used in the study. I n the allocation of animals

, three animals were removed from the study due to the presence of estrus cells durin g pre-dose estrous cycle assessment . An other animal which had estrus cells present on the first day of vaginal smears but no t afterwards was kept in the study and showed no evidence of ovari an tissue present at necropsy

.

3. Animal A ssignment : Animals we re assigned to the test groups noted in Table 1 using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance [ANOVA, Statistical Analysis System (SAS) version 9.1, SAS Institute, Cary, NC].

T ABLE 1. Study Design a



### Test G roup Dose (mg/kg/day) # of Females

Estrogen A gonist A ssay
Vehicle C ontrol (corn oil) 0 8
Piperonyl butoxide Low 320 8
Piperonyl butoxide High 1000 8

17 α

- ethynyl estradiol (EE), R eference E strogen 0.1 8

a Data were obtained from Table 1 on page 15 of the study report.



## 4. Dose S election Rationale : In a range finding study 1 

## , immature female rats were orally dosed with piperonyl butoxide at 200 to 1000 mg/kg (in 200 mg/kg increments) for 14 days. Following three days of dosing, no clinical signs of toxicity or changes in body weights

were observed in animals dosed at ≤ 1000 mg/kg compared to the vehicle controls.

T herefore doses of 1000 and 320 mg/kg/day were used for the uterotrophic study.

5. Dose P reparation and Analysis : Dose formulations in corn oil were prepared once prior to

initiation of treatment

. Dose concentrations and h omogeneity were tested by Smithers Viscient LLC (Wareham, MA) for both formulations (64 and 200 mg/mL) prepared b y ILS prior to dose initiation. Three samples were analyzed (top, middle, bottom ) for each concentration level in corn oil. In addition, middle samples from the 200 mg/mL

1 

### Zorrilla, L. (2011). Range Finder Study for Piperonyl Butoxide (PBO) Tier 1 Endocrine Disrupting Screening

P rogram in vivo Mammalian Assays (draft). Unpublished study report prepared by ILS, Inc. Study No. C192

- 500

.In vivo Uterotrophic Assay ( 2011 ) / Page 5 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## formulation were reanalyzed (12 days after preparation) to confirm stability of the

formulations.

Results of D ose Analysis

Homogeneity ( %CV ): 0.908-1.98% (top, middle, bottom)

Stability (% of nominal) : 105-113% (storage conditions not specified)

Concentration (% of nominal): 86.4-88.4% for 64 mg/mL; 99.7-104% for 200 mg/mL

* The first 200 mg/mL formulation prepared was low (73.6-75.0% of nominal), so a second formulation was prepared and analyzed with acceptable achieved

concentrations.

Although the study did not report the storage conditions for the stability samples (i.e., temperature), the analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable

.

6. Dosage A dministration : A nimals were administered the test formulation

, EE or vehicle (corn oil) daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight . Dose volumes were adjusted daily based on the concurrent body weight

measurement.

7. Statist ics : Descriptive statistics (mean, standard deviation and count) were calculated using MS Excel. Final body weight, body weight gain, and tissue weights were analyzed using SAS version 9.1. Studentized residual plots were used to detect possible outliers and Levene's test was used to assess homogeneity of variance. Body weight gain was transformed by multiplicative inverse.

Final body weight, body weight gain, and uterine weights were analyzed by one- way ANOVA followed by pair wise comparisons using a Dunnett’s one tailed t- test (uterine weights) and Dunnett’s two tailed t-test (final body weight and body weight gain). Posit ive controls (EE) were compared to vehicle controls by appropriate t- tests. Statistically significant effects were reported when p<0.05. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations : Cage

- side checks for mortality and morbidity were conducted twice daily (once daily on weekends). Cage

- side observations for clinical signs of toxicity were also conducted 3 hours following dose administration. Detailed physical examinations were performed upon arrival, at allocation to dose groups, daily prior to dos e administration

and at termination.

2. Body W eight : Animals were weighed upon arrival, at randomization, daily prior to dose

administration and at termination.

3. Food Consumption (Optional) : Food consumption was not measured.In vivo Uterotrophic Assay ( 2011 ) / Page 6 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## 4. Necropsy and Measurement of Uterine Weight : On PND 61 (approximately 24 hours after final administration of the test substance) , all animals were euthanized by CO 2 

## asphyxiation and cervical dislocation, and subjected to a gross necropsy. A nimals were

checked for ovarian tissue at necropsy. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just below the cervix in order to

retain the luminal fl uid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was weighed. Subsequently, the uterus w as pierced and blotted to remove the luminal fluid, and the blotted uterus was weighed.

5. Microscopic Examination (Optional) : Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.

2. Clinical S igns of Toxicity : The incidence of clinical observations is presented in Table 2.

A nimals administered corn oil exhibited no abnormal clinical signs 3

- or 24 -hours post-dose

administration

.

At 320 mg/kg piperonyl butoxide, one animal exhibited uncoordinated movements on Day 1

and one animal was lethargic on Day 2; no clinical observations were noted for any o f these

animals on the day of termination. All animals from the 1000 mg/kg/day piperonyl butoxide group exhibited decreased or uncoordinated movements, hunched posture and were lethargic by Day 3 , and one animal also exhibited nasal discharge. On Day 4 a ll animals from the high dose group were noted as normal, except for o ne rat which appeared thin, exhibited hunched posture, le thargy, and had a red stained face, throat, and front paws, and yellow stained abdomen (genital area) prior to euthanasia.

It is noted that signs of decreased movement and/or lethargic behavior were observed in four animals from the positive control (EE) group on Day 3, but all animals were noted as normal prior to termination.In vivo Uterotrophic Assay ( 2011 ) / Page 7 of 9

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### T ABLE 2. Incidence of Clinical Observations in the Estrogen Agonist Assay a



### Observation Dose (mg/kg/day)

Vehicle Control Piperonyl Butoxide
( 320 ) 

### Piperonyl Butoxide
(1000 ) 

### Reference Estrogen

EE ( 0.1 )

#

Observed #

Examined #

Observed #

Examined #

Observed #

Examined #

Observed #

Examined


### Lethargic 0 8 1 8

8 8 3 8 

### Hunched posture 0 8 0 8 0 8



### Decreased or

uncoordinated

movements 0 8 1 8 4 8



### Nasal discharge 0 8 0 8 1 8 0 8 

### Thin appearance 0 8 0 8 2 8 0 8



### Distended abdomen 0 8 0 8 1 8 0 8



### Fur discoloration 0 8 0 8 1 8 0 8



### a Data were obtained from Appendix 4, page s 66

- 68 of the study report.



## B. BODY WEIGHT AND WEIGHT GAIN : Selected body weight and body weight gain data are presented in Table 3. Body weights were decreased (p<0.05 ) by 10% on Day 4 (termination) in the high dose group compared to the vehicle controls, resulting in a decrease (p<0.05) of 240% in overall body weight gain at 1000 mg/kg piperonyl butoxide. Body weights in the low-dose group were comparable to 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).

TABLE 3

. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a



### Study Day Dose (mg/kg/day)

Vehicle Control Piperonyl Butoxide ( 320 ) Piperonyl Butoxide ( 1000 ) Reference Estrogen

EE ( 0.1 )

N Mean SD N Mean SD N Mean SD N Mean SD

1 8 247.8 13.6 8 247.7 13.4 8 248.6 11.3 8 246.8 9.5

2 8 249.9 13.8 8 250.8 14.1 8 241.6 12.8 8 247.7 12.2

3 8 257.5 15.7 8 257.3 12.2 8 236.0 11.5 8 247.0 12.2
4 (termination) 8 258.6 15.5 8 257.5 14.3 8 233.5* (↓10) 17.8 8 241.6 (↓7) 11.5

Body Weight Gain ( Days 1

- 4 ) 8 10.8 2.7 8 9.8 3.3 8

- 15.1* (↓240) 12.2 8

- 5.2 (↓148) 3.4

a Data were obtained from Table 3 on page 19 (and Appendix 5 pages 70

- 71) of the study report ; statistical significance was only determined for BW on Day 4 and overall BWG of piperonyl butoxide treated groups

. Percent differences from controls

, calculated by the reviewers

, are included in parentheses.

N N umber of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05In vivo Uterotrophic Assay ( 2011 ) / Page 8 of 9

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## C. FOOD CONSUMPTION (Optional) : Food consumption was not measured.

D. PATHOLOGY

1. Uterine W eights : U terine (wet and blotted) weight data are presented in Table 4 . Uterine weights in the 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.

The mea n blotted uterine weight for the vehicle control group was <0.04% of terminal body weight, which is consistent with the performance criteria (<0.0 4 %) of the test guideline (O CSPP 890.1600).

T ABLE 4

. Uterine Weights from Estrogen Agonist Assay in SD Rats a



### Parameter Dose (mg/kg/day)

Vehicle Control Piperonyl Butoxide ( 320 ) Piperonyl Butoxide ( 1000 ) Reference Estrogen

EE ( 0.1 )

N Mean SD N Mean SD N Mean SD N Mean SD

Terminal BW (g) 8 258.6 15.5 8 257.5 14.3 8 233.5 17.8 8 241.6 11.5
Wet, absolute ( g) 8 0.0866 0.0113 8 0.0803 0.0177 8 0.0903 0.0100 8 0.3876* (↑348) 0.1617

Wet, relative (%) b 

### 8 0.034 0.006 8 0.031 0.007 8 0.039 0.003 8 0.161 0.069
Blotted, absolute (g) 8 0.0815 0.0111 8 0.0758 0.0172 8 0.0852 0.0092 8 0.2228* (↑173) 0.0420

Blotted, relative (%) b 

### 8 0.032 0.006 8 0.029 0.006 8 0.036 0.003 8 0.092 0.018

a Data were obtained from Table 4 on page 20 of the study report. Percent differences from controls

, calculated by the reviewers

, are included in parentheses.

b Relative weights (except for relative blotted uterine weight of the vehicle controls) were calculated by the reviewers using the individual terminal BW and uterine weights from Appendix 6 page 73 of the study report.

BW B ody weight

N Number of animals in the group

SD Stand ard Deviation

* Significantly different from controls at p<0.05



## 2. Microscopic Examination (Optional) : Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Administration of piperonyl butoxide significantly decreased final body weights and body weight gain at a dose level of 1000 mg/kg, but not at 320 mg/kg. Uterine weights (wet and blotted) were not statistically changed at either dose level compared to the vehicle control. Thus, piperonyl bu toxide was not estrogenic in this validated test system using ovariectomized adult female rats.

B. AGENCY COMMENTS : All animals survived until scheduled termination. Animals administered piperonyl butoxide at 320 mg/kg exhibited abnormal clinical signsIn vivo Uterotrophic Assay ( 2011 ) / Page 9 of 9

PIPERONYL BUTOXIDE / 067501 O CS PP 8 9 0. 1600/ OECD 440



## (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 decreased or uncoordinated movements, hunched posture and were lethargic b y Day 3. On Day 4 all animals from the high dose group were noted as normal, except for o ne rat which exhibited hunched posture, lethargy, a red stained face, throat, and front paws, and yellow stained abdomen (genital area), and appeared thin prior to euthanasia. It is noted that signs of decreased movement and/or lethargic behavior were observed in four animals from the positive control (EE) group on Day 3, but all animals were noted as normal prior to

termination.

Body weights were decreased (p<0.05) by 10% on Day 4 (termination) in the high dose group compared to the vehicle controls, resulting in a decrease (p<0.05) of 240% in overall body weight gain at 1000 mg/kg piperonyl butoxide. Body weights in the low- dose group were comparable to controls th roughout 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 contro ls, with a decrease in overall body weight gain (↓148).

In the cited range -finding study, doses of 1000 mg/kg/day, no signs of toxicity were observed. However, in this study marked decreases in body weight gain was seen in this study. As there were no changes in relative or absolute uterine weight at either dose level, this does not adversely impact the interpretation of the study.

Uterine weights in the 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.

C. STUDY DEFICIENCIES : None