UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

SUBJECT: Piperonyl 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 Table 

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-- -- Immediate Office 

Health Effects Division (7509P) 

THROUGH: JessRowland 

Deputy Director 

~~~~ 

Health Effects Division 

TO: Jolene Trujillo 

Biologist/Chemical Review Manager 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7505P) 

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 Screening Program (EDSP) Tier 1 assays 

submitted in response to the agency's Test Order for piperonyl butoxide: Test Order # CON067501-9. 

Page I of2 

Page 1 of 252
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) 48618805

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

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Data Evaluation Record on the Toxicity of Piperonyl 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-butoxyethoxy)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 

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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'

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Data Evaluation Record on the Toxicity of Piperonyl 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. 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). 

Disclaimer: 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.

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Data Evaluation Record on the Toxicity of Piperonyl 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). 

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Data Evaluation Record on the Toxicity of Piperonyl 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 Effects1,2 in the Amphibian 

Metamorphosis Assay (AMA) with Piperonyl Butoxide. 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental 

Stage 

Hind Limb 

Length3

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 CaCO3) was greater than recommended (40-43 mg/L 

as CaCO3). 

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. 

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Data Evaluation Record on the Toxicity of Piperonyl 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 

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

 EPA MRID Numbers 48673101, 49606701

B. Test Organism

Table 2: General Information About the Test Species and Parental Care.

Parameter Value(s) Details or Remarks Guideline Recommendations Species 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 reported Were parents maintained as in-house stock? 

Yes Adult X. laevis frogs were initially obtained 

from Xenopus I, Dexter, MI. 

EPA recommends that larvae used in the assay 

be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor Screening 

Program 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). 

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Acclimation period for parental frogs (if applicable): 

Not specified

Details on parental feeding: Parental frogs were fed 

approximately 1 Tablespoon of #3 frog food pellets per tank twice a day. 

Food source is from Zeigler Bros., Inc. 

Details on parental health: Parental frogs were in 

good health, displaying no abnormalities or signs of illness. 

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 EPA MRID Numbers 48673101, 49606701

Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations Best single spawn? 

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 spawns evaluated (if applicable): 

3 

Number of eggs sampled per spawn: 

All specimens on a given day 

Embryonic viability checked daily 

NF stage at test initiation 

51 Study authors state that the developmental stage of X. laevis 

tadpoles was determined by using the staging criteria of Nieuwkoop and Faber.

EPA recommends that the definitive study 

be initiated with larvae at Nieuwkoop – 

Faber (NF) developmental stage 51 (17 days post-fertilization).

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Handling at staging Individual larvae were randomly removed from the pooling tank 

by scooping with a small strainer. Animals were carefully handled during this transfer in order to minimize handling stress and to avoid any injury. The developmental stage was then determined by using a binocular dissection microscope. Selected larvae were randomly distributed to exposure treatment tanks and inspected for animals with abnormal 

appearance (e.g., injuries, abnormal swimming behavior, etc.). Overtly unhealthy looking tadpoles were removed from the treatment tanks and replaced with larvae newly selected from the holding tank. The treatment tanks were randomly assigned to 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. 

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Organism Sampling 

 On Day 7 of the study, 5 randomly chosen tadpoles per 

replicate were removed from each test tank and humanely euthanized in 150 to 200 mg/L MS-222, appropriately buffered with sodium bicarbonate. Tadpoles were rinsed in water and blotted dry, followed by body weight determination to the nearest mg. Hind limb length and SVL, along with developmental stage (using a binocular dissection microscope), were determined for each tadpole. At test termination (Day 21), the remaining tadpoles were removed and similarly euthanized. Tadpoles were rinsed in water and blotted dry, followed by body weight determination to the nearest mg. Developmental stage, hind limb length, and SVL were measured for each tadpole. Prior to fixing tadpole tissue for histopathological analyses, blood samples were collected from each buffered MS-222 anesthetized larvae, plasma 

isolated, and stored for possible analysis of thyroid hormones (thyroxine [T4] and triiodothyronine [T3]). In addition, tail fin samples from each specimen were collected and preserved in RNAlater for possible thyroid receptor beta (TRȕ) gene expression analysis. Lab SOPs were referenced for these methods 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). 

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Age at test initiation: 

14 days post-fertilization (dpf) 

Mean total length at test initiation (if reported): 

Not reported Snout to vent length was the first of two endpoints used to 

assess tadpole growth. SVL (millimeters) was used to help 

establish generalized toxicity of the test article. All length measurements were based on digital photographs of the 

surviving organisms from each treatment.

EPA recommends that the DER briefly 

describe the method of measurement. 

Range of total length at test initiation (if reported): 

Not reported 

Was the optional size selection method used? 

No 

Details on larval selection: 

Normal appearing NF stage 51 

In order to guarantee at least 400 stage 51 acceptable larvae at test setup, clutch sizes of ~1,500 embryos were given preference. 

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Loading 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, PA

Iodide concentration in diet (if known): 

Not provided 

Frequency of feeding: 

2 times/day EPA recommends that feeding occur at 

least twice per day.

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Details on feeding regime: 

Feeding regime followed EPA guideline recommendations. 

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 System

Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations Type of exposure: Flow-through EPA recommends the use of a flow-through 

system.

Type of flow-through dilution system (if applicable): 

Continuous flow serial diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Flow-through rate (if applicable): 

25 mL/min Flow rate provided complete volume 

replacement every 2.7 hours. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

Details on toxicant mixing for flow-through 

systems (if applicable): 

Modified Benoit Mini-Diluter Mixing chamber was a continuous mix chamber 

in which stock solution and dilution water were supplied. Baffle ensured complete mixing since the concentration desired was at or below limit 

of water solubility. Concentration prepared in mixing chamber represents high test concentration. 

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 static renewal (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.

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Source of dilution water: Treated municipal tap water Prepared by passing tap water through a 4 filter 

system: a multimedia filter to remove suspended solids; a 10” pre-treatment filter (5μm) to remove any solids; a 3.6ft3 activated 

virgin carbon treatment filter to remove chlorine, ammonia, and higher molecular weight 

organics; and a 5μm polishing filter to remove any carbon particles from the carbon treatment phase. 

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 water analyzed for pesticides, heavy metals, and other contaminants? 

Yes. See Reviewer’s Comments 

Analyzed on January 28, 2011. 

Iodide supplementation in water? 

No If reconstituted water is used or if background 

levels of iodide in natural water are less than 

0.5 μg/L, iodide supplementation is 

recommended. This supplementation is in 

addition to the recommended dietary source of 

iodide (e.g.. in Sera Micron).

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

 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Test 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: 22.5 x 14.0 x 16.5 cmFill volume: 4 LAdditional details on exposure system: 

NA 

Table 5: Summary of Water Quality Characteristics in the Test System. 

Parameter Minimum Maximum Mean 

Measurement Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 132 144 138.25 Weekly EPA recommends hardness 40 to 48 mg/L 

as CaCO3.

pH 7.3 7.9 7.5 3x Weekly 

EPA recommends pH 7.5 ± 1, inter-replicate 

and inter-treatment differentials should not 

exceed 0.5.

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

 EPA MRID Numbers 48673101, 49606701Parameter Minimum Maximum Mean 

Measurement Interval 

Guideline Recommendations 

Dissolved oxygen (mg/L) 6.1 9.9 7.6 3x 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) 21.4 21.9 21.6 Daily 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment differentials 

should not exceed 0.5oC.

Iodide (μg/L) 2 5.1 3.6 Semi-annually 

EPA recommends aquatic iodide range 0.5 – 

10 μg/L (supplemental iodide should not 

exceed 2 μg/L).

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

 EPA MRID Numbers 48673101, 49606701Parameter Minimum Maximum Mean 

Measurement Interval 

Guideline Recommendations 

Ammonia (mg/L) 0.01 0.06 0.04 Bi-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 Not Reported Perchlorate Not Reported Chlorate Not Reported Other -- -- -- -- 

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 EPA MRID Numbers 48673101, 49606701D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the range-finder? 

Historical toxicity data provided in a similar species 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 laevis Life stage: 51 Test duration: 4 days

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 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Additional details: Nominal concentrations 

were 0 (negative control), 0.0001, 0.001, 0.01, 0.1, 1.0, 10.0, and 25.0 mg/L 

Data from the rangefinder showed that the 4-day NOAEC, EC50, and LC50 were 0.1, 

0.22, and 3.16 mg/L, respectively. No morbidity was observed at 0.1 mg/L PBO mg/L test concentration was the highest concentration evaluated in the range finding study that induced <10% mortality and morbidity, 0.1 mg/L PBO was set as the maximum tolerated concentration (MTC). 

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

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 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Method for selecting the highest test concentration in the definitive test:

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 (if applicable):

NA 

Separation 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 concentrations adjusted for purity? 

No 

Indicate the type of values presented for measured concentrations: 

Time-weighted average TWA- 0.00599, 0.0263, and 0.0741 mg 

a.i./L 

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 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Limit 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 reported Frequency of measurement: Samples collected for 

analysis 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 solvent control (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 item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

Was a solvent used? No Solvent type (if applicable): NA 

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 EPA MRID Numbers 48673101, 49606701Parameter Value(s) Details or Remarks Guideline Recommendations Maximum solvent concentration (if applicable): 

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? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 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: None 3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp.69–92. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing 

and Assessment. No. 23. Paris, France.

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Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Piperonyl Butoxide (PBO). 

Treatment ID 

Nominal Concentration (mg a.i./L) 

TWA Measured Concentration (mg a.i./L) 

Mean CV (%) 

Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.001 NA NA EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 20%.

Treatment 1 0.011 0.00599 12.9 NA Treatment 2 0.033 0.0263 12.9 NA Treatment 3 0.100 0.0741 13.3 NA 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: Day 7: NF Stage, normalized HLL, SVL, wet weight 

Day 21: NF Stage, normalized HLL, SVL, wet weight, thyroid histopathology 

 Daily: mortality, clinical signs 

Were raw (individual) data provided? Yes

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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 (Days 7 and 

21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-based larval selection); 

and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also includes thyroid gross 

pathology observations.

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II. RESULTS AND DISCUSSION A. Results 

By 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 Xenopus laevis.

Treatment (mg a.i./L) [TWA-measured]

Larval Mortality 

Day 71 Day 21 

n Mortality # Mortality % n Mortality # Mortality % 

Negative Control 80 0 0 60 1 1.7 0.00599 80 0 0 60 0 0 0.0263 80 0 0 60 1 1.7 0.0741 80 0 0 60 1 1.7 1 Sample 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 median 

values were between 61 and 62, though there were a few non-late stage tadpoles in the control and treatment groups. No asynchronous development was observed. 

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 EPA MRID Numbers 48673101, 49606701Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L) [TWA-measured]

Developmental Stage 

Day 7 Day 21 

n 

Median 

Stage 

# Asynchronous n 

Median 

Stage 

# Asynchronous 

Negative Control 4 54 0 4 62 0 0.00599 4 54 0 4 61 0 0.0263 4 54 0 4 61 0 0.0741 4 54 0 4 61 0 

Day 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 0.0741 

mg a.i./L treatment groups, respectively (Table 11). The study author reported that mean normalized HLL on Day 21 was 0.941 in the negative control 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. 

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 EPA MRID Numbers 48673101, 49606701Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment 

(mg a.i./L) 

[TWA-measured]

Hind Limb Length (HLL) 

Day 7 Day 21 

n 

Mean

(mm) 

±SD 

HLL: 

SVL1

n 

Mean

(mm) 

±SD 

HLL: 

SVL1

Negative Control 4 1.77 0.417 0.153 4 16.33 5.5 0.941 0.00599 4 1.79 0.325 0.171 4 15.07 6.3 0.826 0.0263 4 1.84 0.441 0.180 4 16.25 5.8 0.916 0.0741 4 1.70 0.413 0.163 4 16.91 6.4 0.959 Abbreviations: SD Standard deviation. 1 Summary 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 (Table 12). At 

test 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 (Table 12). 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 treatment group. 

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 EPA MRID Numbers 48673101, 49606701Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWAmeasured]

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 21 Day 7 Day 21 

n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD 

Negative Control 4 11.50 0.96 4 17.62 1.5 4 0.196 0.04 4 0.677 0.17 0.00599 4 10.47 0.93 4 18.50 1.8 4 0.150 0.04 4 0.769 0.25 0.0263 4 10.28 1.11 4 18.14 1.9 4 0.144 0.05 4 0.756 0.23 0.0741 4 10.35 1.02 4 18.09 1.7 4 0.144 0.04 4 0.730 0.22 Abbreviations: SD Standard deviation. 1 Also 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 observed 

in 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./L treatment groups. There were no apparent treatment-related patterns as the incidence in the control and treatment groups was similar. The same 

pattern of mild incidence across the control and all treatment groups was apparent for follicular cell height increase and follicular cell shape. 

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Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWAmeasured] 

Diagnostic Observations1

Severity 

Thyroid Gland 

Hypertrophy 

Thyroid Gland Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 20 20 20 20 15 20 20 

1 20 0 20 0 20 5 20 0 

2 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 

0.00599 0 20 20 20 20 20 16 20 19 

1 20 0 20 0 20 4 20 1 

2 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 

0.0263 0 20 20 20 20 20 13 20 19 

1 20 0 20 0 20 7 20 1 

2 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 

0.0741 0 20 20 20 20 20 15 20 20 

1 20 0 20 0 20 5 20 0 

2 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 

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 EPA MRID Numbers 48673101, 496067011 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD 

No. 82 for reference. 

Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. 

Treatment 

(mg a.i./L) 

[TWA-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 20 20 20 20 15 20 20 20 15 

1 20 0 20 0 20 5 20 0 20 5 

2 20 0 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 20 0 

0.00599 0 20 20 20 20 20 16 20 20 20 16 

1 20 0 20 0 20 4 20 0 20 4 

2 20 0 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 20 0 

0.0263 0 20 20 20 20 20 13 20 20 20 13 

1 20 0 20 0 20 7 20 0 20 7 

2 20 0 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 20 0 

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Treatment 

(mg a.i./L) 

[TWA-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.0741 0 20 20 20 20 20 15 20 20 20 15 

1 20 0 20 0 20 5 20 0 20 5 

2 20 0 20 0 20 0 20 0 20 0 3 20 0 20 0 20 0 20 0 20 0 

1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 

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

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWA-measured] 

Clinical Signs1

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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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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< 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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 EPA MRID Numbers 48673101, 49606701Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Piperonyl Butoxide. 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

Median p Median p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 -- 62 -- -- -- -- -- -- -- -- -- -- 0.00599 54 1.0 61 0.43 33.3 0.08 -13.2 0.14 -- -- -- -- No 0.0263 54 1.0 61 1.0 33.3 0.08 -5.26 0.74 -- -- -- -- No 0.0741 54 1.0 61 1.0 33.3 0.08 2.63 0.95 -- -- -- -- No Statistical Test Mann-Whitney Mann-Whitney Dunnett Dunnett -- -- -- Abbreviations: Diff. Difference. 

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily 

weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p < 0.05. 3 Hind-limb length is normalized to snout-vent length (SVL). 

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Table 17: Growth Endpoints1,2 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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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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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

Species: Xenopus laevis Brine:

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

Material: Piperonyl butoxide Project: Synergist

067501 4863101

067501 48673101

Batch Note:

Sample Note:

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

07d Developmental Stage Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

07d HLL Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

07d Normalized HLL Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

07d SVL Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

07d Wet Weight Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

21d Developmental Stage Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

21d Only LS HLL Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

21d Only LS Normalized HLL Summary

95% LCL 95% UCL %Effect

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

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

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

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

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

Page 42 of 252
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.

Group Control Type Count Mean Min Max Std Err Std Dev CV%

21d Only LS SVL Summary

95% LCL 95% UCL %Effect

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

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

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

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

Group Control Type Count Mean Min Max Std Err Std Dev CV%

21d Only LS Wet Weight Summary

95% LCL 95% UCL %Effect

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

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

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

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

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

Page 43 of 252
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.

Group Control Type

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

Group Control Type

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

Group Control Type

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

Group Control Type

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

Group Control Type

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

Group Control Type

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

Group Control Type

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

Group Control Type

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

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

Page 44 of 252
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.

Group Control Type

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

Group Control Type

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

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

Page 45 of 252
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. 

Page 46 of 252
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).

Page 47 of 252
PIPERONYL BUTOXIDE I 067501 

Androgen Receptor Binding (Rat Prostate Cytosol) (201 1) I Page I of 17 

OCSPP 890. 11 50/ OECD None 

Primary Reviewer: Deborah Smegal 

Health Effects Division, Office of Pesticide Programs 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division, Office of Pesticide Programs 

gn~y)., Date: ~~ ~ 

Signature: , 

Date: G IL 'f/ld""' 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890. l 150 

PC CODE: 06750 1 DP BARCODE: D398558 

TXR#: 0056259 CAS No.: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93.8% a.i.) 

SYNONYMS: 5-[[2-(2-butoxyethoxy)ethoxy] methy l]-6-propyl-1 ,3-benzodioxole 

CITATION: Willoughby, J.A. (20 11). Piperonyl butoxide: androgen receptor binding (rat 

prostate cytosol). CeeTox, Inc., Kalamazoo, Ml. Laboratory Study Number: 

9146V- 100377ARB, November 17, 2011. MRID 486 18801. Unpublished. 

SPONSOR: 

Willoughby, J.A. (2012). Supplemental Information - Laboratory Proficiency 

Data for ERTA assays and Saturation Binding Data for AR and ER Binding 

Assays for Asso1ted Chemicals. CeeTox, Inc., Kalamazoo, MI. September 24, 

20 11. MRID 48843501. Unpublished. 

Consumer Specialty Products Association, Inc. For Piperonyl Butoxide Task 

Force II, 1900 17111 Street, Washington, D.C. 

TEST ORDER#: CON-067501-9 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 486 18801), 

ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a 

competitive binding experiment, which measured the binding of a single concentration of [3H]-

Rl 881 (l nM) in the presence of increasing concentrations (10- 10 to 10- 3 M) ofpiperonyl 

butoxide (93.8% a.i., Batch #El 04110). DMSO was used as the vehicle at a final assay 

concentration of approximately 3.2%. A total of three independent runs were performed, and the 

assay included dexamethasone as a weak positive control, and RI 881 as the ligand reference 

standard. 

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference androgen (Rl 881 ). Saturation binding data were not originally provided in 

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

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

dissociation constant (Kct) for [3T-I]-R 1881 was 1.080±0.690 nM and the estimated Bmitx was 

Page 48 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

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

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

(0.685 to 1.57 nM), and all Bmax values were below the recommended range (7 to 16 fmol/100 

μg protein). Confidence in these numbers is high based on the goodness of fit (R2 = 0.957-

0.984) and the small variation among runs. 

 

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

at the highest concentration (10−3 M) tested in all three runs, therefore the highest concentration 

used for data evaluation was 10−4 M. The estimated average log IC50 was −9.3 M for R1881 and 

−4.4 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for 

dexamethasone was 0.0022% for the two valid runs (excluding the first run). All performance 

criteria were met for two of the three runs. The concentrations prepared for R1881 and 

dexamethasone were lower than the targeted (guideline) levels in the first run because of an error 

in the stock preparation. Therefore, the curve for dexamethasone was not fully defined (bottom 

percent binding was not achieved). The range of concentrations for R1881 in the first run fully 

defined the curve, but the concentration range was off by more than an order of magnitude. An 

IC50 and relative binding affinity (RBA) could not be calculated for piperonyl butoxide as it did 

not competitively bind to the AR. 

The percent labeled R1881 bound to the AR in the presence of piperonyl butoxide was >75% at 

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

concentration, an IC50 and RBA could not be calculated for piperonyl butoxide. Based on the 

results from the three runs, piperonyl butoxide is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Androgen Receptor Binding 

Assay (OCSPP 890.1150). 

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

Assurance statements were provided. 

Page 49 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ 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 Period: May 23, 2011 to November 17, 2011

2. Test substance: Piperonyl Butoxide

Description: Technical; oily yellow liquid

Source: Endura Fine Chemicals, Italy

Batch #: E104/10 (expiry 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

CH3

OC4H9

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 purity: >97%

Specific activity: 85.1 Ci/mmol

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: Dimethyl sulfoxide (DMSO)

Justification for choice of Piperonyl butoxide solutions up to 10−4 M (limit concentration for the assay) can be 

Page 50 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ 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 Rat Ventral Prostate Cytosol: The rat prostate tissue was purchased from 

Charles River Laboratories (location not specified; Batch #210007463). Male Sprague 

Dawley rats (number not reported) were castrated at approximately 90 days of age and were 

euthanized <24 hours later and the ventral prostate tissues collected.

The ventral prostates were 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 supernatants were 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 Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-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 experiment 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 24 of the study report (MRID 48843501). 

On the day of the assay, the specific activity of the stock solution [3H]-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 to 

Page 51 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ 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 nM [

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 assay. Each saturation binding 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 Runa,b

Total Binding Non-Specific Binding Radioligand alone

Tubes 1-24c Tubes 25-48d Tubes 49-72e

[

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, [3H]-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 minutes 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 at 

Page 52 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ 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 three runs were performed.

3. Competitive Binding 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 concentration Sufficient to bind 8.7-9.7% of 1.0 nM 

radioligandb 

Concentration of test substance (as serial dilutions) 10−10 to 10−3 Mc

Incubation Temperature 4 ± 2oC 

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 experiment measures 

the binding of a single concentration of [3H]-R1881 (adjusted specific activity of 82.6 

Ci/mmol for Run 1; 82.5 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 runs. Therefore, the highest concentration of piperonyl butoxide used 

in evaluating the data was 10−4 M. 

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

reviewer was able to calculate from the competitive binding data that the amount of 

cytosolic protein used in the assay contained enough receptor to bind 9-10% of the [3H]-

R1881. 

Dilutions of the test substance, reference standard (R1881), weak positive control 

(dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations 

Page 53 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

shown in Table 4. Each assay consisted of three independent runs on three different days, 

and each run contained three replicates at each concentration plus six replicates of the [

3

H]-

R1881, NSB, and solvent controls, resulting in a total of 84 samples per run. It is noted that 

in Runs 2 and 3 only three replicates of the [3H]-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

DMSO R1881 Dexamethasone Piperonyl Butoxide (Hot Mix)

Tubes 7-12 Tubes 13-18 and 19-33 d Tubes 37-60 Tubes 61-84 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

-- 1×10−10 (1 ×10−11) 1×10−10

a Data were obtained from pages 35-37 of the study report.

b The [

3

H]-R1881, NSB, and solvent controls were run in 6 replicates and each concentration of each chemical 

was run in triplicate for a total of 84 tubes per run. Tubes 1-84 contained 50 μL of triamcinolone acetonide and 

30 μL [3

H]-R1881. Samples were dried, and 300 μL 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 μL 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 Precipitation was observed at 10−3 M, therefore the highest 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 regression- 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.

Page 54 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 17

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control, and the test substance) to the Hill equation formula which incorporated log IC50 as a 

parameter to be estimated. For parameters reported from the saturation binding experiment 

(Kd and Bmax) and competitive binding experiment (log IC50 and RBA), mean 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 average 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

Bmax: maximal binding capacity

Kd: dissociation constant 

IC50: Concentration of the test substance at which 50% of radioligand is displaced from the AR 

by the competitor

Relative Binding Affinity (RBA): (IC50 of R1881 ÷ IC50 of test substance) × 100 

Log RBA: Log10 (IC50 of R1881 ÷ IC50 of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The dissociation constant (Kd) for [3H]-R1881 was 

1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for the 

single batch of prostate cytosol that was prepared. Only one of the three Kd values was 

within the range reported in the EPA validation program (0.685 to 1.57 nM); the mean value 

was in the acceptable range. Confidence in these numbers is based on large variation in Kd

estimates among runs.

Page 55 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 17

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TABLE 5. Saturation Binding Experiment of [3H]-R1881 with Androgen Receptor from Rat Prostate 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.880 0.986 0.893 0.880-0.986

Bmax (nM) 0.010 0.008 0.011 0.010±0.002

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

Kd (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 R2 is reported and the mean ± SE is reported for the other parameters.

R2 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. 

Page 56 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

FIGURE 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment.

 

Page 57 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 17

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FIGURE 2. Scatchard Plots of the Binding of [3H]-R1881 to the Androgen Receptor.

Page 58 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 17

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B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive 

binding experiments 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 runs; 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 IC50 and RBA could not be calculated for piperonyl butoxide. 

The estimated average log IC50 was −9.0 M for R1881 and −4.5 M for the weak positive 

control (dexamethasone). Compared to R1881, the 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. 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 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 

R2 (unweighted) R1881 NR NR NR NA

Dexamethasone NR NR NR NA

Piperonyl butoxide NR NR NR NA

Log IC50 (M) R1881 -9.9 -9.0 -9.0 -9.3±0.30b

Dexamethasone -4.3 -4.4 -4.6 -4.4±0.09b

Piperonyl Butoxide NA NA NA NA

IC50 (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 NA

Log RBA d Dexamethasone -5.6 -4.6 -4.4 -4.9±0.37

Piperonyl Butoxide NA NA NA NA

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 log IC50 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 log IC50 for dexamethasone from the first 

run as -4.3 M. 

b Values are reviewer-calculated from the log IC50 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 

R2 Goodness of fit

RBA Relative binding affinity

Page 59 of 252
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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

FIGURE 3. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Piperonyl Butoxide (Run 1).

Page 60 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

FIGURE 4. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Piperonyl Butoxide (Run 2).

Page 61 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

FIGURE 5. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Piperonyl Butoxide (Run 3).

Page 62 of 252
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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ OECD None

C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was 

functioning properly, each run was evaluated using the criteria shown in Table 7:

TABLE 7. Criteriona Tolerance Limit(s) b Value Yes No

Ligand depletion is minimal. The recommended ratio 

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 (dexamethasone) fitted curve parameters

Top (% binding) 87 to 106 101 to 105 X

Bottom (% binding) −12 to 12 NA; −1 to 7 c X

Hill Slope −1.4 to −0.6 −1.0 to −0.6 X

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.531 to 1.855 Xd

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

a Data were obtained from pages 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 Calculated 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, nonspecific 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 achieved 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 plateaus 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 COMMENTS: In the saturation binding assay, the mean Kd for [3

H]-R1881 

was 1.080 nM, and the mean estimated Bmax was 1.071 fmol/100 μg protein for the single 

batch of prostate cytosol that was prepared. Only one of the three Kd values was within the 

Page 63 of 252
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 17

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1150/ 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 Kd estimates 

among runs. 

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

observed at the highest concentration (10−3 M) in all three runs, therefore the highest 

concentration used for data evaluation was 10−4 M. The estimated average log IC50 was 

−9.3 M for R1881 and −4.4 M for the weak positive control (dexamethasone). Compared to 

R1881, the mean RBA for dexamethasone was 0.0022%. 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 IC50 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. The 

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. 

Page 64 of 252
DATA EVALUATION RECORD 

PIPERONYL 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. 

Page 65 of 252
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).

Page 66 of 252
PIPERONYL BUTOXIDE I 067501 

Primary Reviewer: Chris Schlosser, M.F.S 

Health Effects Division 

Aromatase (Human Recombina nt) Assay (201 I) I Page 1of14 

OCSPP 890. 1200 I OECD None . 

Secondary Rev iewer: Greg Akerman, Ph.D. ___ _ 

Signature: ~--- Date: ff I I >- Signature: ~ ~ 

Health Effects Division Date: ' <;;' /; ( ltrr: 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890. 1200 

PC CODE: 06750 I DP BARCODE: 0 398558 

TXR#: 0056259 CAS No.: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93.8% a.i.) 

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

CITATION: Wilga, P.C. (2011) Piperonyl butoxide: human recombinant aromatase assay. 

CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9146V-100377AROM, 

November 17, 2011. MRID 48618802. Unpublished. 

SPONSOR: Consumer Specialty Products Association, 1900 17111 Street NW, Washington, 

D.C. 

TEST ORDER#: CON-067501-9 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48618802), 

piperonyl butoxide (93.8% a.i. Lot # £104110) was incubated with human recombinant 

aromatase and tritiated androstenedione ([1B3H(N)]-androst-4-ene-3,17-dione; [31-I)ASDN) in 

:Sl % dimethyl sulfoxide (DMSO) at log concentrations of 10- 10 to 10-3 M for 15 minutes to 

assess the potential of piperonyl butoxide to inhibit aromatase activity. 

Aromatase activity was detennined by measuring the amount of tritiated water produced at the 

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

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

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

using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN) in duplicate, and the 

piperonyl butoxide series ( 10- 10 to I 0-3 M) with three replicates per concentration. 

Aromatase activity in the full activity controls was 0.207±0.025 nmol·mg-protein- 1 ·min- 1. The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity. Activity in the background controls ranged from 0.52 to 0. 78% and averaged 0.63% of 

the full activity controls. 

For the positive control substance (4-0H ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log ICso for 4-0H ASDN 

averaged - 7.13 Mand the Hill slope was -1.02. 

Page 67 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

For piperonyl butoxide, the two highest tested concentrations (10−3

 and 10−3.5 M) showed 

precipitation, and the results for the lowest test concentration (10−10 M) were considered by the 

study author to be a statistical outlier (≤51% of the control); therefore, the 10−4 and 10−9 M test 

concentrations were selected as the highest and lowest test concentrations to be used for study 

interpretation. For piperonyl butoxide, the lowest portion of the response curve across runs was 

greater than 75% activity at all soluble concentrations. Aromatase activity was 95.9 % and 

85.9% of the control at 10−9 and 10−4 M, respectively. 

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

The assay satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 

890.1200). 

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

Assurance statements were provided. 

Page 68 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Piperonyl butoxide

Description: Technical; oily yellow liquid

Source: Endura Fine Chemicals, Bologna, Italy

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

Purity: 93.8%

Volatility: Not reported

Storage conditions: Room temperature (e.g. ambient)

Stability: Not 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 #: 51-03-6

Structure:

O

O

O

O

CH3

OC4H9

2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8

Source: Steraloids, Inc. (Catalog # A6030-100)

Batch # (expiration date): L1712 (April 2016)

Purity: 99.8%

3. Radiolabeled Substrate: 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 Control: 4-hydroxyandrostenedione (4-OH ASDN) 

CAS # 566-48-3

Source: Sigma (Catalog # F2552)

Batch # (expiration date): 081K2133 (March 2015)

Purity: 99.6%

5. Solvent (Vehicle Control): 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%

Page 69 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes

Source: BD GentestTM, Woburn, MA (Catalog # 456260)

Lot # (expiration date): 03897 (February 2014)

Protein concentration: 7.4 mg/mL 

Cytochrome C reductase activity: 290 nmol∙mg-protein−1∙min−1

Aromatase activity: 6.0 pmol/pmol P450/min

B. METHODS

1. Assay Components 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 volatile 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 formulated in DMSO. 

Dilutions were prepared such that the target concentrations 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 GentestTM, 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. 

TABLE 1. Assay Components and Conditions

Assay Factor Values

0.1M sodium phosphate buffer (pH 7.4)

Microsomal Protein 0.004 mg/mL

NADPH 0.3 mM

[3H]ASDN 100 nM

Propylene Glycol 5%

Temperature 37°C

Incubation Time 15 min

2. Suitability Assessments: The protein concentration of the microsomes (Lot #19701) was 

supplied by the vendor as 7.4 mg/mL. Aromatase activity of the microsomes was also 

provided by the vendor as 6.0 pmol/min/ pmol P450. The minimum 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∙mgprotein−1∙min−1. 

Page 70 of 252
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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

3. Aromatase Assay: 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 end 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 3H2O in the aqueous fraction was quantified for each assay tube by LSC, and 

aromatase activity was reported in units of pmol∙mg-protein−1∙min−1. 

4. Demonstration of Proficiency: It was stated that proficiency testing of the 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 Proficiency: 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/mgprotein/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 of Proficiency of New Technician for Conducting 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 −0.92

Top (%) 90 110 97.9 104.1

Bottom (%) −5 +6 0.2 2.4

Log IC50 (M) −7.3 −7.0 −7.2 −7.0

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

b. Proficiency Chemicals: It was stated that the proficiency of the laboratory was 

demonstrated prior to running any test chemicals by conducting three full scale test runs on 

each of the proficiency chemicals as listed in Table 3. The data were not included in the 

study report. It was stated that the four proficiency chemicals were correctly classified as 

non-inhibitor or inhibitor. 

Page 71 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / 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 Activity with Test Chemical(s): The 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 piperonyl 

butoxide, defining the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for each Test Runa

Sample Type Repetitions 

(Tubes) Description Reference or Chemical (M)

Full Activity Control 4 All test componentsb 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 1c, d 3 All test components plus Piperonyl butoxide 1×10−3

Piperonyl butoxide Conc 2c, e 3 All test components plus Piperonyl butoxide 1×10−3.5

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

Piperonyl butoxide Conc 4c, f 3 All test components plus Piperonyl butoxide 1×10−4.5

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

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

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

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

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

Piperonyl butoxide Conc 10c, 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, [3H]ASDN, and NADPH. 

c Test chemical. 

d Highest 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 Runs #2 and 3. 

h Lowest concentration in Run #1.

Page 72 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

C. DATA ANALYSIS

1. Raw Data: 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). 

TABLE 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. Statistical Methods: 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 outlier 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 substance 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. 

Page 73 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / 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% activityb

Equivocal

Average lowest portion of curves across runs is greater 

than 75% activityb

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 Figure 5. 

Page 74 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Piperonyl butoxide on Aromatase Activity (as percent of control) from Independent 

Runsa

Chemical

Concen. 

(Log M) # Runs Overall Meanb Overall SDb Overall SEMb Overall % CVb

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 89.71 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. 42, 44, and 46 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 were 

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 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 Figure 5. 

Page 75 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

FIGURE 1. Inhibition Response Curves for Piperonyl butoxide and 4-OH ASDN from 

Run 1.

FIGURE 2. Inhibition Response Curves for Piperonyl butoxide and 4-OH ASDN from 

Run 2.

Page 76 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

FIGURE 3. Inhibition Response Curves for Piperonyl butoxide and 4-OH ASDN from 

Run 3.

FIGURE 4. Mean Inhibition Response Curves for Piperonyl butoxide and 4-OH ASDN.

Page 77 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / OECD None

FIGURE 5. Combined Response of Means and Runs 1-3 for Piperonyl 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 IC50 and Hill slope estimates were not determined 

for piperonyl butoxide. For 4-OH ASDN, the estimated log IC50 averaged −7.13 M and the 

slope was −1.02 (Table 8). The variation in the positive control values was acceptable 

(<15% CV).

TABLE 8. Effect of Piperonyl butoxide on Aromatase Activity (as Percent of Control) From Independent 

Runsa

Chemical Run 1 Run 2 Run 3 Meanb SDb %CVb

Log IC50 (M)

Piperonyl butoxide NA NA NA NA NA NA

4-OH ASDN −7.0 −7.2 −7.2 −7.13 0.12 −2

Hill Slope

Piperonyl butoxide NA NA NA NA NA NA

4-OH ASDN −1.17 −0.92 −0.96 −1.02 0.13 −13

a Data 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 Not applicable

Page 78 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 13 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / 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 butoxide 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 inhibitor.

Aromatase activity in the full activity controls was 0.207±0.025 nmol∙mg-protein−1∙min−1. 

The response of each full activity control within a run was between 90 to 110% of the 

average full activity. Activity in the background controls ranged from 0.52 to 0.78% and 

averaged 0.63% of the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase results were within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and %CV 

for replicates of each concentration within runs. The estimated log IC50 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 

concentrations, 10−9 and 10−4

 M, was 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 these concentrations 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 concentrations 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. A 

Page 79 of 252
Aromatase (Human Recombinant) Assay (2011) / Page 14 of 14

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1200 / 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 concentration for Runs 2 and 3 should have been included in 

data analyses.

Page 80 of 252
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 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Sandra Hastings Date: 3/30/2012

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. 

Page 81 of 252
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).

Page 82 of 252
PIPERONYL BUTOXID E / 067501 

Estrogen Recc1Hor Binding (Rat Uterine Cytosol) (20 11) I Page I or 18 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Chris Schlosser, M.F.S. Signatur~~~- Hcalth Effects Division Date: ~LI ( 

Seconda ry Reviewer: __ G_e__.g_.__ e_nn--"'-an ..... ,'"""P ..... h._.=-D ...... ______ Sign a turc: ,4;; '}'-- H ca Ith Effects Division Date: <:;;; r/t~ 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); 

OCSPP 890. 1250 

PC CODE: 06750 l DP BARCODE: 0 398558 

TXR# : 0056259 CAS No.: 5 1-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93.8% a.i.) 

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

CITATION: Willoughby, J.A. (2011 ). Piperonyl butoxide: estrogen receptor binding (rat 

uterine cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 

9 146Y- 100377ERB, November 17, 20 11. MRID 48618803. Unpublished. 

Willoughby, J.A. (2012). Supplemental lnfonnation -Saturation Binding 

Proficiency Information provided by CeeTox. CeeTox, Inc., Kalamazoo, MI. 

July 19, 2012. MRID 4884350 I. Unpublished. 

S PONSOR: Consumer Specia lty Products Association, Inc. For Piperonyl Butoxide Task 

Force II, 1900 I 71

h Street, Washington D.C. 

TEST ORDER#: CON-06750 1-9 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48618803), 

uterine cytosol from Sprague-Dawley rats was used as the source of ER to conduct binding 

experiments. A saturation binding experiment was conducted to demonstra te that the ER in the 

rat uterine cytosol was present in reasonable numbers and was functioning with appropriate 

affinity for the radiolabeled reference estrogen prior to routinely conducting ER competitive 

binding experiments. The competitive binding experiment was conducted lo measure the binding 

of a single concentration of [3H} l 7P-estradiol ( 1 nM) in the presence of increasing 

concentrations ( 10- 11 M to 10- 3 M) of piperonyl butox ide (93.8% a.i., Batch #E 104/ I 0). OMSO 

was used as the vehicle at a final concentration of approximately 2%. A total of three runs were 

performed, and each run included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and I 7P-estradiol as the natural ligand reference 

material. 

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

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. Data from two separate saturation binding assays were 

Page 83 of 252
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ 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, the Kd for [

3

H]-17βestradiol was 0.081±0.006 nM, and the estimated Bmax was 52.70±1.05 fmol/100 μg protein. For 

the batch of cytosol prepared on July 10, 2011, the Kd for [

3

H]-17β-estradiol was 0.303±0.090 

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

within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to the 

data, the concavity observed with the data sets from the July 2011 cytosol preparations may 

indicate issues with ligand depletion.

In the competitive binding experiment, the estimated mean log IC50 was −9.2 M for the natural 

ligand (17β-estradiol) and −5.6 M for the weak positive control (19-norethindrone). Compared 

to the natural ligand, the mean RBA was 0.026% for 19-norethindrone. 

Precipitate was observed at 10−3 M piperonyl butoxide in the first two runs; therefore, data from 

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

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

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

3

H]-17β-estradiol 

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

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

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

Receptor Binding Assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Estrogen Receptor Binding 

Assay (OCSPP 890.1250).

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

Assurance statements were provided. 

Page 84 of 252
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 18

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ 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 (expiry 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

CH3

OC4H9

3. Non-labeled ligand: 17β-estradiol

Supplier: Sigma-Aldrich (St. Louis, MO)

Catalog #: E8875

Lot #: 110M0138V

Purity: 100%

CAS #: 50-28-2

4. Radioactive ligand: [

3

H]-17β-estradiol

Supplier: Not reported

Catalog #: Not reported

Batch#: Not reported

Radiochemical purity: 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 #: 66-22-4

Page 85 of 252
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ 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 Rat Uterine 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) + Protease 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) Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-17β-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 Experimenta

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). 

Page 86 of 252
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 18

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

On the day of the assay, the specific activity of the stock solution [3H]-17β-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 achieve 

the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of nonlabeled 17β-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 saturation binding 

experiments conducted in July 2011 (Note: typically 50 ± 10 μg protein per tube). Each 

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 three 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 Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned 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-minute 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 

minutes 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]-17β-estradiol, followed by vortexing, and centrifugation for 10 minutes at 

1000 x g. Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. The 

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

with ethanol. 

3. Competitive Binding Experiment: A summary of the experimental conditions for the 

competitive binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat uterine cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 11.5-13.3% of radioligandb 

Concentration of test substance (as serial dilutions) 10−11 to 10−3 Mc

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 36-38 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 protease 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 the day of the assay, the specific activity of the stock solution [3H]-

17β-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+PI 

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 prepared 

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 binding (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. 

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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 pages 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-labeled 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; precipitate 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 solvent (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+PI 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]-17β-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. 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 17β-estradiol, the 

positive control, and the test substance) to the Hill equation formula which incorporated log 

IC50 as a parameter to be estimated. For parameters reported from the saturation binding 

experiment (Kd and Bmax) and competitive binding experiment (log IC50 and RBA), mean 

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 regression: 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.

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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 was 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 response 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%. 

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

test chemical concentration of 10−6M 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”

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b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

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

IC50: concentration of the test substance (M) at which 50% of the radioligand is displaced 

from the receptor 

Relative Binding Affinity (RBA %): (IC50 of 17β-estradiol ÷ IC50 of test substance) × 100 

Log RBA: Log10(IC50 of 17β-estradiol ÷ IC50 of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Figure 1 illustrates the non-specific, specific, 

and total binding curves for [3H]-17β-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 Scatchard plots that illustrate the binding of [3

H]-17β-estradiol to the estrogen 

receptor for the two saturation binding assays. 

Saturation binding experiment parameters are presented in Table 5. For the Septenber 27, 

2010 batch of cytosol, the Kd for [

3

H]-17β-estradiol was 0.081±0.006 nM, and the estimated 

Bmax was 52.70±1.05 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd for 

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

For the July 10, 2011 batch of cytosol, the Kd for [

3

H]-17β-estradiol was 0.303±0.090 nM, 

and the estimated Bmax was 33.59±0.70 fmol/100 μg protein for the prepared rat uterine 

cytosol. The Kd 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. 

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TABLE 5. Saturation Binding Experiment of [

3

H]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

Cytosol Prep Sept 27, 2010

R2 (unweighted) 0.991 0.986 0.979 0.979-0.991

Bmax (nM) 0.063 0.061 0.061 0.061±0.001

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

Kd (nM) 0.082 0.087 0.074 0.081±0.006

Cytosol Prep Jul 10, 2011

R2 (unweighted) 0.992 0.993 0.955 0.955-0.992

Bmax (nM) 0.029 0.029 0.027 0.029±0.001

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

Kd (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 R2 is reported and the mean ± SEM is reported for the other parameters.

R2 Goodness of fit for curve calculated for specific binding.

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FIGURE 1. Binding of [3H]-17β-estradiol to the Estrogen Receptor during the Saturation 

Binding Experiment. Representative of the runs conducted in October 2010 

and July 2011 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-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 experiments 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 the estimated mean log IC50 and RBA were not calculated for 

piperonyl butoxide. 

The estimated mean log IC50 was −9.2 M for the natural ligand (17β-estradiol) and −5.6 M 

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

RBA was 0.026% for 19-norethindrone. Confidence in these numbers is high due to the 

small variation. As the lowest average percent binding in the presence of piperonyl 

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butoxide was >75% 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 Piperonyl Butoxide with Estrogen Receptor from Rat Uterine 

Cytosol a

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

R2 (unweighted) 17β-estradiol NR NR NR NA

19-norethindrone NR NR NR NA

Piperonyl butoxide NR NR NR NA

Log IC50 (M) 17β-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

IC50 (M) c 17β-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. 

R2

 Goodness of fit

RBA Relative binding affinity

NR Not reported

NA Not applicable

TABLE 7. Binding Classification of Piperonyl 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 <0.5

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

FIGURE 3. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Piperonyl Butoxide, Unlabeled E2, 19-Norethindrone or 

Octyltriethoxysilane. Run 1 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

FIGURE 4. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Piperonyl Butoxide, Unlabeled E2, 19-Norethindrone or 

Octyltriethoxysilane. Run 2

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

FIGURE 5. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Piperonyl Butoxide, Unlabeled E2, 19-Norethindrone or 

Octyltriethoxysilane. Run 3

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay functioned

properly, each run was evaluated using the criteria shown in Table 8: 

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No

17β-estradiol fitted curve parameters

Loge 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 (Log10(M)−1) −1.1 to −0.7 −1.0 X

Weak Positive control (19-norethindrone) fitted curve parameters b

Loge 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 (Log10(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]-17β-estradiol from the ER on 

average across all concentrations

≤25% ≤19.6% X

a Data were obtained from pages 26, 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

The curve for the reference material showed that increasing concentrations of unlabeled 

17β-estradiol displaced [3H]-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 positive 

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 “noninteractive” in all three valid independent runs and thus has a final classification of “noninteractive” 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 piperonyl 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1250/ OECD None

butoxide. For the September 27, 2010 batch of cytosol, the Kd for [

3

H]-17β-estradiol was 

0.081 nM, and the estimated Bmax was 52.70 fmol/100 μg protein for the prepared rat 

uterine cytosol. For the July 10, 2011 cytosol, the Kd for [

3

H]-17β-estradiol was 0.303 nM, 

and the estimated Bmax was 33.59 fmol/100 μg protein. The Kd for each run was within the 

expected range of 0.03 to 1.5 nM. Confidence in these numbers is high based on the 

goodness of fit and the small variation among runs. Although the Scatchard plots fit straight 

lines to 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, 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, therefore the mean log IC50 and RBA were not calculated.

The estimated mean log IC50 was −9.2 M for the natural ligand (17β-estradiol) and −5.6 M 

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

RBA was 0.026% for 19-norethindrone. All performance criteria were met for 17βestradiol, 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 >75% 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.

Page 100 of 252
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 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:

Michelle Sharpe-Kass, M.S Date: 7/13/2012

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 7/16/2012

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. 

Page 101 of 252
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).

Page 102 of 252
PIPERONYL B TOXID E / 067501 

Estrogen Receptor Transcriptional Activation (2011) Page I of 15 

OCSPP 890.1300/ O ECD 455 

Primary Reviewer: Chris Schlosser, M.F.S. 

Health Effects Division 

Secondary Reviewer: Minerva Mercado, Ph.D. 

Health Effects Division 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); 

OCSPP 890. 1300; OECD 455. 

PC CODE: 06750 I DP BARCODE: D398558 

TXR#: 0056259 CAS No.: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl Butoxide (93.8% a.i.) 

SYNONYMS: S-[[2-(2-butoxyethoxy)ethoxy ]methyl]-6-propyl-1 ,3-benzodioxole 

CITATION: Willoughby, J.A. (2011) Pipcronyl butoxide: estrogen receptor transcriptional 

activation (human cell line (l lcLa-9903)). CeeTox, Inc., Kalamazoo, Ml. 

Laboratory Repo11 No.: 9146V-100377ERTA, November 17, 20 11. MRID 

48618804. Unpublished. 

SPONSOR: 

Willoughby, J.A. (2012). Supplemental Information - Laboratory Proficiency 

Data for ERTA assays and Sa1ura1ion Binding Data for AR and ER Binding 

Assays for Assorted Chemicals. CceTox, Inc., Kalamazoo, MI. July 19, 20 12. 

MRID 48843501. Unpublished. 

Consumer Specialty Products Association, Inc., 1900 - 17111 Street NW, Suite 

300, Washington, DC 

TEST ORDER #: CON-06750 1-9 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional acti vation assay (MRID 

48618804), hERa-HeLa-9903 cells cultured in vitro were exposed to piperonyl butoxide (93.8% 

a.i., Lot# El 04/10) al logari thmically increasing concentrations from 10- 11 to l o-.i Min 0.1 % 

DMSO for approx imately 24 hours in two independent runs. The experiments were performed 

using 96-well plates and each piperonyl butoxide concentration was tested in 6 wells/plate in 

each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce reporter 

(luciferase) gene products. Luciferase expression in response to activation of the estrogen 

receptor was measured using a proprietary luciferase assay. 

Piperonyl butoxide was tested up to the limit of cytotoxicity, which was noted at 10-.i M in one 

run. At concentrations up to 10- 5 M, the relative transcriptional activation (RTA) of piperonyl 

butoxide was :::;2.8%. 

Page 103 of 252
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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

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

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

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

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 16.5-24.3% PC. Piperonyl butoxide was only able to reach a maximum of 

1.9-2.8% PC when tested up to the highest concentration possible based on cytotoxicity. 

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

estrogen receptor transcriptional activation in this test system. 

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. 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ 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, MW 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: Not reported (NR)

Volatility: NR

Stability: ~2 years

Storage conditions: Ambient

CAS #: 51-03-6

Structure:

O

O

O

O

CH3

OC4H9

3. Reference substances

17β-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 concentration):

0.1%

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B. METHODS

1. Cell Culture: Stably-transfected hERα-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 Eagles Minimum 

Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10% 

dextran-coated charcoal-treated fetal bovine serum (source not reported), in an incubator 

under 5% CO2 at 37 oC. Upon reaching 75-90% confluence, cells were subcultured 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 Assays: For each test, cells were plated in a 96-well plate 

(estrogenic activity of plastic not reported) at a density of about 1×104 cells/100 μL 

medium/well and allowed to attach for 3 hours. Growth 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. Main assay runs 1 and 2 were conducted on June 14 and June 16, 

2011, respectively. Transcriptional 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 10−10 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 cells at passage 15, 25 and 28, respectively. 

Based on assay dates and passage numbers, it is unlikely that the cell used for proficiency 

testing were from the same frozen stock as cells used in the main assay. Each of the 

following chemicals 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 ---

17α-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 0.01b, 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 0.1b and 1 mM

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5 Positive Cytotoxic at 100μM. PCmax approx. 50% of PC. 

Binds to hERα 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 = RPCmax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCmax 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 ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable.

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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 10−15 to 10−8 Strong estrogen

17α-estradiol 57-91-0 10−13 to 10−6 Weak estrogen

Corticosterone 50-22-6 10−11 to 10−4 Negative compound

17α-methyltestosterone 58-18-4 10−12 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 estrogen 

receptor transcriptional activation if the RPCMax < PC10 in at least 2 of 2 runs. Log EC50

and Hill slope values are calculated only if a positive response is observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = 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 

PC50 = 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 

RPCMax = 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 

PCMax = concentration of a test chemical inducing the RPCMax

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 was

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.

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TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Piperonyl 

butoxidea 

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. POSITIVE 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 2a. In the proficiency tests, the reference chemicals 17β-estradiol, 

17α-estradiol and 17α-methyltestosterone were tested concurrently with each run of the 

assay (Table 2b). In the first run, the responsiveness of 17β-estradiol indicated decreased 

sensitivity to strong agonists, and the response to 17α-methyltestosterone showed an 

increased responsiveness to very weak agonists; despite the minor deviations this run is 

considered acceptable. Run 2 was inadequate as the PC50 could not be calculated for 17αmethyltestosterone indicating a decreased sensitivity to very weak agonists. Run 3 was

acceptable as 17β-estradiol and 17α-methyltestosterone performed within the expected 

range, but the Hill Slope for 17α-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, the 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.

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TABLE 2a. Proficiency Chemicals 

Compound Expected Response Lab Response

Run 1 Run 2 Run 3

Diethylstilbestrol Positive Positive Positive NA

17α-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 2b. 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 PC50 −11.4 to −10.1 −9.6 −11.3 −10.6 Run 1

Log PC10 <−11 −11.5 −12.5 −12.1 X

Log EC50 −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 PC50 −9.6 to −8.1 −8.3 −9.4 −8.7 X

Log PC10 −10.7 to −9.3 −9.3 −10.5 −9.9 X

Log EC50 −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 PC50 −6.0 to −5.1 −6.2 NC −5.2 Run 1, 2

Log PC10 −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 PC50, 

log PC10, log EC50, 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 17αestradiol, there was variability in the response curves for 17β-estradiol and 17α-estradiol and 

the lack of responsiveness of the cells to 17α-methyltestosterone. 

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. The normalized luminescence data indicates that the 

response of the cells in Run 2 was approximately 85-90% lower than in Run 1 at 

concentrations 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 PC50 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 RPCMax was 16.5% for the first run and 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

24.3% for the second run. Cytotoxicity was observed at the highest concentrations tested of 

corticosterone and 17α-methyltestosterone. 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Yes No

17β-estradiol

Log PC50 −11.4 to −10.1 −10.2 −10.7 X

Log PC10 <−11 −11.7 −11.9 X

Log EC50 −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 PC50 −9.6 to −8.1 −8.6 −8.7 X

Log PC10 −10.7 to −9.3 −9.8 −9.7 X

Log EC50 −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 PC50 −6.0 to −5.1 NA b NA X

Log PC10 −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 pages 12 and 23 of the study report. 

b NA = not applicable; 17α-methyltestosterone did not achieve 50% activation at up to 10−5 M.

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

FIGURE 1. Relative Transcription Activation (RTA) of the Natural Ligand, 

17β-estradiol, Compared to the Positive Control, 1 nM 17β-estradiol.

Mean ± SD are presented (n=6).

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

FIGURE 2. Relative Transcription Activation (RTA) of the Weak Estrogen, 

17α-estradiol, Compared to the Positive Control, 1 nM 17β-estradiol.

Mean ± SD are presented (n=6).

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

FIGURE 3. Relative Transcription Activation (RTA) of the Very Weak Estrogen, 

17α-methyltestosterone, Compared to the Positive Control, 1 nM 

17β-estradiol.

Mean ± SD are presented (n=6).

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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). The 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 PC50 (50% of the maximum response) for 1nM 

E2 in this assay was 616950 and 27067 in Runs 1 and 2, respectively, and the PC10 (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 170620 72 1233900 170620

2 850 319 54983 21606 65 54133 21606

a Data were obtained from pages 31-32 of the study report.

b Positive control was 17β-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 RPCMax was 1.9% for 

the first run and 2.8% for the second run, and the associated PCMax was 10−4 M for the first 

run and 10−7 M for the second run. Because the RPCMax < PC10 in both runs, piperonyl 

butoxide was considered negative for estrogen receptor transcriptional activation in this test 

system. 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Transcriptional Activation (RTA) of Piperonyl Butoxidea

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 EC50 NA NA

Hill Slope NA NA

RPCMax 1.9 2.8

PCMax 10−4 10−7

PC50 NA NA

PC10 NA NA

a Data were obtained from pages 21-22 of the study report. 

* Cytotoxicity observed (55.6% viability)

NA = Not Applicable

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ OECD 455

FIGURE 4. Relative Transcription Activation (RTA) of Piperonyl Butoxide Compared to 

the Positive Control, 1 nM 17β-estradiol.

3. Performance Criteria: While the Log PC50, Log PC10, Log EC50, and Hill slope values for 

the concurrent reference chemicals fell within or near the acceptable ranges (Table 3), the 

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PIPERONYL BUTOXIDE / 067501 OCSPP 890.1300/ 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 concentrations tested. 

Piperonyl butoxide is not an agonist of the human estrogen receptor alpha (hERα) 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 decreased sensitivity of the assay to very weak agonists. Although the 

conditions of this assay were not optimal to detect very weak activity, piperonyl butoxide 

responses were similar to those of the negative control corticosterone and not comparable to 

the responses of 17α-methyltestosterone, which was able to reach a maximum of 16.5-

24.3% PC. Piperonyl butoxide was only able to reach a maximum of 1.9-2.8% PC when 

tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < 

PC10 in both assay runs, piperonyl butoxide was considered negative for estrogen receptor 

transcriptional activation in this test system. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

• Cytotoxicity was observed in cells treated with two of the reference compounds 

(corticosterone and 17α-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. 

• The 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. 

Page 117 of 252
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 

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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). 

Disclaimer: 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 

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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 25.5o

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 (17ȕ-estradiol and testosterone) were 

not measured in this study. 

All validity and performance criteria were met in this study. 

Page 3 of 47 

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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 Effects1,2 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 Yes3 No No No NA NA NA NA 

0.0944 Yes No No No No No Yes3 No No No NA NA NA NA 

Abbreviations: E2 17ȕ-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: 

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1. The study author’s analysis of male VTG resulted in <0.1 x 106 Pg/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 

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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. 

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B. Test organism: 

Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes EPA recommends that fish be from a single laboratory stock.

Were acclimation conditions same as definitive 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortalities were <5% prior to preexposure. 

Fish did not receive any treatment for disease during acclimation period. Behavioral abnormalities or clinical signs 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/day Details on feeding: Food was withheld 12 

hours prior to tissue sampling. 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age 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 test initiation (if determined): 2.53 

±20%=2.02-3.04 

A sub-sample of fish (not defined) used to 

stock aquaria for pre-exposure phase were weighted. Mean and range calculated from 

Pre-test Weights in report appendix, p. 101. 

EPA recommends thata 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 g 

Individual weights within ± 20% of the estimated mean 

Mean weight of females at test initiation (if determined): 1.89 

±20%=1.51-2.27 

A sub-sample of fish (not defined) used to stock aquaria for pre-exposure phase were weighted. Mean and range calculated from Pre-test Weights in report appendix, p. 101. 

Range of individual weights (females) at test initiation (if determined): Range: 1.54-2.31 g 

Five females over 20% of mean – up to 22.2% over 

Mean length of males at test initiation (if determined): 

Not reported 

Mean length of females at test initiation (if determined): 

Not reported 

Duration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical 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: 2 Number of females per tank: 4 Pre-exposure fecundity: At least 15 

eggs/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: None 

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C. Exposure System

Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Continuous flow-through. EPA recommends the use of a flow-through system. As 

noted in the Corrections and Clarifications document1 , 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.2

Flow-through rate: 2.7 L/hr or at least 6.5 

volume additions/test vessel/day. 

Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor Screening 

Program 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). 

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flowthrough systems: Flow-through diluter system was used and the 4 replicate test 

vessels. Treatment recoveries prior to test initiation were not reported. Flow splitting accuracy was not reported. 

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, carbon 

and 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 for pesticides, heavy metals, and other contaminants? 

Yes 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test 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 of 

4-in diameter PVC pipe per tank 

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-inches Additional details on exposure system: None Table 5: Summary of Water Quality Characteristics in the Test System.

Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Temperature (°C) 24.4 25.5 25 Daily 

EPA recommends temperature 25±1oC; inter-replicate and intertreatment differentials should not 

exceed 1oC.

pH 7.3 8.1 7.7 3X/week EPA recommends pH 6.5 to 9.0.

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Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Dissolved oxygen (mg/L) 6.7 9.2 7.5 3X/week 

EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3) 32 44 38 Weekly 

EPA recommends total alkalinity >20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 136 152 142 Weekly Total organic carbon (mg C/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 <0.06 Bi-monthly 

EPA recommends that unionized 

ammonia in the dilution water be <1 

Pg/L. 

Residual chlorine (mg/L) <0.01 <0.01 <0.01 Bi-monthly 

EPA recommends that residual 

chlorine in dilution water be <10 

Pg/L. 

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Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Conductivity (PS/cm) Not reported Not reported Not reported 

Study authors state: “Water quality characteristics of the dilution water were monitored bimonthly for pH, dissolved oxygen (DO), conductivity, hardness, alkalinity, ammonia, residual chlorine (oxidants); and at least annually for iodide (I-), polyaromatic hydrocarbons (PAHs), pesticides, and metals. The dilution water was analyzed for pesticides, PAHs, and metals on January 28, 2011. All water quality measurements cited above met the U.S. EPA criteria for aquatic toxicity test culture/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. 

1 Means 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 at 

2 to 5.1 Pg/L (mean 3.6 Pg/L, measured semi-annually). 

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D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes Four-day range finding studies 

were conducted with adult fathead minnow, studying mortality and morbidity (defined as abnormal development or unusual behavioral reaction to exposure). Study concentrations were selected based on these four studies. 

EPA recommends conducting a range-finder if 96-hour LC50

data for the fathead minnow are unavailable. 

If yes, what was the method for determining the highest test concentration in the range-finder? 

 Experimentally-derived MTC of 0.1 mg/L based on morbidity and mortality 

4-day LC50 was 12.9 mg/L, EC50

was 1.6 mg/L EC50 based on moderate gill 

hemorrhage and abnormal 

swimming 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) 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Life 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 and 25.0 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 LC50, may be 

selected as the highest exposure concentration in the 21-day 

test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the definitive 

test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Method for selecting the highest test concentration in the definitive test:

Maximum tolerated concentration based on morbidity and mortality observations in the range finder to ensure no overt toxic response 

Also considered using 0.33x the lower confidence interval of the 4-day EC50 of 1.6, (0.6-4.4) mg 

a.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 LC50), whichever 

concentration is lowest.

Reference study citation (if applicable):

Not reported 

Separation of test concentrations: 0 (control), 0.011, 

0.033, and 0.1 mg a.i./L 

EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or ten-fold) 

is scientifically acceptable1 . 

Number of test concentrations: 3 EPA recommends a minimum of 3 concentrations 

and a control, plus solvent control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean time-weighted average 

Limit 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Level 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 complete block 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 solvent control (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 item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Was a solvent used? No Solvent type (if applicable): NA Maximum solvent concentration (if applicable): 

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? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 (light:dark).

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp.69–92. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing 

and Assessment. No. 23. Paris, France.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Light 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 exposure duration 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 fertilization 

success measurement but do not give details on the magnification or stage of development when fertilization was determined. Study authors reference the EPA guideline as the method used to map nuptial tubercles. Immediately after anesthetization of an individual fish, length and weight measurements were taken, and fresh tissues (e.g., gonads) collected. During necropsy, rapid gonad fixation in modified Davidson’s fixative was performed to prevent autolysis and cellular deterioration. Using a syringe, approximately 0.5 mL of modified Davidson’s fixative was gently applied to the gonads in situ. 

Approximately 90 seconds following the application of fixative, the liquid fixative within the abdomen was removed with a gauze sponge, and the gonads excised. The fixed gonads were removed and weighed (fixed weight to the nearest 0.1 mg) to determine the GSI (GSI=100% x gonad wt/body wt). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Gonadal stage was determined based on a scale that was comparable to that provided 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 equal proportions, 2.5=mature sperm predominate, 3.0=germinal epithelium thinner than Stage 2.5, 3.5=germinal epithelium barely apparent and large coalescing tubule lumina are filled with spermatozoa, 4.0=not used. Findings were recorded in an electronic data recording system. The pathology report was provided. Blood was collected from the caudal artery/vein with a heparinized microhematocrit capillary tubule. Plasma was separated from the blood via centrifugation (approximately 3 minutes at 15,000 x g) and stored with protease inhibitors at -75oC to -85oC until analyzed for VTG. Sex steroids were not analyzed in the present study. Non-detected (ND) samples were reported in data sets as < detection limit (DL). The measurement of VTG in plasma samples was performed using a commercial ELISA kit (Biosense, Bergen, Norway). The stock solutions were clear and colorless with no precipitate. 

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Table 8: Summary of Treatment Concentrations in Fish Short-Term Reproduction Assay with PBO.

Treatment ID 

Nominal Concentration 

(mg a.i./L) 

Mean Time-weighted 

Concentration 

(mg a.i./L) 

Mean CV 

(%) 

Details or Remarks – 

Mean Measured 

Concentrations (mg 

a.i./L) 

Guideline Recommendations 

Control (dilution water only) 

0 <LOQ NA NA 

EPA recommends that test item concentrations be 

maintained at a coefficient of variation (CV) 

20%.

Treatment 1 0.011 0.0101 12.8 0.0103 Treatment 2 0.033 0.0348 3.0 0.0351 Treatment 3 0.10 0.0944 3.7 0.0952 Abbreviations: CV Coefficient of variation. 

LOQ=0.0010 mg a.i./L. 

E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sex 

characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology, and plasma vitellogenin 

were evaluated.

Were raw (individual) data provided? Yes

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EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical 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 behavior. 

II. RESULTS AND DISCUSSION 

A. Results 

Mean male and female survival was 100% at all treatment levels (Table 9). 

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Table 9: Adult Fish Survival in Fathead Minnow (Pimephales promelas). 

Treatment 

(mg a.i./L) 

[TWA measured] 

Males Females 

n1 # Surviving % Survival n1 # Surviving % Survival 

Control (<LOQ) 8 8 100 16 16 100 

0.0101 8 8 100 16 16 100 

0.0348 8 8 100 16 16 100 0.0944 8 8 100 16 16 100 

1 Total 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 1.20 g in the control 

and 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, 61.6 and 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, 0.0348 and 0.0944 

mg a.i./L treatment levels, respectively. 

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Table 10: Size at Test Termination in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Control (<LOQ) 4 2.36 0.14 4 1.24 0.10 NA 62.1 3.0 NA 52.6 1.7 

0.0101 4 2.29 0.25 4 1.37 0.20 NA 64.9 1.8 NA 53.1 2.3 

0.0348 4 2.17 0.38 4 1.26 0.01 NA 61.6 2.3 NA 50.9 1.1 0.0944 4 1.96 0.19 4 1.20 0.05 NA 58.0 2.0 NA 51.5 1.3 

Abbreviations: NA Not applicable. SD Standard deviation. 

LOQ=0.0010 mg a.i./L. 

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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 (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured]

Fecundity1 Fertilization Success2

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 (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured]

Males Females 

n Median Tubercle 

Score1 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 GSI1

(%) 

±SD n Mean GSI1

(%) 

±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 (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured]

Males Females 

N Median Stage1 n Median Stage2

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.

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured] 

Diagnostic Observations1

Severity 

Increased Proportion of 

Spermatogonia 

Presence of Testis-Ova 

Increased Testicular 

Degeneration 

Duct Mineralization 

Interstitial cell (Leydig) 

hypertrophy/ 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 8 5 NA NA 8 7 8 7 8 8 

1 8 1 NA NA 8 1 8 1 8 0 

2 8 0 NA NA 8 0 8 0 8 0 3 8 0 NA NA 8 0 8 0 8 0 4 8 2 NA NA 8 0 8 0 8 0 

0.0101 0 8 1 NA NA 8 7 8 6 8 7 

1 8 4 NA NA 8 0 8 0 8 1 

2 8 1 NA NA 8 1 8 2 8 0 3 8 0 NA NA 8 0 8 0 8 0 4 8 2 NA NA 8 0 8 0 8 0 

0.0348 0 8 5 NA NA 8 5 8 7 8 8 

1 8 1 NA NA 8 3 8 1 8 0 

2 8 1 NA NA 8 0 8 0 8 0 3 8 1 NA NA 8 0 8 0 8 0 4 8 0 NA NA 8 0 8 0 8 0 

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Treatment 

(mg a.i./L) 

[TWA measured] 

Diagnostic Observations1

Severity 

Increased Proportion of 

Spermatogonia 

Presence of Testis-Ova 

Increased Testicular 

Degeneration 

Duct Mineralization 

Interstitial cell (Leydig) 

hypertrophy/ 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.0944 0 8 4 NA NA 8 3 8 7 8 8 

1 8 2 NA NA 8 4 8 1 8 0 

2 8 0 NA NA 8 1 8 0 8 0 3 8 2 NA NA 8 0 8 0 8 0 4 8 0 NA NA 8 0 8 0 8 0 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

LOQ=0.0010 mg a.i./L. 

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion of 

Spermatogonia 

Increased Vascular or 

Interstitial Proteinaceous 

Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 NA NA NA NA NA NA NA NA 8 7 

1 NA NA NA NA NA NA NA NA 8 1 

2 NA NA NA NA NA NA NA NA 8 0 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

0.0101 0 NA NA NA NA NA NA NA NA 8 4 

1 NA NA NA NA NA NA NA NA 8 2 

2 NA NA NA NA NA NA NA NA 8 2 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

0.0348 0 NA NA NA NA NA NA NA NA 8 6 

1 NA NA NA NA NA NA NA NA 8 2 

2 NA NA NA NA NA NA NA NA 8 0 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

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Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion of 

Spermatogonia 

Increased Vascular or 

Interstitial Proteinaceous 

Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.0944 0 NA NA NA NA NA NA NA NA 8 5 

1 NA NA NA NA NA NA NA NA 8 2 

2 NA NA NA NA NA NA NA NA 8 1 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

LOQ=0.0010 mg a.i./L. 

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Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas). 

Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte Atresia 

Perifollicular Cell 

Hyperplasia/ Hypertrophy 

Decreased Yolk Formation 

Aggregates of macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 16 6 NA NA NA NA NA NA 

1 16 2 NA NA NA NA NA NA 

2 16 2 NA NA NA NA NA NA 3 16 6 NA NA NA NA NA NA 4 16 0 NA NA NA NA NA NA 

0.0101 0 16 5 NA NA NA NA NA NA 

1 16 3 NA NA NA NA NA NA 

2 16 0 NA NA NA NA NA NA 3 16 8 NA NA NA NA NA NA 4 16 0 NA NA NA NA NA NA 

0.0348 0 16 8 NA NA NA NA NA NA 

1 16 3 NA NA NA NA NA NA 

2 16 2 NA NA NA NA NA NA 3 16 3 NA NA NA NA NA NA 4 16 0 NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte Atresia 

Perifollicular Cell 

Hyperplasia/ Hypertrophy 

Decreased Yolk Formation 

Aggregates of macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

0.0944 0 16 5 NA NA NA NA NA NA 

1 16 2 NA NA NA NA NA NA 

2 16 2 NA NA NA NA NA NA 3 16 7 NA NA NA NA NA NA 4 16 0 NA NA NA NA NA NA 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

LOQ=0.0010 mg a.i./L. 

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Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Interstitium/multifocal inflammation Egg Debris in Oviduct 

Granulomatous 

Inflammation 

Decreased Post-Ovulatory 

Follicles 

n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 NA NA 16 15 16 14 16 11 

1 NA NA 16 0 16 1 16 2 

2 NA NA 16 1 16 0 16 1 3 NA NA 16 0 16 1 16 2 4 NA NA 16 0 16 0 16 0 

0.0101 0 NA NA 16 16 16 14 16 13 

1 NA NA 16 0 16 2 16 1 

2 NA NA 16 0 16 0 16 0 3 NA NA 16 0 16 0 16 2 4 NA NA 16 0 16 0 16 0 

0.0348 0 NA NA 16 16 16 15 16 11 

1 NA NA 16 0 16 1 16 3 

2 NA NA 16 0 16 0 16 0 3 NA NA 16 0 16 0 16 2 4 NA NA 16 0 16 0 16 0 

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Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Interstitium/multifocal inflammation Egg Debris in Oviduct 

Granulomatous 

Inflammation 

Decreased Post-Ovulatory 

Follicles 

n Incidence n Incidence n Incidence n Incidence 

0.0944 0 NA NA 16 16 16 16 16 13 

1 NA NA 16 0 16 0 16 1 

2 NA NA 16 0 16 0 16 1 3 NA NA 16 0 16 0 16 1 4 NA NA 16 0 16 0 16 0 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

LOQ=0.0010 mg a.i./L. 

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Mean male VTG was <0.1 x 106 ng/mL at all treatment levels and mean female VTG was 25.9x106, 

25.8x106

, 26.0x106 and 26.1x106 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 (Pimephales 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 106 NA 4 25.9x106 0.263x106

0.0101 4 <0.1 x 106 NA 4 25.8x106 0.573x106

0.0348 4 <0.1 x 106 NA 4 26.0x106 0.364x106

0.0944 4 <0.1 x 106 NA 4 26.1x106 0.313x106

Abbreviations: NA Not applicable. SD Standard deviation. 

LOQ=0.0010 mg a.i./L. 

Mean plasma testosterone and plasma 17ȕ-estradiol in male and females were not measured (Table 20). 

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Table 20: Plasma Sex Steroids in Fathead Minnow (Pimephales promelas). Not measured.

Treatment 

(mg a.i./L) 

[TWA 

measured]

Plasma Testosterone (T) Plasma 17ȕ-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 (Pimephales 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 pd0.05 (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. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with PBO. 

Treatment 

(mg a.i./L) 

[TWA measured]

Tubercle Score4 GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative control (<LOQ) 54 NA 0 NA No 0 NA NA NA NA NA 

0.0101 58 0.010 -20.9 0.653 No NA NA NA NA NA NA 

0.0348 55 0.906 -4.0 0.995 Yes3 NA NA NA NA NA NA 0.0944 56 0.174 7.8 0.968 Yes3 NA NA NA NA NA NA 

Statistical Test Dunnett’s Dunnett’s NA NA NA NA Abbreviations: Diff. Difference. E2 17ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. 

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Increased testes degeneration at mid (marginally significant) and high concentrations. 4 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. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with PBO. 

Treatment 

(mg a.i./L) 

[TWA measured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

% Diff. p 

% 

Diff 

P Median p 

% 

Diff. 

p 

Effect? 

(Yes/No) 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Negative control 

(<LOQ) 

0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA 

0.0101 6.8 0.054 0.2 0.243 0 NA 17.1 0.673 No -0.7 0.843 NA NA NA NA 

0.0348 14.0 0.005 0 1.00 0 NA 4.7 0.988 No 0.3 0.983 NA NA NA NA 0.0944 18.7 0.0005 -0.1 0.852 0 NA 7.8 0.948 No 0.6 0.892 NA NA NA NA 

Statistical Test 

JonckheereTerpstra Dunnett’s NA Dunnett’s NA Dunnett’s NA NA 

Abbreviations: Diff. Difference. E2 17ȕ-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

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

LOQ=0.0010 mg a.i./L. 

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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction 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 0.978 -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 

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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 Pg 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 106

 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. 

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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. 

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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

Species: Pimephales promelas Brine: Not Applicable

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

Material: Piperonyl Butoxide Project: Insecticide

in-life dates not reported in study; 067501 48687701

067501 48687701

Batch Note:

Sample Note:

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

13-4760-3726 Fecundity 10.1 34.8 18.75 NA Jonckheere-Terpstra Step-Down Test

11-6044-2142 Fecundity 10.1 34.8 18.75 9.19% Dunnett Multiple Comparison Test

07-1481-5287 FemaleBodyWt 94.4 >94.4 NA 16.5% Dunnett T3 Multiple Comparison Test

18-7888-2608 FemaleGSI 94.4 >94.4 NA 48.4% Dunnett Multiple Comparison Test

13-5904-8490 FemaleLength 94.4 >94.4 NA 5.89% Dunnett Multiple Comparison Test

20-0951-1690 FemaleVTG 94.4 >94.4 NA 2.71% Dunnett Multiple Comparison Test

03-7035-4533 Fertility 94.4 >94.4 NA 0.31% Dunnett Multiple Comparison Test

16-6147-4066 MaleBodyWt 34.8 94.4 57.32 NA Jonckheere-Terpstra Step-Down Test

03-5486-3646 MaleBodyWt 94.4 >94.4 NA 20.6% Dunnett Multiple Comparison Test

10-1798-4877 MaleGSI 94.4 >94.4 NA 57.4% Dunnett Multiple Comparison Test

02-2355-7914 MaleLength 94.4 >94.4 NA 7.08% Dunnett Multiple Comparison Test

02-4393-0165 MaleMedianTubercleScore <10.1 10.1 NA 5.74% Dunnett Multiple Comparison Test

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 165 of 252
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.

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

Fecundity Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 33.5 32.6 34.4 33 34.3 0.278 0.556 1.66% 0.0%

10.1 4 35.8 31.2 40.4 33.4 39.9 1.44 2.88 8.04% -6.79%

34.8 4 38.2 36.4 40 37 39.7 0.569 1.14 2.98% -14.0%

94.4 4 39.8 36.4 43.2 37.2 42.4 1.07 2.15 5.4% -18.7%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.24 1.08 1.4 1.1 1.33 0.0512 0.102 8.25% 0.0%

10.1 4 1.38 1.06 1.69 1.22 1.66 0.0977 0.195 14.2% -10.9%

34.8 4 1.26 1.25 1.27 1.25 1.26 0.0025 0.005 0.4% -1.41%

94.4 4 1.21 1.13 1.28 1.14 1.26 0.025 0.05 4.15% 2.82%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 10.1 6.36 13.8 6.61 11.7 1.18 2.35 23.3% 0.0%

10.1 4 11.8 7.6 16 8.8 14.2 1.33 2.65 22.4% -17.1%

34.8 4 10.6 5.46 15.7 7.79 14.9 1.61 3.21 30.4% -4.68%

94.4 4 10.9 7.87 13.9 9 13.4 0.95 1.9 17.4% -7.8%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 52.6 50 55.3 50.3 54.3 0.841 1.68 3.2% 0.0%

10.1 4 53.1 49.5 56.6 50.8 56 1.12 2.24 4.22% -0.81%

34.8 4 51 49.3 52.7 49.8 52.3 0.538 1.08 2.11% 3.18%

94.4 4 51.5 49.4 53.6 50 53 0.645 1.29 2.51% 2.18%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 2.59E+7 2.55E+7 2.63E+7 2.57E+7 2.63E+7 1.31E+5 2.63E+5 1.01% 0.0%

10.1 4 2.58E+7 2.48E+7 2.67E+7 2.54E+7 2.66E+7 2.87E+5 5.74E+5 2.23% 0.68%

34.8 4 2.60E+7 2.55E+7 2.65E+7 2.57E+7 2.63E+7 1.47E+5 2.94E+5 1.13% -0.29%

94.4 4 2.61E+7 2.57E+7 2.65E+7 2.58E+7 2.64E+7 1.25E+5 2.50E+5 0.96% -0.58%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

Fertility Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 99 98.7 99.2 98.8 99.1 0.0641 0.128 0.13% 0.0%

10.1 4 99.1 99.1 99.2 99.1 99.2 0.0292 0.0585 0.06% -0.2%

34.8 4 98.9 98.6 99.3 98.6 99.1 0.119 0.238 0.24% 0.0%

94.4 4 98.9 98.6 99.1 98.7 99.1 0.0851 0.17 0.17% 0.08%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 2.36 2.13 2.58 2.24 2.55 0.0699 0.14 5.93% 0.0%

10.1 4 2.3 1.9 2.69 2.13 2.65 0.123 0.246 10.7% 2.65%

34.8 4 2.18 1.56 2.79 1.69 2.63 0.192 0.384 17.7% 7.74%

94.4 4 1.97 1.67 2.26 1.77 2.19 0.0937 0.187 9.53% 16.6%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.26 0.829 1.68 1.01 1.6 0.134 0.268 21.3% 0.0%

10.1 4 0.993 0.752 1.23 0.88 1.2 0.0756 0.151 15.2% 20.9%

34.8 4 1.2 0.57 1.84 0.78 1.62 0.2 0.399 33.1% 3.98%

94.4 4 1.35 0.448 2.26 0.86 1.85 0.284 0.569 42.0% -7.77%

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 166 of 252
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.

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 62.1 57.4 66.8 58 65 1.48 2.95 4.76% 0.0%

10.1 4 64.9 61.9 67.8 62.5 67 0.921 1.84 2.84% -4.43%

34.8 4 61.6 57.9 65.3 59.5 64.5 1.16 2.32 3.77% 0.81%

94.4 4 58 54.8 61.2 57 61 1 2 3.45% 6.64%

C-μg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 53.9 52.7 55.1 53 54.5 0.375 0.75 1.39% 0.0%

10.1 4 58 56.3 59.7 56.5 59 0.54 1.08 1.86% -7.66%

34.8 4 54.5 49.8 59.2 51.5 57.5 1.47 2.94 5.4% -1.16%

94.4 4 56.1 55.4 56.9 55.5 56.5 0.239 0.479 0.85% -4.18%

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 167 of 252
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.

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 168 of 252
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.

C-μg ai/L Control Type

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

C-μg ai/L Control Type

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

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 169 of 252
DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 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. 

Page 170 of 252
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).

Page 171 of 252
PIPERONYL B TOXIDE/067501 

P rimary Reviewer: Stephen C. Dapson. Ph.D. 

Senior· Pharmacologist, RAB6, RED (7509P) 

Secondary Reviewer: Orel! Akerman. Ph.D. 

Health Effects Division 

/11 vi vo Hershberger Assay (20 11 )/ Page I of 13 

OCSPP 890. 1-'00/ OECD -'-'1 

Signature: ~<2o& J...,- SJ:> 

Signa

Date: 

ture: ,/S ~ 

Date: ~ l \ 9' ( 1, .; 

Template version 10120 11 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 44 1 

PC CODE: 067501 DP BARCODE: D3985 58 

TXR#: 0056259 CAS#: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93.8% a.i.) 

SYNO NYMS: 5-[[2-(2-butoxyetho.>ry)ethoxy]methylj-6-propyl-l .3-benzodioxole 

CITATION: Zorilla, L. (20 11 ). The Hershberger Bioassay for Piperonyl Butoxide. 

Integrated Laboratory Systems, Inc .. Durham, NC. Laboratory Project Study 

lD: C192-200. November 10, 20 11. Unpublished. EPA MRID Number 

48618805. 

SPONSOR: Consumer pecialty Products Association Inc. for the Piperonyl Butoxide Task 

Force IL 1667 K Street NW, Suite 300, Washington. DC 20006 

TEST ORDER #: CON-067501-9 

EXECUTIVE SUMMARY: In a Hershberger assay (MRJD 48618805) screening for 

androgeni c activity, piperonyl butoxide (93.8% a. i .. Lot# E104-IO) in corn oil was administered 

daily via oral gavage to groups of 58- to 59-day old, castrated male Sprague Dawley rats 

(8/group) at dose levels of 0 (vehicle). 320. or I 000 (limit dose) mg/kg/day. An androgenic 

positive control group consisting of eight castrated rats exposed to 0.4 mg/kg/day testosterone 

propionate (TP) in corn oil by subcutaneous (s.c.) injection was also included. To screen for 

potential anti-androgenic activity. piperonyl butoxide in corn oil was administered daily via oral 

gavage to 58- to 59-day old, castrated male Sprague Dawley rats (8/group) at dose levels of I 00. 

320. or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 

mg/kg/day by s.c. injection. The negative control group consisted of eight castrated male rats 

dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the anti-androgeni c positive control 

group consisted or eight castrated male rats dosed orall y with flutan1ide (FT) in corn oil at 3 

mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

Page 172 of 252
In vivo Hershberger Assay (2011)/ Page 2 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ 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 110%). 

Animals in the TP group had increases (p≤0.05) in accessory sex organ weights as follows: 

1799% in seminal vesicles; 997% in ventral prostate; 242% in levator ani-bulbocavernosus 

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

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

and glans penis (26% vs. 22%). The maximum guideline-specified %CV was slightly exceeded 

for the Cowper’s gland in the 1000 mg/kg/day piperonyl butoxide group (58% vs. 55%). All 

other %CV values were less than the maximum recommended values.

In the anti-androgen assay, no adverse, treatment-related findings were observed on mortality, 

clinical signs, or accessory sex organ weight. Terminal body weight was decreased (NS) by 4% 

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

decreased (p<0.05) by 36% at 320 mg/kg/day and decreased (p<0.05) by 62% at 1000 

mg/kg/day. The only significant decrease in androgen-sensitive tissue weights observed in 

animals dosed with piperonyl butoxide + TP, was a 21% (p≤0.05) decrease in LABC weight at 

1000 mg/kg/day. 

Animals dosed with TP +FT had decreased (p<0.05) overall body weight gains (↓40%). 

Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT in the anti-androgen assay had 

decreased (p≤0.05) accessory sex organ weights as follows: 90% in seminal vesicles; 85% in 

ventral prostate; 56% in LABC; and 66% in Cowper’s gland. The maximum guideline-specified 

%CV was exceeded for the glans penis in the negative control and for the ventral prostate in the 

TP + FT positive control. All other %CV values were less than the maximum recommended 

values.

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Piperonyl butoxide was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

Page 173 of 252
In vivo Hershberger Assay (2011)/ Page 3 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

COMPLIANCE: 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 23, 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 20 days at 1-10°C

CAS #: 51-03-6

Structure:

O

O

O

O

CH3

O CH3

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 applicable

Page 174 of 252
In vivo Hershberger Assay (2011)/ Page 4 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

6. Test Animals:

Species: Rat (castrated males)

Strain: Sprague Dawley (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 heattreated 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 tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

22-24oC

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 activity 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 significant 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 significant 

differences in group means at study initiation.

Page 175 of 252
In vivo Hershberger Assay (2011)/ Page 5 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

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) 1000 + 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 antiandrogen 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 received 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, 20111); 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.

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In vivo Hershberger Assay (2011)/ Page 6 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

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. Dosage 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 weight 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 ttests. Since one tissue was significantly 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 at 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 were 

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PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

weighed, euthanized by CO2 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 of 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%). 

Page 178 of 252
In vivo Hershberger Assay (2011)/ Page 8 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body 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 (1000) 

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 7c 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 57.1*

(↑110)

11.4 8 30.0 10.5 7 1.1*

(↓96)

9.9

a Data were obtained from Table 6 on page 23 and Appendix V on page 114 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 1000 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. 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. 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 AntiAndrogen 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 

(1000 + 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 7c 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 115 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 Standard Deviation

* Significantly different from vehicle controls at p<0.05.

C. FOOD CONSUMPTION (Optional): Food consumption was not determined. 

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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 recommended 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

(1000)

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-androgen 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 1000 mg/kg/day, the LABC weight 

was decreased (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; 56% in LABC; and 66% in Cowper’s gland. The 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. 

Page 180 of 252
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PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

Table 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 

(1000 + 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 46c

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 20c 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 

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.

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 

glans penis, Cowper’s glands, seminal vesicle, ventral prostate and LABC weights 

compared 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 increase 

androgen dependent tissue weights compared to the weights of these tissues in the vehicle 

control animals.

Page 181 of 252
In vivo Hershberger Assay (2011)/ Page 11 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

In the antagonist assay, final body weights of animals administered 100, 320, or 1000 mg/kg 

of PBO were not statistically different as compared 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 reduction 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 on 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: seminal 

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. 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. 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 accessory 

Page 182 of 252
In vivo Hershberger Assay (2011)/ Page 12 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ OECD 441

sex organ; therefore, the finding was considered incidental. 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 vesicles, 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 weights. 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, the 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. This higher CV did not alter the conclusions of the anti-androgen 

assay. 

Page 183 of 252
In vivo Hershberger Assay (2011)/ Page 13 of 13

PIPERONYL BUTOXIDE/067501 OCSPP 890.1400/ 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, LABC, 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 the 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 the 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 significance 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 biologically 

significant. Therefore, co-administration of corn oil with TP had no effect on the androgendependent tissue weights. 

Page 184 of 252
DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 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. 

Page 185 of 252
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).

Page 186 of 252
PIPERONYL B TOXIDE/ 06750 1 

Primary Reviewer: Yung G. Yang. Ph.D. 

Risk Assessment Branch VI, Health Effects Division 

Secondary Reviewer: Eli zabeth Mendez. Ph.D. 

Health Effects Division 

female Pubertal Assay (20 11 ) I Page I of 16 

OC PP 890.1.tSO/ OECD None 

Sign a tu re: --H-----..~-=---.R"--.9-

Da te: -:::::::="' r~S::""'D'7"( S igna tu re ..... · -~ll"'l'OQllll~r;tri'oW!f"

D ate: ----IH-'+-'-~-

DATA EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890. 1450; OECO None. 

PC CODE: 067501 DP BARCODE: D398558 

TXR#: 0056259 CAS#: 51 -03-6 

TEST MATERIAL (PURJTy): Piperonyl butoxidc (93.8% a.i.) 

SYNONYMS: 5-l[2-(2-butoxyethoxy )ethoxy] methyl]-6-propyl- 1.3-benzod ioxole 

CITATION: Zorilla, L. (20 11 ). Puberta l development and thyroid function in intact 

juvcnile/peripubertal female and male rats with piperonyl butoxide. Integrated 

Labo ratory Systems, Inc., Durham, NC. Laboratory Proj ect Study ID: 

C J 92-300, November 10, 20 l l. MRID 486 18806. Unpublished. 

PONSOR: Piperonyl Bmoxide Task Force 11 c/o Conswner Specialty Products Assoc iatio n. 

1667 K Street NW, Suite 300. Washington. DC 

TEST ORDER#: CON-067501 -9 

EXECUTIVE SUMMARY: In a fernaJe pubertal assay (MRlD 486 18806), 16 female Sprague 

Dawley rats/dose group were treated daily via oral gavage with piperonyl butoxide (93.8% a.i., 

Batch/lot# E l04-I 0) in corn oil at doses of 0 (vehicle). 500, or 1000 (l imit dose) mg/kg/day from 

post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily 

beginni ng on PND 22. and weight at day of attainment was recorded. Following sacrifice on 

PND 42 or 43. tota l serum levels of thyroxine (T.1) and thyroid stimulati ng hormo ne (TSH) levels 

were determined using a radioimmunoassay: serum was also analyzed for levels of se lected 

clinical chemistry parameters. Adrenal. kidneys. liver, pituitary. thyroid. and urogenital organs 

were weighed. and microscopic examinations were performed on the ovaries. uterus, thyroid, and 

kid neys. 

No treatment-related cffocts were observed on mortality or clinical signs of toxicity. There were 

also no treatment-re lated effects on the weights of pituitary. uterus. or thyroid. With regards to 

reproductive development, no treatment-related effects were observed on the age at YO, we ight 

at YO. and number of animals anaining VO. mean age at first vaginal estrus, mean cycle length. 

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

corpora lutea. 

At 500 and 1000 mg/kg/day, the final adjusted body we ight was decreased (p<0.05) by 5 and 

7%, respectively. and the adjusted body we ight ga in was decreased (p<0.05) by 9 and I 0%, 

Page 187 of 252
Female Pubertal Assay (2011) / Page 2 of 16

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1450/ OECD None

respectively. In addition, the adjusted and relative liver weights were increased (p<0.05) dosedependently by 15-35% and 22-44% at 500 and 1000 mg/kg/day, respectively, and the 

unadjusted liver weight was increased (p<0.05) by 36% at 1000 mg/kg/day. Compared to the 

controls, increases (p<0.05) were also noted in the relative kidney weight (↑6%) and unadjusted, 

adjusted, and relative adrenal weights (↑20-29%) at 1000 mg/kg/day, and the adjusted weights 

for ovaries were decreased (p<0.05) by 16%. Although significant weight changes were 

observed in the kidneys and ovaries, no treatment-related histopathological effects were observed 

in these organs. 

Although uterine weights were not affected by treatment, selected histological findings were 

noted in uteri of rats in the high dose group. At 1000 mg/kg/day, moderate atrophy of the 

myometrium, uterine stroma, and endometrial glands was noted in 2 of 14 rats, mild atrophy of 

the endometrial gland was observed in another two rats, and mild luminal dilation was observed 

in 2 of 14 rats; compared to 0 of 16 rats in the control group. 

Although no treatment-related effects were noted on serum levels of T4 or TSH and thyroid 

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

epithelial cell height with a concomitant decrease in the colloid area. Follicular cell height was 

Grade 1 in controls compared to Grades 1–3 at 500 mg/kg/day and Grades 3–5 at 1000 

mg/kg/day. Follicular colloid area was Grades 4–5 in the controls compared to Grades 3–4 at 

500 mg/kg/day and Grades 1–3 at 1000 mg/kg/day.

The doses tested were adequate since the high dose was the Limit Dose (1000 mg/kg/day). 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450).

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

Assurance statements were provided. 

Page 188 of 252
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 16 – November 10, 2011

2. Test Substance: 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 20 days at 1-10 °C

CAS #: 51-03-6

Structure:

O

O

O

O

CH3

O CH3

3. Vehicle: Corn oil

4. Test Animals:

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. STUDY DESIGN

1. In-Life Dates: Start: April 22, 2011 End: August 9, 2011 

2. Mating: Time-mated pregnant dams were received from the supplier on gestation Day 8. 

Litters were standardized to 8 pups, with equal numbers of males and females, between 

PND 3 and 5. 

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3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a 

procedure that stratified 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 Females

Control 0 16

Low 500 16

High 1000 16

a Data were obtained from Table 1 on page 14 of the study report.

4. Dose Selection 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/day for 14 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 for females was selected since 

1000 mg/kg/day is the limit dose. 

 

5. Dose Preparation and Analysis: Dose formulations were prepared two times during the 

study by mixing appropriate amounts of test substance with corn oil. Samples of dose 

formulations from all preparations were analyzed for homogeneity 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 Dose 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.

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6. Dosage Administration: All doses were administered once daily by gavage in a volume of 

5 mL/kg of body weight. The dose formulations were administered for 21 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 then 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 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 VO / PPS

Age at first estrus

Cycle length

Ovarian counts (follicles, cysts, corpora lutea)

Homogeneous data sets:

One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed 

t-tests

Tissue weights

Relative tissue weights (liver, kidneys, pituitary and

adrenals)

Hormone levels

Clinical chemistry levels

Two-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

Two-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.

Cycling status

Percent of animals cycling regularly

Chi-square analysis

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant,

followed 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.

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C. METHODS

1. Mortality and Clinical 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 Weight: Animals were weighed at randomization, daily prior to dosing, and at 

termination. 

3. Vaginal Opening: 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. Estrous Cyclicity: 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 mean age at first 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 immediately 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 T4 and TSH levels were determined using 

radioimmunoassays. Samples were assayed in duplicate and in conjunction with multiple 

quality control samples. 

b. Clinical Chemistry: 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. Reference ranges were provided from published 

literature (Table 10 on page 30 of the MRID). The following CHECKED (X) parameters 

were examined. 

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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 Guideline 890.1450.

c. Organ Weights 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 890.1450

+ Histopathological examination required based on guideline 890.1450

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; 

following fixation in 10% neutral buffered formalin, the thyroid was dissected from the 

trachea before transfer to alcohol storage. All collected tissues were routinely processed, 

sectioned, stained with hematoxylin and eosin, and examined microscopically. 

Two 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. 

Page 193 of 252
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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, mortality 

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 1000 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 

groups were similar to control. However, when adjusted for initial body weight on PND 21, 

the final body weights were decreased (p<0.05) by 5% and 7% in the 500 and 1000 

mg/kg/day groups, 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 groups, respectively. Age 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 VO, and final 

body weight in the control group were within the acceptable range provided in the Guideline 

(890.1450), however, the following CVs were 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%). 

Page 194 of 252
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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 

(%) Nb Mean SD

CV 

(%) Nb 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 weightc 

(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 43, the last day all body weights were collected.

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

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: Organ 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 1000 

mg/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. Increases (p<0.05) in relative kidney weight (↑6%) 

and unadjusted, adjusted, and relative adrenal weights (↑20-29%) were also noted at 1000 

mg/kg/day compared to the controls, and the 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 glands weight (34.6 mg; acceptable range 38.34-48.84 mg). The %CVs for 

liver (15.7%; maximum 13.13%) and ovaries (24.0%; maximum 23.20%) were also outside 

the acceptable ranges.

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TABLE 3. Organ Weights at Necropsya

Organ 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 (%)

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 NRb NA NA 15 NRb NA NA 15 NRb 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 21 

A Adjusted for body weight on PND 21 

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: Estrous 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. 

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TABLE 4. Estrous Cyclicitya

Dose Level 

(mg/kg/day) N

Mean Age at 

First Vaginal 

Estrus (PND)

Mean Cycle 

Length 

(days)

Cycling 

(%)

Regularly 

Cycling 

(%)

Cycle Status at Necropsy (# Females) b

Diestrus Proestrus Estrus

Not 

Cycling

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

1000 14 37.2 3.8 100 71.43 6 1 6 0

a Data were obtained from Tables 6 and 7 on pages 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 T4 or TSH, or on the other evaluated clinical 

chemistry parameters. The results for serum T4 in the control group were within the 

acceptable range of the performance criteria provided in the Guideline (890.1450). 

TABLE 5. Hormone Levels and Clinical Chemistrya

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 T4, 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) 15 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) 15 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*(↓24) 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 0.1 185.2

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TABLE 5. Hormone Levels and Clinical Chemistrya

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) 15 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 dose-dependent 

increase in the thyroid gland follicular epithelial cell height and concomitant with a decrease 

in the colloid area. Follicular cell height was Grade 1 in controls compared to Grades 1–3 at 

500 mg/kg/day and Grades 3–5 at 1000 mg/kg/day. Follicular colloid area was Grades 4–5 

in the controls compared to Grades 3–4 at 500 mg/kg/day and Grades 1–3 at 1000 

mg/kg/day. 

TABLE 6. Thyroid Gland Follicular Cell Height and Colloid Areaa

Grades Vehicle Control Piperonyl butoxide

500 mg/kg/day

Piperonyl butoxide

1000 mg/kg/day*

# of animals 

examined 16 14 c 15 

Follicular cell heightb

1 16 (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 areab

1 0 0 1 (7%)

2 0 0 1 (7%)

3 0 1 (7%) 13 (87%)

4 8 (50%) 13 (93%) 0

5 8 (50%) 0 0

a Data were obtained from Table 12 on page 32 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.

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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 effects were observed in the uterus at 500 mg/kg/day. However, at 1000 

mg/kg/day, 2 of 14 rats had moderate 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 Kidneya

Findings Vehicle Control Piperonyl butoxide

500 mg/kg/day

Piperonyl butoxide

1000 mg/kg/day

# 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 pages 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. 

Page 199 of 252
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 Luteaa

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

Antretic Follicles 16 10.1 3.3 9.9 15 10.4 3.7 35.6 15 12.9 5.2 40.3

Follicular Cysts 16 0.03 0.11 366.7 15 0.03 0.11 366.7 15 0.02 0.06 300.0

Corpora 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 11 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, relative kidney and adrenal weights were significantly increased compared to 

control animals. There 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, and 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 animals 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 T4 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. Histopathological changes that occurred in the 

thyroid gland in the high (1000 mg/kg) dose group are a sensitive indicator of thyroid gland 

modulation. 

Page 200 of 252
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 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, and the weights of pituitary, uterus, or thyroid. Treatment-related 

effects were 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 T4 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, 

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) 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, 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 for these findings. 

In the thyroid, there were no dose-related effects on serum T4 or TSH levels; however, there 

was a dose-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%. 

Page 201 of 252
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 mean adrenal weight (34.6 mg) was below the performance criteria acceptable 

range (38.34-48.84 mg). 

Page 202 of 252
DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 890.1500, Male 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. 

Page 203 of 252
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).

Page 204 of 252
PIPERONYL llUTOXIDE I 067501 

Prima ry Reviewer: Patience Browne. Ph.D. 

Risk Assessment Branch VI, Health Effects Division 

Secondary Reviewer: John Liccione. Ph.D. 

Health Effects Division 

Male Pubertal Assay (20 11) I Page I of 16 

OCSPP 890. 1500/ OECO None 

Signat

natur

Date: ure: 

e: ~ 

~ = ... 

Date: :;;,:(.,. os12011 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay: OCSPP 890.1500; OECD None. 

PC CODE: 067501 OP BARCODE: 0 398558 

TXR#: 0056259 CAS No.: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93.8% a.i.) 

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

CITATION: Zorilla. L. (20 11 ) Pubertal developmental and thyroid function in intact 

juvenile/peripubertal female and male rats with piperonyl butoxide. 

Integrated Laboratories, Inc., Durham, NC. Laboratory Study No.: C l92-300. 

November 10, 20 11. MRID 48618806. Unpublished. 

SPONSOR: Piperonyl 13utoxide Task Force 11 , c/o Consumer Specialty Products 

Association, 1667 K Street NW. Suite 300. Washington DC 

TEST ORDER#: CON-06750 1-9 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48618806), 16 male SpragueDawley (Crl:CD®(SD) IGS) rats/dose group were treated daily via oral gavage with piperonyl 

butoxide (93.8% a.i., Lot #: E104-IO) in corn oil at doses o l' O, 500. or 1000 (limit dose) 

mg/kg/day from post-natal day (P D) 23 to 53/54. The animals were examined for preputial 

separation (PPS) daily beginning on P D 30, and body weight at day of attainment was 

recorded. Following sacrifice on PND 53/54. total serum testosterone, thyroxine (T4), and 

thyroid stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal , 

liver. thyroid. pituitary, and urogenital organ weights were recorded, and microscopic 

examinations of the testes, epididymides, thyroid, and kidneys were performed. 

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

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

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

at PPS, weight al PPS, and scrum levels of T..1 and TSI-1. There were also no treatment-related 

histopathological findin gs noted in the testes, cpididymides, or kidneys at either dose. 

General growth was decreased in a dose dependent manner. The final body weight (adjusted for 

body weight on PND 23) was decreased by 5% (not statisticall y significant [NS]) and 9% 

Page 205 of 252
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 

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

shortest in the control (15/15 rats at Grades 1-2) and tallest at 1000 mg/kg/day (14/15 at Grades 

3-5), and colloid area was largest in the control (15/15 at Grades 4-5) and smallest at 1000 mg 

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

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

among treated and control groups. 

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

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

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

26% (p<0.05), and adjusted and unadjusted weights of the seminal vesicles with coagulating 

gland (with/without fluid) were decreased by 21-35% (p<0.05). 

The dose concentrations used in the study are considered adequate based on the 11% decrease in 

adjusted body weight grain observed at 1000 mg/kg/day, which also is the dose limit for the 

assay.

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500).

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

statements were provided. 

Page 206 of 252
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 Directors: Zorilla, L.

Other Personnel: Not reported

Study Period: March 16, 2011 through November 10, 2011

2. Test Substance: Piperonyl butoxide

Description: Pale, oily liquid

Source: Endura Fine Chemicals, Bologna, Italy

Lot #: E104-10 (retest date of July 2012)

Purity: 93.8%

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

CH3

OC4H9

3. Vehicle: Corn oil

4. Test Animals:

Species: Rat (males only)

Strain: Sprague-Dawley (Crl:CD®(SD) IGS)

Age/Mean Weight 

at Study Initiation: PND 23 / 46.0-75.9 g 

Source: Charles River Laboratories International, Inc. (Raleigh, NC)

Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and 

hardwood bedding; 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 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was stated 

that the 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 

Conditions:

Temperature:

Humidity:

Air changes:

Photoperiod:

21-23°C

32-63% 

Not reported

14 h light / 10 h dark

B. STUDY DESIGN

1. In-Life Dates: Start: April 23, 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. 

Page 207 of 252
Male Pubertal Assay (2011) / Page 4 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

3. Animal Assignment: Following weaning on PND 21, animals 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 are presented in Table 1. 

TABLE 1. Study design a

Test group Dose (mg/kg/day) # of Males

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 Selection 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. The high dose selected for males was 1000 mg/kg/day, which is 

the limit dose.

5. Dose Preparation 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. The mean results of the homogeneity analyses were used to demonstrate 

achieved concentrations. 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 based on a separate 

study;2 however, no data were submitted to allow independent verification.

Results

Stability (% of time zero): Not reported

Concentration (% of nominal): 95.6–108.2% 

Homogeneity (%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.

Page 208 of 252
Male Pubertal Assay (2011) / Page 5 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

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 Administration: Dose 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

transformed (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 pairwise 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:

One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed 

t-tests

Tissue weights

Relative tissue weights (liver, kidneys, pituitary and

adrenals)

Hormone levels

Clinical chemistry levels

Two-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

Two-way analysis of covariance (ANCOVA) with PND 23 

body weight (weight on first day of administration) as the 

covariable. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests.

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant,

followed 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.

Page 209 of 252
Male Pubertal Assay (2011) / Page 6 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

C. METHODS

1. Mortality and Clinical 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 Weight: 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 CO2 inhalation and death confirmed by 

cervical dislocation. For animals surviving to scheduled termination, animals were 

humanely euthanized by decapitation at 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: T4, 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 Chemistry: The following CHECKED (X) parameters were examined.

Page 210 of 252
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 Alkaline 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 890.1500.

c. Organ Weights 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

XX Testes (left and right separately)*+ XX Thyroid a*+

XX 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*

XX Kidneys (paired)*+

Gross lesions and masses

a Thyroid was weighed post-fixation.

* Weights required based on guideline 890.1500. 

+ Histopathological examination required based on guideline 890.1500. 

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% 

histology 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. 

Page 211 of 252
Male Pubertal Assay (2011) / Page 8 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

II. RESULTS

A. Mortality: One control rat and one 1000 mg/kg/day rat were found dead on Days 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 observed at 1000 mg/kg/day 

included: (i) one rat was thin on Day 3; (ii) one rat had an ungroomed appearance on Day 4; 

and (iii) six 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. 

General 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 differences 

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 not 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. 

Page 212 of 252
Male Pubertal Assay (2011) / Page 9 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

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<0.01. 

*** Significantly different from controls at p<0.001. 

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%; all other androgen-dependent tissue 

weights were similar to controls at this dose. At 1000 mg/kg/day, the following decreases 

(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 epididymis (↓10%).

Page 213 of 252
Male Pubertal Assay (2011) / Page 10 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

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 criteria 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: 22.32). 

TABLE 3. Organ Weights at Necropsy a

Organ

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

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***

(↑15)

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 --- ---

Page 214 of 252
Male Pubertal Assay (2011) / Page 11 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ

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

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 pages 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 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.

E. Clinical Chemistry and Hormone Levels: Clinical chemistry and hormone 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 

serum T4 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 serum thyroid hormone (T4 and TSH) levels among 

treated and control groups. However, the following Guideline criteria for control levels were 

Page 215 of 252
Male Pubertal Assay (2011) / Page 12 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

not met: (i) mean serum T4 level of 3.65 μg/dL (acceptable range: 4.056–7.376); and mean 

serum TSH level of 2.25 ng/mL (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 T4, 

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/mL)

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 5.6

Page 216 of 252
Male Pubertal Assay (2011) / Page 13 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

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 cells 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. Incidence 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

(500 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

(1000 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. Examined

Page 217 of 252
Male Pubertal Assay (2011) / Page 14 of 16

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1500/ OECD None

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 Kidneya

Findings

Dose Level (mg/kg bw/day)

Vehicle Control Piperonyl butoxide (500) Piperonyl butoxide (1000)

# Observed # Examined # Observed # Examined # Observed # Examined

Epididymides

Minimal inflammation 9 15 7 14 7 15

Thyroid

Ectopic thymus 1 14 1 13 0 15

Minimal C-cell hyperplasia 1 14 1 13 0 15

Kidneys

Minimal basophilic tubules 5 15 8 14 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 pages 145–173 of the study report. No abnormal effects were noted in the testis.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Administration 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 serum levels of T4 and TSH. There 

were also no treatment-related histopathological findings noted in the testes, epididymides, 

or kidneys at either dose.

General growth was slightly attenuated in a dose dependent manner. Decreases in body 

weights and body weight gains were noted; however, the exact magnitudes of these effects 

were 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. 

Page 218 of 252
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 the 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, because of the discrepancy in initial mean weights, adjusted tissue weights were 

required for meaningful comparisons. At 500 mg/kg/day, adjusted 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 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%); (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, the 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 TSH level of 2.25 ng/mL (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, respectively; 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 vesicles with coagulating gland (with/without fluid), and thyroid 

colloid area. 

Page 219 of 252
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: 

• %CV=7.1 for age at PPS (top of 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) 

Page 220 of 252
DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 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. 

Page 221 of 252
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).

Page 222 of 252
PI PERONYL B TOXID E / 067501 

Primary Reviewer: Yung G. Yang. Ph.D. 

Risk Assessment Bra nch VI, Health Effects Division 

Secondary Reviewer: John Liccione. Ph.D. 

Health Effects Division 

Steroidogenesi Assay (20 11 ) I Page I of 19 

OCSPP 890. 1550/ OECD None 

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 06750 I DP BARCODE: 0398558 

TXR#: 0056259 CAS No.: 51 -03-6 

TEST MATERIAL (PURITY): Piperonyl Butoxide (93.8% a.i.) 

SYNONYMS: 5-[[2-(2-butoxyethoxy)ethoxy]methyl]-6-propyl-l .3-benzodioxole; PBO 

CITATION: Wagner, H. (2011). Piperonyl butoxide: H295R steroidogenesis assay. 

CeeTox. Inc .. Kalamazoo, M l. Laboratory Study No. 9146Y-100377STER, 

November 17, 2011. MRJD 48618807. Unpublished. 

SPONSOR: Piperonyl Butoxide Task Force IL c/o Consumer Specialty Products 

Assoc iation. Inc., 1900 - l 7111 Street NW. Suite 300. Washington, DC. 

TEST ORDER #: CO -067501-9 

EXECUTIVE SUMMARY: [n a steroidogenesis assay (MRID 48618807). H295R cells 

cultured in vitro in 24-well plates were incubated with piperonyl butoxide, (PBO; 93.8% purity. 

Batch #E104/ JO) at log concentrations from 0.0001to100 μM in tripli cate for 48 hours. 

Dimethyl sulfoxide (DMSO) was used as the vehicle. at a final concentration of 0.05%. 

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

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

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

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

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

estradiol and testosterone production. 

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

that the laboratory proficiency data was previously dctem1ined for this batch or cells; however, 

details of a previous proficiency determination. including estimates of EC;;os for prochloraz and 

forskolin were not included in Lhe study report. Data from the highest tested PBO concentration. 

I 00 μM. were not evaluated due to precipitation that was observed post-exposure. 

All guideline acceptability requirements were met, including lack of cytotoxicity, adequate 

production of testosterone and estradiol, acceptable reproducibility (low %CY), and appropriate 

induction and inhibition with positive controls. 

Page 223 of 252
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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, four of the six PBO concentrations in the fourth 

run exhibited statistically significant decreases (0.87- to 0.91-fold) in testosterone production, 

but these decreases were not dose related and are not considered an effect of PBO exposure. 

Statistically significant increases in estradiol production (1.11- to 1.31-fold) were observed in 

three test runs (with a non-significant increase of 1.45-fold in a fourth test run) at the PBO 

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

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

decreases are not considered an effect of PBO exposure since no effect was observed in the 

previous three runs. 

Based on the hormone responses in at least three independent runs, PBO treatment resulted in 

statistically significant and reproducible increases in estradiol production, but did not result in 

statistically significant and reproducible alterations in testosterone production.

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550).

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

Assurance statements were provided. 

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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. Haines, Scientist

F. Wong, Scientist

J. Burgam, Associate Scientist

L. Blakeman, Associate Scientist

Study Period: May 23, 2011 to November 17, 2011

2. Test Substance: Piperonyl Butoxide (PBO)

Description: Technical 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

CH3

OC4H9

3. Positive Control (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 Control (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 #: 67747-09-5

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5. Solvent/Vehicle Control: 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; Lot #7635054). Cells were maintained in DMEM/Ham-F12 with 15 mM HEPES, 

sodium bicarbonate, ITS+Premix, and 2.5% Nu-Serum in a 5% CO2 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. 22R-Hydroxycholesterol (10 μM) 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 × 105 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 5

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X Frozen cells cultured for 4 additional passages

Total number of passages does not exceed 10 (not reported)

B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: 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

H5]-testosterone and [2H5]-estradiol as internal standards. The extracts were analyzed 

using HPLC/MS-MS with positive ion multiple reaction monitoring. The method MDLs for 

testosterone and estradiol were 100 and 10 pg/mL, respectively, for a 300-μL portion of 

medium.

c. Laboratory Proficiency Test: Laboratory proficiency testing was 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 

μM. Mastermix solutions with PBO were prepared by 1:2000 dilutions in supplemented 

medium containing 10 μM 22R-hydroxycholesterol. 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 μM 

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 μM for 

PBO. Final concentrations of DMSO and ethanol in the medium were 0.05% and 0.025% 

(v/v), respectively.

3. Cell Plating and Preincubation: 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 μM) was 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 × 105 cells/mL. Plates were incubated 

in a 5% CO2 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 × 105 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. 

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TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Piperonyl Butoxide (Final 

Concentrations 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 Schematic for the QC Plate for Positive Controls (Final Concentrations in μM)a

1 2 3 4 5 6

A Blankb Blank Blank Backgroundc Background Background

B DMSO DMSO DMSO DMSO + MeOHd DMSO + MeOH DMSO + MeOH

C Forskolin 1μMe Forskolin 1μMe Forskolin 1μMe 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 were 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 Viability/Cytotoxicity Assay: Cell viability was determined with the MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test described 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% CO2 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.

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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 20-minute 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 was considered evidence of cytotoxicity.

6. Hormone Measurement System: 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 recovery 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 exhibited ≥20% cytotoxicity, or where precipitation was observed, were 

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. Statistical 

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 the 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-Wallis test, followed by Dunn’s test, to 

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evaluate statistical significance between the treatment and control groups. These analyses 

are considered acceptable. 

II. RESULTS

A. TEST COMPOUND: The hormone concentrations after exposure to PBO, and the folddifference 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 precipitation 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 based 

on absolute hormone concentrations in all four test runs (calculated by the reviewers) 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. Statistically 

significant 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 μM) exhibited statistically significant decreases in estradiol 

production (similar to those observed with testosterone); however, these 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 

48a Hours.a

Nominal 

Concentration 

(μM)

Run 1 Run 2 Run 3 Run 4 Run 1 Run 2 Run 3 Run 4 Meanb ± SDb

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 0.02

0.001 2507 2623 1864 2357 0.99 0.99 0.95 0.88* 0.98 0.02

0.01 2441 2566 1932 2331 0.96 0.97 0.99 0.87* 0.97 0.02

0.1 2684 2673 1952 2498 1.06 1.01 1.00 0.93 1.02 0.03

1 2498 2712 2015 2429 0.99 1.02 1.03 0.91* 1.01 0.02

10 2552 2996 2030 2583 1.01 1.13 1.03 0.97 1.06 0.06

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 0.03

0.001 163 235 156 201 0.99 0.92 0.98 0.8* 0.96 0.04

0.01 157 237 160 199 0.96 0.93 1.00 0.79* 0.96 0.04

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TABLE 3. Mean (±SEM) Hormone Concentrations Following Treatment with Piperonyl Butoxide for 

48a Hours.a

Nominal 

Concentration 

(μM)

Run 1 Run 2 Run 3 Run 4 Run 1 Run 2 Run 3 Run 4 Meanb ± SDb

0.1 165 239 163 224 1.01 0.94 1.02 0.89* 0.99 0.04

1 167 269 175 216 1.02 1.06 1.1 0.86* 1.06 0.04

10 214 369 207 281 1.31* 1.45 1.30* 1.11* 1.35 0.08

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

* Statistically significant (p≤0.05)

FIGURE 1. Change in Testosterone Production Relative to PBO Concentration in 

Test Run 1. 

Page 231 of 252
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FIGURE 2. Change in Testosterone Production Relative to PBO Concentration in 

Test Run 2.

FIGURE 3. Change in Testosterone Production Relative to PBO Concentration in 

Test Run 3.

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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.

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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.

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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 chemical 

plates; methanol exposure reduced cell viability to 5.1-11.4%.

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TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or 

Piperonyl Butoxide for 48 Hours.a 

Compound Concen. 

(μM)

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 9.10 1.98 5.60 2.51 11.4 1.99 5.10 0.14

Forskolinb 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.

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FIGURE 10. MTT Cell Cytotoxicity Results From Test Run 2.

FIGURE 11. MTT Cell Cytotoxicity Results From Test Run 3.

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FIGURE 12. 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. Minimum 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 basal hormone production increase criteria in SC of

≥5-fold for testosterone and ≥2.5-fold for estrogen above 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-fold; however, when the background levels are 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 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 the % 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 met the 

performance criteria recommended by the Guideline (≤30%). 

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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 ± SD b

Testosterone (pg/mL) Fold Difference

Background NA 1056 1135 1073 1262 0.49 0.45 0.57 0.54 0.51 0.05

Blank NA 2174 2581 1873 2304 1.01 1.03 1.00 0.99 1.01 0.02

DMSO NA 2143 2511 1874 2339 1.00 1.00 1.00 1.00 1.00 --

Forskolinc 1 2930 3764 2595 3120 1.37d 1.50 d 1.38 d 1.33 d 1.40 d 0.07

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 0.02

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 0.08

a Data were obtained from page 23 and 24 of the study report. 

b Calculated by the reviewers.

c Forskolin concentration was 3.33 μM 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 1.82-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 μM. These changes were considered 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 concentration (10 μM). 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 μM. These decreases were considered to be random effects unrelated to 

test chemical treatment. 

B. AGENCY COMMENTS: Laboratory proficiency testing was not conducted in the current 

study because proficiency data was reportedly generated previously for the batch of cells

used in this study. However, the details and results of this previous proficiency test, 

including estimates of EC50s 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 three 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 containing 100 μM PBO, the highest concentration of PBO in the assay deemed 

Page 239 of 252
Steroidogenesis 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 used in the current assay. 

The QC plate results met the minimum 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 μM 

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 are 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 

estradiol 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 variability data for the QC plates, 

based on the between plate % CVs for the absolute hormone concentrations in the SC wells, 

yielded % 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 concentration 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 

concentrations 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 were 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 μM) exhibited statistically significant decreases (0.78- to 0.89-fold) in 

estradiol production, but are not considered an effect of PBO exposure. 

The 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 μM. This was not confirmed at the next 

highest concentration (100 μM) 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 current 

Page 240 of 252
Steroidogenesis Assay (2011) / Page 19 of 19

PIPERONYL BUTOXIDE / 067501 OCSPP 890.1550/ OECD None

batch of cells, including estimates of EC50s 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. 

Page 241 of 252
DATA EVALUATION RECORD

PIPERONYL BUTOXIDE

Study Type: OCSPP 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: 4/13/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 242 of 252
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).

Page 243 of 252
PIPERONYL B TOXIOE/ 06750 I 

In vfro Uterotropbic Assay (2011 ) I Page I or9 

OCSPP 890. 1600/ OEC D 440 

Primary Reviewer: Yung G. Yang, Ph.D. Signature: V'~ G <r/-a 

Risk Assessment Branch VI, Health Effects Division Date: r/,,1/go1'.<' 

Secondary Reviewer: _ .....;J:;...;:e=s=-s-=- R=o'""'"w.:....:cl=an;.:..;d""--_____ _ _ __ Signature: ~a..;.~ 

Health Effects Division Date: 5:1J..7 I1 ) 

Template version 09/2011 

DAT A EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat): OCSPP 890.1600; OECO 440 

PC CODE: 06750 I DP BARCODE: 0 398558 

TXR#: 0056259 CAS#: 51-03-6 

TEST MATERIAL (PURITY): Piperonyl butoxide (93 .8% a.i.) 

SYNONYMS: 5-[2-(2-butoxyethoxy)ethoxymethyl]-6-propyl-J ,3-benzodioxole 

CITATION: Zorrilla. L. (2011 ). The Uterotrophic Assay for Piperonyl Butoxide (PBO). 

Integrated Laboratory Systems. Inc., Durham, NC. Laboratory Project Study No.: 

Cl92-IOO. November 18, 201 J (revised report date). MRfD 48618808. 

Unpublished. 

SPONSOR: Consumer Specialty Products Association, Inc. For Piperonyl Butoxide Task 

Force II. 1667 K Street NW. Suite 300. Washington, D.C. 

TEST ORDER #: CON-06750 1-9 

EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRlD 48618808) conducted to screen 

for potential estrogenic activity, piperonyl butoxide (93.8% a.i., Batch #El 04/ 10) in corn oil was 

administered da ily via oral gavage to groups of eight ovariectomized female Sprague Dawley 

rats at dose levels of 0 (vehicle). 320 or I 000 (limit dose) mg/kg/day on post-natal days (PND) 

58-60. A positive control group was treated with a daily dose of I 7a-ethynyl estradiol (EE) at 

0.1 mg/kg/day by oral gavage. Body weights were determined daily. All animals were 

terminated and necropsied on PND 61 approximately 24 hours after the final dose administration 

to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. Two animals administered piperonyl butoxicle 

at 320 mg/kg exhibited abnonnal clinical signs (uncoordinated movements or lethargy) during 

dosing, but all animals were noted as normal prior to termination. All animals dosed with IOOO 

mg/kg piperonyl butoxide group exhibited abnormal clinical signs (decreased or uncoordinated 

movements, hunched posture, lethargy, and/or nasal discharge) by Day 3 of dosing. At 

termination, all high close animals were noted as normal, except for one rat which exhibited 

multiple signs of toxicity. 

Body weights were decreased (p<0.05) on Day 4 (l 10%) in the high dose group compared to the 

vehicle controls. resulting in a decrease (p<0.05) in overall body weight gain (BWG: 1240%). 

Body weights in the low-dose group were comparable to the vehicle controls throughout the 

duration of the assay. Statistical significance was not determined for the body weights and BWG 

Page 244 of 252
In vivo Uterotrophic Assay (2011) / Page 2 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ 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 the vehicle controls, with a decrease in overall body weight 

gain (↓148). 

Uterine weights in all piperonyl butoxide treated groups were comparable to the vehicle controls. 

Absolute wet and blotted uterus weights for the positive control (EE) group were increased 

(p<0.05) by 348% and 173%, respectively, as expected. 

No statistically significant changes were seen in uterine weight in this assay. Piperonyl butoxide 

is negative in the uterotrophic assay. 

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. 

Page 245 of 252
In vivo Uterotrophic Assay (2011) / Page 3 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ 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 18, 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

CH3

OC4H9

3. Reference Estrogen: 17α-ethynyl estradiol (EE)

Supplier: Sigma-Aldrich (St. Louis, MO)

Lot/Batch #: 090M1241V (expiry 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 Animals:

Species: Rat (ovariectomized)

Strain: Sprague Dawley, Crl:CD®(SD) IGS

Age/weight at dose initiation: 58 days old/ 223.2 – 270.8 g females

Source: Charles River Laboratories International, Inc. (Raleigh, NC).

Housing: 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:

Humidity: 

Air changes:

Photoperiod:

22-24oC

33-57% 

Not reported

12 hrs light/ 12 hrs dark

Acclimation period: 8 days

Page 246 of 252
In vivo Uterotrophic Assay (2011) / Page 4 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ OECD 440

B. STUDY DESIGN 

1. In Life Dates: Start: April 8, 2011 End: April 11, 2011 

2. Study Design: Sexually mature ovariectomized female rats (ovariectomized 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 would not be used in the study. In the allocation of animals, three 

animals were removed from the study due to the presence of estrus cells during pre-dose 

estrous cycle assessment. Another animal which had estrus cells present on the first day of 

vaginal smears but not afterwards was kept in the study and showed no evidence of ovarian 

tissue present at necropsy. 

3. Animal Assignment: Animals were 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]. 

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females

Estrogen Agonist Assay

Vehicle Control (corn oil) 0 8

Piperonyl butoxide Low 320 8

Piperonyl butoxide High 1000 8

17α-ethynyl estradiol (EE), Reference Estrogen 0.1 8

a Data were obtained from Table 1 on page 15 of the study report.

4. Dose Selection Rationale: In a range finding study1, 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. 

Therefore doses of 1000 and 320 mg/kg/day were used for the uterotrophic study. 

5. Dose Preparation and Analysis: Dose formulations in corn oil were prepared once prior to 

initiation of treatment. Dose concentrations and homogeneity were tested by Smithers 

Viscient LLC (Wareham, MA) for both formulations (64 and 200 mg/mL) prepared by 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 

Program in vivo Mammalian Assays (draft). Unpublished study report prepared by ILS, Inc. Study No. C192-500. 

Page 247 of 252
In vivo Uterotrophic Assay (2011) / Page 5 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ OECD 440

formulation were reanalyzed (12 days after preparation) to confirm stability of the 

formulations. 

Results of Dose 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 Administration: Animals 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. Statistics: 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). Positive 

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 dose administration

and at termination.

2. Body Weight: Animals were weighed upon arrival, at randomization, daily prior to dose 

administration and at termination.

3. Food Consumption (Optional): Food consumption was not measured. 

Page 248 of 252
In vivo Uterotrophic Assay (2011) / Page 6 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ 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 CO2

asphyxiation and cervical dislocation, and subjected to a gross necropsy. Animals 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 fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) 

was weighed. Subsequently, the uterus was 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 Signs of Toxicity: The incidence of clinical observations is presented in Table 2. 

Animals 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 of 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 all animals from the 

high dose group were noted as normal, except for one rat which appeared thin, exhibited 

hunched posture, lethargy, 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. 

Page 249 of 252
In vivo Uterotrophic Assay (2011) / Page 7 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ OECD 440

TABLE 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, pages 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 Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05

Page 250 of 252
In vivo Uterotrophic Assay (2011) / Page 8 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ OECD 440

C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY

1. Uterine Weights: Uterine (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 mean blotted uterine weight for the vehicle control group was <0.04% of terminal body 

weight, which is consistent with the performance criteria (<0.04%) of the test guideline 

(OCSPP 890.1600). 

TABLE 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 Body weight

N Number of animals in the group

SD Standard 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 butoxide 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 signs 

Page 251 of 252
In vivo Uterotrophic Assay (2011) / Page 9 of 9 

PIPERONYL BUTOXIDE/ 067501 OCSPP 890.1600/ 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 by 

Day 3. On Day 4 all animals from the high dose group were noted as normal, except for one 

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 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). 

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 

Page 252 of 252