UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 64
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 64
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: O-PHENYLPHENOL (o-PP) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 64
O-PHENYLPHENOL FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

I. Introduction.......................................................................................4 

II. Sources of Scientific Data and Technical Information......................................5 

A. EDSP Tier 1 Screening Assays............................................................5 

B. Other Scientifically Relevant Information (OSRI)......................................5 

III. Weight of Evidence (WoE) Evaluation.......................................................5 

A. EDSP Tier 1 Screening Assays.............................................................8 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9 

1. Effects on Estrogen Pathway.....................................................9 

2. Effects on Androgen Pathway..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17 

IV. Committee’s Assessment of Weight of Evidence..........................................20 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20 

1. Tier 1 in vivo Assays.............................................................21 

2. OSRI...............................................................................22 

B. Estrogen Pathway...........................................................................24 

C. Androgen Pathway.........................................................................24 

D. Thyroid Pathway...........................................................................25 

E. Conclusions.................................................................................25 

V. EDSP Tier 2 Testing Recommendations....................................................25 

VI. References.......................................................................................26 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for o-Phenylphenol...........................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol..............................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for o-Phenylphenol...........................14 

 Table 4. Thyroid Pathway for o-Phenylphenol.....................................................18 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................30 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................42 

APPENDIX 3. References Not Utilized in the o-Phenylphenol WoE Analysis......................55 

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O-PHENYLPHENOL FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

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O-PHENYLPHENOL FINAL 

iv 

Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

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O-PHENYLPHENOL FINAL 

v 

Abbreviation Terminology 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

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O-PHENYLPHENOL FINAL 

vi 

Abbreviation Terminology 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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O-PHENYLPHENOL FINAL 

Page 1 of 56 

Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether o-phenylphenol (o-PP) interacts with E, A or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) that 

also resulted in general systemic toxicity or overt toxicity. 

On September 11, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) of the potential interaction of o-PP 

with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in 

this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in 

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited 

OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For o-PP, for the estrogen pathway, the Tier 1 in vitro estrogen receptor (ER) binding assay was 

equivocal results and positive for ER activation (Routledge and Sumpter, 1997 and Miller et al., 

2001). Additionally o-PP inhibited aromatase activity and increased estradiol production in the 

Tier 1 aromatase and steroidogenesis assays, respectively. However, in the mammalian in vivo

assays, there was no convincing evidence for an interaction with the estrogen pathway. The 

uterotrophic assay was negative and in the female pubertal assay, the sole effect observed was a 

decrease in the percent regularly cycling animals at the high dose compared to the controls which 

occurred in the presence of systemic toxicity. Due to the lack of redundancy of responses across 

the mammalian assays, there is insufficient evidence for a potential interaction in vivo in 

mammals. 

However, there are indications of potential effects on the reproductive status in fish that included 

decreases in female vitellogenin, fecundity and fertility in the fish short-term reproduction assay 

(FSTRA); however, there were no effects on gonadal somatic index (GSI) or gonadal 

histopathology. In another fish reproduction study with the same species as the FSTRA, 

decreases in egg production were observed. These in vivo effects, along with indications of 

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O-PHENYLPHENOL FINAL 

Page 2 of 56 

potential in vitro effects including ER activation and altered steroidogenesis, suggest that o-PP 

may potentially interact with the estrogen pathway in fish. 

For the androgen pathway, there was evidence of potential interaction in the in vitro androgen 

receptor (AR) binding assay and conflicting evidence in the steroidogenesis assay where a 30% 

increase in testosterone production at 10 μM o-PP and a 30% decrease at 100 μM o-PP. Studies 

published in the open literature showed o-PP was not an androgen agonist in the MDA-kb2 cell 

assay, however an anti-androgen effect of o-PP was observed with this cell line. The Hershberger 

assay was negative and in the male pubertal assay, decreases seen in a number of androgendependent tissues were observed in the presence of overt toxicity. In the FSTRA, decreases in 

fertility were observed, however, no other androgen-related effects were observed in this study 

or in the OSRI fish reproduction study. There were no androgen-related effects observed in the 

Part 158 mammalian studies. Therefore, there is no convincing evidence for potential interaction 

with the androgen pathway. 

For the thyroid pathway, there was no evidence of potential interaction of o-PP. In female 

pubertal assay, there were no treatment-related effects on thyroid hormones [T4 and thyroid 

stimulating hormone (TSH)], thyroid weights or thyroid histopathology. In the male pubertal 

assays the only thyroid-related effect observed was a decrease in serum T4 levels at all doses. In 

the amphibian metamorphosis assay (AMA), no treatment-related finding were observed except 

for a significant reduction in Day 21 normalized hindlimb length; however, given the lack of 

effects on developmental stage or thyroid histopathology, this finding alone is not considered to 

be sufficient evidence of a potential interaction of the thyroid pathway in amphibians. No thyroid 

effects were noted in any of the Part 158 mammalian studies. 

In summary, there is no convincing evidence of potential interaction with the estrogen, androgen 

or thyroid pathways in vivo in mammals. There is also insufficient evidence of a potential 

interaction of the thyroid pathway in amphibians. However, there is evidence of potential 

interaction with the estrogen pathway in non-mammalian species as suggested by the decreases 

in reproductive output in the FSTRA and fish OSRI. These findings in the fish assays along with 

the results in the in vitro assays, suggest potential interaction with the estrogen pathway in fish. 

There is no convincing evidence of a potential interaction with the androgen pathway in fish. 

 

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

recommended for o-PP since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. Effects on fecundity/fertility were observed at 876 μg 

a.i./L in the FSTRA with effects on reproduction reported at 36 and 293 μg a.i./L in the fish 

OSRI assay (which was similar to the FSTRA in test design). Currently, there are no acceptable 

40 CFR Part 158 chronic toxicity data for fish. At this time, based on the reproductive responses 

observed in the fish assays and the in vitro assays, EDSP Tier 2 Medaka Extended OnePage 10 of 64
O-PHENYLPHENOL FINAL 

Page 3 of 56 

Generation Reproduction Test (MEGORT) is recommended to better understand potential 

reproductive effects in fish. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are not available for o-PP; however, were requested under the final workplan for the 

registration review for o-PP (USEPA 2014). The type of data obtained from Part 158 avian 

reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential 

reproductive effects to birds from o-PP exposure. Relative to the EDSP, additional testing is not 

recommended. 

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O-PHENYLPHENOL FINAL 

Page 4 of 56 

I. Introduction 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether O-phenylphenol (o-PP)interacts with E, A or T hormone 

pathways, the number and type of effects induced, the magnitude and pattern of 

responses observed across studies, taxa and sexes were considered. Additionally, the 

conditions under which effects occur were considered, in particular, whether or not 

endocrine-related responses occurred at dose(s) that also resulted in general systemic 

toxicity or overt toxicity. 

On September 11, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of o-PP with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Ortho-phenylphenol (o-PP) has a water solubility of 700 mg/L. Due to a vapor pressure of 

2×10−3

 mm Hg at 25°C, o-PP is anticipated to exist solely as a vapor if released to the 

atmosphere. The octanol/water partition coefficient (log Kow) is 3.3 and a bioconcentration 

factor of 51 in fish. o-PP is stable and persistent in abiotic aqueous medium, but is not stable 

photolytically. It is immobile in soils, but has a potential for volatilization from moist soils. 

The available information considered in determining the potential interaction of o-PP with the E, 

A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant 

information (OSRI) such as general toxicity studies and other published articles. These data are 

summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, 

using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 study recommendations are presented in Section V. 

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O-PHENYLPHENOL FINAL 

Page 5 of 56 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 Guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on o-PP (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

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

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

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

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

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

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

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

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

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Page 6 of 56 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

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

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

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

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

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

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, under what 

conditions did the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

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

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the 

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Page 7 of 56 

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

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

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

 For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by o-PP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

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

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

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

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether or not a substance has the potential to interact with the 

E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the 

text for each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

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O-PHENYLPHENOL FINAL 

Page 8 of 56 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for o-PP are shown below in 

Table 1. 

Table 1. Tier 1 Screening Assays for o-Phenylphenol.

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48672604)) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied by OSRI 

(Routledge and Sumpter, 1997 and 

Miller et al., 2001) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48672603) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48672601) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48672608) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48672605) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48672602) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48686601) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID No. 48686602) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48672606) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48916901) 

Page 16 of 64
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Page 9 of 56 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for o-PP to interact with the estrogen pathway is summarized in Table 2. The 

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table 

also includes HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

In the Tier 1 in vitro assays, aromatase activity was inhibited in a dose-dependent manner at 10−4

and 10−3 M in the aromatase assay, and dose-dependent induction of estradiol production was 

observed in the steroidogenesis assay at concentrations of 10-5 and 10-4 M (2.6- and 4.0-fold, 

respectively). 

In the Tier 1 binding assay, o-PP appeared to displace estradiol from the ER in a concentrationdependent manner in all three runs, however the binding curve exhibited steep Hill slopes, 

suggesting some other type of interference instead of direct competition for ER binding. Blair et 

al., 2000 reported negative results for ER binding assay in rat uterine cytosol. In OSRI used to 

satisfy the Tier 1 ERTA assay, no estrogenic activity was seen in ER-related transcription in 

yeast expressing the human ERα gene (Routledge and Sumpter, 1997 and Miller et al., 2001). 

o-PP was negative in the Tier 1 in vivo uterotrophic assay. In the female pubertal rat assay, the 

percent regularly cycling animals in the high-dose (900 mg/kg/day) was decreased (28.6% vs. 

86.7% controls), and there was a dose-dependent tendency to spend more than 4 consecutive 

days in diestrus (1/15 control, 2/15 low, 3/15 mid, 7/15 high dose), leading to 4 animals not 

cycling (i.e. spending all the time in diestrus) at the high dose. The cyclicity effects were 

observed in the presence of systemic toxicity including kidney histological changes and increases 

in serum urea nitrogen. There were no other estrogen-related effects in this study. There were no 

estrogen-related effects observed in the Part 158 mammalian toxicity studies. In a study by 

Kaneda, 1978, fetal resorptions were increased at a dose that was overtly toxic (600 mg/kg/day). 

In the FSTRA, while fecundity or fertility were not significantly (p>0.05) decreased at the high 

concentration (876 μg a.i. /L; ↓47.5 and 32%, respectively), they were considered to be 

biologically meaningful decreases. Additionally, female VTG concentration also was decreased 

(p<0.05; 35.5%) at the high-treatment level. There were no other treatment-related effects 

observed. In an OSRI, non-guideline fish reproduction study with fathead minnow (which was 

similar to the FSTRA), while control fecundity was observed to be variable in the study, data 

suggested a decrease in fecundity (total number of eggs per female) at 36 and 293 μg/L. There 

were no treatment-related effects on GSI, VTG induction, or growth (length and weight). 

Gonadal histology or plasma sex steroids were not analyzed in this study (reportedly due to no 

evidence of VTG induction). 

Page 17 of 64
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Page 10 of 56 

Table 2. Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

EDSP Tier 1 Assays 

ER Binding (MRID 48672604) E ERTA Requirement satisfied by OSRI (Routledge and Sumpter, 1997, and Miller et al., 2001; see Aromatase (MRID 48672608) P Steroidogenesis (MRID 48672601) P Uterotrophic (MRID 48672605) N Female Pubertal Rat (MRID 48686601) N N N N P (H)4 N FSTRA (MRID 48672606) NE N N N P5

↓35.

5% 

(H) 

OSRI 

ER Binding (Blair et al., 2000) N ERTA (Routledge and Sumpter, 1997) P 
O-PHENYLPHENOL FINAL 

Page 11 of 56 

Table 2. Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

ERTA (Miller et al., 2001) P Subchronic toxicity (Dog; MRID 41656401) 

 NE NE N NE 

Developmental toxicity (Rat; Kaneda et al., 1978) 

 N 

Developmental toxicity (Rat; MRID 00164362) 

 NE N 

Developmental toxicity (Rabbit; MRID 41925003) 

N N 

Two-generation reproduction (Rat; MRID 41729401) 

 NE N N N N 

Two-generation reproduction (Rat; MRID 43928801) 

 NE N N N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 43954301) 

 NE N N NE 

Carcinogenicity (Mouse; MRID 43545501) 

 NE NE N NE 
O-PHENYLPHENOL FINAL 

Page 12 of 56 

Table 2. Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for o-Phenylphenol1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

Chronic toxicity (Dog; MRID 41656401) 

 NE N N N 

Freshwater fish reproduction (Fathead minnow; MRID 48136404) 

 N NE NE P (MH, H)7 N 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not a

Changes in weight are absolute unless otherwise indicated. LW: Liver weight; ADN: Adrenal; BUN: Blood Urea Nitrogen; ALT: AHisto: Histopathology. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

details see Section IV. A 

4. The percent of females that were regularly cycling was decreased from 86.7% to 28.6% in the high-dose group. 

 5. Fertility success was decreased 32% at the high-dose and fecundity was decreased 47%; while not significantly different (p>0.05), 

biologically significant. 

 6. While survival was 79% in the highest treatment concentration (876 μg a.i./L), one test vessel was observed to have 100% mortalit

7 total mortalities at this concentration which may indicate a tank-related issue and not treatment-related mortality. There were no 

signs of toxicity or significant effects on body weight or length. 

7. Decreased egg production (total number of eggs per female) at the mid-high and high concentration. 

 E Equivocal 

P Positive findings (in vitro)/No effect (in vivo) N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
O-PHENYLPHENOL FINAL 

Page 13 of 56 

2. Effects on Androgen Pathway 

The potential for o-PP to interact with the androgen pathway is summarized in Table 3. The 

various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for androgenic, anti-androgenic or HPG axis effects. This 

table also includes HPG-relevant findings from the data evaluated as OSRI. Effects that 

occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

o-PP was positive in the Tier 1 in vitro androgen receptor (AR) binding assay with an estimated 

mean Log IC50 of −3.749 Log M. In a published AR transcriptional activation (ARTA) study by 

Orton et al. (2011), o-PP was not an androgen agonist in the MDA-kb2 cell assay, however an 

anti-androgen effect of o-PP was observed in this assay (IC20= 3.43 μM). The Tier 1 

steroidogenesis assay showed conflicting results with a 30% increase in testosterone production 

at 10 μM, and a 30% decrease at 100 μM. 

o-PP was negative in the Tier 1 in vivo Hershberger assay. In the male pubertal assay, no 

treatment-related effects were observed on serum testosterone concentrations. The adjusted age 

of attainment of preputial separation (PPS) was increased by 1.6 days in the high-dose group 

(45.1 vs. 43.5 days control), but this change from control was not statistically significant. 

Numerous androgen-dependent tissues had decreased mean weights at the high dose when 

compared to corresponding controls. Unadjusted and adjusted pituitary weights were decreased 

in the high-dose animals (15% and 10%, respectively). All these effects, however, were seen 

only in the presence of overt toxicity that manifested as decreases in body weight and systemic 

effects including increases in liver and adrenal weights, increased serum blood urea nitrogen and 

histopathological lesions of the kidneys. The only androgen-related effect observed in the Part 

158 mammalian toxicity studies was an increase in absolute (34%) and relative (46%) testes 

weights observed in high-dose males in the chronic toxicity study in rats, however this effect cooccurred with 10-12% decreased body weight (compared to controls) throughout the exposure 

time and 11% decreased body weights at termination. 

In the FSTRA, fertility was decreased 32% at the high treatment level, however, there were no 

other effects observed in male fish. Additionally, there were no male effects noted in the nonguideline freshwater fish reproduction study. 

Page 21 of 64
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Page 14 of 56 

Table 3. Androgenic/Anti-Androgenic Pathway for o-Phenylphenol

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for o-Phenylphenol1Study Type/ Literature Citation 

AR Binding/Activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin (VTG) 

EDSP Tier 1 Assays 

AR Binding (MRID 48672603) P Steroidogenesis (MRID 48672601) P4 Hershberger (MRID 48672602) N Male Pubertal Rat (MRID 48686602) N N N N N N N N FSTRA (MRID 48672606) N N N Frt: (H)5 N 

OSRI 

ARTA (Orton et al., 2011) P Subchronic toxicity (Dog; MRID 41656401) 

 NE N NE N NE NE 

Dominant Lethal (Mouse; Kaneda et al., 1978) 

 N 
O-PHENYLPHENOL FINAL 

Page 15 of 56 

Table 3. Androgenic/Anti-Androgenic Pathway for o-Phenylphenol

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for o-Phenylphenol1Study Type/ Literature Citation 

AR Binding/Activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin (VTG) 

Two-generation reproduction (Rat; MRID 41729401) 

N N NE N N 

Two-generation reproduction (Rat; MRID 43928801) 

N N NE N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 43954301) 

 N N NE N NE 

Carcinogenicity (Mouse; MRID 43545501) 

N N NE N NE 

Chronic toxicity (Dog; MRID 41656401) 

 N N NE N N 

Freshwater fish reproduction (Fathead minnow; MRID 48136404) 

N NE NE N N 

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

(↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not app

Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SDorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans peweight; LW: Liver weight; ADN: Adrenal weight; BUN: Blood Urea Nitrogen; Histo: Histopathology 
O-PHENYLPHENOL FINAL 

Page 16 of 56 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for th

X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by 

details see Section IV. A 

 4. A 30% increase in testosterone production was observed at 10 μM o-PP; however a 30% decrease at 100 μM o-PP 

 5. Fertility success was decreased 32% at the high-dose and was not statistically significant but was considered biologically mean

 6. While survival was 79% in the highest treatment concentration, one test vessel was observed to have 100% mortality, account

mortalities at this concentration which may indicate a tank-related issue and not a treatment effect on mortality. There were no 

signs of toxicity or significant effects on body weight or length. 

 P Positive findings  N Negative findings (in vitro)/No effect (in vivo)  NE Not examined 
O-PHENYLPHENOL FINAL 

Page 17 of 56 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

pubertal female and male rat assays, and the AMA (frog). 

The potential for o-PP to interact with thyroid regulation are summarized in Table 4. The 

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

No effects on thyroid weight or histopathology were observed in the in vivo male and female 

pubertal assays. TSH concentrations were not affected, but serum T4 concentrations were 

decreased in all treatment groups in the male pubertal assay; however the T4 changes at the 

highest dose males occurred in the presence of overt toxicity. 

In the AMA, o-PP did not alter the median Nieuwkoop-Faber (NF) developmental stage at Day 7 

or 21 relative to the negative control. There were no treatment related effects on thyroid 

histopathology. There were no significant (p>0.05) effects on Day 7 normalized hind-limb length 

(HLL), but a significant decrease of 8% was observed in HLL on Day 21 at the high test 

concentration. There were also no significant (p>0.05) differences in snout-to-vent length (SVL) 

and body weight. 

No thyroid effects were noted in any of the Part 158 mammalian studies. 

Page 25 of 64
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Page 18 of 56 

Table 4. Thyroid Pathway for o-Phenylphenol

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for o-Phenylphenol1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and HistopathologySerum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed 3 

EDSP Tier 1 Assays 

Female Pubertal Rat 

(MRID 48686601) N N N N N 

LW:↓9% 

ADN:↓13% 

BUN:↑23% 

ALT: ↑2X 

Kidney: Histo 

(H) 

N 

Male Pubertal Rat 

(MRID 48686602 ) N N 

↓15, 

23% 

(L, M) 

N N 

LW: ↑21%; 

ADN: ↑16% 

BUN: ↑24% 

Kidney: Histo 

 (H) 

↓12% 

BW 

 (H) 

AMA (Frog; MRID 

48916901) N 

Day 21 

HLL: 

↓8% 

(H) 

N N 

OSRI 

Subchronic toxicity 

(Dog; MRID 41656401) NR N NE X 

(M, H) N 

Two-generation 

reproduction (Rat; 

MRID 41729401) 

NE NE NE X X 

(H) 

Two-generation 

reproduction (Rat; 

MRID 43928801) 

NE NE NE X 

(H) 

X 

(H) 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 43954301) 

N N NE X X 

(H) 

Carcinogenicity 

(Mouse; MRID 

43545501) 

NE N NE X 

X 

(L, M, 

H)

Chronic toxicity (Dog; 

MRID 41656401) N N N X 

(M, H) N 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates 

that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute 

unless otherwise indicated. BW: Body weight; LW: Liver weight; ADN: Adrenal weight; BUN: Blood Urea 

Nitrogen; Histo: Histopathology. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic 

toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

Page 26 of 64
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Page 19 of 56 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI 

is indicated by an X in this column. For details see Section IV. A 

N Negative findings 

NE Not examined 

NR Not reported 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

pathways) as well as recommendations regarding Tier 2 testing.. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

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

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

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

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

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tier 1 assays stated that “In summary, 

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

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

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

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

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

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

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

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

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1. Tier 1 in vivo Assays 

In the Hershberger assay, the majority of high-dose (1000 mg/kg/day) animals (6/7 in the 

androgenic portion and 5/7 in the anti-androgenic portion) displayed some or all of the following 

clinical signs: clear or red perioral soiling, perineal soiling (urine and/or feces), decreased 

activity, noisy respiration, and soft feces. At necropsy, one animal in each high-dose group 

(androgenic and anti-androgenic portions) was found to have thickening of the non-glandular 

mucosa of the stomach. Adrenal, liver and kidney weights of treatment groups at necropsy were 

comparable to the control group. 

In the androgen agonist portion of the Hershberger assay, mean body weight was decreased by 

15% on Day 7 in the high-dose group; however, mean body weights at the other determinations 

were only slightly decreased (NS; ↓6-8%). Food consumption in this group also was decreased 

by approximately 30-40% between Days 1-7, but was comparable to the negative control during 

Days 7-11. Body weights and food consumption in the lower dose groups were comparable to 

controls throughout the duration of the study. 

In the anti-androgen portion of the Hershberger assay, body weights in o-PP-treatment groups 

were generally comparable to the control group; mean food consumption was decreased 20% in 

the high-dose animals, with no treatment effects at the lower doses. 

In the uterotrophic assay, body weights in the treated groups were comparable to control 

throughout the duration of the study. The only organ assessed was the uterus, therefore 

additional information on systemic toxicity for this assay is not available. 

In the female pubertal rat assay, mean body weights generally were similar across all dose 

groups at all times measured. At the high dose (900 mg/kg/day), relative (but not absolute) liver 

weight was increased 9%, and adjusted (but not absolute) adrenal weights were decreased 13%; 

histological analysis of this organs is not available. Kidney weights of treated groups were not 

different from the control group, however the high dose group had elevated serum nitrogen urea 

levels (23%) compared to controls and histological examination found increased incidence of 

collecting duct epithelium hypertrophy (0/15 control, low and mid dose vs. 3/14 high dose), 

multifocal tubule degeneration (4/15 control, 9/15 low and mid, 12/14 high), multifocal tubule 

dilation (1/15 control, 0/15 low, 1/15 mid, 9/14 high), and focal tubule necrosis with 

inflammation (0/15 controls, low and mid doses vs. 2/14 high dose). 

In the male pubertal assay, mean terminal body weight was decreased by 12% in the high-dose 

group (900 mg/kg/day); body weights were not affected in the lower dose groups. Mean relative 

and adjusted (but not absolute) liver weights were increased (21% and 10%, respectively) at the 

high dose. Mean absolute adrenal weights were decreased at the low, mid and high doses (11, 11 

and 16%, respectively), but relative and adjusted weights were not different from controls. 

Kidney weights of treated groups were not different from the control group, however the high 

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Page 22 of 56 

dose group had elevated serum nitrogen urea levels (27%) compared to controls and histological 

examination found increased incidence of collecting duct epithelium hypertrophy (0/15, 1/15, 

0/15 and 5/14) and multifocal tubule dilation (1/15, 0/15, 2/15, 11/14); there was also increased 

severity (but not increased incidence) of multifocal tubule degeneration (very slight vs. slight). 

In the FSTRA, there were no clinical signs of toxicity or significant (p>0.05) effect on body 

weight or length. In the highest treatment concentration (0.876 mg a.i/L, mean measured time 

weighted average), mortality was by 31% in the high-dose females and was 28% overall across 

both genders. One test vessel was observed to have 100% mortality, accounting for 6 of the 7 

total mortalities at this concentration. It was reported that these mortalities may have been tank 

related and not treatment related since other tanks in this treatment concentration did not have 

similar results. In the AMA, there were no treatment-related effects on survival [(only a single 

mortality was observed at the highest treatment concentration (1.92 mg a.i/L, mean measured 

time weighted average)] and there were no notable clinical signs of toxicity reported. 

2. OSRI 

In a subchronic toxicity study in dogs, dose-related emesis was noted in all treated animals with 

the high-dose group exhibiting the most severity. Body weight gains in all study animals were 

variable, but treated animals generally gained less (or lost) weight during the study period. In a 

developmental toxicity study in rats, there were no reported clinical observations related to 

treatment. Body weight gains during GDs 6-16 were decreased in the medium- and the highdose groups, with slight overall body weight gain (GDs 6-21) decreases in both groups. In a 

developmental toxicity study in rabbits, ten deaths occurred during the study. At least four of 

these deaths, in the high-dose group, were considered to be treatment-related, and an abortion at 

the high dose also may have been related to treatment. Clinical signs (incidences of decreased 

feces and perineal soiling) may have been related to treatment in the medium-dose, as well as the 

high-dose, group. 

In a two-generation reproduction study in rats, there were no treatment-related clinical signs of 

toxicity in any treated groups (adults and pups), and no treatment-related increase in mortality, 

although the death of one medium-dose animal probably was due to potentially treatment-related 

kidney failure. Body weights were decreased (↓6-13%) during the pre-mating periods in highdose F0 and F1 parents, as well as during the rest period between the F1a and F1b, and the F2a 

and F2b, breedings. Although body weights were occasionally decreased in females during the 

gestation and lactation periods, these decreases were generally <10% and body weight gains 

were not affected. Food consumption also was decreased in the high-dose F0 and F1 groups 

during the rest periods between breedings. 

In another two-generation reproduction study in rats, one P generation high-dose male died due 

to treatment-related kidney failure. Additionally at this dose, an increase in the incidence of 

urine staining was noted in the P and F1 generation males. This finding was associated with 

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Page 23 of 56 

treatment-related gross and microscopic lesions of the urinary tract in these animals. Decreased 

body weights during pre-mating were observed in high-dose P females, but not males, with 

greater decreases (generally ≥10%) in high-dose F1 males and females. Body weight decreases 

continued in the females during gestation and lactation, but the decreases were generally <10%. 

In the chronic toxicity/carcinogenicity study in rats, there was no treatment-related effect on 

mortality observed. There was an increase in abnormal color urination and red staining in highdose males (402 mg/kg/day), brown staining in medium- and high-dose females (248 and 647 

mg/kg/day), and urine staining in high-dose males and all treated females. Slight body weight 

decreases were noted in the medium-dose animals in the two-year oncogenicity group (no 

decrease in the one-year interim sacrifice group) at Weeks 13, 52, and 78, with weights 

comparable to control by Week 104. Body weight gains in the medium-dose animals were 

generally >90% of control. Body weights in the high-dose animals were decreased <10% in 

animals in the one-year interim sacrifice group, but were decreased ≥10% in high-dose animals 

in the two-year carcinogenicity group at all determinations. Body weight gains in the high-dose 

animals were decreased by 11-26%, compare to the control. Food consumption was generally 

decreased in the two-year carcinogenicity group at the high-dose, with sporadic decreases in the 

medium-dose animals. 

In a chronic toxicity/carcinogenicity study in mice, there was no treatment-related effect on 

mortality and no clinical signs of toxicity. Body weights were decreased in the medium- and 

high-dose males (500 and 1000 mg/kg/day) by Day 147, with decreases ≥10% in the high-dose 

group. Overall body weight gains were also decreased by 16 and 27% in the medium- and highdose males, with no effects on body weight or body weight gain in the low-dose males (250 

mg/kg/day). Consistent, dose-dependent decreases in body weights were also observed in 

females in all treatment groups, with the decreases being ≥10% at selected intervals in each 

group. Overall body weight gains were also decreased by 10, 25, and 38% in the low-, mediumand high-dose females during the study. 

In a chronic toxicity study in dogs, there was no treatment-related mortality. Dose-related 

emesis was noted in all treated animals, with the high-dose group exhibiting the most severity 

(emesis was generally similar to control at the two lower doses). Body weight gains in the highdose females were decreased at all time-points through study termination. Body weight gain 

decreases observed in low- and medium-dose females at Days 92 and 176 were not present at 

study termination. 

In a published, non-Guideline developmental study in rats, (Kaneda et al., 1978), ataxia was 

observed after dosing in dams administered o-PP at 300, 600, and 1200 mg/kg/day. The duration 

of ataxia was reported as lasting several hours, with severity increasing in a dose-related manner. 

Ten of eleven dams died after 3-9 days of treatment in the high-dose group. No clinical signs of 

toxicity were noted at the low dose (150 mg/kg/day). Body weight gains on GDs 9, 12, 15 

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O-PHENYLPHENOL FINAL 

Page 24 of 56 

and 20 were decreased in the medium-low and medium-high dose groups. Body weight gain 

also was decreased in the single surviving animal in the high-dose group. 

In a non-guideline freshwater fish reproduction toxicity study with fathead minnows 

(Pimephales promelas), no mortalities occurred during the exposure period. There were no 

reported clinical signs of toxicity or significant effects on length or weight. 

B. Estrogen Pathway 

The Tier 1 in vitro ER binding assay was equivocal and positive for ER activation (Routledge 

and Sumpter, 1997 and Miller et al., 2001). Additionally, o-PP inhibited aromatase activity and 

increased estradiol production in the Tier 1 aromatase and steroidogenesis assays, respectively. 

However, in the mammalian in vivo assays, there was no convincing evidence for an interaction 

with the estrogen pathway. The uterotrophic assay was negative, and in the female pubertal 

assay the sole effect observed was a decrease in the percent regularly cycling animals at the high 

dose compared to the controls which occurred in the presence of systemic toxicity. Due to the 

lack of redundancy of responses across the mammalian assays, there is insufficient evidence for 

a potential interaction in vivo in mammals. 

However, there are indications of potential effects on the reproductive status in fish that included 

decreases in female vitellogenin, fecundity and fertility in the FSTRA; there were no effects on 

GSI or gonadal histopathology. In another fish reproduction study with the same species as the 

FSTRA, decreases in egg production were observed. These in vivo effects, along with 

indications of potential in vitro effects including ER activation and altered steroidogenesis, 

suggest that o-PP may potentially interact with the estrogen pathway in fish. 

C. Androgen Pathway 

There was evidence of potential interaction with the androgen pathway in the in vitro AR 

binding assay and conflicting evidence in the steroidogenesis assay where a 30% increase in 

testosterone production at 10 μM o-PP and a 30% decrease at 100 μM o-PP. Studies published 

in the open literature showed o-PP was not an androgen agonist in the MDA-kb2 cell assay, 

however an anti-androgen effect of o-PP was observed with this cell line. 

The Hershberger assay was negative, and in the male pubertal assay, decreases seen in a number 

of androgen-dependent tissues were observed in the presence of overt toxicity. In the FSTRA, 

decreases in fertility were observed, however, no other androgen-related effects were observed in 

this study or the OSRI fish reproduction study. There were no androgen-related effects observed 

in the Part 158 mammalian studies. Therefore, there is no convincing evidence for potential 

interaction with the androgen pathway.

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O-PHENYLPHENOL FINAL 

Page 25 of 56 

D. Thyroid Pathway 

There was no evidence of potential interaction of o-PP with the thyroid pathway. In female 

pubertal assay, there were no treatment-related effects on thyroid hormones (T4 and TSH), 

thyroid weights or thyroid histopathology. In the male pubertal assays the only thyroid-related 

effect observed was a decrease in serum T4 levels at all doses. In the AMA, no treatment-related 

finding were observed except for a significant reduction in Day 21 normalized hindlimb length; 

however, given the lack of effects on developmental stage or thyroid histopathology, this finding 

alone is not considered to be sufficient evidence of a potential interaction of the thyroid pathway 

in amphibians. No thyroid effects were noted in any of the Part 158 mammalian studies. 

E. Conclusions 

!

The conclusion of the WoE evaluation is that o-PP demonstrates no convincing evidence of 

potential interaction with the estrogen, androgen or thyroid pathways in vivo in mammals. There 

is also insufficient evidence of a potential interaction of the thyroid pathway in amphibians. 

However, there is evidence of potential interaction with the estrogen pathway in non-mammalian 

species as suggested by the decreases in reproductive output in the FSTRA and fish OSRI. These 

findings in the fish assays along with the results in the in vitro assays, suggest potential 

interaction with the estrogen pathway in fish. There is no convincing evidence of a potential 

interaction with the androgen pathway in fish. 

 

V. EDSP Tier 2 Testing Recommendations 

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

recommended for o-PP since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

Effects on fecundity/fertility were observed at 876 μg a.i./L in the FSTRA with effects on 

reproduction reported at 36 and 293 μg a.i./L in the fish OSRI assay (which was similar to the 

FSTRA in test design). Currently, there are no acceptable 40 CFR Part 158 chronic toxicity data 

for fish. At this time, based on the reproductive responses observed in the fish assays and the in 

vitro assays, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test (MEOGRT) is 

recommended to better understand potential reproductive effects in fish. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are not available for o-PP; however, were requested under the final workplan for the 

registration review for o-PP (USEPA 2014). The type of data obtained from Part 158 avian 

reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential 

reproductive effects to birds from o-PP exposure. Relative to the EDSP, additional testing is not 

recommended.

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VI. References 

Blair, R.M., Fang, H., Branham, W.S., Hass, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., 

Perkins, R., and Sheehan, D.M. (2000) The estrogen receptor relative binding affinities of 188 

natural and xenochemicals: structural diversity of ligands. Toxicological Sciences 54:138-153. 

Caunter, J.E. and Williams, T.D. (2002) Preventol 0 extra: determination of effects on the 

reproduction of fathead minnow (Pimephales promelas). Unpublished study conducted by 

Brixham Environmental Laboratory, AstraZeneca UK Limited, UK. Study number: AH0603A. 

Study Sponsor: Bayer AG, Germany. (MRID 48135404) 

Coady, K.K. and Sosinski, L.K. (2012) Evaluation of Ortho-Phenylphenol in the Human 

Recombinant Aromatase Assay. Unpublished study conducted by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study 

ID No.: 111007, January 3, 2012. (MRID 48672608) 

Cosse, P.F., Stebbins, K.E., Stott, W.T., Johnson, K.A, and Atkin, L. (1990) OrthoPhenylphenol: Palatability/probe, four week and one-year oral toxicity studies in beagle dogs. 

Unpublished study conducted by DOW Chem. Co., Study ID: K-001024-039, September 24, 

1990. (MRID 41656401) 

Eigenberg, D.A. (1990) Two-Generation dietary reproduction study in rats using 

orthophenylphenol. Unpublished study conducted by Mobay Corp., September 17, 1990. (MRID

41729401; also submitted under MRID 40976501) 

Eigenberg, D.A. and Lake, S.G. (1995) Two-generation dietary reproduction study in SpragueDawley rats using technical grade ortho-phenylphenol. Unpublished study conducted by Bayer 

Corporation, Agriculture Division, Toxicology, Stilwell, KS. Laboratory Study No.: 93-672-

VX, September 28, 1995. (MRID 43928801) 

John, J.A., Murray, F.J., Rao, K.S., and Schwetz, B.A. (1981) Teratological evaluation of 

Orthophenylphenol in rats. Fundamental and Applied Toxicology 1, 282-286. (MRID 00067616 

and 00164362) 

John, J.S., Murray, F.J., Crawford, A.A., Murray, J.S., Pilny, M.K., Schlachter, M.M., and Rao, 

K.S. (1990) Phase 3 Reformat of MRID 00066716/00164362: The effect(s) of orally 

administered orthophenylphenol on rat embryonal and fetal development: Project HET K0001024-33(R). (MRID 92154037) 

Kaneda, M., Teramoto, S., Shingu, A, Shirasu, Y. (1978) Teratogenicity and Dominant-Lethal 

studies with o-Phenylphenol. J. Pesticide Sci. 3:365-370. 

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Page 27 of 56 

LeBaron, M.J., Kan, H.L., Perala, A.W. (2012) Evaluation of Ortho-Phenylphenol (o-PP) in the 

In Vitro Steroidogenesis Assay. Unpublished study conducted by Toxicology & Environmental 

Research and Consulting, Midland, MI. Laboratory Report No.: 111014, Jan 4, 2012. (MRID 

48672601) 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011) Evaluation of orthophenylphenol (oPP) in an in vitro androgen receptor binding assay. Unpublished study conducted by Toxicology 

& Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project ID: 111114, December 19, 2011. (MRID 48672603) 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2012) Evaluation of orthophenylphenol (oPP) in an in vitro estrogen receptor binding assay. Unpublished study conducted by Toxicology 

& Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project ID: 111120, January 4, 2012. (MRID 48672604) 

Lehman C.M., Fiting, J.A., Hutchinson, K.L., Louch, D.W., Malowinski, N.A., McFadden, L.G. 

and Thomas, J. (2012) Ortho-Phenylphenol: The Fish Short-term Reproduction Assay Using the 

Fathead Minnow, Pimephales promelas. Unpublished study conducted by Toxicology and 

Environmental Research and Consulting, Midland, Michigan. Lab Study No.: 111032. Study 

sponsored by o-PP EDSP Consortium, Dow Chemical Company, Midland, Michigan and 

LANXESS Corporation, Pittsburgh, Pennsylvania. Study completed January 04, 2012. (MRID 

48672606) 

Marty, M.S., Andrus, A.K., and Sura, R. (2012) Ortho-Phenylphenol: Pubertal Development and 

Thyroid Function in Intact Juvenile/Peripubertal Male Crl: CD(SD) Rats. Unpublished study 

conducted by Toxicology and Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory Project Study ID No.: 111088; April 13, 2012. (MRID 

48686602) 

Marty, M. S., Marshall, V. A., and Jeong, Y. C. (2012) Ortho-phenylphenol: Hershberger assay 

in castrated male Crl:CD(SD) rats. Unpublished study conducted by Toxicology & 

Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory 

Project Study ID: 111084, January 5, 2012. (MRID 48672602) 

Marty, M. S., Zablotny, C. L., Passage, J. K., and Jeong, Y.C. (2012) Ortho-phenylphenol (oPP): uterotrophic assay in the immature female Crl:CD(SD) rat. Unpublished study conducted by 

Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID: 111086, January 5, 2012. (MRID 48672605) 

Marty, M.S., Zablotny, C.L., and Sura, R. (2012) Ortho-Phenylphenol: Pubertal Development 

and Thyroid Function in Intact Juvenile/Peripubertal Female Crl: CD(SD) Rats. Unpublished 

study conducted by Toxicology and Environmental Research and Consulting, The Dow 

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Page 28 of 56 

Chemical Company, Midland, MI. Laboratory Project Study ID No.: 111087; April 13, 2012. 

(MRID 48686601) 

Miller, D., Wheals, B., Beresford, N., and Sumpter, J. (2001) Estrogenic activity of phenolic 

additives determined by an in vitro yeast bioassay. Environmental Health Perspectives 109: 

133-138. 

Orton, F., Rosivatz, E., Scholze, M., and Kortenkamp, A. (2011) Widely used pesticides with 

previously unknown endocrine activity revealed as in vitro antiandrogens. Environ Health 

Perspect. 119(6):794-800. 

Quast, J.P. and McGuirk, R.J. (1995) Ortho-Phenylphenol: Two-year dietary chronic 

toxicity/oncogenicity study in B6C3Fl mice. Unpublished study conducted by DOW Chem. Co., 

Study ID:K-001024-047, February 1, 1995. (MRID 43545501) 

Rehmann, K., Schramm, K-W., and Kettrup, A.A. (1999) Applicability of a yeast oestrogen 

screen for the detection of oestrogen-like activities in environmental samples. Chemosphere

38(14): 3303-3312. 

Routledge, E. and Sumpter, J. (1997) Structural features of alkylphenolic chemicals associated 

with estrogenic activity. The Journal of Biological Chemistry 272(6): 3280-3288. 

Schisler, M.R. (2012) Data Evaluation Record (DER) for: Evaluation of orthophenylphenol (oPP) in an in vitro androgen receptor binding assay. Unpublished study conducted by Toxicology 

& Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project ID: 111114, December 19, 2011. (MRID 48618303) 

Schisler, M.R. (2012) Data Evaluation Record (DER) for: Evaluation of orthophenylphenol (oPP) in an in vitro estrogen receptor binding assay. Unpublished study conducted by Toxicology 

& Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project ID: 111120, January 4, 2012. (MRID 48618304) 

USEPA (2010) O-Phenylphenol - Report of the Endocrine Disruptor Review Team; Test Order# 

EDSP-064103-225 and 226. December 20, 2010. Office of Chemical Safety and Pollution 

Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

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

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

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

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

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

Page 36 of 64
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Page 29 of 56 

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

Agency, Washington, DC. 

USEPA (2014) ortho-Phenyl Phenol (oPP) and Salts Final Work Plan. Registration Review 

Initial Docket Case Number 2575. March 2014. Office of Chemical Safety and Pollution 

Prevention. U.S. Environmental Protection Agency, Washington, DC. EPA-HQ-OPP-2013-

0524-0005. 

Wahle, B.S. and W.R. Christenson (1996) Technical Grade ortho-Phenylphenol: A Combined 

Chronic Toxicity/ Oncogenicity Study in the Rat. Unpublished study conducted by Bayer 

Corporation, Stillwell, KS. Study ID 92-272-SC. (MRID 43954301) 

Wahle, B.S. and W.R. Christenson (1996) Supplemental Submission to Bayer Toxicology Report 

No. 7908 (EPA MRID 43954301). Unpublished study conducted by Bayer Corporation, 

Stillwell, KS. Study ID 92-272-SC. (MRID 44832201) 

Wahle, B.S. and W.R. Christenson (1996) Supplemental Submission to Bayer Toxicology Report 

No. 7908 (EPA MRID 43954301). Unpublished study conducted by Bayer Corporation, 

Stillwell, KS. Study ID 92-272-SC. (MRID 44852701) 

Zablotny, C.L., Breslin, W.J., and Kociba, R.J. (1991) Ortho-Phenylphenol (o-PP): Gavage 

teratology study in New Zealand White Rabbits. Unpublished study conducted by DOW Chem. 

Co. Study ID: K-001024-045, April 23, 1991. (MRID 41925003) 

 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 48916901) of ortho-phenylphenol (99.9% purity) on amphibian 

metamorphosis of the African clawed frog (Xenopus laevis) was studied under flow-through 

conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80/control and 

treatment group; 20/replicate) were exposed to nominal concentrations of 0 (negative control), 

0.024, 0.12, 0.60, and 3.0 mg a.i./L. The time-weighted average (TWA), measured 

concentrations were <0.00514 (<LOQ; negative control), 0.0175, 0.0673, 0.352, and 1.92 mg 

a.i./L. The test system was maintained at 21.3-22.8°C and a pH of 7.0-7.6. 

A single mortality was observed in the high treatment level during the study. Over the course of 

the exposure, a single tadpole in the mid-low treatment level was observed resting on the surface, 

and a single tadpole in the high treatment level was observed resting on the bottom. There were 

no other signs of clinical toxicity. 

Ortho-phenylphenol did not significantly affect (p>0.05) Day 7 normalized (for snout-vent 

length) hind-limb length (HLL) at any treatment level; however, Day 21 HLL of non-late stage 

tadpoles (NF<61) was significantly reduced (Jonckheere, p = 0.006) by 11% at the high 

treatment level relative to the negative control. Day 21 normalized HLL was also significantly 

reduced (Jonckheere, p = 0.036) by 8% at the high treatment level relative to the negative 

control. Among late-stage tadpoles (NF>61), there were no significant effects on normalized 

HLL or growth endpoints compared to the negative control. There was no significant (p>0.05) 

acceleration or delay of median NF developmental stage at Day 7 or Day 21 in any treatment 

group relative to the negative control. No asynchronous development was observed throughout 

the test. Mild follicular cell hypertrophy was observed in the negative control and all treatment 

groups, though there was no apparent treatment-related effect. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48672603), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which measured the binding of a single concentration of [3

H]-R1881 (1 nM) in the presence of 

increasing concentrations (logarithmic increase from 10−10 to 10−3

 M) of o-phenylphenol (o-PP, 

99.9% purity, Lot #CHHYDD0051). Ethanol was used as a vehicle at a final assay concentration 

of <3%. A total of three runs were performed, and each run included dexamethasone as a weak 

positive control, and R1881 as the ligand reference standard. 

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Prior to conducting the AR competitive binding experiments, a saturation binding experiment 

was conducted to determine if the AR in the cytosol preparation was present in reasonable 

numbers and functioning with appropriate affinity for the radiolabeled ligand. Data from a 

saturation binding experiment were not provided in the study report; however the results from a 

single run of the saturation binding experiment were included in a study profile (MRID 

48618303) submitted by the test order recipient. In the saturation binding experiment, the 

maximum binding capacity (Bmax) was 3.245 fmol/100 μg protein and the dissociation constant 

(Kd) was 0.4641 nM. Although these values were slightly below the range of values from the 

validation studies, the results were highly reproducible and all other performance criteria and the 

responses in the competitive binding assays indicated acceptable performance of the assay. The 

Scatchard plot indicated a linear response across the concentrations of ligand added. Nonspecific 

binding as a percent of total binding was less than 20% across the entire concentration range in 

the saturation binding assays (range 6.2-19.8%, with the exception of the high concentration (10 

nM) in one assay, which was 24.6%). 

In the competitive binding experiment, o-PP reduced the mean specific binding of the 

radiolabeled ligand in a concentration-dependent manner in all three runs. The maximum 

binding reduction occurred at the highest concentration tested (10−3 M), with specific binding 

being reduced to 6.3-7.4%. Specific binding was also reduced at 10−4 M (62.8-68.8%), but not at 

concentrations ≤10−5 M (92.8-103.7% binding). Specific [3H]-R1881 binding was reduced to 

<50% at the highest concentrations tested in all three runs. 

The estimated mean log IC50 was −3.749 M for o-PP compared to that of R1881 (−8.970 M) and 

the weak positive control of −4.418 M. The mean RBA was 0.0006% for o-PP compared to 

0.0028% for the positive control. Confidence in these numbers is high due to the small variation. 

No precipitation of the test compound was reported. The solvent control responses indicated no 

drift in the study assay. All performance criteria were met. 

Based on the results from the three runs, o-PP is classified as a Binder in the Androgen Receptor 

Binding Assay.

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48672608), o-phenylphenol (o-PP, 99.9% a.i., 

lot# CHHYDD0051) was incubated with human recombinant aromatase and tritiated 

androstenedione ([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in 1% ethanol for 15 

minutes to assess the potential of o-PP to inhibit aromatase activity. o-PP was tested at 

logarithmic concentrations from 10–10 M to 10−3

 M in 3 independent runs. 

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

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

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

included a full activity control, a background activity control, a positive control series (10–10 to 

10–5) with a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test chemical 

series (10–10 to 10–3) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.080 to 0.159 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.124±0.029 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 2.38% to 7.88% and 

averaged 5.7% of the full control activity. The response of the full activity controls and 

background controls was acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.108±0.054 

nmol·mg-protein−1

·min−1 at the lowest tested concentration (10−10 M) and 0.002±0.004 nmol·mgprotein-1·min−1

 at the highest tested concentration (10−5 M). These results were generally within 

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

coefficient of variation for replicates of each concentration, with the exceptions that the overall 

%CV for the highest three concentrations (51.2, 36.2, and 129%) exceeded the recommended 

limit of 15%. The estimated log IC50 for 4-OH ASDN averaged −7.1 M and the Hill slope was 

−1.17. 

For o-PP, aromatase activity averaged 0.124±0.031 nmol·mg-protein−1·min−1 at the lowest tested 

concentration (10−10 M) and 0.007±0.013 nmol·mg-protein−1

·min−1 at the highest successfully 

tested concentration (10−3 M). The overall % CV for the 10−3 and 10−8 M concentrations (69% 

and 22%, respectively) exceeded the recommended limit of 15%. The data for o-PP were 

modeled for all runs, and the goodness of fit (r2

) values were 0.87-0.98. The best fit Hill slope 

and log IC50 values for o-PP across the three independent runs did not differ significantly. 

The lowest portion of the response curve was less than 50% activity at highest two 

concentrations tested. Aromatase activity was 53% at 10–4 M, and decreased to 6% at 10−3

 M oPP. The average log IC50 and Hill slope for o-PP were −3.97 M and −0.99, respectively. 

Based on the data from the average response curve, o-PP is classified as an Inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48672604) for o-phenylphenol (o-PP), uterine 

cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding 

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experiment which measured the binding of a single concentration of [3H]-17β-estradiol (1 nM) in 

the presence of increasing concentrations (logarithmic increase from 10−10 to 10−3 M) of o-PP 

(99.9% purity, Lot # CHHYDD0051). Ethanol was used as the solvent vehicle at a final 

concentration of <3%. A total of three runs were performed, and each run included 19-

norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-βestradiol as the natural ligand reference material. 

Saturation binding experiments were conducted first to demonstrate that the ER in the uterine 

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

the radioligand. No data from the saturation binding experiment were provided in the study 

report; however, the results from a single run of the saturation binding experiment were included 

in a study profile (MRID 48618304) submitted by the test order recipient. The results from the 

single saturation binding run showed a maximum binding capacity (Bmax) of 59.28 fmol/100 μg 

protein with the dissociation constant (Kd) of 0.1032 nM. Nonspecific binding as a percent of 

total binding was 1.7-8.6% across the concentration range. 

In the competitive binding experiment, the estimated mean log IC50 was −8.943 M for 17βestradiol and −5.454 M for the weak positive control. The mean RBA was 0.032% for the weak 

positive control. All performance criteria were met. o-PP reduced the mean specific binding of 

the radiolabeled ligand in a concentration-dependent manner in all three runs (declining to <50% 

in Runs 1 and 2); however, the o-PP binding curve exhibited steep Hill slopes (−2.262 to 

−4.588), indicating that the data did not fit the four-parameter, nonlinear regression model for 

analysis of one-site binding. The mean estimated log IC50 (n=2) was −3.192 M for o-PP; an 

RBA value was not determined. 

Based on the results from the three runs, o-PP is classified as Equivocal in the Estrogen Receptor 

Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

Requirement satisfied by OSRI (Routledge and Sumpter, 1997, and Miller et al., 2001); see 

Appendix 2 for summaries. 

Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48672606) of ortho-phenylphenol (o-PP) with 

fathead minnows (Pimephales promelas) was conducted under flow-through conditions. Adult 

fish (16 spawning groups; 2 males and 4 females in each group; 5.5 months old) were exposed to 

o-PP at nominal concentrations of 0 (negative control), 0.02, 0.20, and 2.0 mg a.i./L; timePage 41 of 64
O-PHENYLPHENOL FINAL 

Page 34 of 56 

weighted average (TWA), measured concentrations were <0.00495 (<LOQ), 0.0158, 0.0755, and 

0.876 mg a.i./L. The test system was maintained at 24.6 to 25.5o

C and a pH of 7.2 to 7.4. 

Overall survival was significantly decreased (p<0.05) by 29% at the high treatment level (75% 

survival in males and 69% survival in females) compared to the negative control with 100% 

survival in the negative control and all other treatment groups. Six of seven deaths occurred in a 

single tank over two days, potentially indicating a tank-related issue (the high treatment level 

was well below any acute toxicity level). o-PP exposure was not associated with increases or 

decreases in body weight and/or length in either sex at any treatment level (p>0.05). With the 

exception of a single lethargic male in the high treatment group, no effects on secondary sex 

characteristics or clinical signs were observed for any treatment group compared to the negative 

control. 

In the negative control, spawning did not occur at least every 4 days in three of the four 

replicates. Mean fecundity was 13.9 eggs/female/day/replicate (range: 7.3-20.1); fertilization 

success in the negative control group averaged 97.2% (range: 92.4-99.7%). Two of the four 

negative control replicates were below the minimum fecundity criterion of 15 

eggs/female/day/replicate at 7.3 and 9.2 eggs/female/reproductive day. o-PP did not significantly 

affect (p>0.05) fecundity or fertility compared to the negative control. However, the variances in 

the high treatment group for fecundity and fertilization success were high (7.3±6.38 

eggs/female/day/replicate and 66.0±57.13%, respectively); while neither value was significantly 

different (p>0.05) from the negative control, both were markedly decreased by 47.5 and 32%, 

respectively, compared to the negative control, and these differences were considered 

biologically significant. 

Female vitellogenin (VTG) was significantly reduced (p<0.05) by 35.5% relative to the negative 

control at the high treatment level; male VTG was unaffected by o-PP treatment. There were no 

other significant effects on endpoints (e.g., nuptial tubercle score, gonado-somatic index (GSI), 

or gonadal staging). There were no treatment-related histopathological observations. In females, 

there was an increase in the incidence and severity of oocytes atresia in treated fish compared to 

the negative control; however, the effects lacked a concentration-response relationship. Plasma 

testosterone and 17β-estradiol levels were not measured. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48672602) screening for androgenic activity, orthophenylphenol (o-PP; 99.9% a.i., Lot # CHHYDD0051) in corn oil was administered daily via 

oral gavage (4 mL/kg) to groups of seven 55-day old, castrated male Sprague Dawley rats at 

dose levels of 0 (vehicle), 50, 250, or 1,000 (limit dose) mg/kg/day. An androgenic positive 

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control group consisted of seven castrated rats exposed to 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, o-PP (99.9% a.i., batch/lot # CHHYDD0051) in 

corn oil was administered daily via oral gavage (4 mL/kg) to groups of seven 55-day old, 

castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 50, 250, or 1,000 mg/kg/day in 

conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of seven castrated rats exposed to 0.4 

mg/kg/day TP and 3 mg/kg/day flutamide (FT). TP alone was used as the anti-androgenic 

negative control. 

For both components of the assay, body weights and food consumption were determined daily. 

The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the 

final dose administration. At necropsy, the five androgen-dependent tissues, adrenal glands, 

kidneys, and liver were collected and weighed. 

All animals survived until scheduled termination. One animal in the high dose group was 

observed to have mottled lungs at necropsy, indicating a gavage error; therefore, the results for 

this animal were excluded from the study. There were no treatment-related clinical signs in 

animals dosed at ≤250 mg/kg/day in either assay. In both assays, the majority of high-dose 

animals (6/7 androgen assay; 5/7 in anti-androgen assay) had some or all of the following 

observations: clear or red perioral soiling, perineal soiling (urine and/or feces), decreased 

activity, noisy respiration, and soft feces. At necropsy, one animal in each high dose group (both 

assays) was found to have thickening of the nonglandular mucosa of the stomach. 

In the androgen agonist assay, body weights were decreased (p<0.05) by 15% on Day 7 in the 

1,000 mg/kg/day group compared to controls; however, body weights at the end of the study 

were similar to controls. Food consumption in this group was decreased (p<0.05) by 42% during 

Days 4-7. Body weights and body weight gains in the 50 and 250 mg/kg/day groups were 

comparable to controls throughout the duration of the study; food consumption was increased 

(p<0.05) in the 50 mg/kg/day group on Days 1-4 (↑6%). In the positive control group, 

cumulative body weight gains were increased (p<0.05) on Days 1-4 (↑45%), Days 1-7 (↑50%), 

and Days 1-11 (↑36%); food consumption was increased (p<0.05) in this group on Days 1-4 

(↑14%), Days 4-7 (↑14%), and Days 7-11 (↑12%). There were no treatment-related effects on 

weights of the accessory sex organs/tissues, adrenal glands, kidneys, or liver for any o-PPtreatment group. Animals in the TP group had increased (p<0.05) accessory sex organ weights 

as follows: 1760% in seminal vesicles; 1117% in ventral prostate; 226% in levator anibulbocavernosus (LABC); 806% in Cowper’s gland; and 67% in glans penis. Adrenal glands, 

kidney, and liver weights in the positive control group were comparable to the vehicle control. 

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In the anti-androgen assay, body weights and body weight gains in the o-PP treatment groups 

and positive control group were comparable to the negative control over the duration of the 

study. In the 1,000 mg/kg/day group, food consumption was decreased (p<0.05) by 19% during 

Days 4-7. Food consumption in the 50 and 250 mg/kg/day groups and the positive control group 

was similar to controls over the duration of the study. At 1,000 mg/kg/day, the weights for the 

ventral prostate were decreased (p<0.05) by 28% compared to the controls. However, there were 

no treatment related effects on the weights of the seminal vesicles, LABC, Cowper’s glands, 

glans penis, adrenal glands, kidneys, or liver for any o-PP-treatment group. Animals dosed with 

TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as follows: 83% 

in seminal vesicles; 77% in ventral prostate; 46% in LABC; 61% in Cowper’s gland; and 18% in 

glans penis. Adrenal glands, kidney, and liver weights in the positive control group were 

comparable to the vehicle control. 

Percent CVs for the vehicle control and the 1,000 mg/kg/day treatment groups for both assays 

were compared to the performance criteria in the guidelines. All %CV values were less than the 

maximum recommended values, with the exception of the %CV for Cowper’s glands for the 

control group in the androgen agonist assay (CV = 88%; maximum recommended CV = 55%) 

and the %CV for LABC for the high dose group in the anti-androgen assay (CV = 22%; 

maximum recommended CV = 20%). 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. o-PP was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48686601), 15 Sprague Dawley Crl:CD(SD) rats/dose group 

were treated daily via oral gavage with O-phenylphenol (o-PP; 99.9% purity, Lot No. 

CHHYDD0051) in corn oil at doses of 0 (vehicle), 50, 250, or 900 mg/kg/day from post-natal 

day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 

22, and the body weight at day of attainment was recorded. Following sacrifice on PND 42, 

thyroxine (T4) and thyroid stimulating hormone (TSH) levels were analyzed by 

radioimmunoassay or chemiluminescent immunoassay. Liver, pituitary, adrenals, and urogenital 

organ weights were recorded, and microscopic examinations were performed on the ovaries, 

uterus, thyroid, and kidneys. 

All animals survived until scheduled termination, with the exception of one animal in the highdose group (death due to gavage error). Additionally, no treatment-related clinical signs of 

toxicity were observed in animals for any dose groups. 

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Mean body weights were similar across all dose groups on PND 21. There were no effects on 

age at attainment of VO for any dose of o-PP. Initial and terminal body weights, body weight 

gains, and body weight at attainment of VO were similar across all dose groups. However, the 

900 mg/kg/day group weighed 8% less than control animals at PND 35. The 900 mg/kg/day 

group gained 10% to 13% less than control between PND 22-35, but these decreases were not 

statistically (NS) different from control animals. High-dose rats gained more weight than control 

animals during the PND 35-42 interval, such that body weight gains were only slightly decreased 

(3.2%, NS) over the entire dosing period (PND 22-42). 

At 900 mg/kg/day, modest decreases (p<0.05) were noted in adjusted pituitary (↓12%) and 

adrenal (↓13%) weights, and a minor increase (p<0.05) of 9% was noted in relative liver weight. 

Relative pituitary weights were decreased (p<0.05) by 10%. There were no dose-related effects 

on the weights of kidneys, ovaries, uterus, or thyroid. 

There were no effects of treatment on mean age at first estrus or mean cycle length. In the highdose animals, the percent cycling was decreased (NS) relative to the control from 93.3% to 

64.3%, and the percent regularly cycling was decreased (p<0.05) from 86.7% to 28.6%. 

However, the estrous cycle status at necropsy (diestrus, proestrus, estrus, not cycling) was 

generally similar across all groups.

Serum urea nitrogen (BUN) and phosphorus levels were increased (p<0.05) in the 900 

mg/kg/day dose group (↑23.1 and ↑14.1%, respectively). These increases were considered 

treatment-related, and were associated with treatment-related kidney effects. Significantly 

increased alanine aminotransferase activity (ALT; p<0.05; 102%) was also considered treatmentrelated. 

o-PP was not associated with any significant treatment-related changes in thyroid histopathology. 

At 900 mg/kg/day, increased incidences of the following histological lesions were noted in the 

kidney: (i) multifocal renal tubular dilatation (12/14 treated animals, very slight and slight 

severity vs. 4/15 controls, very slight severity); (ii) multifocal collecting duct epithelium 

hypertrophy (3/14 treated animals, very slight severity vs. 0/15 controls); (iii) multifocal renal 

tubule dilatation (9/14 treated animals, very slight to slight severity vs. 1/15 controls, very slight 

severity; and (iv) slight focal renal tubular necrosis, with accompanying inflammation (2/14 

treated animals vs. 0/15 controls). 

The doses tested were adequate based on the increased serum ALT, BUN, phosphorus, renal 

lesions, and effects on estrous cyclicity seen at the high dose (900 mg/kg/day). 

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Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48686602), 16 Sprague Dawley Crl:CD(SD) rats/dose group 

were treated daily via oral gavage with O-phenylphenol (o-PP; 99.9% purity, Lot No. 

CHHYDD0051) in corn oil at doses of 0 (vehicle), 50, 250, or 900 mg/kg/day from post-natal 

day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on 

PND 30, and the body weight at day of attainment was recorded. Following sacrifice on PND 

53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were 

analyzed by radioimmunoassay or chemiluminescent immunoassay. Liver, pituitary, adrenal and 

urogenital organ weights were recorded, and microscopic examinations were performed on the 

testes, epididymides, thyroid, and kidneys. 

All animals survived until scheduled termination, with the exception of four animals. The cause 

of death for three of these animals (one in each o-PP-dose group) was considered to be gavage 

error. The cause of death of the fourth animal (high-dose group) was not determined, but was 

not attributed to o-PP treatment. Additionally, no treatment-related clinical signs of toxicity 

were observed in animals for any dose groups. Adjusted terminal body weights were decreased 

by 8% in the 900 mg/kg/day group, due to a treatment-related effect on body weight gain (↓13%; 

p<0.05) over the duration of the study. Body weights and body weight gains were not affected at 

≤250 mg/kg/day. The adjusted age of attainment of PPS was delayed by 1.6 days (not 

significant; NS) in the 900 mg/kg/day group compared to the control (45.1 vs. 43.5 days). 

Delayed PPS appeared to be related to the decreased terminal body weight and body weight gain 

observed in the high-dose group. 

Significant (p<0.05) organ weight changes after administration of o-PP included increased 

relative liver weight at 250 mg/kg/day (↑8%), and adjusted and relative liver weights at 

900 mg/kg/day (↑10% and ↑21%, respectively). Compared to the control, unadjusted and 

adjusted pituitary weights were decreased by 15% and 10%, respectively, in the 900 mg/kg/day 

group, and unadjusted adrenal weights were decreased by 11% at ≤250 mg/kg/day and by 16% at 

900 mg/kg/day. However, the most relevant measures are the adjusted weights, and only minor 

increases (p<0.05) were noted in the liver and adrenal at 900 mg/kg/day. 

Additionally, decreased (p<0.05) organ weights at 900 mg/kg/day included: unadjusted weights 

of seminal vesicles + coagulating glands with and without fluid (↓23% and ↓18%, respectively); 

unadjusted and adjusted ventral prostate and LABC weights (↓17-20% and ↓16-18%, 

respectively); unadjusted weights for the left and right epididymides (↓10% and ↓9%, 

respectively); and unadjusted and adjusted weights for the left testis (↓9% and ↓7%, 

respectively). As adjusted weight decreases were observed in three androgen sensitive tissues, 

these effects were considered treatment-related. There were no dose-related effects on the 

weights of kidneys, dorsolateral prostate, right testis, or thyroid. 

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No treatment-related effects were observed on serum testosterone or TSH concentrations. Serum 

T4 concentrations were decreased (p<0.05) at all dose levels by 15-23%, but without clear dosedependence. There were no gross or histological lesions of the thyroid. At 900 mg/kg/day, an 

apparent treatment-related effect was observed on serum urea nitrogen concentrations, which 

corroborated the renal histological evidence. Significantly increased alanine aminotransferase 

(ALT) activity (↑95%) was considered treatment-related, but the 32% increase in aspartate 

aminotransferase activity (AST) was a slight response and not adverse. The 40% decreases 

(p<0.05) in gamma glutamyl transferase (GGT) activities at 250 and 900 mg/kg/day are 

considered slight and were not dose-dependent. 

No treatment-related findings were observed in the right testes or right epididymides at any o-PP 

dose. However, in the kidneys, there were increased incidences of renal tubular dilatation 

(combined focal and multifocal, very slight and slight severity) at 900 mg/kg/day (12/14 

animals) compared to controls (5/15 animals). Increased incidences of collecting duct 

epithelium hypertrophy (combined focal and multifocal, very slight severity) were also observed 

at 900 mg/kg/day (5/14) compared to controls (0/15). Although the incidences of renal tubular 

degeneration were similar in high-dose and control animals, the severity was increased at the 

high dose; the incidence of slight multifocal tubular degeneration was 3/14 at 900 mg/kg/day 

compared to 0/15 in controls. 

The high dose of 900 mg/kg/day was sufficient based on the 8% decrease in the adjusted 

terminal body weight, the 13% decrease overall body weight gain, the 7-17% decreases in 

androgen-sensitive organ weights, the 94% increase in serum ALT, and increased incidences 

and/or severity of renal lesions. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48672601), H295R cells cultured in vitro in 24-well plates 

were incubated with ortho-phenylphenol (o-PP, 99.9% a.i., Lot# CHHYDD0051) at log 

concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) 

was used as a vehicle, at a final assay concentration of 0.1%. 

Testosterone and estradiol levels were measured by LC/APPI-MS/MS. A Quality Control (QC) 

plate was run concurrently with each independent run of a test chemical plate to demonstrate that 

the assay responded properly to positive control agents at two concentration levels. The positive 

controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol 

and testosterone production. 

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No precipitation was observed at o-PP concentration up to 100 μM, and o-PP was not cytotoxic 

to the H295R cells. Cell viability for the QC and test plates was ≥95.4% for o-PP and the 

reference standards. 

Four independent test runs of the steroidogenesis assay with o-PP were conducted, but the fourth 

was not statistically evaluated as three acceptable independent assays for o-PP had been 

completed. The minimum basal hormone production levels in the solvent control (SC) wells 

were acceptable for testosterone, but basal production of estradiol was below the recommended 

40 pg/mL level. However, basal estradiol production was still >26 pg/mL which is >2.5-fold the 

minimum detection limit (MDL), as per Guideline recommendation. Forskolin at 10 μM 

induced testosterone and estradiol production as required (≥2-fold and ≥7.5-fold, respectively), 

and 1 μM prochloraz inhibited testosterone production by >50% as required; however, inhibition 

of estradiol production (40%) was below the required 50%. 

The intra- and inter-plate variability (%CV) was reported to be 0.5-24.4% for the testosterone 

and estradiol in the test runs, with the exception of 0.01 μM o-PP in Run 3 where the intraplate 

CV was 39.9%, due to increased responsiveness. 

Conflicting results were observed for testosterone production, with a statistically significant 30% 

increase at 10 μM o-PP and a 30% decrease at 100 μM o-PP. Statistically significant increases 

in estradiol production were observed at 10 μM o-PP (2.6-fold) and 100 μM o-PP (4.0-fold). 

These effects on estradiol were reproducible and concentration-dependent in all three runs. 

Based on the hormone responses in the three independent runs, o-PP treatment resulted in 

statistically significant and reproducible increases in estradiol and testosterone production; 

however, the alterations in testosterone production were inconsistent in direction of the response. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48672605) conducted to screen for potential estrogenic activity, 

ortho-phenylphenol (o-PP; 99.9% a.i., Lot # CHHYDD0051) in corn oil was administered daily 

via oral gavage to groups of six immature female Sprague Dawley rats at dose levels of 0 

(vehicle), 50, 250, or 1000 (limit dose) mg/kg/day on post-natal days (PND) 19-21. A positive 

control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 10 μg/kg/day by oral 

gavage. Body weights were determined daily. All animals were examined for precocious 

vaginal opening, and then terminated and necropsied on PND 22 approximately 24 hours after 

the final dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No treatment-related clinical findings or 

precocious vaginal opening were observed in o-PP dosed animals. Overall body weight gains 

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were decreased (p<0.05) by 25% in the 1000 mg/kg/day group. Body weights in the 50 and 250 

mg/kg/day dose groups and the positive control group were comparable to controls throughout 

the duration of the study. Uterine weights in the o-PP treatment 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 897% and 432%, respectively, as expected. 

No statistically significant changes were seen in uterine weight in this assay. o-PP is negative in 

the uterotrophic assay. 

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of o-PP with the endocrine system are summarized below. Given the 

breadth of toxicity data available on o-PP, the only Part 158 studies discussed in this appendix 

are those that fulfill current guideline requirements and are directly relevant to assessing 

potential endocrine effects (i.e., multiple-generation rodent reproductive studies, development 

studies, chronic toxicity studies, etc.). 

AR Transcriptional Activation (ARTA) 

In a published study by Orton et al. (2011), several chemicals, including o-PP (>97% purity) 

were tested for endocrine-disrupting effects in an in vitro ARTA (MDA-kb2) assay. The 

chemicals were dissolved in ethanol to prepare stock solutions (final ethanol concentration was 

not reported). The MDA-kb2 assay is conducted with human breast cancer cells stably 

transfected with a fire-fly luciferase reporter gene that is driven by androgen-response elementcontaining promoter. 

The MDA-kb2 cell concentration was 1×105

 cell/mL in phenol red-free Leibowitz-15 medium 

with 10% (charcoal-stripped) fetal calf serum. Cells were seeded and allowed to attach for 24 

hours, followed by exposure to eight serial dilutions of o-PP (in range of 0.01 to 100 μM) with or 

without 0.25 nM dihydrotestosterone (DHT) for an additional 24 hours. On each plate, the 

controls included media, ethanol, DHT co-exposure (0.25 nM), DHT serial dilutions (0.002-

10 nM), and flutamide (0.013-8 μM) or procymidone (0.005-3.2 μM) serial dilutions. All 

concentrations were tested in duplicate with two plates. 

In the MDA-kb2 cell assay, o-PP was negative as an androgen agonist, but an anti-androgen IC20

of 3.43 μM was determined for o-PP. o-PP was not cytotoxic at the tested concentrations (up to 

50 μM); therefore, the concentration required to yield cytotoxicity was greater than the 

concentration associated with anti-androgenicity. 

ERTA/ER Binding/Cell Proliferation 

In a published study, Blair et al. (2000) o-PP was tested in an ER competitive binding assay to 

determine ER affinity. The source of the ER for this assay was rat uterine cytosol. Chemicals 

were first screened at a high concentration and those that were positive for binding were further 

tested by using a range of concentrations to generate a binding curve and calculate the IC50 for 

each chemical. o-PP (99% purity) was reported to be negative for ER binding at a concentration 

of 1×10−4

 M. 

In a published study, Routledge and Sumpter (1997) evaluated 29 alkylphenolic compounds, 

including o-PP (99% purity) and other isomers of phenylphenol, for estrogenic activity with an 

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in vitro yeast assay. The yeast strain employed was stably transfected to express the human ERα

(hERα) gene together with expression plasmids (containing estrogen responsive elements and the 

lac-Z reporter gene encoding the enzyme β-galactosidase). Chlorophenol red-β-Dgalactopyranoside (CPRG) was used as the chromogenic substrate to measure receptor binding 

activity. o-PP, serially diluted in ethanol (final ethanol concentration was not reported), was 

evaluated at concentrations of 800 μM to 200 nM; solvent and 17β-estradiol controls were 

included in the assay. The estrogenic activity of the different isomers of phenylphenol (4-, 3-, 

and 2-phenylphenol) indicated that the most active derivative, 4-phenylphenol, was 20,000 times 

less potent than 17β-estradiol. The study authors reported that each successive movement of the 

phenyl group around the phenolic ring resulted in an approximately 7-fold reduction in activity. 

o-PP (2-phenylphenol) was the least active isomer and was estimated by the reviewers (based on 

4-phenylphenol and a 14-fold reduction in activity) to be 280,000 times less potent than 

17β-estradiol. 

In a published study, Rehmann et al. (1999) tested thirty compounds, including o-PP (purity not 

reported), for estrogenic activity with recombinant yeast cells expressing a fusion protein 

containing the hER binding domain and the GAL4 DNA binding domain to activate transcription 

of the β-galactosidase reporter gene. o-PP was tested at 10 nM, 10 μM, and 1 mM in three 

independent experiments and was reported to be negative for hER activation in this test system. 

In a published study, Miller et al. (2001) evaluated 73 phenolic additives, including o-PP (≥98% 

purity), for estrogenic activity with an in vitro yeast assay. The yeast strain employed was stably 

transfected to express the hERα gene. Chlorophenol red-β-D-galactopyranoside (CPRG) was 

used as the chromogenic substrate to measure receptor binding activity. Stock solutions of the 

test chemicals were serially diluted in ethanol for the assay (final ethanol concentration was not 

reported); 17β-estradiol and solvent controls were included with each assay. Each chemical was 

tested at least twice to ensure consistency of the data. The ED50 (effective dose) was calculated 

by using the optical density that gave a 50% response relative to the reference standards. The 

ED50 for 17β-estradiol was 2.0 × 10−10 M (±0.22 × 10−10 M; n=14). o-PP exhibited weak 

estrogenic activity (1/2,000,000 relative to 17β-estradiol) in the in vitro yeast assay. 

In a published study, Soto et al. (1997) developed a method to extract and separate 

xenoestrogens from ovarian estrogens in human serum, followed by determination of the 

cumulative xenoestrogen load (as estradiol equivalents) in the E-SCREEN bioassay. The 

chemicals tested included o-PP (purity not reported) and its meta- and para- isomers (4-, 3-, and 

2-phenylphenol). The test substances were initially identified and ranked by estrogenic effect 

using the E-SCREEN bioassay, after which those with more potent activity were used in 

development of the test method and in further testing for cumulative effects using the bioassay. 

In the E-SCREEN assay, cloned MCF-7 cells were plated with the experimental medium 

containing estradiol (E2) or a range of concentrations of the test compounds dissolved in ethanol 

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or DMSO. The assay was terminated 6 days after initiation (late exponential phase) by removing 

the medium from the wells and addition of a cell-lysing solution. Cell yield achieved at different 

concentrations of the test substances compared to the hormone free control was determined by 

counting cell nuclei. Estrogenic activity of the test chemicals was assessed by determining the 

relative proliferative potency (RPP) and the relative proliferative effect (RPE). The RPE 

indicated whether or not the test compound induced a proliferative response quantitatively 

similar to the one obtained with E2 (full agonist, RPE=100) or a proliferative yield lower than 

that obtained with E2 (partial agonist). In the initial E-SCREEN assay, o-PP was shown to have 

minimal estrogenic activity and was a partial agonist (RPE = 30% and RPP = 0.0001%) at a test 

concentration of 100 μM (the lowest concentration needed for maximal effect). 

In a published study, Itoh et al. (2000) investigated the estrogenic effects of sixteen chemicals, 

including o-PP (purity not reported), with the MVLN assay, which utilizes an MCF-7 derivative 

that has been stably transfected with the Vit-Luc reporter gene. The MVLN cell line expresses 

the endogenous ER of MCF-7 and contains an exogenous estrogen-responsive reporter 

(luciferase); therefore, the estrogen-specific transcription activity of a test chemical is directly 

related to the activity of luciferase measured in the lysate of treated MVLN cells. 

In the luciferase assay, MVLN cells were withdrawn from estrogen six days prior to passage and 

then plated in medium and fed treatment media containing the test chemical in ethanol (tested 

concentrations were not reported, solvent concentration <1%). Relative luciferase activity (%) 

was calculated against 17β-estradiol (1 nM) as the standard, and relative estrogenic potency was 

determined as the ratio between the minimal concentration of 17β-estradiol needed for a 

maximal response and the minimal dose of the test chemical needed to achieve a maximal 

response. The relative estrogenic activity of a test chemical in solution was estimated as the 

product of the relative estrogenic potency and the relative luciferase activity. In the MVLN 

assay, o-PP had a relative estrogenic potency of 0.0054 compared to 1 nM 17β-estradiol, and a 

relative luciferase activity of 23%. The relative estrogenic activity of o-PP was calculated to be 

0.00012. 

Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150) 

In a subchronic toxicity study in dogs (MRID 41656401), o-PP (99.77% purity) dissolved in 

peanut oil was administered by gastric intubation to Beagle dogs (2/sex/dose group) at doses of 

0, 100, 200, or 400 mg/kg/day for four weeks; the 400 mg/kg/day dose was reduced to 300 

mg/kg/day on Day 5 due to excessive emesis. Organ weights were determined for the kidneys 

and liver; histopathological examinations were conducted on these tissues as well as the ovaries, 

testes, pituitary, thyroid, adrenals, urinary bladder, epididymis, prostate, mammary gland, and 

cervix. 

Dose-related emesis was noted in all treated animals with the high-dose group exhibiting the 

most severity; occurrence of emesis by animal-days for males/females was 0/0, 2/3, 12/11 and 

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15/17 for the control, low- , mid- and high-dose groups, respectively. Body weight gains in all 

study animals were variable, but treated animals generally gained less (or lost) weight during the 

study period. No treatment-related differences were noted in food consumption. No treatmentrelated differences were noted in the ophthalmological examination, urinalysis, hematology, or 

clinical chemistry results. Finally, there were no treatment-related changes in the absolute or 

relative weights of the liver and kidneys, with no differences noted in gross or microscopic 

pathology. 

The LOAEL is 200 mg/kg/day based on increased emesis and reduced body weight gain, and the 

NOAEL is 100 mg/kg/day. 

Prenatal Development in Rodents (Rat; OSCPP 870.3700) 

In a developmental toxicity study in rats (MRID 00067616/00164362; MRID 92154037), o-PP 

(99.69% purity) was administered by gavage to mated SD rats (37/control group; 27/low- and 

medium-dose groups; and 26/high-dose group) at doses of 0 (cottonseed oil), 100, 300, or 700 

mg/kg/day on GDs 6-15, inclusive. The rats were sacrificed at GD 21 and the number and 

position of live, dead, and resorbed fetuses, and staining of apparent non-pregnant uteri, were 

determined along with liver weights. All fetuses were weighed, measured, sexed, and examined 

for gross external alterations and cleft palate. One-third of the fetuses in each litter were 

examined for visceral soft tissue alterations by dissection; heads were examined after sectioning. 

The remaining two-thirds of the fetuses in each litter were examined for skeletal alterations. 

There were no reported clinical observations related to treatment. Body weight gains during 

GDs 6-16 were decreased (NS) by 9 and 21% in the medium- and the high-dose groups, 

respectively, with overall body weight gain (GDs 6-21) decreases of 9 and 7%. Food 

consumption and food efficiency were slightly reduced in the medium- and high-dose groups 

during the dosing period. Decreased (NS) absolute and relative liver weights were noted in the 

high-dose group (↓8 and 6%, respectively). 

The maternal toxicity LOAEL is 300 mg/kg/day and the NOAEL is 100 mg/kg/day based on 

decreased body weight gain, food consumption, and food efficiency. There were no treatmentrelated changes in the number of corpora lutea, implantation sites, early or late resorptions, 

number of live fetuses/litter, fetal sex ratio, or developmental abnormalities/malformations at any 

dose. The developmental toxicity LOAEL is >700 mg/kg/day and the developmental toxicity 

NOAEL is ≥700 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study in rabbits (MRID 41925003), o-PP (99.88% purity) was 

administered to female New Zealand White rabbits (18/control group; 16/low- and medium-dose 

groups; and 24/high-dose group) by gavage at doses of 0 (corn oil), 25, 100 or 250 mg/kg/day 

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from GDs 7-19, inclusive. On GD 28, all surviving does were sacrificed, and subject to detailed 

necropsy. In addition, the number and position of fetuses, the number of live and dead fetuses, 

the number and position of resorption sites, and the number of corpora lutea in pregnant animals 

were recorded. All fetuses were sexed, weighed, and examined for gross external 

malformations. Each fetus was examined viscerally by fresh dissection and the brain examined 

via coronal section. All carcasses were eviscerated, cleared, and stained for skeletal 

examination. 

Ten deaths occurred during the study; seven animals died spontaneously or were sacrificed in a 

moribund condition, and three animals were sacrificed due to evidence of abortion. At least four 

of these deaths, in the high-dose group, were considered to be treatment-related and the abortion 

at the high dose also may have been related to treatment. A dose-related increase in the 

incidence of blood in the feces, urine, and/or cage pan was noted in the medium- and high-dose 

groups (3/medium and 6/high vs. l/control), and increased incidences of decreased feces and 

perineal soiling may also of have been related to treatment. There were no effects of treatment 

on body weight or body weight gain, or on absolute or relative kidney or liver weight. 

Treatment-related alterations in microscopic kidney structure (e.g., inflammation and tubular 

degeneration) were noted only in the high-dose animals. There appeared to be no treatmentrelated effects on the number of corpora lutea, implantation sites, live or dead fetuses, or early or 

late resorptions. Mean fetal weight values, fetal viability, and fetal sex ratio were similar 

between control and treated groups. There were no statistical or biologically-significant, 

treatment-related differences in the incidence of any fetal malformation or variation in any of the 

dose groups. 

The maternal LOAEL is 100 mg/kg/day, based on treatment-related microscopic kidney 

alterations, and the maternal NOAEL is 25 mg/kg/day. The LOAEL for developmental toxicity 

is not determined, and the NOAEL is ≥250 mg/kg/day. 

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800) 

In a two-generation reproduction study in rats (MRIDs 40976501 and 41729401), o-PP (99.86% 

and 99.47% purity; Weeks 0-38 and 38-study termination, respectively) was administered to CD 

SD rats (35/sex/dose group) in the diet at nominal doses of 0 (acetone and corn oil), 40, 140, and 

490 mg/kg/day (actual intakes 36, 125, and 457 mg/kg/day). The F0 parental animals were given 

the test diets 15 weeks prior to mating to produce the F1 offspring and continued throughout 

mating, gestation, and lactation, with further exposure of the offspring after weaning. The F1 

parental animals were not mated until 10 weeks after they were selected from the F1b litters 

(selected at weaning, 21 days of age). Treatment continued throughout mating, gestation, and 

lactation, with further exposure of the offspring after weaning. The estrous cycle was examined 

from vaginal smears taken for three weeks from F0 and F1 (10/dose) prior to mating. The 

estrous cycle was classified as leukocyte (diestrus), proestrus, proestrus/cornified, and cornified 

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(estrus). The organs weighed at sacrifice included the liver, kidneys, testes, and ovaries. Gross 

and microscopic pathological examinations were conducted on these organs as well as the 

pituitary, urinary bladder, epididymis, prostate, seminal vesicles, vagina, uterus, and cervix. 

There were no treatment-related clinical signs of toxicity in any of the treated groups, including 

adults and pups. There was no treatment-related increase in mortality; however, one animal in 

the medium-dose group probably died as a result of uremia or kidney failure that may be 

treatment-related. Body weights were decreased (p<0.05) during the pre-mating periods in highdose F0 males and females (↓7-8% and 6-7%, respectively) and the high-dose F1 males and 

females (↓11-12% and 7-12%, respectively). Decreased body weights were also reported for the 

high-dose groups (both F0 and F1) during the rest period between the F1a and F1b (↓8-12% and 

6-7% in males and females, respectively) and the F2a and F2b (↓12-13% and 9-10% in males 

and females, respectively) breedings. Although body weights were occasionally decreased 

(p<0.05) in females during the gestation and lactation periods, these decreases were generally 

<10% and body weight gains were not affected. Food consumption also was decreased 

(significance not reported) in the high-dose F0 and F1 groups during the F1a - F1b and F2a - F2b 

rest periods. 

No treatment-related effects were observed in the reproductive performances (male or female 

mating indices, male or female fertility indices, gestation index, gestation length, live birth index, 

viability index, lactation index, parturition, or fetal sex ratio). There was no treatment-related 

effect on estrus cycling measured in the F0 and F1 rats during the 14-day period prior to 

breeding, and no treatment-related changes in absolute or relative weights of the testes or ovaries 

were seen in the F0 and the F1 adults. Relative kidney weights were increased (p<0.05) in highdose F0 and F1 males, with no change in absolute weights. Other changes in kidney, liver, and 

gonad weights were not considered to be related to treatment (only kidney data was provided). 

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, vagina, cervix, and pituitary gland of the F0 and F1 adults. 

Increased incidences of renal calculi (p<0.05) in high-dose F0 males and in urinary bladder 

calculi (NS) in the medium- and high-dose F0 males and the high-dose F1 males were observed. 

In addition, there was an increased (p<0.05) incidence of transitional cell hyperplasia in the 

urinary bladder in high-dose F0 males and females and in high-dose F1 males. 

At the high-dose, body weights were decrease (p<0.05) by 13 and 18% on PNDs 14 and 21, 

respectively, for the F1b pups, by 6 and 12% on PNDs 14 and 21 for the F2a pups, and by 12% 

on PND 21 for the F2b pups. 

The parental NOAEL is 40 mg/kg/day (36 mg/kg/day actual), and the parental LOAEL is 

140 mg/kg/day [125 mg/kg/day actual; based on decreases in body weight, increases in relative 

kidney weights, in urinary bladder and kidney transitional cell hyperplasia, in renal hemorrhage, 

and in pyelonephritis starting at the high dose, and on an increase in urinary bladder calculi 

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starting at the medium dose (gross observations only)]. The reproductive NOAEL is 

490 mg/kg/day (457 mg/kg/day actual; HDT), and the LOAEL is not determined. The 

developmental NOAEL is 140 mg/kg/day, and the developmental LOAEL is 490 mg/kg/day 

based on decreased body weight in pups. 

In a subsequent two-generation reproduction study in rats (MRID 43928801), o-PP (≥99.5% 

purity) was administered continuously in the diet to SD rats (30/sex/dose group) at nominal 

doses of 0, 20, 100, or 500 mg/kg/day for two consecutive generations. Test material 

concentrations were adjusted weekly during the pre-mating period based on group mean body 

weight and food consumption for each dose group. During the gestation and lactation periods, 

the females from each dose group received the same dietary concentrations that were supplied 

during the last week of pre-mating. The actual doses during the lactation phase were 

approximately twice the doses received during the pre-mating and gestation phases because the 

dose was not adjusted and food consumption increased. The P generation animals were exposed 

to the test diets, beginning at approximately eight weeks of age, for ten weeks prior to mating to 

produce the F1a litter. After a two-week rest period, the P animals were mated again to produce 

the F1b litter. The F1b offspring (30/sex/dose group) selected to be parents of the next 

generation were fed the same test diet concentrations as their parents for twelve weeks prior to 

mating to produce the F2 litters. The organs weighed at sacrifice included the kidneys, testes, 

and ovaries. Gross and microscopic pathological examinations were conducted on these organs 

as well as the pituitary, urinary bladder, epididymis, coagulating gland, prostate, seminal 

vesicles, vagina, uterus, and cervix. 

One P generation high-dose male was found dead on Day 173 due to kidney failure; this death 

was attributed to treatment. Five other rats found dead and six euthanized in extremis were 

attributed to other non-treatment causes. Additionally at this dose, an increase in the incidence 

of urine staining was noted in the P and F1 generation males. This finding was associated with 

the gross and microscopic lesions of the urinary tract in these animals. 

In the high-dose P generation females, pre-mating body weights were decreased (p<0.01; ↓5-7%) 

on Days 21, 42, and 70. Additionally, overall body weight gains (Days 0-175, male or 0-70, 

female) were decreased (NS) in the P generation males and females (↓12 and 19%, respectively). 

In the F1 generation, body weights were decreased (p<0.01) throughout the entire pre-mating 

period in males and females (↓9-13%, males and ↓7-12%, females, respectively). Overall body 

weight gains also were decreased (NS) in the F1 generation males and females (↓10 and 8%, 

respectively). No differences in overall (GD 0-20) body weight gains were noted in either 

female generation during gestation. Body weights were decreased (p<0.01 or 0.05) in the highdose P and F1 generation females throughout the gestation periods for the F1a, F1b, F2a, and 

F2b litters (↓5-9%, except for GD6 during gestation of the F2b litter; ↓11%). These decreases 

were considered a continuation of the lower body weights observed during pre-mating in the P 

and F1 generation females. Body weights were decreased (p<0.01 or 0.05) in the P generation 

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high-dose females throughout lactation for both the F1a and F1b litters (↓6-9%). Additionally in 

the high-dose F1 generation females, body weights were decreased (p<0.01) during LD 0-14 of 

the F2a litter and decreased (p<0.05) during LD 0-7 of the F2b litter (↓8%). At this dose, 

absolute food consumption was increased during lactation for both litters in both generations. 

Treatment-related microscopic lesions noted in the urinary tract of the high-dose P generation 

males included: (i) simple hyperplasia, nodular/papillary hyperplasia, calculi, and chronic 

inflammation in the urinary bladder; (ii) chronic active inflammation and debris in the renal 

pelvis in the kidney; and (iii) dilation and hyperplasia in the ureter. Similar findings were 

observed in the high-dose F1 generation males. No organ weight changes due to treatment were 

observed. Gross necropsy findings were limited to an increased incidence of urinary bladder 

calculi in the high-dose P and F1 generation males. No treatment-related histopathological 

lesions were seen in the testes, epididymides, seminal vesicle, prostate, coagulating gland, 

ovaries, uterus, cervix, vagina, or pituitary glands of the P and F1 adults. In addition, no adverse 

effects of treatment were noted at the low or medium dose in either generation. 

The LOAEL for parental toxicity is 500 mg/kg/day based on decreased body weights, body 

weight gains, and effects on the urinary tract (bladder, kidneys and ureter) in males. The 

NOAEL for parental toxicity is 100 mg/kg/day. 

There were no compound-related effects on estrous cycle duration or the number of days until 

insemination in either generation. No treatment-related effects on the mating, fertility, or 

gestation indices were observed in either generation. There were no effects of treatment on 

gestation duration in either generation. The LOAEL for reproductive toxicity is not observed, 

and the NOAEL for reproductive toxicity is 500 mg/kg/day. 

No clinical signs of toxicity were observed in the pups of either generation from birth to PND 21. 

No treatment-related effects on the number of litters, number stillborn, litter size, sex ratio, or 

live birth, viability, or lactation indices were observed in either generation. There were no 

treatment-related gross lesions noted in the pups. At the high dose, overall (PND 1-21) pup body 

weight gains were decreased (NS) in both generations compared to control (↓12-14%). Body 

weights at this dose were decreased (p<0.01 or 0.05) in the F1 generation on PND 21 (↓10-12%) 

and in the F2 generation on PNDs 14 (↓6-7%) and 21 (↓11-12%). No treatment-related effects 

were noted at the low or medium dose in the pups. The LOAEL for offspring toxicity is 

500 mg/kg/day based on decreased pup body weights and body weight gains. The NOAEL for 

offspring toxicity is 100 mg/kg/day. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat; OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity study in rats (MRIDs 43954301, 44832201, 44852701), oPP (≥99.5% purity) was administered in the diet to CDF [F-344]/BR rats (50/sex/dose group) at 

nominal concentrations of 0, 800, 4000 and 8000 ppm in males and 0, 800, 4000, and 

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10,000 ppm in females (equivalent to 39, 200, and 402 mg/kg/day for males and 49, 248, and 

647 mg/kg/day for females) for two years. An interim-sacrifice group of twenty animals/sex for 

control and high-dose groups and ten animals/sex for the low- and medium-dose groups were 

sacrificed at twelve months. At study termination, surviving rats in all groups were sacrificed for 

gross and histopathological evaluation. The organs weighed at sacrifice included the adrenals, 

liver, kidneys, testes, ovaries, and thyroid. Gross and microscopic pathological examinations 

were conducted on these organs as well as the pituitary, urinary bladder, epididymis, prostate, 

seminal vesicles, mammary gland, uterus, and vagina. 

There was no treatment-related effect on mortality observed in this study. There was an increase 

in abnormal color urination and red staining in high-dose males, brown staining in medium- and 

high-dose females, and urine staining in high-dose males and all treated females. Body weights 

in the medium-dose animals were decreased (p<0.05) in males and females in the two-year 

oncogenicity group (no decrease in the one-year interim sacrifice group) at Weeks 13, 52, and 78 

(↓3-5%), with weights comparable to control at Week 104. Body weight gains in the mediumdose animals were generally >90% of control. Body weights in the high-dose animals were 

decreased (p<0.05) in males and females in the one-year interim sacrifice group (↓7-9%) and 

two-year oncogenicity group at Weeks 13, 52, 78, and 104 (↓9-15%). Body weight gains in the 

high-dose animals were decreased by 11-26%, compared to the control. Food consumption was 

generally decreased in the two-year carcinogenicity group at the high-dose (↓4-11%), and was 

decreased sporadically in the medium-dose animals (↓≤8%). 

Organ weight effects at study termination in high-dose males included decreased (p<0.05) 

absolute and relative liver weight (↓19 and 10%, respectively) and absolute kidney weight 

(↓13%), and increased (p<0.05) absolute and relative testes weights (↑34 and 46%, respectively). 

No related pathology was observed to account for organ weight differences, except for the 

kidneys. No treatment-related changes were seen in absolute or relative weights of the ovaries, 

thyroid, or the adrenal glands. 

Gross pathological observations at study termination included an increase in observed masses in 

the urinary bladder of high-dose males and an increased incidence of kidneys with pitted zones 

in high-dose females. Non-neoplastic effects observed included increased incidences of renal 

calculi in high-dose males at the 12-month sacrifice (kidneys) and the 24-month study 

termination (urinary bladder). Increased hyperplasia of the urinary bladder was present at the 

12- and 24-month sacrifices in high-dose males (and high-dose females at study termination), 

along with an increase in congestion, hemorrhage, mineralization, and necrosis of the urinary 

bladder at study termination in high-dose males. High-dose females had an increase in 

hyperplasia of the kidney, as well as increased infarct, acute inflammation and mineralization of 

the kidney. High-dose males and females also had an increase in cysts of the kidney at study 

termination. Relevant neoplastic observations were confined to the urinary bladder with an 

increased incidence of papilloma and transitional cell carcinomas in high-dose males noted at the 

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12-month sacrifice and the 24-month study termination in both the medium- and high-dose males 

(transitional cell carcinoma). These findings were not present in females. No treatment-related 

effects on histopathology were noted in the testes, epididymides, seminal vesicle, prostate, 

ovaries, uterus, vagina, mammary glands, thyroid, pituitary, or adrenal glands at any dose. 

The systemic toxicity LOAEL is 4000 ppm (200 mg/kg/day for males and 248 mg/kg/day for 

females) based on decreased body weight and body weight gain, decreased food consumption 

and food efficiency, and increased clinical and gross pathological signs of toxicity. The systemic 

toxicity NOAEL is 800 ppm (39 mg/kg/day for males and 49 mg/kg/day for females).

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a carcinogenicity study in mice (MRID 43545501), o-PP (99.88% purity) was administered to 

B6C3F1 albino mice (60/sex/dose group) in the diet at concentrations equivalent to doses of 0, 

250, 500, and 1000 mg/kg/day for 24 months. Interim sacrifices were conducted after twelve 

months of dosing with 10 mice/sex/dose group. At study termination, all surviving mice in all 

groups were sacrificed for gross and histopathological evaluation. The organs weighed at 

sacrifice included the adrenals, liver, kidneys, and testes. Gross and microscopic pathological 

examinations were conducted on these organs as well as the pituitary, thyroid, urinary bladder, 

epididymis, prostate, seminal vesicles, mammary gland, uterus, and vagina. 

There was no treatment-related effect on mortality in the interim-sacrifice or oncogenicity 

groups, and no clinical signs of toxicity. Decreased (p<0.05) body weights were observed in the 

high-dose males by Day 147 with decreases ranging from 3-9% between Days 147-287. Further 

body weight decreases (p<0.05) reached 10% in the high-dose males at Day 315, and ranged 

from 10-18% from Day 315-728 (study termination). Decreased (p<0.05) body weights were 

observed in the medium-dose males by Day 231 with decreases ranging from 4-8% between 

Days 231-728. Overall body weight gains were decreased by 16 and 27% in the medium- and 

high-dose males; no effects on body weight or body weight gain occurred in the low-dose males. 

Intermittent decreases (p<0.05) in body weights were noted in all dose groups in female mice 

through Day 91. Consistent body weights decreases (p<0.05) were observed in the high-dose 

females by Day 147 ranging from 6-8% between Days 147-231. Further body weight decreases 

(p<0.05) reached 11% in the high-dose females at Day 259, and ranged from 11-25% from Day 

259-728. Consistent body weights decreases (p<0.05) were observed in the medium-dose 

females by Day 343 with decreases ranging from 7-9% between Days 343-427; further body 

weight decreases (p<0.05) ranged from 10-18% from Day 455-728. Generally non-significant 

body weight decreases were present in the low-dose females (≤6%) between Days 119-623; 

greater decreases (p<0.05; ↓9-11%) were observed at Days 651-707, with a decrease in body 

weight (NS, ↓6%) at Day 728. Overall body weight gains were decreased by 10, 25, and 38% in 

the low-, medium- and high-dose females during the study. No consistent, or discernible, 

treatment-related effects on food consumption or food efficiency were observed during the study. 

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No consistent, treatment-related findings were observed in the hematology, clinical chemistry, or 

urinalysis findings. There were increases (p<0.05 or NS) in absolute and relative liver weights at 

the interim (12-month) and terminal (24-month) sacrifices in all treated males and females; 

increases were non-dose-dependent, except for interim-sacrifice females, and the increases were 

greater after the 12-month treatment compared to the 24-month treatment. Absolute adrenal 

weights were increased in the treated males, and decreased in treated females, in a non-dosedependent manner at the terminal sacrifice. Spleen weights (absolute and relative) in males and 

females were reduced in all treated groups (except low-dose males); increases in females were 

non-dose-dependent. No treatment-related changes were seen in absolute or relative weights of 

the testes at any dose. 

Non-neoplastic observations showed an accentuated lobular pattern of the liver of all treated 

animals. There was an increased incidence in hepatic tumors in high-dose males at the interim 

sacrifice, and in medium- and high-dose males at the terminal sacrifice in the form of 

hepatocellular adenomas; similar tumors with a lesser incidence were observed in the mediumand high-dose females at the terminal sacrifice. No treatment-related effects on histopathology 

were noted in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, mammary 

glands, thyroid, pituitary, or adrenal glands at any dose. 

A NOAEL was not observed. The systemic toxicity LOAEL is ≤250 mg/kg/day, based on 

increased liver weights, and decreased spleen weights, and gross observations in the livers of all 

treated animals.

Chronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.4100) 

In a chronic toxicity study in dogs (MRID 41656401; 1990), o-PP (99.77% purity) was 

administered via gastric intubation to Beagle dogs (2/sex/dose group) at doses of 0 (peanut oil), 

30, 100, or 300 mg/kg/day for one year. Organ weights determined included adrenals, kidneys, 

liver, ovaries, testes, pituitary, and thyroid; histopathological examinations were conducted on 

these tissues as well as the urinary bladder, epididymis, prostate, mammary gland, and cervix. 

Two dogs in the high-dose group died on study due to gavage error. Dose-related emesis was 

noted in all treated animals, with the high-dose group exhibiting the most severity (emesis was 

generally similar to control at the two lower doses). Body weight gains in the high-dose females 

were decreased (NS; ↓7-16%) at all time-points through study termination. Body weight gain 

decreases in low- and medium-dose females at Days 92 and 176 were not observed at study 

termination. Food consumption was slightly increased (NS; 8-13%) in the high-dose group, with 

a lower food efficiency in the high-dose females. No treatment-related differences were noted in 

the ophthalmological examination, urinalysis, hematology, or clinical chemistry results. There 

were no treatment-related changes in the absolute or relative weights of the adrenals, kidneys, 

liver, ovaries, testes, pituitary, and thyroid. There were also no treatment-related effects on gross 

or microscopic pathology, including in the testes, epididymides, prostate, ovaries, uterus, 

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mammary glands, thyroid, pituitary, or adrenal glands at any dose, and no evidence for 

oncogenicity due to o-PP treatment. 

The LOAEL is 300 mg/kg/day based on increased emesis, reduced body weight gain, and 

reduced food efficiency, and the NOAEL is 100 mg/kg/day. 

Developmental Toxicity (Rat) and Dominant Lethal (Mouse) Studies

In a published non-Guideline study, Kaneda et al. (1978) dosed pregnant Wistar rats (14-weeks 

of age) with o-PP (99.7%) at 0, 150, 300, 600, and 1200 mg/kg/day in distilled water with 5% 

gum arabic by gavage on GDs 6 through 15; dams were sacrificed on GD 20. The number of 

dams assigned to each dose group was not stated directly; apparently, the control, low-, and 

medium-low dose groups had twenty animals each, with eighteen animals in the medium-high 

dose group and eleven in the high-dose group. The uterine contents were examined for 

implantation sites and live or dead fetuses; live young were inspected externally and weighed 

individually. The pups were divided into two groups; half of the pups were fixed in 95% alcohol 

and prepared for staining with alizarin red S for skeletal examination and the remaining pups 

were fixed in Bouin's fluid for internal examination according to a modified method of Wilson. 

Ataxia was observed after dosing in dams administered o-PP at ≥300 mg/kg/day; the duration of 

ataxia was reported as lasting several hours with severity increasing in a dose-related manner. 

Ten of eleven dams died after 3-9 days of treatment in the high-dose group; no clinical signs of 

toxicity were noted at the low dose (150 mg/kg/day). Body weight gains on GDs 9, 12, 15, 

and 20 were decreased in the medium-low (p<0.05, 0.01, or 0.001; ↓17-28%) and medium-high 

(p<0.001; ↓46-62%) dose groups. Body weight gain also was decreased in the single surviving 

animal in the high-dose group (not statistically assessed). No treatment-related changes were 

seen in the number of implantation sites, resorption rates, or number of live fetuses/litter in the 

low and medium-low dose groups. Fetal resorptions were increased (p<0.01; 25.7% vs. 13.9% 

control) at the medium-high dose, and pup weights were decreased (p<0.01) by 6-8%. Fetal 

resorptions were further increased (not statistically assessed; 38.5%) in the one remaining highdose dam, with further decreases in pup weights. Soft tissue and skeletal examinations did not 

reveal any treatment-related effects. 

The same authors dosed male C3H mice (10-weeks of age; 15/dose group) with o-PP at 0, 100 or 

500 mg/kg/day in distilled water with 5% gum arabic for 5 consecutive days. Immediately after 

the administration, each male was mated with two untreated virgin females. After one week, the 

females were removed and replaced with two other untreated virgin females (this mating 

procedure was repeated for six weeks). Females were sacrificed on GD 12 or 13, and scored for 

the numbers of corpora lutea, implants, living embryos, and early or late embryonic deaths. The 

frequency of dominant-lethal mutations was calculated. No increase was seen in the frequency 

of induced dominant-lethal mutations at any dose or mating period. The mean numbers of 

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corpora lutea, implants, live embryos per pregnancy, and incidences of early embryonic deaths in 

the treated groups were not different from control. 

Freshwater Fish Reproduction (Fathead minnow) 

The 21-day short-term reproduction assay of 2-phenylphenol (o-PP) with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions (MRID 48135404). Adult 

fish, 6 spawning groups (1 male and 1 female in each group; ca. 6 months old), were exposed to 

2-phenylphenol (99.9% purity) at nominal concentrations of 0 (control), 1.0, 5.0, 50.0 and 500 

μg a.i./L; average (MEAN)-measured concentrations were <LOQ (not reported), <LOQ, 4.03, 

36, and 293 μg a.i./L. A positive control (17α ethynylestradiol; 10 ng/L) was also included. The 

test system was maintained at 24.7 to 25.5o

C and a pH of 7.50 to 7.92. 

The study consisted of a 21-day pre-exposure phase followed by a 21-day exposure phase. The 

same 6 spawning groups were used for each phase. In the pre-exposure phase, the study 

measured fecundity (as represented by the number of spawnings, number of eggs, and number of 

eggs per brood) and fertility (as represented by egg hatch) to establish fish with a record of 

reproductive success. Fish that did not have sufficient egg production or egg hatch during the 

pre-exposure phase were excluded from the exposure phase. This resulted in variable n sizes 

across the control and treatment groups (n size is 3, 4, 4, 4, 3, and 4 for the negative control, 

positive control, <LOQ, 4.03, 36, and 293 μg/L treatment groups, respectively). In the exposure 

phase, the study measured fecundity (same as above), fertility (same as above), plasma 

vitellogenin (VTG) and gonadosomatic index (GSI). 

 Mortality was observed in the study with the highest number of mortalities occurring in the 

negative control. All mortalities occurred during the pre-exposure phase. No significant effects 

were observed in fertility, plasma VTG or GSI for either the male or female fish. Mean number 

of eggs per female was 274 in the control, and 220, 268, 166, 94 and 15.5 in the positive control, 

low, mid-low, mid-high and highest treatment group, respectively. There was a significant 

decrease of 65.7% and 94.3% in measures of fecundity (as represented by the number of eggs per 

female) in the 36 μg/L (p = 0.0081) and 293 μg/L (p = 0.0013) treatment groups compared to the 

control. It is noted that there were only 3 breeding pair replicates available in the negative 

control during the exposure phase and variability in fecundity output was observed (total 

number of eggs per female in control was 328, 0 and 495 eggs/female). However, in the highest 

treatment (n=4 replicates), total number of eggs ranged from 0-22, and was 131, 151 and 0 in the 

three replicates in the mid-high treatment replicates (n=3). 

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APPENDIX 3: References Not Utilized in the o-Phenylphenol WoE Analysis 

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

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

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

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

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

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

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

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

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

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

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for O-Phenylphenol but were not utilized due to one or more of the following reasons: 

1) the article was not available in English; 2) the compound of interest was not used in the study; 

3) the test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

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

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

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Caspers, N. (2001) Life cycle test with water fleas-Daphnia Magna. Testing laboratory: Bayer 

AG, WG-UWS, Institute of Environmental Analysis Evaluation, D-51368 Leverkusen. Study 

number, 51 A/88. Study Sponsor: Bayer AG. (MRID 48135402) 

Caspers, N. (2001) Preventol 0 extra daphnia magna reproduction test. Testing laboratory: Bayer 

AG, WG-UWS, Institute of Environmental Analysis Evaluation, D-51368 Leverkusen. Study 

number, 1092 A/01 DL. Study Sponsor: Bayer AG. (MRID 48135403) 

Itoh, S., Ueda, H., Naasaka, T., Nakanishi, G., and Sumitomo, H. (2000) Evaluating variation of 

estrogenic effect by drinking water chlorination with the MVLN assay. Water Science and 

Technology 42 (Nos. 7-8): 61-69. 

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O-PHENYLPHENOL FINAL 

Page 56 of 56 

NTP Technical Report: Toxicology and carcinogenesis studies of orthophenylphenol (CAS No. 

90-43-7) alone and with 7,12-Dimethylbenz(a)anthracene (CAS No. 57-97-6) in Swiss CD-1 

mice (dermal studies). NTP TR 301 (1986). 

Soto, A., Fernandez, M., Luizzi, M., Oles Karasko, A., and Sonnenschein, C. (1997) Developing 

a marker of exposure to xenoestrogen mixtures in human serum. Environmental Health 

Perspectives 105(3): 647-654. 

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