### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, 

## D.C. 20460



### MEMORANDUM

Date: 6/29/2015 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### SUBJECT: o-phenylphenol: Data Evaluation Records (DERs) for EDSP Tier 1 Assays

PC Code: 064103


### Decision No.: 461010

, 464741 

### Petition No.: NA DP 

### Barcode: D398785, D401755 

### Registration No.: NA 

### Regulatory Action: NA 

### Risk Assessment Type: NA


### TXR No.: 0055304


### MRID No.: See Table 

### Case No.: NA 

### CAS No.: 90-43-7 

### 40CFR: NA



### FROM: Greg Akerman

, Ph.D.

## ~ 

# ,Af---

Immediate Office
Health E ffects Division (7509P)



### THROUGH: Jess Rowland

# ~-v-51--­ Deput y Director
Health Effects Division



### TO: Jolene Trujillo Biologist / Chemical Review Manager
Risk Management and Implementation Branch V Pesticide Re-evaluation Division (7505P)

I. 

### ACTION REQUESTED Ver

. Apr

. 20 I 0



### The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects
Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency ' s Test Order for o-phenylphenol : Test Order# CON064103-27

.

Page I of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for o-phenylphenol.

III. MRID Table

Chemical: o-phenylphenol PC Code: 064103 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48916901
890.1150 Androgen Receptor Binding (Rat Prostate) 48672603
890.1200 Aromatase Assay (Human Recombinant) 48672608
890.1250 Estrogen Receptor Binding 48672604
890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) NA

890.1350 Fish Short-Term Reproduction 48672606
890.1400 Hershberger (Rat) 48672602
890.1450 Female Pubertal (Rat) 48686601
890.1500 Male Pubertal (Rat) 48686602
890.1550 Steroidogenesis (Human Cell Line – H295R) 48672601
890.1600 Uterotrophic (Rat) 48672605 NA= Not Applicable (requirement satisfied by ot her scientifically relevant information (OSRI))Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 1 of 75

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 398784

OECD Data Point 231
EPA MRID 48916901 (revised report); 48672607 (original)

EPA Guideline 890.1100
Amphibian Metamorphosis Assay (Frog)

Test Material: ortho -Phenylphenol Purity (%): 99.9%

Common Name o Phenyl Phenol
Chemical Name IUPAC (1,1’-Biphenyl)-2-ol

CAS Name Not reported

CAS No. 90-43-7

Synonyms Preventol O Extra

EPA PC Code 064103

Primary Reviewer: John Marton Signature:

Staff Scientist, Cambridge Environmental, Inc. Date: 01/13/2013

Secondary Reviewer: Teri S. Myers Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 01/29/2013

Primary and Final Additional Reviewer: Robin Sternberg Signature :

USEPA/OCSPP/OPP/EFED/ERB1 Date: 02/22/2013, 05/27/2015

Additional Reviewer: Ed Scollon Signature: No longer with EPA

USEPA/OCSPP/OPP/EFED/ERB4 Date:

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126
Date: 2015.06.01 16:45:16 -04'00'Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 2 of 75

DER Template Version: 22 September 2011 CITATION: Lehman, C.M., K. Hutchinson, J.A. Fiting, and J. Thomas. 2011. Ortho -Phenylphenol: The Amphibian

Metamorphosis Assay Using the African Clawed Frog, Xenopus laevis . Unpublished study performed by

Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Laboratory report number 111018. Study sponsored by OPP EDSP Consortium, Dow Advanced Materials, The

Dow Chemical Company, Midland, Michigan, and LANXESS Corporation, Materials Protection Products,

Pittsburgh, Pennsylvania. Study completed October 14, 2011.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven
screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the
estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths

of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay,

where available, and redundancy across the battery. Thus, the results of each individual assay should not be

considered in isolation but rather should be considered in the context of other assays in the battery as well as
Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact
with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with
the findings in the OSRI, should be undertaken (refer to the WoE Document).

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

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

Guidelines, nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 3 of 75

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay 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.

The study met all validity and performance criteria with the exception that the coefficients of variation (CVs) for
the TWA-measured concentrations exceeded 20% (range: 42.7 to 72.2%). This deviation did not impact the

interpretation of the study.

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

Guideline 890.1100).Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 4 of 75

DER Template Version: 22 September 2011 Results Synopsis:

Test organism NF stage at test initiation: 51
Test organism total length at test initiation (optional): Not reported

Test type: Flow-through

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

Metamorphosis Assay (AMA) with Ortho-Phenylphenol (Non-Late Stage Tadpoles Only)

Treatment

(mg a.i./L)

[TWA-m easured] NF Developmental

Stage Hind Limb

Length 3 Asynchronous

Development Thyroid Gross

and

Histopathology

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

0.0175 No No No No No No No

0.0673 No No No No No No No

0.352 No No No No No No No

1.92 No No No Yes No No No

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

otherwise specified.

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

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

board-certified pathologist.

3 Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 5 of 75

DER Template Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in U.S. EPA (2009)

Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1100,
Amphibian Metamorphosis (Frog), EPA 740-C-09-002; OECD (2009),

OECD Guideline for the Testing of Chemicals, The Amphibian Metamorphosis

Assay, OECD 231 Adopted 7 September 2009. Supporting guidance

documents were: “Standard Guide for Conducting the Frog Embryo
Teratogenesis Assay- Xenopus” (ASTM, 2004); “Standard Guide for

Conducting Acute Toxicity Tests on Test Materials with Fishes,
Macroinvertebrates, and Amphibians” (ASTM, 2007); and, “Normal Table of

Xenopus laevis (Daudin)” (Nieuwkoop and Faber, 1994). The following

deviations from OCSPP and OECD guidelines were noted:

1. The coefficients of variation for the four treatment levels ranged from 42.7 to 72.2%, exceeding the

guideline performance criterion of ≤ 20%.

2. The storage conditions of the test material were not provided.

3. Details pertaining to parental frogs ( i.e. , acclimation conditions and period, feeding, health) were not

provided.

4. The reported water hardness (64-78 mg/L as CaCO 3 ) exceeded the recommended range of 40-48

mg/L as CaCO 3 .

These deviations do not impact the interpretation of the study.

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

statements were provided. This study was conducted in compliance with the

following GLP Standards: OECD Series on Principles of Good Laboratory

Practice and Compliance Monitoring, Number 1, OECD Principles on Good
Laboratory Practice (as revised in 1997) ENV/MC/CHEM(98)17; and, USData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 6 of 75

DER Template Version: 22 September 2011

EPA FIFRA GLPs Title 40 CFR, Part 160- Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA), Good Laboratory Practice Standards, Final Rule.

A. Test Material ortho -Phenylphenol

Description: White solid

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

vapor pressure of test compound, expiration date.

Lot No./Batch No. : CHHYDD0051 (Lot #)

Purity: 99.9%

Impurities: None reported

Stability of Compound: Time-weighted average concentrations yielded recoveries of 72 to 115% of

nominal with coefficients of variation of 42-72%. Test concentrations were

substantially decreased from Day 0 to Day 21, accounting for the large range

in recoveries. The range in measured concentrations were attributed to the

affinity of the test material to bind to glass tanks, food, tadpoles and excreted

organic matter

Storage Conditions of

Test Chemicals: Not reportedData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page7of75 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be

identified.Species scientific name: Xenopus laevisSpecies strain (if stated):Not reported1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP),Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page8of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?YesBreeding pairs, from which tadpoles wereobtained, were acquired from XenopusExpress (Brooksville, Florida). EPA recommends that larvae used in the assay be derived from in -house adults.Were parental acclimationconditions same as definitivetest?Not specifiedAcclimation period for parentalfrogs (if applicable):Not specifiedDetails on parental feeding:Not specifiedDetails on parental health:Not specifiedTable 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Not specified EPA and OECD recommend that the best 2 –Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page9of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns evaluated (ifapplicable):Not specified 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn

(i.e., the spawns are not co-mixed)Number of eggs sampled perspawn:NANF stage at test initiation51 EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 (≤ 17 days post-fertilization).Age at test initiation:~14 dayspost-fertilization (dpf)Mean total length at test initiation(if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?NoData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page10of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on larval selection:Not describedNo details were provided on selection ofindividual tadpoles used in the test.Adult males and females were injectedwith human chorionic gonadotropin theday before the desired breeding andplaced in 10-gallon glass aquaria. Adultswere removed after breeding and eggmasses were distributed over severalhatching tanks such that the embryoswere not overcrowded. Afterapproximately 4 days, tadpoles were freeswimming and were transferred to rearingvessels (10 tadpoles/L). Tadpoles werereared until NF stage 51.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page11of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLoading rate (rearing density):10 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static - renewal exposure systems.Type of food:Sera Micron EPA recommends Sera Micron ® throughout

pre - exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should

dem onstrate equal performance to Sera

Micron ® .Source of food:Sera North America,Inc., Montgomeryville,PennsylvaniaIodide concentration in diet (ifknown):50μg/gFrequency of feeding:2 times/day EPA recommends that feeding occur at least

twice per day.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page12of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime:5 days post hatch to NFstage 51: 30mg/animal/dayDuring exposure,increased from 30mg/animal/day (days0-4) to 80mg/animal/day (days15-21)Daysmg Seramicron/Tank0-4305-7408-105011-147015-2180 It is recommended that food rations during the

pre - exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study

Day 0 - 4) to 80 mg/larva/day in the last week of the test (Study Day 15 -21).C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page13of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem (if applicable):Continuous flow Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):32 ± 3 mL/minThis flow-rate provided a complete volumeturnover approximately every 2.7 hours.Day14 analytical recoveries had droppedsubstantially from day 7 values. Therefore, inan attempt to increase recovery rates, theflow ofstock solutions to the test chamberswas increased by ~70% on day 16. Recommended flow-through rate is 25 mL/min (complete volume replacement ca.
every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page14of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):For each treatment level,acontinuous-flow syringepump continuouslydelivered a concentratedtest substance stocksolution at a designatedrate from a stock vessel toa stainless steel mixingchamber where it mixedwith dilution water; no testsubstance stock wasadded for the control. Recommended toxicant mixing for

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

test syst em is recommended; 4) The recommended flow splitting accuracy is

within 10%.Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends

24 -hr renewal; renewal period is recommended not to exceed 72 hours.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page15of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration?None reported EPA recommends maintaining dissolved oxygen concentrations > 40% air saturation

(> 3.5 mg/L) . Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.Source of dilution water:Laboratory dilution waterwas Lake Huron watersupplied to The DowChemical Company by theCity of Midland WaterTreatment Plant. Thewater was obtained fromthe upper Saginaw Bay ofLake Huron offWhitestone Point and waslimed and flocculated withferric chloride. Prior to use EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV a nd tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test

guideline.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page16of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsin the laboratory, thewater was sand-filtered,pH-adjusted with gaseousCO2, carbon-filtered, andUV-irradiated.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes, all below levels ofconcernIodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 μ g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g. in Sera Micron).Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page17of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:Glass sealed togetherwith clear siliconeadhesive EPA and OECD recommend that

water - contact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:30 x 14.5 x 20 cmFill volume:~5.2 LAdditional details on exposuresystem:NAData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page18of75 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMean1MeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)64 78 71Weekly in the controland highesttreatment groupreplicates EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH7.07.67.3Weekly in allreplicate test vessels EPA recommends pH 7.5 ± 1,

inter -replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)7.1 8.8 8.2Weekly in allreplicate test vessels EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature(°C)21.3 22.822.4Weekly in allreplicate test vesselsand continuously inone control vessel EPA recommends temperature 22±1 o C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o C.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page19of75 DER Template Version: 22 September 2011ParameterMinimumMaximumMean1MeasurementIntervalGuideline RecommendationsIodideNotreported EPA recommends aquatic iodide range 0.5

– 10 μ g/L (supp lemental iodide should not

exceed 2 μ g/L).AmmoniaNot reported General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia, should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously. OECD recommends that DO and temperature be measured at least weekly and that pH and hardness be measured at least at theFluorideNot detectablePerchlorateNot detectableChlorateNot detectableAlkalinity (mg/L as CaCO3)Conductivity (μmhos/cm)361594822641181Weekly in the controland highesttreatment groupreplicatesData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page20of75 DER Template Version: 22 September 2011ParameterMinimumMaximumMean1MeasurementIntervalGuideline Recommendations beginning and end of the test.1Not reported by study author; reviewer calculatedD. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in therange-finder?No toxicity data wereavailable to Xenopusand the reportedsolubility was 700 mg/Lat 20°C.Amaximumconcentration of 25 mga.i./L was selected. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of to xicity (e.g., < 10% mortality), whichever concentration is lowest.Species: Xenopus laevisData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page21of75 DER Template Version: 22 September 2011Life stage:Not specifiedTest duration:4 daysAdditional details:A non-GLP, 96-hr,static renewal test wasconducted in generalaccordance with ASTMmethods. Nominalconcentrations were 0(negative control), 1.6,3.1, 6.3, 12.5, and 25mg a.i./L. After 96hours, completemortality was observedin the two highesttreatment groups,yielding an LC50of 8.87mg a.i./L.Dividing the LC50by 3 yieldedapproximately 3.0 mg a.i./L, which wasset as the highest concentration in thedefinitive AMA.Behavioral abnormalities ( e.g., laying onthe bottom, floating near the top) wereobserved in the 1.3 and 3.1 mg a.i./Ltreatment groups, an additional lowconcentrationwas included to ensure thatan adequate number of treatments did notexhibit overt toxicological effects.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page22of75 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Range-finder; 1/3 of theLC50 EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., < 10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of test concentrations:0.2 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:4 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page23of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAre nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Time-weighted averageLimit of quantification (LOQ):0.00514 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:Days 0, 7, 14, 17, and21 It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week

thereafter.Number of replicates in control:4 EPA recommends 4 replicates.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page24of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):NA EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80 (20/rep)Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006).Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page25of75 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs darkcool white fluorescent bulbs EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’s surface:0.759-0.893 kluxmeasured weekly EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NA4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page26of75 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Ortho-Phenylphenol.Treatment IDNominalConcentration(mg a.i./L)Time-WeightedAverageMeasuredConcentrationa(mg a.i./L)Mean CV1(%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNAPer the study report, thedecline inconcentrations waslikely due toincreasedbiomatter and enhancedbiodegradationthroughout the test. EPA and OECD recommend that test item concentrations be maintained at a coefficient of

variation (CV)≤ 20%.Treatment 10.0240.017542.7Treatment 20.120.067368.8Treatment 30.600.35272.2Treatment 43.0 1.92 57.1Abbreviations:CVCoefficient of variation.1As reported by study authorE. ObservationsBiological Endpoints:Day 7- NF stage, hind-limb length (HLL), snout-vent length (SVL), HLL normalized by SVL, wet weightData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page27of75 DER Template Version: 22 September 2011Day 21- NF stage, HLL, SVL, normalized HLL, wet weight, histopathologyDaily- mortality and sub-lethal effectsWere raw (individual) data provided?Yes EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-based lar val selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results

No mortality was observed in the control. Only a single mortality was observed in the 1.92 mg a.i./L
treatment group ( Table 9 ).

Table 9: Larval Mortality in Xenopus laevis

.

Treatment

(mg a.i./L)

[TWA-measured] Larval Mortality

Day 7 1 Day 21

n 2 Mortality # Mortality % n 2 Mortality # Mortality %

Negative Control 80 0 0 60 0 0

0.0175 80 0 0 60 0 0

0.0673 80 0 0 60 0 0

0.352 80 0 0 60 0 0

1.92 80 0 0 60 1 1.3

1 Sample size and mortality values at Day 7 prior to interim sacrifice.

2 Total number of tadpoles per replicateData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 On Day 7, NF stages ranged from 53 to 55 with the majority of tadpoles (63%) at NF stage 54 ( Table

10 ). No asynchronous development was observed.

Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment

(mg a.i./L)

[TWA-measured] Developmental Stage

Day 7 Day 21

n 1 Median

Stage 2 # Asynchronous n 1 Median

Stage 2 # Asynchronous

Negative Control 4 54.5 0 4 59.5 3 0

0.0175 4 54 0 4 59.5 0

0.0673 4 54 0 4 59 0

0.352 4 54 0 4 59.5 0

1.92 4 54 0 4 59 0

1 Number of replicate tanks

2 Not reported by study author unless indicated; reviewer-calculated

3 Reported as median stage 59 by study authorData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 Day 7 mean normalized HLL ranged from 0.13 to 0.14 across the treatment groups ( Table 11).

Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment

(mg a.i./L)

[TWA-measured] Hind Limb Length (HLL)

Day 7 Day 21 2

n 3 Mean 4

(mm) ±SD 4 HLL:

SVL 1 n 3 Mean 4

(mm) ±SD 4 HLL:

SVL 1

Negative Control 4 2.71 0.16 0.144 4 18.59 1.06 0.610

0.0175 4 2.53 0.25 0.135 4 18.84 0.85 0.622

0.0673 4 2.72 0.04 0.141 4 18.18 0.95 0.608

0.352 4 2.59 0.31 0.136 4 18.00 0.99 0.597

1.92 4 2.41 0.15 0.131 4 16.52 1.26 0.564

Abbreviations: SD Standard deviation.

1 Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).

2 Results based on non-late stage tadpoles (NF < 61) only.

3 Number of replicate tanks

4 Not reported by study author; reviewer-calculatedData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page31of75 DER Template Version: 22 September 2011Day 7 wet weight ranged from 0.563 g in the TWA 1.92 mg a.i./L group to 0.632 g in the negative control (Table 12). Day 21 mean wet weight rangedfrom 2.06 to 2.30 g (Table 12) across the treatment groups.Table 12: Larval Growth in Xenopus laevis.Treatment(mg a.i./L)[TWAmeasured]Snout-Vent Length (SVL)Body Weight1Day 7Day 212Day 7Day 212n3Mean(mm)±SDn3Mean(mm)±SDn3Mean(g)±SDn3Mean(g)±SDNegative Control419.21.12430.50.21340.6320.07742.300.1130.0175418.70.924430.30.28540.5940.06842.200.1030.0673419.20.360430.01.2940.6290.03542.170.1900.352419.10.516430.30.84240.6140.05042.300.1171.92418.40.262429.41.1640.5630.05042.060.231Abbreviations:SDStandard deviation.1Also referred to as “wet weight” in the test guideline.2Results based on non-late stage tadpoles (NF < 61) only.3number of replicate tanksData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 The study authors found no treatment-related histopathological changes in the thyroid gland in any of
the treatment groups ( Table 13 ). There was no evidence of any remarkable glandular atrophy or

hypertrophy of follicular cell hyperplasia in any of the thyroid glands examined across all treatment groups.
The incidence of tall columnar cells lining the follicles (follicular cell hypertrophy) did not show any

treatment-related differences and was interpreted to be within normal limits at all treatment concentrations.

Further, there was no evidence of any inflammatory or degenerative changes in the thyroid glands

examined from all groups including the control. All other histopathological criteria, such as the overall size

of the gland, the follicular lumen area, amount and type of colloid, and the follicular cell type and

arrangement, of all tadpoles exposed to increasing treatment concentrations were comparable to those

of the controls.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment

(mg a.i./L)

[TWAmeasured] Diagnostic Observations 1

Severity Thyroid Gland

Hypertrophy Thyroid Gland

Atrophy Follicular Cell

Hypertrophy Follicular Cell

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Negative Control 0 20 20 20 20 20 11 20 20

1 20 0 20 0 20 9 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0175 0 20 20 20 20 20 13 20 20

1 20 0 20 0 20 7 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0673 0 20 20 20 20 20 13 20 20

1 20 0 20 0 20 7 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.352 0 20 20 20 20 20 12 20 20

1 20 0 20 0 20 8 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1.92 0 20 20 20 20 20 9 20 20

1 20 0 20 0 20 11 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild,

2=Moderate, 3=Severe. See OECD No. 82 for reference.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page34of75 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.01750NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.06730NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.3520NANANANANANANANANANAData Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page35of75 DER Template Version: 22 September 2011Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA1.920NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANAAbbreviations:NANot applicable.1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page36of75 DER Template Version: 22 September 2011All control tadpoles appeared normal and healthy throughout the test (Table 15). One tadpole in the 1.92 mg a.i./L treatment group was observed restingon the bottom and one tadpole in the 0.0673 mg a.i./L treatment group was observed resting on the surface. No other effects were noted.Table 15: Clinical Signs in Xenopus laevis.Treatment(mg a.i./L)[measured]Clinical Signs1Typen2IncidenceNegative ControlNo effects80--0.0175No effects80--0.0673Resting on surface801 (1.3%)0.352No effects80--1.92Resting on bottom791 (1.3%)1Note that asynchronous development (unable to stage) is reported previously inTable.2Total number of tadpoles per replicateData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

The appropriate units of statistical analyses were the measures of central tendency from the replicate test

vessels. The statistical significance of all tests was judged at the 0.05 significance level, with the exception

of the Shapiro-Wilk test, which was judged at the 0.01 significance level.

For Day 7 data, statistical analyses of the continuous data set, including normalized HLL, wet weight,

and SVL were analyzed with the Jonckheere-Terpstra test in a step down manner when the data were

consistent with a monotonic dose-response. When these endpoints were non-monotonic, the data were

assessed for normality using the Shapiro-Wilk test and variance homogeneity using the Levene test. When

non-normality and variance heterogeneity was observed, normalizing and/or variance stabilizing

transformations were applied. When the data were normally distributed with homogenous variances, then

a significant treatment effect was determined using Dunnett’s test. When the data were normally

distributed with heterogeneous variance, the Mann-Whitney-Wilcoxon U test was used. When no

normalizing transformation was found, the Mann-Whitney-Wilcoxon U test using a Bonferroni-Holm

adjustment was used. HLL was normalized by SVL to account for the effects of growth. Mortality was

assessed at Day 21 using Yates’s chi-square pairwise test and Cochran-Armitage linear trend test. A

significant treatment effect for developmental stage was determined from the step-down application of

the multi-quantal Jonckheere-Terpstra test from the 20 th to the 80 th percentile.

In this study, ≥ 20% of the tadpoles had reached NF stage 60 in the 0.0673 and 0.352 mg a.i./L

treatment groups by Day 21. Therefore, to assess growth effects due to the chemical treatments while

taking into account the effect of late stage development on wet weight, SVL, and normalized HLL, these
data were analyzed using a two-factor ANOVA with a nested variance structure (tank nested within

treatment) for each of these three endpoints at Day 21. Data for tadpoles were first separated into two
groups according to NF stage (i.e., ≤ 60 “early” or ≥ 61 “late”. Then, the interaction between stage (either

late or early) and test concentration was tested to determine whether the increased developmental stage

influenced the endpoints. Furthermore, two additional ANOVA tests were conducted by “slicing” the

interaction term into the two developmental stage groups. These slices were not statistically significantData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 so comparisons of the least square means between the treatment groups and control within each “slice”

were made and a Bonferroni-Holm adjustment was applied.

Throughout the entire exposure period, there was a single mortality in the highest treatment group. Further,

only two tadpoles exhibited behavioral abnormalities, one in the TWA 1.92 mg a.i./L group and one in

the 0.0673 mg a.i./L group. On Day 7, there were no signs of either advanced or delayed development,

asynchronous development, or growth effects. Tadpoles in the TWA 1.92 mg a.i./L treatment group

exhibited delayed development on Day 21, though no other significant effects were observed and there

were no apparent treatment-related trends in histopathological findings.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Mortality did not exceed 1.3% in any treatment group and was therefore not

statistically analyzed. None of the Day 7 growth or development endpoints exhibited a monotonic trend
and all endpoints except developmental stage were normally distributed (Shapiro-Wilks, α = 0.01) with

equal variance (Levene’s, α = 0.01) and were therefore analyzed with Dunnett’s test. Day 7

developmental stage was analyzed using the Mann-Whitney test.

Late-stage tadpoles were present in each control and treatment replicate on Day 21 with the total number

of late-stage tadpoles representing 13, 15, 22, 25, and 12% of surviving tadpoles in the control and

0.0175, 0.0673, 0.352, and 1.92 mg a.i./L treatment groups, respectively. Therefore, separate analyses

were conducted for Day 21 late-stage and non-late stage tadpoles.

Non-Late Stage Tadpoles (NF < 61)

Day 7 developmental stage was not monotonic and did not meet the assumptions of parametric statistics

and was therefore analyzed using the Mann-Whitney test. Day 21 wet weight was not monotonic, but
was normally distributed with equal variance, and was therefore analyzed using Dunnett’s test. Day 21

SVL, HLL, and normalized HLL exhibited monotonic trends and were therefore analyzed using the

Jonckheere-Terpstra test.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 Late-Stage Tadpoles (NF ≥ 61)

Day 21 normalized HLL and growth endpoints were analyzed separately for late-stage tadpoles. All

endpoints did not exhibit a monotonic response, but met the assumptions of parametric statistics and were

therefore analyzed using Dunnett’s test.

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

statistically significant at p < 0.05.

Conclusions:

Day 7

There were no significant effects on any Day 7 development or growth endpoints in any treatment group

relative to the negative control.

Day 21- Non-Late-Stage Tadpoles

There were no significant effects on Day 21 developmental stage, wet weight, or SVL. Day 21 HLL was
significantly reduced at the highest dose relative to the negative control (11% reduction; Jonckheere, p = 0.006). Similarly, Day 21 normalized HLL was also significantly reduced at the highest dose (8%

reduction; Jonckheere, p = 0.036).

Day 21- Late-Stage Tadpoles

Among late-stage tadpoles, there were no significant effects on normalized HLL or growth endpoints.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page40of75 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Ortho-Phenylphenol (Non-Late Stage).Treatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54NA59.5NANANANANANANANANANo0.0175540.6259.51.0-4.090.792.010.81NANANANANo0.0673540.22590.620.181.0-0.290.67NANANANANo0.352540.6259.51.0-4.260.76-2.010.35NANANANANo1.92540.22590.18-7.280.35-7.540.04NANANANANoStatistical TestMann-WhitneyMann-WhitneyDunnett’sJonckheereNANANAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weightedby the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page41of75 DER Template Version: 22 September 2011Table 17: Growth Endpoints1,2in the AMA with Ortho-Phenylphenol (Non-Late Stage).Treatment(mg a.i./L)[TWA-measured]Snout-Vent LengthBody WeightDay 7Day 21Day 7Day 21% Diff. p% Diff. p% Diff. p% Diff. pNegative Control----------------0.0175-2.600.73-0.700.19-6.840.66-4.390.780.06730.001.0-1.760.33-0.471.0-5.740.600.352-0.391.0-0.840.32-2.610.980.081.01.92-4.300.34-3.760.06-10.840.30-10.30.16Statistical TestDunnett’sJonckheereDunnett’sDunnett’sAbbreviations:Diff.Difference.1Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.Data Evaluation Record on the Toxicity of ortho-Phenylphenol to Amphibians, Metamorphosis AssayEPA MRID Number 48916901 (revised report); 48672607 (original)Page42of75 DER Template Version: 22 September 2011Table 17: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Ortho-Phenylphenol (Late Stage).Treatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p% Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative ControlNANANANANANA----NANANANANo0.0175NANANANANANA-3.540.73NANANANANo0.0673NANANANANANA4.730.52NANANANANo0.352NANANANANANA-0.441.0NANANANANo1.92NANANANANANA6.510.32NANANANANoStatistical TestNANANADunnett’sNANANAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weightedby the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 Table 19: Growth Endpoints 1,2 in the AMA with Ortho-Phenylphenol (Late Stage).

Treatment

(mg a.i./L)

[TWA-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

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

Negative Control NA NA NA NA NA NA NA NA

0.0175 NA NA -1.45 0.97 NA NA -7.87 0.81

0.0673 NA NA -1.23 0.98 NA NA -4.90 0.95

0.352 NA NA 0.39 1.0 NA NA 0.69 1.0

1.92 NA NA -8.15 0.079 NA NA -26.97 0.052

Statistical Test NA Dunnett’s NA Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

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

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

E. Study Deficiencies

Although there were deviations from the guideline, as noted in the Section I. Materials and Methods section

of the DER, the study met all validity and performance criteria with the exception that the CVs for

time-weighted average, measured concentrations exceeded 20%. These deviations do not impact the

interpretation of the study.

F. Reviewer’s Comments

The reviewer’s results included both late-stage and non-late stage tadpoles, whereas the study authors

only analyzed non-late stage tadpoles. Therefore, the reviewer’s results are reported in the Executive

Summary of this DER.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 The in-life portion of the definitive toxicity test was conducted from April 19 to May 10, 2011

This study report was originally MRID 48672607; the revised report is MRID 48916901 and was
submitted to satisfy EFED’s request for replicate tank data to provide independent statistical verification

of the results.

III. REFERENCES

ASTM Standard E1439 (2004). Standard Guide for Conducting the Frog Embryo Teratogenesis Assay -
Xenopus (FETAX). ASTM International, West Conshohocken, Pennsylvania. DOI:

10.1520/E1439-98R04. www.astm.org.

ASTM Standard E729 (2007). Standard Guide for Conducting Acute Toxicity Tests on Test Materials with

Fishes, Macroinvertebrates, and Amphibians. ASTM International, West Conshohocken, Pennsylvania.

DOI: 10.1520/E0729-96R07 www.astm.org

.

Bentley, R.E. (1976). Acute toxicity of Dowicide CO antimicrobial to bluegill ( Lepomis macrochirus ) and

rainbow trout ( Salmo garidneria ). Bionomics EG&G Environmental Consultants, Wareham,

Massachusetts. Unpublished Report sponsored by The Dow Chemical Company, Midland, Michigan.

Birk, K.H., Dixon, T.S., Harfmann, R., and Johnson, R. (2011). Characterization of Preventol O Extra

( ortho -Phenylphenol) Lot # CHHYDD0051 for use as a Test Substance in a Toxicology Study. ML

AL-20110000666. Report of The Dow Chemical Company, Midland, Michigan.

Gonsior, S.J. and Tryska, T.J. (1997). Evaluation of the biodegradability of o -Phenylphenol. Report of

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

Gonsior, S.J., Rhinehart, W.L., Spence, M.W., and Bailey, R.E. (1984). Biodegradation of o -Phenylphenol

in river water and activated sludge. J. Agric. Food Chem. 32 (3), 593-596.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

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DER Template Version: 22 September 2011 Hollander, M. and Wolfe, D.A. (1973). Nonparametric Statistical Methods . John Wiley & Sons, New York.

Nieuwkoop, P.D. and Faber, J. (1994). Normal Table of Xenopus laevis (Daudin). Garland Publishing, New

York.

Organization for Economic Cooperation and Development OECD (2007). Guidance Document on Amphibian

Thyroid Histology. Environmental Health and Safety Publications. Series on Testing and Assessment. No.

82. Paris

Organization for Economic Cooperation and Development OECD (2009). OECD Guideline for the Testing

of Chemicals: The Amphibian Metamorphosis Assay. OECD 231 Adopted 7 September 2009.

United States Environmental Protection Agency (USEPA) (2009). Endocrine Disruptor Screening Program
Test Guidelines OPPTS 890.1100: Amphibian Metamorphosis (Frog). EPA 740-C-09-002, October

2009.Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
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DER Template Version: 22 September 2011 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Non-Late Stage Tadpoles Only (NF < 61)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.966 0.661 0.265 0.896 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.63 0.08 0.04 12.22 0.51, 0.75 Dose1 4 0.59 0.06 0.03 10.27 0.49, 0.68 Dose2 4 0.63 0.04 0.02 5.63 0.57, 0.69 Dose3 4 0.62 0.05 0.03 8.46 0.53, 0.70 Dose4 4 0.56 0.05 0.02 8.84 0.48, 0.64

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.63 0.54 0.73 . .
Dose1 0.60 0.51 0.65 93.16 6.84
Dose2 0.62 0.60 0.68 99.53 0.47
Dose3 0.60 0.58 0.69 97.39 2.61
Dose4 0.56 0.50 0.62 89.16 10.84

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.06 0.411

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.63 . 0.63 . . . . . .
Dose1 0.59 0.664 0.61 0.363 . . . . .
Dose2 0.63 1.000 0.61 0.388 0.849 . . . .
Dose3 0.62 0.981 0.61 0.402 0.960 0.997 . . .
Dose4 0.56 0.296 0.56 0.070 0.968 0.500 0.697 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-valueData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 47 of 75

DER Template Version: 22 September 2011 4 4.29 0.368

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.63 . .
Dose1 0.60 0.494 0.193
Dose2 0.62 0.678 0.385
Dose3 0.60 0.678 0.304
Dose4 0.56 0.235 0.052

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.06 0.411

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.63 . -0.61 . . . . . .
Dose1 -0.59 0.664 -0.61 0.820 . . . . .
Dose2 -0.63 1.000 -0.61 0.850 0.849 . . . .
Dose3 -0.62 0.981 -0.61 0.865 0.960 0.997 . . .
Dose4 -0.56 0.296 -0.61 0.874 0.968 0.500 0.697 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.29 0.368

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -0.63 . .
Dose1 -0.60 0.494 0.807
Dose2 -0.62 0.678 0.615
Dose3 -0.60 0.678 0.696
Dose4 -0.56 0.235 0.948

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 48 of 75

DER Template Version: 22 September 2011 test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.853 0.006 8.750 <.001 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.50 0.58 0.29 1.06 53.58, 55.42 Dose1 4 54.25 0.50 0.25 0.92 53.45, 55.05 Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 54.25 0.50 0.25 0.92 53.45, 55.05 Dose4 4 54.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 54.50 54.00 55.00 . .
Dose1 54.00 54.00 55.00 99.54 0.46
Dose2 54.00 54.00 54.00 99.08 0.92
Dose3 54.00 54.00 55.00 99.54 0.46
Dose4 54.00 54.00 54.00 99.08 0.92

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.05 0.415

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 54.50 . 54.50 . . . . . .
Dose1 54.25 0.800 54.25 0.239 . . . . .
Dose2 54.00 0.286 54.13 0.135 0.905 . . . .
Dose3 54.25 0.800 54.13 0.139 1.000 0.905 . . .
Dose4 54.00 0.286 54.00 0.067 0.905 1.000 0.905 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.16 0.385

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 49 of 75

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 54.50 . .
Dose1 54.00 0.624 0.247
Dose2 54.00 0.223 0.059
Dose3 54.00 0.624 0.159
Dose4 54.00 0.223 0.062

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.05 0.415

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -54.50 . -54.20 . . . . . .
Dose1 -54.25 0.800 -54.20 0.906 . . . . .
Dose2 -54.00 0.286 -54.20 0.927 0.905 . . . .
Dose3 -54.25 0.800 -54.20 0.937 1.000 0.905 . . .
Dose4 -54.00 0.286 -54.20 0.943 0.905 1.000 0.905 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.16 0.385

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -54.50 . .
Dose1 -54.00 0.624 0.753
Dose2 -54.00 0.223 0.941
Dose3 -54.00 0.624 0.841
Dose4 -54.00 0.223 0.938

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent length (mm) )Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 50 of 75

DER Template Version: 22 September 2011 TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.966 0.664 2.432 0.093 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 19.20 1.12 0.56 5.85 17.41, 20.99 Dose1 4 18.70 0.95 0.47 5.07 17.19, 20.21 Dose2 4 19.20 0.36 0.18 1.85 18.63, 19.77 Dose3 4 19.13 0.48 0.24 2.50 18.36, 19.89 Dose4 4 18.38 0.25 0.12 1.36 17.98, 18.77

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 19.15 17.90 20.60 . .
Dose1 18.55 17.80 19.90 97.40 2.60
Dose2 19.10 18.90 19.70 100.00 0.00
Dose3 19.00 18.70 19.80 99.61 0.39
Dose4 18.35 18.10 18.70 95.70 4.30

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.06 0.412

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 19.20 . 19.20 . . . . . .
Dose1 18.70 0.726 19.01 0.422 . . . . .
Dose2 19.20 1.000 19.01 0.450 0.858 . . . .
Dose3 19.13 1.000 19.01 0.466 0.915 1.000 . . .
Dose4 18.38 0.335 18.38 0.082 0.966 0.505 0.591 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.92 0.296

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-valueData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 51 of 75

DER Template Version: 22 September 2011 Ctrl 19.15 . .
Dose1 18.55 0.678 0.282
Dose2 19.10 1.000 0.558
Dose3 19.00 1.000 0.537
Dose4 18.35 0.346 0.103

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.06 0.412

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -19.20 . -18.92 . . . . . .
Dose1 -18.70 0.726 -18.92 0.790 . . . . .
Dose2 -19.20 1.000 -18.92 0.821 0.858 . . . .
Dose3 -19.13 1.000 -18.92 0.838 0.915 1.000 . . .
Dose4 -18.38 0.335 -18.92 0.848 0.966 0.505 0.591 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.92 0.296

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -19.15 . .
Dose1 -18.55 0.678 0.718
Dose2 -19.10 1.000 0.442
Dose3 -19.00 1.000 0.463
Dose4 -18.35 0.346 0.897

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 52 of 75

DER Template Version: 22 September 2011 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.955 0.458 3.128 0.047 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 2.71 0.16 0.08 5.98 2.45, 2.97 Dose1 4 2.53 0.25 0.12 9.69 2.14, 2.92 Dose2 4 2.72 0.04 0.02 1.59 2.65, 2.79 Dose3 4 2.59 0.31 0.15 11.87 2.10, 3.08 Dose4 4 2.41 0.15 0.08 6.35 2.16, 2.65

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 2.71 2.56 2.86 . .
Dose1 2.54 2.28 2.76 93.36 6.64
Dose2 2.71 2.68 2.78 100.37 -0.37
Dose3 2.50 2.34 3.02 95.57 4.43
Dose4 2.46 2.18 2.52 88.75 11.25

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.68 0.207

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 2.71 . 2.71 . . . . . .
Dose1 2.53 0.549 2.63 0.336 . . . . .
Dose2 2.72 1.000 2.63 0.359 0.682 . . . .
Dose3 2.59 0.818 2.59 0.275 0.993 0.890 . . .
Dose4 2.41 0.150 2.41 0.032 0.903 0.234 0.702 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 6.05 0.196

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 2.71 . .
Dose1 2.54 0.346 0.124
Dose2 2.71 1.000 0.413Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 53 of 75

DER Template Version: 22 September 2011 Dose3 2.50 0.678 0.272
Dose4 2.46 0.066 0.039

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4
Jonckheere Dose4

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.68 0.207

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -2.71 . -2.59 . . . . . .
Dose1 -2.53 0.549 -2.59 0.864 . . . . .
Dose2 -2.72 1.000 -2.59 0.890 0.682 . . . .
Dose3 -2.59 0.818 -2.59 0.903 0.993 0.890 . . .
Dose4 -2.41 0.150 -2.59 0.911 0.903 0.234 0.702 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 6.05 0.196

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -2.71 . .
Dose1 -2.54 0.346 0.876
Dose2 -2.71 1.000 0.587
Dose3 -2.50 0.678 0.728
Dose4 -2.46 0.066 0.961

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes ConclusionData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 54 of 75

DER Template Version: 22 September 2011 Test Stat P-value Test Stat P-value
0.933 0.179 2.443 0.092 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.14 0.01 0.00 5.67 0.13, 0.15 Dose1 4 0.14 0.01 0.01 8.83 0.12, 0.15 Dose2 4 0.14 0.00 0.00 1.00 0.14, 0.14 Dose3 4 0.13 0.01 0.01 9.34 0.11, 0.15 Dose4 4 0.13 0.01 0.00 5.40 0.12, 0.14

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.14 0.13 0.15 . .
Dose1 0.13 0.12 0.15 95.91 4.09
Dose2 0.14 0.14 0.14 100.18 -0.18
Dose3 0.13 0.12 0.15 95.74 4.26
Dose4 0.13 0.12 0.14 92.72 7.28

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 0.96 0.459

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.14 . 0.14 . . . . . .
Dose1 0.14 0.785 0.14 0.402 . . . . .
Dose2 0.14 1.000 0.14 0.429 0.881 . . . .
Dose3 0.13 0.761 0.13 0.242 1.000 0.865 . . .
Dose4 0.13 0.353 0.13 0.087 0.954 0.503 0.962 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.21 0.266

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.14 . .
Dose1 0.13 0.494 0.193
Dose2 0.14 0.889 0.615
Dose3 0.13 0.494 0.320
Dose4 0.13 0.235 0.072Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 55 of 75

DER Template Version: 22 September 2011 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 0.96 0.459

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.14 . -0.14 . . . . . .
Dose1 -0.14 0.785 -0.14 0.826 . . . . .
Dose2 -0.14 1.000 -0.14 0.855 0.881 . . . .
Dose3 -0.13 0.761 -0.14 0.870 1.000 0.865 . . .
Dose4 -0.13 0.353 -0.14 0.879 0.954 0.503 0.962 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.21 0.266

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -0.14 . .
Dose1 -0.13 0.494 0.807
Dose2 -0.14 0.889 0.385
Dose3 -0.13 0.494 0.680
Dose4 -0.13 0.235 0.928

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.857 0.007 0.750 0.573 USE NON-PARAMETRIC TESTSData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 56 of 75

DER Template Version: 22 September 2011 ********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose1 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose2 4 59.25 0.50 0.25 0.84 58.45, 60.05 Dose3 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose4 4 58.75 0.50 0.25 0.85 57.95, 59.55

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 59.50 59.00 60.00 . .
Dose1 59.50 59.00 60.00 100.00 0.00
Dose2 59.00 59.00 60.00 99.58 0.42
Dose3 59.50 59.00 60.00 100.00 0.00
Dose4 59.00 58.00 59.00 98.74 1.26

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.42 0.276

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 59.50 . 59.50 . . . . . .
Dose1 59.50 1.000 59.50 0.583 . . . . .
Dose2 59.25 0.914 59.38 0.475 0.965 . . . .
Dose3 59.50 1.000 59.38 0.491 1.000 0.965 . . .
Dose4 58.75 0.206 58.75 0.046 0.341 0.700 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.76 0.312

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 59.50 . .
Dose1 59.50 1.000 0.500
Dose2 59.00 0.624 0.246
Dose3 59.50 1.000 0.414
Dose4 59.00 0.177 0.056

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 57 of 75

DER Template Version: 22 September 2011 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.42 0.276

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -59.50 . -59.30 . . . . . .
Dose1 -59.50 1.000 -59.30 0.779 . . . . .
Dose2 -59.25 0.914 -59.30 0.811 0.965 . . . .
Dose3 -59.50 1.000 -59.30 0.828 1.000 0.965 . . .
Dose4 -58.75 0.206 -59.30 0.838 0.341 0.700 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.76 0.312

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -59.50 . .
Dose1 -59.50 1.000 0.500
Dose2 -59.00 0.624 0.754
Dose3 -59.50 1.000 0.586
Dose4 -59.00 0.177 0.944

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.959 0.517 1.541 0.241 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.IntervalData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 58 of 75

DER Template Version: 22 September 2011 Ctrl 4 2.30 0.11 0.06 4.89 2.12, 2.48 Dose1 4 2.20 0.10 0.05 4.52 2.04, 2.36 Dose2 4 2.17 0.19 0.09 8.76 1.87, 2.47 Dose3 4 2.30 0.12 0.06 5.08 2.11, 2.49 Dose4 4 2.06 0.23 0.12 11.21 1.70, 2.43

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 2.32 2.16 2.40 . .
Dose1 2.22 2.06 2.29 95.61 4.39
Dose2 2.17 1.99 2.34 94.26 5.74
Dose3 2.27 2.19 2.47 100.08 -0.08
Dose4 2.12 1.74 2.27 89.75 10.25

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.57 0.232

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 2.30 . 2.30 . . . . . .
Dose1 2.20 0.779 2.22 0.300 . . . . .
Dose2 2.17 0.599 2.22 0.321 0.999 . . . .
Dose3 2.30 1.000 2.22 0.332 0.886 0.755 . . .
Dose4 2.06 0.156 2.06 0.033 0.751 0.883 0.263 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.53 0.339

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 2.32 . .
Dose1 2.22 0.346 0.124
Dose2 2.17 0.346 0.121
Dose3 2.27 0.889 0.426
Dose4 2.12 0.156 0.103

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all testsData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 59 of 75

DER Template Version: 22 September 2011 Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.57 0.232

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -2.30 . -2.21 . . . . . .
Dose1 -2.20 0.779 -2.21 0.865 . . . . .
Dose2 -2.17 0.599 -2.21 0.891 0.999 . . . .
Dose3 -2.30 1.000 -2.21 0.904 0.886 0.755 . . .
Dose4 -2.06 0.156 -2.21 0.911 0.751 0.883 0.263 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.53 0.339

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -2.32 . .
Dose1 -2.22 0.346 0.876
Dose2 -2.17 0.346 0.879
Dose3 -2.27 0.889 0.574
Dose4 -2.12 0.156 0.897

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.971 0.781 4.785 0.011 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 30.54 0.21 0.11 0.70 30.20, 30.87 Dose1 4 30.32 0.28 0.14 0.94 29.87, 30.78 Dose2 4 30.00 1.29 0.65 4.32 27.94, 32.06Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 60 of 75

DER Template Version: 22 September 2011 Dose3 4 30.28 0.84 0.42 2.78 28.94, 31.62 Dose4 4 29.39 1.16 0.58 3.94 27.55, 31.23

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 30.61 30.22 30.69 . .
Dose1 30.27 30.04 30.71 99.30 0.70
Dose2 29.87 28.89 31.37 98.24 1.76
Dose3 30.14 29.45 31.39 99.16 0.84
Dose4 29.70 27.73 30.43 96.24 3.76

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.03 0.425

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 30.54 . 30.54 . . . . . .
Dose1 30.32 0.990 30.32 0.437 . . . . .
Dose2 30.00 0.800 30.14 0.340 0.984 . . . .
Dose3 30.28 0.981 30.14 0.352 1.000 0.990 . . .
Dose4 29.39 0.237 29.39 0.054 0.573 0.858 0.613 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.61 0.461

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 30.61 . .
Dose1 30.27 0.494 0.193
Dose2 29.87 1.000 0.330
Dose3 30.14 0.494 0.320
Dose4 29.70 0.103 0.063

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.03 0.425Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 61 of 75

DER Template Version: 22 September 2011 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -30.54 . -30.10 . . . . . .
Dose1 -30.32 0.990 -30.10 0.831 . . . . .
Dose2 -30.00 0.800 -30.10 0.860 0.984 . . . .
Dose3 -30.28 0.981 -30.10 0.874 1.000 0.990 . . .
Dose4 -29.39 0.237 -30.10 0.883 0.573 0.858 0.613 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.61 0.461

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -30.61 . .
Dose1 -30.27 0.494 0.807
Dose2 -29.87 1.000 0.670
Dose3 -30.14 0.494 0.680
Dose4 -29.70 0.103 0.937

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.945 0.303 0.139 0.965 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 18.59 1.06 0.53 5.72 16.89, 20.28 Dose1 4 18.84 0.85 0.42 4.49 17.49, 20.19 Dose2 4 18.18 0.95 0.48 5.22 16.67, 19.70 Dose3 4 18.00 0.99 0.49 5.49 16.43, 19.58 Dose4 4 16.52 1.26 0.63 7.62 14.51, 18.52Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 62 of 75

DER Template Version: 22 September 2011 Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 18.60 17.28 19.88 . .
Dose1 18.93 17.87 19.65 101.38 -1.38
Dose2 18.36 16.88 19.14 97.84 2.16
Dose3 17.98 16.95 19.12 96.87 3.13
Dose4 16.89 14.71 17.58 88.87 11.13

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 3.09 0.048

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 18.59 . 18.71 . . . . . .
Dose1 18.84 0.989 18.71 0.656 . . . . .
Dose2 18.18 0.948 18.18 0.376 0.892 . . . .
Dose3 18.00 0.839 18.00 0.289 0.778 0.999 . . .
Dose4 16.52 0.040 16.52 0.007 0.041 0.202 0.295 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.47 0.113

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 18.60 . .
Dose1 18.93 1.000 0.500
Dose2 18.36 0.494 0.190
Dose3 17.98 0.678 0.131
Dose4 16.89 0.103 0.006

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4
Jonckheere Dose4

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 3.09 0.048

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trendData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 63 of 75

DER Template Version: 22 September 2011 Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -18.59 . -18.03 . . . . . .
Dose1 -18.84 0.989 -18.03 0.850 . . . . .
Dose2 -18.18 0.948 -18.03 0.877 0.892 . . . .
Dose3 -18.00 0.839 -18.03 0.891 0.778 0.999 . . .
Dose4 -16.52 0.040 -18.03 0.899 0.041 0.202 0.295 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.47 0.113

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -18.60 . .
Dose1 -18.93 1.000 0.500
Dose2 -18.36 0.494 0.810
Dose3 -17.98 0.678 0.869
Dose4 -16.89 0.103 0.994

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.947 0.327 0.497 0.738 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.61 0.03 0.02 5.50 0.56, 0.66 Dose1 4 0.62 0.03 0.02 5.18 0.57, 0.67 Dose2 4 0.61 0.05 0.02 7.76 0.53, 0.68 Dose3 4 0.60 0.03 0.02 5.84 0.54, 0.65 Dose4 4 0.56 0.02 0.01 3.91 0.53, 0.60

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.61 0.57 0.65 . .
Dose1 0.63 0.58 0.65 102.01 -2.01Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 64 of 75

DER Template Version: 22 September 2011 Dose2 0.63 0.54 0.64 99.71 0.29
Dose3 0.59 0.56 0.64 97.99 2.01
Dose4 0.57 0.53 0.58 92.46 7.54

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.61 0.223

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.61 . 0.62 . . . . . .
Dose1 0.62 0.963 0.62 0.684 . . . . .
Dose2 0.61 1.000 0.61 0.587 0.978 . . . .
Dose3 0.60 0.963 0.60 0.413 0.855 0.992 . . .
Dose4 0.56 0.232 0.56 0.053 0.180 0.413 0.656 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.64 0.227

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.61 . .
Dose1 0.63 0.494 0.807
Dose2 0.63 0.889 0.670
Dose3 0.59 0.494 0.354
Dose4 0.57 0.156 0.036

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences)
Jonckheere Dose4

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.61 0.223

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-valuesData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 65 of 75

DER Template Version: 22 September 2011 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.61 . -0.60 . . . . . .
Dose1 -0.62 0.963 -0.60 0.735 . . . . .
Dose2 -0.61 1.000 -0.60 0.769 0.978 . . . .
Dose3 -0.60 0.963 -0.60 0.787 0.855 0.992 . . .
Dose4 -0.56 0.232 -0.60 0.798 0.180 0.413 0.656 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.64 0.227

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -0.61 . .
Dose1 -0.63 0.494 0.193
Dose2 -0.63 0.889 0.330
Dose3 -0.59 0.494 0.646
Dose4 -0.57 0.156 0.964

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

Day 21- Late Stage Only:

ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.857 0.007 0.750 0.573 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose1 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose2 4 59.25 0.50 0.25 0.84 58.45, 60.05 Dose3 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose4 4 58.75 0.50 0.25 0.85 57.95, 59.55

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 59.50 59.00 60.00 . .
Dose1 59.50 59.00 60.00 100.00 0.00
Dose2 59.00 59.00 60.00 99.58 0.42Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 66 of 75

DER Template Version: 22 September 2011 Dose3 59.50 59.00 60.00 100.00 0.00
Dose4 59.00 58.00 59.00 98.74 1.26

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.42 0.276

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 59.50 . 59.50 . . . . . .
Dose1 59.50 1.000 59.50 0.583 . . . . .
Dose2 59.25 0.914 59.38 0.475 0.965 . . . .
Dose3 59.50 1.000 59.38 0.491 1.000 0.965 . . .
Dose4 58.75 0.206 58.75 0.046 0.341 0.700 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.76 0.312

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 59.50 . .
Dose1 59.50 1.000 0.500
Dose2 59.00 0.624 0.246
Dose3 59.50 1.000 0.414
Dose4 59.00 0.177 0.056

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 15 1.42 0.276

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 67 of 75

DER Template Version: 22 September 2011 Ctrl -59.50 . -59.30 . . . . . .
Dose1 -59.50 1.000 -59.30 0.779 . . . . .
Dose2 -59.25 0.914 -59.30 0.811 0.965 . . . .
Dose3 -59.50 1.000 -59.30 0.828 1.000 0.965 . . .
Dose4 -58.75 0.206 -59.30 0.838 0.341 0.700 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.76 0.312

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -59.50 . .
Dose1 -59.50 1.000 0.500
Dose2 -59.00 0.624 0.754
Dose3 -59.50 1.000 0.586
Dose4 -59.00 0.177 0.944

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.960 0.581 2.054 0.142 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.59 0.12 0.06 7.70 1.39, 1.78 Dose1 4 1.46 0.31 0.16 21.21 0.97, 1.96 Dose2 4 1.51 0.09 0.05 6.27 1.36, 1.66 Dose3 4 1.60 0.24 0.12 15.23 1.21, 1.99 Dose4 3 1.16 0.16 0.09 13.57 0.77, 1.55

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.57 1.48 1.73 . .
Dose1 1.49 1.08 1.80 92.13 7.87
Dose2 1.51 1.42 1.60 95.10 4.90
Dose3 1.59 1.32 1.89 100.69 -0.69
Dose4 1.25 0.98 1.26 73.03 26.97Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 68 of 75

DER Template Version: 22 September 2011 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.48 0.092

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.59 . 1.59 . . . . . .
Dose1 1.46 0.807 1.52 0.396 . . . . .
Dose2 1.51 0.954 1.52 0.423 0.997 . . . .
Dose3 1.60 1.000 1.52 0.438 0.877 0.971 . . .
Dose4 1.16 0.052 1.16 0.010 0.343 0.222 0.087 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.01 0.136

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1.57 . .
Dose1 1.49 0.678 0.282
Dose2 1.51 0.346 0.190
Dose3 1.59 0.889 0.463
Dose4 1.25 0.100 0.050

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4
Jonckheere Dose4

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.48 0.092

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.59 . -1.48 . . . . . .
Dose1 -1.46 0.807 -1.48 0.844 . . . . .Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 69 of 75

DER Template Version: 22 September 2011 Dose2 -1.51 0.954 -1.48 0.871 0.997 . . . .
Dose3 -1.60 1.000 -1.48 0.886 0.877 0.971 . . .
Dose4 -1.16 0.052 -1.48 0.882 0.343 0.222 0.087 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.01 0.136

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1.57 . .
Dose1 -1.49 0.678 0.718
Dose2 -1.51 0.346 0.810
Dose3 -1.59 0.889 0.537
Dose4 -1.25 0.100 0.950

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.967 0.720 1.221 0.346 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 26.41 0.76 0.38 2.88 25.20, 27.62 Dose1 4 26.03 1.82 0.91 7.01 23.12, 28.93 Dose2 4 26.08 0.46 0.23 1.76 25.35, 26.81 Dose3 4 26.51 0.73 0.37 2.76 25.35, 27.67 Dose4 3 24.26 1.36 0.79 5.61 20.87, 27.64

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 26.24 25.70 27.45 . .
Dose1 25.75 24.10 28.50 98.55 1.45
Dose2 26.12 25.50 26.60 98.77 1.23
Dose3 26.45 25.88 27.28 100.39 -0.39
Dose4 24.35 22.85 25.57 91.85 8.15

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-testData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 70 of 75

DER Template Version: 22 September 2011 Numerator df Denominator df F-stat P-value
4 14 2.14 0.129

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 26.41 . 26.41 . . . . . .
Dose1 26.03 0.968 26.21 0.475 . . . . .
Dose2 26.08 0.982 26.21 0.506 1.000 . . . .
Dose3 26.51 1.000 26.21 0.523 0.970 0.982 . . .
Dose4 24.26 0.079 24.26 0.015 0.288 0.260 0.117 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 6.77 0.148

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 26.24 . .
Dose1 25.75 0.676 0.281
Dose2 26.12 0.678 0.385
Dose3 26.45 1.000 0.646
Dose4 24.35 0.100 0.111

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.14 0.129

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -26.41 . -25.94 . . . . . .
Dose1 -26.03 0.968 -25.94 0.801 . . . . .
Dose2 -26.08 0.982 -25.94 0.831 1.000 . . . .
Dose3 -26.51 1.000 -25.94 0.848 0.970 0.982 . . .
Dose4 -24.26 0.079 -25.94 0.846 0.288 0.260 0.117 . .Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 71 of 75

DER Template Version: 22 September 2011 ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 6.77 0.148

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -26.24 . .
Dose1 -25.75 0.676 0.719
Dose2 -26.12 0.678 0.615
Dose3 -26.45 1.000 0.354
Dose4 -24.35 0.100 0.889

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.965 0.681 2.204 0.121 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 23.95 0.90 0.45 3.74 22.53, 25.37 Dose1 4 22.75 0.53 0.27 2.33 21.91, 23.59 Dose2 4 24.82 1.22 0.61 4.91 22.88, 26.76 Dose3 4 23.92 0.97 0.48 4.05 22.38, 25.46 Dose4 3 23.43 0.42 0.24 1.79 22.39, 24.47

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 23.68 23.20 25.25 . .
Dose1 22.73 22.15 23.40 94.99 5.01
Dose2 24.81 23.46 26.20 103.63 -3.63
Dose3 23.86 22.95 25.00 99.86 0.14
Dose4 23.60 22.95 23.73 97.82 2.18

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.94 0.059Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 72 of 75

DER Template Version: 22 September 2011 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 23.95 . 23.95 . . . . . .
Dose1 22.75 0.215 23.83 0.500 . . . . .
Dose2 24.82 0.467 23.83 0.532 0.034 . . . .
Dose3 23.92 1.000 23.83 0.549 0.374 0.609 . . .
Dose4 23.43 0.855 23.43 0.305 0.848 0.286 0.947 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 8.90 0.064

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 23.68 . .
Dose1 22.73 0.103 0.022
Dose2 24.81 0.346 0.768
Dose3 23.86 0.889 0.712
Dose4 23.60 0.615 0.599

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.94 0.059

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -23.95 . -23.35 . . . . . .
Dose1 -22.75 0.215 -23.35 0.892 . . . . .
Dose2 -24.82 0.467 -24.11 0.503 0.034 . . . .
Dose3 -23.92 1.000 -24.11 0.520 0.374 0.609 . . .
Dose4 -23.43 0.855 -24.11 0.540 0.848 0.286 0.947 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all testsData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 73 of 75

DER Template Version: 22 September 2011 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 8.90 0.064

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -23.68 . .
Dose1 -22.73 0.103 0.978
Dose2 -24.81 0.346 0.232
Dose3 -23.86 0.889 0.288
Dose4 -23.60 0.615 0.401

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - OPP ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.961 0.588 2.506 0.089 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.91 0.01 0.01 1.58 0.89, 0.93 Dose1 4 0.88 0.05 0.03 6.13 0.79, 0.96 Dose2 4 0.95 0.05 0.02 4.82 0.88, 1.03 Dose3 4 0.91 0.06 0.03 6.84 0.81, 1.00 Dose4 3 0.97 0.04 0.02 3.94 0.87, 1.06

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.91 0.89 0.93 . .
Dose1 0.87 0.82 0.95 96.46 3.54
Dose2 0.96 0.90 1.00 104.73 -4.73
Dose3 0.91 0.84 0.97 99.56 0.44
Dose4 0.97 0.93 1.01 106.51 -6.51

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.32 0.108

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOECData Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 74 of 75

DER Template Version: 22 September 2011 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.91 . 0.92 . . . . . .
Dose1 0.88 0.732 0.92 0.714 . . . . .
Dose2 0.95 0.518 0.92 0.749 0.203 . . . .
Dose3 0.91 1.000 0.92 0.767 0.906 0.617 . . .
Dose4 0.97 0.318 0.92 0.770 0.127 0.990 0.418 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.19 0.126

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.91 . .
Dose1 0.87 0.346 0.124
Dose2 0.96 0.235 0.847
Dose3 0.91 1.000 0.680
Dose4 0.97 0.100 0.934

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
4 14 2.32 0.108

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.91 . -0.89 . . . . . .
Dose1 -0.88 0.732 -0.89 0.771 . . . . .
Dose2 -0.95 0.518 -0.93 0.358 0.203 . . . .
Dose3 -0.91 1.000 -0.93 0.371 0.906 0.617 . . .
Dose4 -0.97 0.318 -0.97 0.076 0.127 0.990 0.418 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 7.19 0.126Data Evaluation Record on the Toxicity of ortho -Phenylphenol to Amphibians, Metamorphosis Assay
EPA MRID Number 48916901 (revised report); 48672607 (original)

Page 75 of 75

DER Template Version: 22 September 2011 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -0.91 . .
Dose1 -0.87 0.346 0.876
Dose2 -0.96 0.235 0.153
Dose3 -0.91 1.000 0.320
Dose4 -0.97 0.100 0.066

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

# DATA EVALUATION RECORD

O-PHENYLPHENOL (OPP)

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

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672603)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 05/15/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/29/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/29/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).0-PHENYLPHENOL/ 064103 Androgen Receptor Binding (Rat Prostate Cytoso l) (2011) I Page I of 13 OCSPP 890.1150/ OECD None



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D.
Health Effects Division, Office of Pesticide Programs Secondary Reviewer: Greg Akerman, Ph.D.
Health Effects Division, Office of Pesticide Programs Signature: 

# f)**r6l~~d** Date:**6~ ~**  

### Signature:

# ~**~** Date:

____**6...........,!1~ ._,_l.,_** **-+ \**  _ _ _ 

# , 

# . :;. Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol) ; OCSPP 890.1150

PC CODE: 064103 DP BARCODE: D398785

TXR#: 0055304 CAS No.: 90-43-7

TEST MATERIAL (PURITY): Ortho-phenylphenol (o-phenylphenol ; 99

. 9%)

SYNONYMS: (1

, 1 ' -Biphenyl)-2-ol, OPP

, Prevento! 

### 0 Extra

CITATION: LeBaron

, M.J.

, Schisler

, M.R.

, and Visconti

, N.R. ( 2011)

. Evaluation of orthophenylphenol (OPP) in an i n vitro androgen receptor binding assay. Toxicology
& 

### Environmental Research and Consulting

, The Dow Chemical Company

, Midland

, MI. Laboratory Project ID: 111114

, December 19

, 2011. MRID
48672603. U npublished

.

SPONSOR: OPP EDSP Consortium

, The Dow Chemical Company

, Midland, MI and
LANXESS Corporation

, 111 RIDC Park West Drive

, Pittsburgh

, PA

TEST ORDER#: CON-064103-27

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48672603)

,
v entral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competiti v e 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

- 1 0



### to 10

- 3 

### M) of o-phenylphenol (OPP

, 99.9% purity, Lot # CHHYDD0051). Ethanol was used as a

v ehicle 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 R 1881 as the ligand reference standard.

Prior to conducting the AR competitive binding experiments

, a saturation binding experiment

wa s 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 (Bm ax) 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

v alidation 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. NonspecificAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### binding as a percent of total bi nding was less than 20% across the entire concentration range in the saturation binding assa ys (range 6.2-19.8%, with the excep tion of the high concentration (10 nM) in one assay, which was 24.6%).

In the competitive binding experiment, OPP reduced the mean specific binding of the radiolabeled ligand in a con centration-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%. Specifi c 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 [ 3 

### H]-R1881 binding was reduced to <50% at the highest concentrations tested in all three runs.

The estimated mean log IC 50 

### was

− 3.749 M for OPP compared to that of R1881 (

− 8.970 M) and the weak positive control of

− 4.418 M. The mean RBA was 0.0006% for OPP compared to 0.0028% for the positive control. Confidence in thes e numbers is high due to the small variation. No precipitation of the test compound was reporte d. The solvent control responses indicated no drift in the study assay. All pe rformance criteria were met.

Based on the results from the three runs, OPP is classified as a Binder in the Androgen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided. All phases of the study were conducted in compliance with FIFRA GLP Standards except that concentration checks for the positive control substances used in the study were not performed.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting

Location: The Dow Chemical Company, Midland, MI Study Director: M.R. Schisler
Other Personnel: M.J. LeBaron, Lead Scientist N.R. Visconti, Research Biologist B.B. Gollapudi, Technical Reviewer Study Period: July 27, 2011 to December 19, 2011



### 2. Test substance: O-phenylphenol (OPP)

Description: Solid
Source: Lanxess Deutschland GmbH , Leverkusen, Germany
Lot #: CHHYDD0051 Purity: 99.9% Solubility: Soluble in ethanol up to 30 mM; ca . 0.5-0.6 g/L in water at 20°C Volatility: ca . 0.0007 mbar (@ 20oC) Stability: 2 years shelf life Storage conditions: Ambient
CAS #: 90-43-7 Molecular weight: 170.2 g/mol
Structure: OH



### 3. Non-labeled ligand: R1881 Supplier: Perkin-Elmer, Boston, MA Catalog #:
Lot #: Not reported
614156 Purity: >97%
CAS #: 965-93-5



### 4. Radioactive ligand: [ 3 

### H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog and Lot #: NET590250UC (Lot # 614814) Date of production: July 1, 2010
Date of use: July 28, 2011 to August 4, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol
Concentration of stock: 1.0 mCi/mL



### 5. Positive control: Dexamethasone
Supplier: Sigma, St. Louis, MO Catalog #
Lot #: Not reported
BCBC9269 Purity: 98.9%
CAS # : 50-02-2



### 6. Solvent/vehicle control: Ethanol

Justification for choice of None providedAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

solvent:
Final Concentration: < 3%



### B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River Laboratories (Wilm ington, MA). Male Sprague Dawley rats (number not reported) were castrated at a pproximately 90 days of age and euthanized approximately 24 hours later. The ventral pros tate tissues were collected and stored at approximately

− 80°C until use, and were processed as a batch and used for multiple studies.

The cytosol was prepared by adding low-sa lt TEDG buffer [0.01 M Tris buffer, 10% glycerol, 1 mM sodium molybdate, 1.5 mM EDTA] and 1 mM phenylmethylsulfonyl fluoride (PMSF) with dithiothre itol (DTT)] at pH 7.4 to the ve ntral prostate tissues at 10 mL/g of tissue. The tissues were minced, hom ogenized on ice, and cen trifuged for 30 min at 30,000 × g at 4°C. The supernatant was collect ed, pooled from all tissues, aliquoted (amounts not reported) and stored at

− 80°C until used. Protein concentration of the cytosol prepared for this study was determined to be 6.566 mg/mL using th e Pierce BCA method (Thermo Scientific Pierce Research Lab, Rock ford, IL) compatible with DTT in the TEDG buffer.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-R1881 was performed to demonstrate that the AR was present in reasonabl e concentrations and had the appropriate affinity for the R1881 ligand. The conditions for the satura tion binding experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand (as serial dilutions) 0.25-10 nM Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM Optimization of receptor concentration Sufficient to bind 8.6-9.0% b of radioligand at 0.25 nM Temperature ~2-8 oC
Incubation time ~16 hours Composition of assay buffer (TEDG) Tris 10 nM (pH 7.4)

EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1.0 mM

DTT 1.0 mM a Data were obtained from page 9 of the study profile (MRID 48618303). b As recommended in the test guideline (le ss than 25-35% of the radiolabeled R1881).



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

### H]-R1881 was not adjusted for decay over time, and serial diluti ons in TEDG buffer were prepared to achieve the final concentrations in cytosol of 0.25, 0.5, 0.7, 1.0, 1.5, 2.5, 5.0, and 10.0 nM to determine total binding. To determine non-spec ific binding, solutions of non-labeled R1881Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### were prepared in a similar manner to achieve co ncentrations that were 100-fold greater than each respective radiolabeled concentration, result ing in final concentra tions in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, the radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

### H]-R1881 was measured. For each batch of cytosol, the optimal protein concentration was determined by calculating specific binding to differing amounts of protein per tube, using 0.25 nM radiolabeled R1881. The optimal protein co ncentration was determined to be 1.97 mg protein/assay tube, which resulted in the bi nding of 8.6-9.0% of th e total radioactivity added. As indicated in the guideline fo r acceptable assay performance the receptor concentration bound less than 25 to 35% of th e radiolabeled R1881. Cytosolic protein used in this assay was thawed fresh for this experi ment at ~4°C and maintained at ~4°C during the binding assay. Each run c ontained three concurrent repli cates at each concentration, resulting in the 72 samples depicted in Table 2.

Table 2. Saturation Binding Experiment Run a ,b
Total Binding Non-Specific Binding Radioligand alone
Tubes 1-24 c Tubes 25-48 d Tubes 49-72 e [ 3 H]-R1881 Final conc. (nM) [ 3 H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 H]-R1881 Initial conc. (nM) [ 3 H]-R1881 (μL)

0.25 0.25 25 10 7.5
0.50 0.50 50 10 15
0.70 0.70 70 10 21
1.0 1.0 100 10 30
1.5 1.5 150 10 45
2.5 2.5 250 100 7.5
5.0 5.0 500 100 15
10 10 1000 100 30 a Data were obtained from page 10 of the study profile (MRID 48618304). b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. Samples were dried, and 300 μl of prostate cytosol were added. d Tubes 25-48 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. R1881 was added in a 100-fold mola r excess of [ 3 H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 H]-R1881 or 7.5, 15, 21, or 30 μL of 100 nM [ 3 H]-R1881 without cytosol or other component s to determine the total counts added.



### Following addition of triamcinolone acetate, [ 3 

### H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated for approximately 16 hours at 2-8°C. Samples were maintained at temperatures of 4°C except during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, and the supernatant was aspirated and discarded. The samples were washed 3 times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, pellets were then extracted by addition of 2 ml ethanol. The samples were vortexed 3 times at 5 minute intervals. Samples were maintain ed on ice at all times between vortexing. Each ethanol supernatant was then decanted into a scintillation vial, and the radiation was quantified by liquid scintillation counting. A total of 4 runs were performed on 2 batches ofAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### cytosol with similar results. For the batch of cytosol used for the competitive assay, 2 runs were performed, which had highly similar bi nding profiles. Fina l determination of acceptable AR binding assay performance wa s primarily based on guideline suggested standards for the competitive binding assay, although the saturation binding parameters were evaluated.

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

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 4.3-5.1% of 1.0 nM radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 M Incubation Temperature 4-8 oC
Incubation time Overnight Composition of assay buffer Tris 0.01 M (pH 7.4)

EDTA 1.5 mM Glycerol 10% (v/v) Phenylmethylsulfonyl fluoride (PMSF) with DTT 1 mM

Sodium molybdate 1 mM Triamcinolone acetonide 60 μM a Data were obtained from pages 16-17 and 20-21 of the study report. b Data obtained from pages 39, 41 and 43 of the study report. Protein concentrations (μg/tube) were not reported.



### The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a singl e concentration of [ 3 

### H]-R1881 (specific activity of 85.1 Ci/mmol) to the AR in the presence of increasing concentrations of a test substance.

Ethanol was used as the solvent vehicle, and the solubility of the test material in the vehicle and assay buffer was evaluated visu ally. No precipitation was noted.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (ethanol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days. Each run contained duplicate blanks, and three replicates at each concentration of the solvent blank, NSB, reference standard, weak positive control, and test chemical resulting in a total of 77 samples per run. In addition, duplicate blanks followed by six replicates [ 3 

### H]- R1881 only (for total binding cal culations) were run the day before each analysis run (the day of preparation of sample tubes).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a b

Solvent Control Reference standard We ak positive control Test Chemical None Ethanol R1881 Dexamethasone OPP
Tubes 3-5 and 72-74 Tubes 6-23 and 75-77 c Tubes 24-47 Tubes 48-71 Tubes 1-2

-- 1×10

− 3 1×10

− 3
-- 1×10

− 4 1×10

− 4

-- 1×10

− 5 1×10

− 5

1×10

− 7 1×10

− 6 1×10

− 6

1×10

− 8 1×10

− 7 1×10

− 7

1×10

− 9 1×10

− 8 1×10

− 8

1×10

− 10 1×10

− 9 1×10

− 9

1×10

− 11 1×10

− 10 1×10

− 10 a Data were obtained from pages 18-20, and 39-44 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 77 tubes per run. Tubes 3-77 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 H]-R1881. Samples were dried, and 300 μL of prostate cytosolwere added. Tubes 3-77 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tube s 6-8 and 75-77 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Six tubes analyzed the day prio r to each run analysis contained only 30 μL of [ 3 H]-R1881 to determine ligand activity. c Tubes 6-8 and 75-77 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881.



### Sample tubes were stored overn ight at 4-8°C in the dark to allow the reaction to reach equilibrium, bound R1881 was separated from free R1881 by washing with buffer and extraction with ethanol, followed by scintillation counting of bound [ 3 

### H]-R1881.

4. Data Analysis: The top and bottom of the curve, Hill Slope and inhibition concentration (IC 50 

### ) were calculated with nonlin ear regression analysis by us ing Graph Pad Prism v.5.0 (GraphPad Software, Inc., La Jolla, CA) and means were calculated for each run and for the composite three runs.



### 5. Definitions

a. Classification of test material

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

Binder: The average curve for the test chemical across runs crosses 50% of radioligand bound, and the Hill Slope is between

− 0.6 to

− 1.4.

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

− 0.6 to

− 1.4).

Non-Binder: The average lowest portion of curves acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), or the data do not fit the model.

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

− 6 

### M and the binding curve does not cross 50%, the chemical is judged to be untestable.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### b. Descriptors for receptor binding

B max 

### : maximal binding capacity
K d 

### : dissociation constants IC 50 

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

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

### of R1881 ÷ IC 50 

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

### (IC 50 

### of R1881 ÷ IC 50 

### of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding data were not provided in the study report; however, a su mmary data for the two saturation binding runs were provided in the study profil e (MRID 48618303) and the information is provided in Table 5. The dissociation constant (K d 

### ) for [ 3 

### H]-R1881 was 0.4641, and the estimated B max 

### was 0.6392 nM for the batch of prostate cytosol used to evaluate competitive binding. The mean and individual K d 

### values were below the range reported in the EPA validation program (0.685 to 1.57 nM ). Confidence in these numbers is high according to the goodness of fit (R 2 

### = 0.9871-0.9937) and the small variation among the

two runs.

TABLE 5. Saturation Binding Experiment of [ 3 H]-R1881 with Androgen R eceptor from Ra t Prostate Cytosol a

Parameter Run 1 Run 2 Run 3 Mean b

R 2 (unweighted) 0.994 0.987 ND 0.987-0.994

B max (nM) 0.062 0.066 ND 0.064

B max (fmol/100 μ g protein) 3.146 3.343 ND 3.245

K d (nM) 0.436 0.492 ND 0.464 a Data were obtained from page 15 of the study profile (MRID 48618303). b The range of R 2 is reported and the mean is reported for the other parameters.
R 2 Goodness of fit for curve calculated for specific binding ND not determined



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

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

### H]-R1881 to the androgen receptor.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### FIGURE 1. Binding of [ 3 

### H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment.

FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-R1881 to the Androgen Receptor.

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters are presented in Table 6 and shown graphically in Figures 3 through 6.

OPP reduced the mean specific binding of the radiolabeled ligand in a concentrationdependent manner in all three runs. The maximum binding inhibition occurred at the highest concentration of OPP tested (10 − 3 

### M), with specific binding being reduced to 6.3-Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### 7.4%. Specific binding was also reduced at 10 − 4 

### M (62.8-68.8%), but not at OPP concentrations ≤ 10

− 5 

### M (92.8-103.7% binding).

The estimated mean log IC 50 

### was

− 3.749 M for OPP compared to that of R1881 of

− 8.970 M and the weak positive control of

− 4.418 M. The mean RBA was 0.0006% for OPP compared to 0.0028% for the weak positive cont rol. Confidence in these numbers is high due to the small variation. No precipitation of the test co mpound was reported. The solvent control responses indicated no drift in the study assay. Repeat runs were unnecessary.

TABLE 6. Competitive Binding Assay of O PP with AR from Rat Prostate Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

r 2 (unweighted) R1881 1.000 1.000 0.9999 0.9999-1.000 Dexamethasone 0.9995 0.9997 0.9995 0.9995-0.9997
OPP 0.9976 0.9986 0.9989 0.9976-0.9989 Log IC 50 (M) R1881 -8.956 -8.958 -8.994 -8.970 ± 0.012
Dexamethasone -4.416 -4.382 -4.455 -4.418 ± 0.021
OPP -3.701 -3.687 -3.860 -3.749 ± 0.055
IC 50 (M) R1881 1.106 × 10 -9 1.101 × 10 -9 1.014 × 10 -9 1.072 × 10 -9 (± 0.030)
Dexamethasone 3.838 × 10 -5 4.154 × 10 -5 3.504 × 10 -5 3.818 × 10 -5 (± 0.188)
OPP 1.992 × 10 -4 2.058 × 10 -4 1.381 × 10 -4 1.810 × 10 -4 (± 0.216) Log RBA b Dexamethasone -4.540 -4.577 -4.539 -4.552 ± 0.012
OPP -5.222 -5.301 -5.155 -5.226 ± 0.042 RBA (%) Dexamethasone 0.0029 0.0027 0.0029 0.0028 ± 0.0001
OPP 0.0006 0.0005 0.0007 0.0006 ± 0.0001 a Data were obtained from pages 32-33 of the study report. b The range of R 2 is reported and the mean ± SE is reported for the other parameters. Log RBA and SE values are reviewer-calculated, as well as mean va lues reported for OPP. Some reviewer-calculated means differed slightly from study author-calculated means reported on p. 32 of the study report. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. SE Standard Error NA Not applicable

r 2 Goodness of fit RBA (%) relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### FIGURE 3. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and OPP (Run 1).

FIGURE 4. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and OPP (Run 2).

FIGURE 5. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and OPP (Run 3).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### FIGURE 6. Mean Percentage R1881 Bound to the Androgen Receptor in the Presence of OPP in Three Competitive Binding Assays.

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

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

Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 H]- R1881 added per assay tube. ≤ 15% 4.3 to 5.1 X

Test chemical Top (% binding) 80 to 115 92.98 to 102.41 X
R1881 fitted curve parameters Top (% binding) 82 to 114 98.8 to 102.9 X Bottom (% binding)

− 2.0 to 2.0 -1.7 to 0.4 X Hill Slope

− 1.2 to

− 0.8 -1.020 to -0.9690 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 99.3 to 103.6 X Bottom (% binding)

− 12 to 12 -0.6 to 0.5 X Hill Slope

− 1.4 to

− 0.6 -0.9968 to -0.9603 X Saturation Binding Experiment K d (nM) 0.685 to 1.57 NR X Non-specific binding (%) ≤ 10.0 7.09 to 7.96 c X a Data were obtained from pages 32 and 39-44 of the study report. b These values represent ranges from the validation study. c Values reported for the three NSB tubes at the beginning of the run; does not includ e the three NSB tubes at the end of the run. NR Not reported



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

### H]-R1881 in a manner consistent with one-site binding, as indicated by a descent from ca . 90% to 10% binding over approximately an 81-fold increase in concentration of R1881 (i.e., covering approx imately 2 log units). Examination across theAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 13 O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None



### runs indicated consistency of the Hill slope , placement along the X-axis, and top and bottom plateaus.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the combined responses in each of three independent androgen receptor binding assays, it was determined that ortho-phenylphenol (OPP) treatment resulted in alterations in the binding of the referen ce radiolabeled androgen (R1881) at the highest two con centrations tested, includi ng the assay limit (1 mM, 10 -3 

### M), while no appreciable effect was seen at any other lower concentrations (10 -10 

### to 10 -3 

### M). When compared to the weak positive (dexamet hasone), OPP was less effective at displacing R1881 as determined by the lower relative binding affinity. The results of the in vitro androgen receptor binding assay using rat prosta te cytosol indicate that, under the conditions of this study, OPP was determined to be positive for androgen receptor binding at concentrations greater than 10 -4 

### M.

B. AGENCY COMMENTS: The saturation binding data were reported in a study profile submitted by the test order recipient. Only two saturation binding runs were conducted rather than the three runs recommended in th e test guideline. The dissociation constant (K d 

### ) for [3H]-R1881 was 0.4641, and the estimated B max 

### was 0.6392 nM for the batch of prostate cytosol used to evaluate competitive binding.

In the competitive binding experiment, OPP reduced the mean specific binding of the radiolabeled ligand in a con centration-dependent manner in all three runs. The maximum binding inhibition occurred at the highe st concentration of OPP 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 OPP concentrations ≤ 10

− 5 

### M (92.8-103.7% binding). OPP is classified as a “binder” in the AR assay as specific binding was reduced to <50% at the highest concentrations tested in all three runs.

The estimated mean log IC 50 

### was

− 3.749 M for OPP compared to that of R1881 (

− 8.970 M) and the weak positive control (

− 4.418 M). The mean RBA was 0.0006% for OPP 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 repor ted. The solvent control responses indicated no drift in the study assay. All performa nce criteria were met for the competitive binding experiment.

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

 Only two saturation binding runs were perfor med instead of three runs as recommended in the test guideline.

 The specific activity of the [ 3 

### H]-R1881 stock solution was no t checked prior to use on each assay day.

 The protein concentration ( μ g/tube) used in the assay was not reported.

# DATA EVALUATION RECORD

O-PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672608)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 4/30/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/14/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/29/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/29/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).0-PHENYLPHENOL I 064103 Aromatase (Human Recombinant) Assay (2012)/ Page I of 12 OCSPP 890.1200 I OECD None



### Primary Reviewer: Ayaad Assaad, D

. V.M.

, Ph.D.


### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division Signature:
Date: Signature:
Date:

DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890

. 1200 Template version 08/2011



### PC CODE: 064103 

### DP BARCODE: D398785



### TXR#: 0055304 

### CAS No.: 90-43- 7



### TEST MATERIAL (PURITY): 0-Phenylphenol (99.9%)



### SYNONYMS: Ortho-Phenylphenol; OPP; Preventol 0 Extra ; (1

, 1 ' -Biphenyl)-2-ol



### CITATION: Coady

, K.K.

, and Sosinski

, L.K. (2012) Evaluation of Ortho-Phenylphenol in
the Human Recombinant Aromatase Assay. Toxicology & 

### Environmental
Research and Consulting

, The Dow Chemical Company, Midland

, MI. Laboratory Project Study ID No.: 111007

, January 3

, 2012. MRID 48672608.
U npublished.



### SPONSORS: OPP EDSP Consortium, Dow Microbial Control, The Dow Chemical Company

, Midland

, MI

LANXESS Corporation

, Materials Protection Products

, 111 RIDC Park West Drive

, Pittsburgh

, PA



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In an 

### in vitro aromatase (CYP 19) assay (MRID 48672608)

, o-phenylphenol (OPP

, 99.9 % a.i.

, lot# CHHYDD0051) was incubated with human recombinant

aromatase and tritiated androstenedione**([1~- H(N)]-androst-4-ene-3** **17-dione;** 

### [3H]ASDN) in
1

% 

### ethanol for 15 minutes to assess the potential of OPP to inhibit aromatase activity

. OPP was tested at logarithmic concentrations from 10

- 1 0 M to 10

- 3 

### Min 3 independent runs.

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

- 1 0 to 1 

# o

- 5 ) 

### with a known inhibitor (4-hydroxyandrostenedione ; 4-0H ASDN)

, and the test chemical Series (1 o

- IO 

### to 1 o

- 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 2.3 8% to 7 .88% andAromatase (Human Recombinant) Assay (2012)/ Page 2 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### 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 curv e, bottom of the curv e, Hill slope, log IC 50 

### , and coefficient of variation for replicates of each c oncentration, 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 IC 50 

### for 4-OH ASDN averaged

− 7.1 M and the Hill slope was

− 1.17.

For OPP, 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 recommen ded limit of 15%. The data for OPP were modeled for all runs, and the goodness of fit (r 2 

### ) values were 0.87-0.98. The best fit Hill slope and log IC 50 

### values for OPP across the three independe nt runs did not diffe r significantly.

The lowest portion of the response curve wa s 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 IC 50 

### and Hill slope for OPP were

− 3.97 M and

− 0.99, respectively.

Based on the data from the average response curv e, OPP is classified as an Inhibitor of aromatase activity in this assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.Aromatase (Human Recombinant) Assay (2012)/ Page 3 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: O-phenylphenol (OPP) Description: No physical description provided Source: Lanxess Deutschland GmbH , Leverkusen, Germany Lot #: CHHYDD0051 (expiry date April 14, 2011) Purity: 99.9% Volatility: Not provided Storage conditions: Ambient Stability: Not provided Solvent: Ethanol Solubility (in test solvent): Up to 0.1 M Highest Concentration Tested: 1 × 10

− 3 M Stock Solution Preparation Methodology: Dissolved the test material in ethanol, then prepared serial dilutions for dosing Molecular weight: 170.2 CAS #: 90-43-7 Structure: OH



### 2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8 Source (catalog #): Steraloids Inc., Newport, RI (A6030-100) Lot/Batch # (expiration date): L1627 (expiration date not provided) Purity: 98.36%



### 3. Radiolabeled Substrate: 1- β [ 3 

### H(N)]-Androst-4-ene-3,17-dione; ([ 3 

### H]ASDN) Source (catalog #): Perkin Elmer Life and Analytical Sciences, Boston, MA Lot/Batch # (expiration date): 3615304 (expiration date not provided) Radiochemical Purity (Supplier): >97% Specific activity: 20.7 Ci/mmol Radiochemical Purity (In-lab determination): Not reported



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)

CAS #: 566-48-3 Source (catalog #): Sigma-Aldrich, St. Louis, MO (F2552) Lot/Batch # (expiration date): 081K2133 (expiration date not provided) Purity: 99.6%



### 5. Solvent (Vehicle Control): Ethanol Lot/Batch # (expiration date): 72596PMV (expiration date not provided) Justification for choice of solvent: Adequate solubility at the specified concentrations Concentration (% of total volume in assays): 1%



### 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes Source (catalog #): Gentest, Woburn, MA (456260) Lot/Batch # (expiration date): 03897 (expiration date not provided)
Protein concentration: Not provided Cytochrome C reductase activity: Not provided Aromatase activity: 6.0 pmol/pmol P450/minAromatase (Human Recombinant) Assay (2012)/ Page 4 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### B. METHODS

1. Assay Components and Preparations: A mixture of non-labe led and radiolabeled [ 3 

### H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 2 μM, and the amount of tritiu m added to each incubation tube yielded a predicted radioactivity of 1.0 μ Ci/mL.

OPP stock solutions were prepared such that the total volume of each OPP formulation used per assay was no more than 1% of the total assay volume to minimize the potential for the solvent to inhibit the enzyme. Selection of the solvent, ethanol, was based on the test chemical’s solubility.

A stock solution of the positive control substance, 4-OH ASDN, was formulated in ethanol. Fresh dilutions of the stock solution were prepar ed in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (10

− 5 

### to 10

− 10 

### M; Table 4) were achie ved by the addition of 20 μ L of the dilution for a fina l assay volume of 2 mL.

Human recombinant microsomes were purchas ed from Gentest (Woburn, MA), and stored at approximately –80oC for an unspecified ti me period. Microsomes were portioned into individual vials based on th e protein concentration of th e batch (0.004 mg/mL microsomal protein per tube) and stored at approximately –80oC for an unspecified time period.

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

TABLE 1. Assay Components and Conditions Assay Factor Values a 0.1M sodium phosphate buffer (pH 7.4)
Microsomal Protein 0.004 mg/mL
NADPH 6 mM [ 3 H]ASDN 2 μM Propylene Glycol 100 μL Temperature 37°C
Incubation Time 15 min a Data obtained from pages 11-14 of study report.



### 2. Suitability Assessments: It was stated that the protei n concentration was determined on each day the aromatase assay was run. The deta ils of the protein determinations were not reported, except that the analyses were c onducted in accordance with SOP No. TOX-BMT192.05 (Quantitation of Proteins). Microsomes were diluted w ith phosphate buffer such that approximately 0.004 mg/mL protein was present in the final reaction solution. Aromatase activity in each lot of human recombinant microsomes was determined to demonstrate the presence of sufficient activity for analysis of OPP. The minimum aromatase activity in the full activity controls was determ ined to be 0.080 nmol·mg-protein

− 1 

### ·min

− 1 

### , which is slightly below the minimum recommended aromatas e activity of 0.1 nmol·mg-protein

− 1 

### ·min

− 1 

### . The four full activity controls for the second run were <0.1 nmol·mg-protein

− 1 

### ·min

− 1 

### . However,Aromatase (Human Recombinant) Assay (2012)/ Page 5 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### eight other measurements reported were ≥ 0.10 nmol·mg-protein

− 1 

### ·min

− 1 

### , and the mean aromatase activity for the full activity controls was 0.124 nmol·mg-protein

− 1 

### ·min

− 1 

### ; therefore, sufficient aromatase activity was present.

3. Aromatase Assay: Each assay run contained 4 tube s for the full enzyme activity and background activity controls, respectively, and full concentration curves for the positive control (duplicate) and test substance (triplicate). The aromatase assay was conducted according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10), except that all assay tubes were centrifuged at 1000 rpm for 10 minutes for each of the 3 methylene chloride washes.

The amount of 3 

### H 2 

### O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol·mg-protein –1 

### ·min –1



### .
4. Demonstration of Proficiency:

a. Positive Control

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

 Mean aromatase activity in the absence of an inhibito r was at least 0.1 nmol·mgprotein

− 1 

### ·min

− 1



### .

 Mean background control activity was ≤ 10% of the full activity control.

 The percent CV between repl icates within each sample type and concentration was <15%.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data for slope, t op and bottom percent met the criteria as listed in section (i) of OCSPP 890.1200.

TABLE 2. Performance Criteria for the Positive Control a Parameter Lower Limit Criteria Upper Limit Crit eria Actual Lower Limit Actual Upper Limit Slope

− 1.2

− 0.8

− 1.2

− 1.1 Top (%) 90 110 92 104 Bottom (%)

− 5 +6 0.072 2.8 Log IC 50 (M)

− 7.3

− 7.0

− 7.2

− 7.0 a Data obtained from page 27 of study report.



### b. Proficiency Chemicals: Assays to classify proficiency chemicals (econazole, fenarimol, nitrofen, and atrazine) were not performed.Aromatase (Human Recombinant) Assay (2012)/ Page 6 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations

Econazole 24169-02-6 No data No data
Fenarimol 60168-88-9 No data No data
Nitrofen 1836-75-5 No data No data
Atrazine 1912-24-9 No data No data



### 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of a test ch emical per run, in triplicate, was tested during three independent runs.

No insolubility of OPP was reported at any concentration up to the highest (10 –3 

### M), and the lowest concentration tested was 10 –10 

### M. The full enzymatic activity was obtained at the two lowest concentrations of the test chemi cal to define the top of the concentrationresponse curve.

TABLE 4. Test Chemical Study Design for each Test Run a

Sample Type Repetitions (Tubes) Description Reference or Test Chemical (M) Full Activity Control 4 All test components b plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A
4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10

− 5

4-OH ASDN Conc 2 2 All test components plus 4-OH ASDN 1×10

− 6

4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10

− 6.5

4-OH ASDN Conc 4 2 All test components plus 4-OH ASDN 1×10

− 7

4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10

− 7.5

4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10

− 8

4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10

− 9

4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10

− 10

OPP Conc 1 c 3 All test components plus OPP 1×10 –3

OPP Conc 2 c 3 All test components plus OPP 1×10 –4

OPP Conc 3 c 3 All test components plus OPP 1×10 –5

OPP Conc 4 c 3 All test components plus OPP 1×10 –6

OPP Conc 5 c 3 All test components plus OPP 1×10 –7

OPP Conc 6 c 3 All test components plus OPP 1×10 –8

OPP Conc 7 c 3 All test components plus OPP 1×10 –9

OPP Conc 8 c 3 All test components plus OPP 1×10 –10 a Data were obtained from page 12 of the study report. b The complete assay contained buffer, pr opylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH. c Test chemical.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatase activity (nmol·mg-protein

− 1 

### ·min

− 1 

### ) and percent control for the positive control and OPP. The following raw data and calculated endpoints for each run were include d in the report (Table 5).Aromatase (Human Recombinant) Assay (2012)/ Page 7 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol·mg protein

− 1 ·min

− 1 X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations a Data were obtained from pages 43-45 of the study report.
DPM Disintegrations per minute



### 2. Statistical Methods: Statistical analyses and graphica l displays were conducted using Graph Pad Prism (Version 4.0, La Jolla, CA). Basic statistical analyses were performed on the data, which included means of replicates, standard deviation of the mean, relative standard deviation, and coefficient of variation. The Hill slope and log IC 50 

### values across three independent runs were compared base d on a one-way random effects analysis of variance, treating runs as random effects.

The response curve was fitted by nonlinear regressi on analysis. Model fits were carried out using a 4-parameter regression model. For each run, percent of full activity control were plotted versus logarithm (base 10) of the test chemical concen tration or 4-OH ASDN concentration. Each run was plot ted with the data’s best fit cu rve. Additionally, the average inhibition response curve across all runs was also plotted.

3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

TABLE 6. Interpret ation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

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

Data do not fit model --- a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

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

− 2.0, the result is classified as equivocal. b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically determined to be untestabl e in the aromatase assay.Aromatase (Human Recombinant) Assay (2012)/ Page 8 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.080 to 0.159 nmol·mg-protein

− 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 

### . The response of each full activity control was between 90 to 110% of the averag e full activity, with th e exception of the 3 rd 

### replicate in Run 2 (89.0%) and 3 rd 

### replicate in Run 3 (83.1%). Activity in the background controls ranged from 2.38% to 7.88% and aver aged 5.7% of the full control activity. The response of the full activity controls and background controls was acceptable.

B. POSITIVE CONTROL: 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·mg-protein -1 

### ·min

− 1 

### at the highest tested concentration (10

− 5 

### M). The mean aromatase activity of the pos itive control (expresse d as % full control activity) for each concentration tested across a ll three runs is presented in Table 7, along with the overall standard deviation, standard error of the mean, and %CV. Inhibition response curves for the positive control from each run and the average of all runs are shown in Figure 1. These results were generally with in the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 

### , and coefficient of varia tion for replicates of each concentration.

TABLE 7. Effect of OPP on Arom atase Activity (as percent of co ntrol) from Independent Runs a

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

4-OH ASDN (positive control) − 5 3 1.61 2.07 1.20 129.0

− 6 3 5.79 2.10 1.21 36.2

− 6.5 3 17.75 9.08 5.24 51.2

− 7 3 40.90 1.94 1.12 4.8

− 7.5 3 76.10 9.65 5.57 12.7

− 8 3 88.80 5.69 3.28 6.4

− 9 3 98.46 4.21 2.43 4.3

− 10 3 99.44 8.76 5.06 8.8

OPP

− 3 1 5.57 3.86 2.23 69.3

− 4 3 52.88 4.04 2.33 7.6

− 5 3 90.11 2.40 1.39 2.7

− 6 3 103.67 5.22 3.02 5.0

− 7 3 94.91 2.35 1.36 2.5

− 8 3 85.37 18.74 10.82 22.0

− 9 3 98.54 1.09 0.63 1.1

− 10 3 99.81 2.47 1.43 2.5 a Values were calculated by the reviewers based on data provided on pages 43-45. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of VarianceAromatase (Human Recombinant) Assay (2012)/ Page 9 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### FIGURE 1. Inhibition Response Curves for 4-OH ASDN.

C. TEST SUBSTANCE: For OPP, aromatase activity averaged 0.124±0.031 nmol·mgprotein

− 1 

### ·min

− 1 

### at the lowest tested concentration (10

− 10 

### M) and 0.007±0.013 nmol·mgprotein

− 1 

### ·min

− 1 

### at the highest tested concentration (10

− 3 

### ). The mean aromatase activity of OPP (expressed as % full control activity) for ea ch concentration tested across all three runs is presented in Table 7 (above ), along with the overall st andard deviation and %CV. Inhibition response curves for OPP from each r un are shown in Figure 2, and the average inhibition response curve across all runs is shown in Figure 3.Aromatase (Human Recombinant) Assay (2012)/ Page 10 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### FIGURE 2. Inhibition Response Curves for OPP From Each Test Run.

FIGURE3. Mean Inhibition Response Curve for OPP.Aromatase (Human Recombinant) A ssay (2012)/ Page 11 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### The effect of OPP on the inhi bition of aromatase activity is presented in Table 8. The estimated log IC 50 

### ranged from -4.0 to -3.9 log M fo r OPP and averaged -3.97 M compared to the positive control 4-OH ASDN, which aver aged -7.1 M. The average slope of the concentration response curve was -0.99 for O PP and -1.17 for 4-OH ASDN. Confidence in these numbers is high due to the generally small variation.

TABLE 8. Effect of OPP on Arom atase Activity (as Percent of Co ntrol) From Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SEM %CV Log IC 50 (M) o-phenylphenol -4.0 -3.9 -4.0 -3.97 0.03 1.46
4-OH ASDN -7.1 -7.0 -7.2 -7.1 0.06 1.41 Hill Slope o-phenylphenol -1.2 -0.81 -0.95 -0.99 0.11 20.0
4-OH ASDN -1.1 -1.2 -1.2 -1.17 0.03 4.95 a Data were provided on pages 27-28 of the study report. Me an, SD, and %CV were calculated by the reviewers based on these data. SEM Standard error of the mean CV Coefficient of Variance NA Not applicable. Values for OPP were not suitable for modeling.



### Based on the data from the average response curv e and the criteria listed above in Table 7, the results support the conclusion that OPP inhi bits aromatase activity under the conditions of this assay.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: The average response from three independent runs, of the human recombinant aromatase assay with OPP fit the four parameter regression model to yield an inhibition curve which resulted in greater than 50% inhibi tion at the highest concentration tested (10

− 3 

### M). Therefore, under the conditions of this study, OPP is classified as an inhibitor of aromatase activity.

B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 0.080 to 0.159 nmol·mg-protein

− 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 

### . The response of each full activity control was between 90 to 110% of the averag e full activity, with th e exception of the 3 rd 

### replicate in Run 2 (89.0%) and 3 rd 

### replicate in Run 3 (83.1%). Activity in the background controls ranged from 2.38% to 7.88% and aver aged 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·mg-protein -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 IC 50 

### , and coefficient of variation for replic ates of each con centration, with the exceptions of the overall %CV for the highe st three concentrations (51.2, 36.2, and 129%) exceeded the recommended limit of 15%.Aromatase (Human Recombinant) A ssay (2012)/ Page 12 of 12 O-PHENYLPHENOL / 064103 OCSPP 890.1200 / OECD None



### For OPP, 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 tested concentration (10

− 3 

### M). The overall % CV for the 10

− 3 

### and 10

− 8 

### M concentrations (69% and 22%, respectively) exceeded the recommende d limit of 15%. The data for OPP were modeled for all runs, and the goodness of fit (r 2 

### ) values were 0.87-0.98. For test material curves, the best fit Hill slope and log IC 50 

### values 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 IC 50 

### and Hill slope for OPP were

− 3.97 M and

− 0.99, respectively.

For 4-OH ASDN, the estimated log IC 50 

### averaged

− 7.1 M and the Hill slope was

− 1.17. Confidence in the mean log IC 50 

### and mean Hill slope for the positive control is high due to the small variation (1.41% and 4.95% CV, respectively).

Based on the data from the average response curv e, OPP is classified as an Inhibitor of aromatase activity in this assay.

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

 Proficiency data for the positive control and recommended proficiency chemicals were not provided.

 Information on the stability of the test subs tance in the stock solution was not reported.

# DATA EVALUATION RECORD

O-PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672604)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 05/18/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/29/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/29/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).0-PHENYLPHENOL/ 064103 Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) I Page 1 of 14 OCSPP 890.1250/ OECD None



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division

# Sign•~:::; 

# fd!J : 7



# i

Signature:

# ~ Date:

___**_o_e~/i'--'-1~ ::r-** **'-'-/~ C:--**  Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC) ; OCSPP 890.1250



### PC CODE: 064103 

### DP BARCODE: D398785



### TXR#: 0055304 

### CAS No.: 90-43-7



### TEST MATERIAL (PURITY): Ortho-phenylphenol (o-phenylphenol; 99.9%)



### SYNONYMS: (l

, l ' -Biphenyl)-2-ol

, OPP

, Prevento! 

### 0 Extra



### CIT A TI ON: LeBaron

, M.J.

, Schisler

, M.R., and Visconti, N.R. (2012). Evaluation of orthophenylphenol (OPP) in an 

### in vitro estrogen receptor binding assay. Toxicology 

### &
Environmental Research and Consulting

, The Dow Chemical Company

, Midland

, MI. Laboratory Project ID : 111120, January 4, 2012

. MRID 48672604. Unpublished.



### SPONSOR: OPP EDSP Consortium

, The Dow Chemical Company

, Midland

, MI and
LANXESS Corporation, 111 RIDC Park West Drive, Pittsburgh

, PA



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48672604) for o-phen y lphenol (OPP)

, uterine cytosol from Sprague Dawley rats was used as the source of ER
for a competitive binding experiment which measured the binding of a single concentration of**[3H]-17~-estradiol** 

### ( 1 nM) in the presence of increasing concentrations (logarithmic increase
from 10

- 1 0 

### to 10

- 3 

### M) of OPP (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-~-estradiol** 

### 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 (Bm ax ) of 59.28 fmol/l 00 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.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



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

### was

− 8.943 M for 17 β

estradiol 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. OPP 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 OPP bindi ng 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 IC 50 

### (n=2) was

− 3.192 M for OPP; an RBA value was not determined.

Based on the results from the three runs, OPP is cl assified as Equivocal in the Estrogen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided. All phases of the study were conducted in compliance with FIFRA GLP Standards except that concentrati on checks for the control substances used in the study were not performed.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 3 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting

Location: The Dow Chemical Company, Midland, MI Study Director: M.R. Schisler
Other Personnel: M.J. LeBaron, Lead Scientist N.R. Visconti, Research Biologist B.B. Gollapudi, Technical Reviewer Study Period: September 21, 2011 to January 4, 2012



### 2. Test substance: O-phenylphenol (OPP)
Description: Solid
Source: Lanxess Deutschland GmbH , Leverkusen, Germany
Lot/Batch #: CHHYDD0051 Purity: 99.9% Solubility: Soluble in ethanol up to 30 mM; ca . 0.5-0.6 g/L in water at 20°C Volatility: ca . 0.0007 mbar (at 20°C) Stability: 2 years shelf life Storage conditions: Ambient
CAS #: 90-43-7 Molecular weight: 170.2 g/mol
Structure: OH



### 3. Non-labeled ligand: 17 β -estradiol
Supplier: Sigma, St. Louis, MO Catalog #: E8875
Lot #: 098K1372 Purity: 100%
CAS #: 50-28-2



### 4. Radioactive ligand: [ 3 

### H]-17 β -estradiol
Supplier: Perkin-Elmer, Boston, MA Catalog #: NET517001MC
Lot#: 639068 Radiochemical purity: >97% Specific activity: 162.9 Ci/mmol
Concentration of stock: 1.0 mCi/mL



### 5. Positive control: 19-Norethindrone
Supplier: Sigma, St. Louis, MO Catalog #: N4128
Lot #: 030M1359 Purity: 99%
CAS #: 68-22-4Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 4 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### 6. Negative control: Octyltriethoxysilane
Supplier: Sigma, St. Louis, MO Catalog #: 440213
Lot #: 72596AMV Purity: 98.58%
CAS #: 2943-75-1



### 7. Solvent/vehicle control: Ethanol

Justification for choice of
solvent: Preferred solvent in guideline

Final Concentration: <3%



### B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Trimmed uterine tissue from 85- to 100-day old female Crl:CD(SD) rats (number unknown), which were ovariectomized approximately 7-10 days prior to being euthanized, was purch ased from Charles River (Wilmington, MA). The uteri were weighed, minced and homogeni zed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) buffer + PMSF (phenyl methylsulfonyl fluoride), then centrifuged for 10 min at 2500 × g at 4oC. Supernatant was transferre d and centrifuged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein concen tration of the cytosol was determined to be 3.573 mg/mL using a protein kit compatible w ith DTT in the TEDG buffer (Pierce BCA method; Thermo Scientific Pierce Research Lab). Cytosol was divided into aliquots (volume not reported) th at were stored at

− 80oC for up to 90 days until use.

2. Saturation (radioligand) Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-17 β -estradiol was reportedly performed to demonstrate that the ER was present in reason able concentrations and had the appropriate affinity for the native ligand. The conditions for the single run of the saturation binding experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat uterine cytosol Concentration of radioligand (as serial dilutions) 0.03-3.0 nM Concentration of non-labeled ligand (100X [radioligand]) 3.0-300 nM Concentration of receptor Sufficient to bind 40.77% of radioligand at 0.03 mM b Temperature ~2-8 oC
Incubation time ~16 hours Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM Glycerol 10%
PMSF 1 mM

DTT 1 mM a Data were obtained from page 9 of the study profile (MRID 48618304). b This value was slightly higher than the suggested range in the test gu ideline; however, all other values including ligand depletion met the performance criteria.



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

### H]-17 β -estradiol was not adjusted for decay over time. Serial dilutions in TEDG + PMSF buffer were prepared toEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 5 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of non-labeled 17 β -estradiol were prepared in a similar manner to achieve concentrations that were 100-fold greater than each respective radi olabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol. The optim al protein concentration was determined to be 0.1191 mg protein/assay tube, which result ed in the binding of 40.77% of the total radioactivity added. This value wa s slightly higher than the suggested range in the guideline, however all other values met th e performance criteria. Cytosoli c protein used in this assay was thawed fresh for this experiment at 4° C and maintained at ~ 4 oC during the binding assay. Each run contained three concurrent rep licates at each concentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a Total binding b Non-specific binding c Radioligand alone d Assay Components Tubes 1-24 Tubes 25-48 Tubes 49-72 350 μL 300 μL --- TEDG + PMSF buffer 50 μL 50 μL 50 μL [ 3 H]-17 β -estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) f 100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from page 10 of the study profile (MRID 48618304). b Total binding = [ 3 H]-17 β -estradiol bound to ER c Non-specific binding = [ 3 H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 H]-17 β -estradiol alone for dpm determ ination at each concentration e Final concentrations of [ 3 H]-17 β -estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. f Final concentrations of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### 3. Competitive Binding Experiment: A summary of the experiment conditions for the competitive binding experiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 6 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat Uterine Cytosol Concentration of radioligand 1 nM

Concentration of receptor Sufficient to bind 6.21-7.60% of radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 M Temperature 4-8  C
Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH not reported)

EDTA 1.5 mM Glycerol 10%
PMSF 1 mM

DTT 1 mM a Data were obtained from pages 17, 21-22, and 42-47 of the study report. b The protein concentration used was 1.191 mg/mL (p. 28).



### Solubility of OPP in ethanol and assay buffer was evaluated visually and no precipitation was noted. On the day of the assay, the speci fic activity of the stock solution [ 3 

### H]-17 β -estradiol was not adjusted for decay over time, and dilu ted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. Uterin e cytosol was used at a 1:2 dilution to achie ve a protein concentration of 1.191 mg/mL that bound 6.21-7.60% of the total radioactivity added. Serial dilutions of the test substance, positive control (19-norethindrone), negative control (octyltriethoxysilane), and reference material (non-labeled 17 β -estradiol) were prepared to achieve the concentrations shown in Table 4. Ea ch assay consisted of three independent runs on three different days. Each run included dupl icate blanks, and triplic ate sets of solvent blank and 0.1 μ M estradiol (non-specific binding, NSB) at the beginning and end of each run, along with triplicate tube s of the reference standard, the weak positive control, negative control, and OPP at each concentration, resul ting in a total of 104 samples per run. In addition, duplicate blanks followed by six replicates of [ 3 

### H]-17 β -estradiol only (for total binding calculations) were run th e day before each analysis run (the day of preparation of sample tubes).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 7 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

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

OPP Positive control Negative co ntrol Reference Chemical

19-Norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol
Tubes 75-98 c Tubes 27-50 c Tubes 51-74 c Tubes 6-26 and 102-104 c

10

− 10 10

− 8.5 10

− 10 Solvent control d

10

− 9 10

− 7.5 10

− 9 10

− 11

10

− 8 10

− 7 10

− 8 10

− 10

10

− 7 10

− 6.5 10

− 7 10

− 9.5

10

− 6 10

− 6 10

− 6 10

− 9

10

− 5 10

− 5.5 10

− 5 10

− 8.5

10

− 4 10

− 4.5 10

− 4 10

− 8

10

− 3 10

− 4 10

− 3 10

− 7 a Data were obtained from pages 35-36 and 42-47 of the study report. b Each tube contains: 10μL of either the test substance, positiv e control, negative contro l, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG + PMSF buffer with [ 3 H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), fo r a total of 500 μL. c Each concentration of each chemical was run in triplicate, for a total of 104 tubes per run. d Solvent is ethanol in Tubes 3-5 and 99-101.



### Tubes were gently vortexed and incubated for 16-20 hours at 4-8  C. To separate bound from free estradiol, hydroxyapatite (HAP) slu rry was added to each tube and vortexed (4 times at 5 minute intervals). Subsequently, the contents of each tube were washed three times as follows: TEDG buffer (+PMSF) was added, vortexed, centrifuged for 10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in each tube to extract the bound [ 3 

### H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 min at 1000 × g . Aliquots of supernatant we re radioassayed by liquid scintillation counting. The temperature was main tained at 4-8 oC thr oughout the assay prior to extraction with ethanol.

C. DATA ANALYSIS: For the competitive binding assay, GraphPad Prism v. 5.0 (GraphPad Software, Inc., La Jolla , CA) was used to c onduct non-linear regression analyses and to fit a curve (for 17 β -estradiol, the positive control, and th e test substance) to the Hill equation formula which incorporated log IC 50 

### as a parameter to be estimated. For parameters reported from the c 

### ompetitive binding experiment (log IC 50 

### and %RBA), m eans were calculated for each run and for the composite three runs.



### 1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually classified as follows:

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

Not interactive = there are usable data points at or above 10 -6 

### M and either the lowest point on the fitted response curve within the range of the da ta is above 75% (i.e., <25% of the radiolabeled estradiol has been displaced from the ER) or aEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 8 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Equivocal up to the limit of concentrations tested = there are no data points at or above a test chemical concentration of 10

− 6 

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

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

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

Run Classification Numerical Value Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of c oncentrations tested “missing”



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

Test Material Classifi cation Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of c oncentrations tested “missing”



### b. Descriptors for receptor binding:

B max 

### : maximum specific binding number (fmol ER/1 00 μg cytosol protein) measures the concentration of active receptor sites
K d 

### : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

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

### of 17 β -estradiol ÷ IC 50 

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

### (IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Figure 1, Figure 2 and Table 5. The test guideline recommends performing three independent runs. Only a single run was performed for O-phe nylphenol. For the prepared rat uterin e cytosol, the K d 

### for [ 3 

### H]-17 β -estradiol was 0.1032 nM. The Bmax was 0.07097 nM, which fell within the expected rang e of 0.03 to 1.5 nM. The data produced a linear Scatchard plot. Confidence in these num bers is high for the run due to the goodness of fit (R 2 

### =0.9617).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 9 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

TABLE 5. Saturation Binding Experiment of [ 3 H]-17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 b Run 2 Run 3 Mean ± SE

R 2 (unweighted) 0.9617 ND ND NA

B max (nM) 0.07097 ND ND NA

B max (fmol/100 μ g protein) 59.28 ND ND NA

K d (nM) 0.1032 ND ND NA a Data were included in the study profile (MRID 48618304) b Only a single run of the saturation binding experiment was conducted on the batch of cytosol used for this competitive binding experiment.
R 2 Goodness of fit for curve calculated for specific binding. ND Not determined NA Not applicable



### FIGURE 1. Binding of [ 3 

### H]-17 β -estradiol to the Estrogen R eceptor during the Saturation Binding Experiment.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 10 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### FIGURE 2. Scatchard Plot of the binding of [ 3 

### H]-17 β -estradiol to the Estrogen Receptor.

B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding experiments are presented graphically in Figures 3-6. The parameters determined from the competitive binding curves (r 2 

### , IC 50 

### , log IC 50 

### , and RBA) are presented in Table 6.

OPP exhibited >50% displacement of radiolab eled estradiol at concentrations of 10 -3 

### M in Runs 1 and 2; however, it was classified as “e quivocal” due to failure to fit the 4-parameter nonlinear regression model for analysis of one -site binding. There were no apparent alterations in radiolabeled 17 β -estradiol binding at O PP concentrations of 10 -10 

### to 10 -4 

### M (92.04-111.98%), with the specific binding of the radiolabeled ligand declining to 47.95- 48.54% at a concentration of 10 -3 

### M in Runs 1 and 2 (55.61% in Run 3). The mean estimated log IC 50 

### was

− 3.192 M for OPP in Runs 1 and 2; however, steep Hill slopes (

− 2.262 to

− 4.588) indicated a poor fit to the model. RBA values were not determined for OPP.

The three reference chemicals performed as expected. The estimated mean log IC 50 

### was

− 8.943 M for 17 β -estradiol and

− 5.454 M for the weak positive control. The mean RBA was 0.032% for the weak positive control. Octyltriethoxysilane had no effect on specific binding of the [ 3 

### H]-ligand (84.94 to 112.41%). Confiden ce in these numbers is high due to the small variation. Additio nal runs were unnecessary.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 11 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

TABLE 6. Competitive Binding Assa y of O-phenylphenol with Estrog en Receptor from Rat Uterine Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

r 2 (unweighted) 17 β -estradiol 0.9997 0.9977 0.9998 NA
19-norethindrone 0.9992 0.9985 0.9997 NA
OPP 0.9968 0.9956 0.9958 NA Log IC 50 (M) 17 β -estradiol

− 8.963

− 8.939

− 8.929

− 8.943 ± 0.010
19-norethindrone

− 5.475

− 5.442

− 5.445

− 5.454 ± 0.011
OPP ~-3.018 c ~-3.365 c NA NA
IC 50 (M) 17 β -estradiol 1.089 × 10

− 9 1.151 × 10

− 9 1.177 × 10

− 9 1.140 × 10

− 9 (± 0.026)
19-norethindrone 3.353 × 10

− 6 3.614 × 10

− 6 3.586 × 10

− 6 3.518 × 10

− 6 (± 0.083)
OPP ~9.588 × 10

− 4 c ~4.314 × 10

− 4 c NA NA Log RBA 19-norethindrone

− 3.488

− 3.497

− 3.484

− 3.489 ± 0.004
OPP NA NA NA NA RBA (%) 19-norethindrone 0.032 0.032 0.033 0.032 ± 0.0003
OPP NA NA NA NA a Data were obtained from pages 35-36 of the study report. b SE values are reviewer-calculated. For means ex pressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. c Approximate estimates as 95% CI estimates were very wide

r 2 Goodness of fit RBA (%) Relative binding affinity NA Not applicable

TABLE 7 Binding Classification of OPP with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d Classification category value b 1 1 1 1 Equivocal a Data were obtained from page 32 of the study report. b Classification category value: Interac tive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Mean of three runs expressed to the tenths place d Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5



### FIGURE 3. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or OPP, Run 1.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 12 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### FIGURE 4. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or OPP, Run 2.

FIGURE 5. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or OPP, Run 3.

FIGURE 6. Mean Percentage [ 3 

### H]-E2 Bound to the Estrogen Receptor in the Presence of OPP in Three Competitive Assays.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 13 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated usi ng the criteria shown in Table 8.

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

17 β -estradiol fitted curve parameters Log e residual SD ≤ 2.35

− 0.1540 to 1.004 X Top (% binding) 94 to 111 100.2 to 101.8 X Bottom (% binding)

− 4 to 1

− 0.9 to

− 0.7 X Hill Slope (Log 10 (M)

− 1 )

− 1.1 to

− 0.7

− 0.9981 to

− 0.9491 X Weak Positive control (19-norethindrone) fitted curve parameters b Log e residual SD NA

− 0.0245 to 0.7309 X Top (% binding) NA 95.2 to 99.7 X Bottom (% binding) NA

− 1.3 to 1.2 X Hill Slope (Log 10 (M)

− 1 ) NA

− 1.205 to

− 1.023 X
Solvent concentration
Ethanol ≤ 3% <3% X Negative control (octyltriethoxysilane) does not displace more than 25% of [ 3 H]-17 β -estradiol from the ER on average across all concentrations ≤ 25% ≤ 15.1% c X

a Data were obtained from pages 35-36 of the study report. b The EPA Guideline does not define a set of tolerance limits for 19-norethi ndrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an accepta ble weak positive control. c Reviewer-calculated. The minimum specific binding of [ 3 H]-17 β -estradiol in the negative control assays was 84.9% (Run 3). NA Not applicable



### Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17 β -estradiol displaced [ 3 

### H]-17 β -estradiol in a manner consistent with one-site binding, as indicated by a descent fr om 91.41-92.79% to 10.11-10.73% binding over approximately an 81-fold increase in concentr ation of the test chemical (i.e., covering approximately 2 Log units).

OPP was tested over a concentration range that fully defined the top of the curve. The percent binding at this top plateau 93.67-108.98% was within 25 percentage points of the value for solvent control 98.25-99.77% and the lowest concentrati on of the estradiol standard 99.34-100.03%. Examination across th e runs indicated steep Hill slopes (

− 2.262 to

− 4.588) for OPP, and curves without well-defined bottom plateaus.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the combined responses in each of three independent estrogen receptor binding assays, it was determ ined that ortho-phenylphenol (OPP) had no appreciable effect in the binding of the reference estrogen (17 β -estradiol) at any concentration up to 10

− 4 

### M. At the guideline limit concentration (10

− 3 

### M) a reproducible decrease in 17 β -estradiol binding was observed; however , the calculated Hill slope indicated a non-specific displacement of the radioligand (i .e., excessively steep) and a poor fit to theEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 14 of 14 O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None



### model, consistent with a false positive re sponse in this assay. The results of the in vitro estrogen receptor binding assay using rat uterine cytosol indi cate that, under the conditions of this study, OPP was determined to be equivocal for estrogen receptor binding.

B. AGENCY COMMENTS: The results from the single saturation binding run showed a maximum binding capacity (B max 

### ) of 59.28 fmol/100 μg protei n with the dissociation constant (K d 

### ) 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, OPP reduced the mean specific binding of the radiolabeled liga nd in a concentration-dependent manner in all three runs (declining to < 50% in Runs 1 and 2). The mean estimated log IC 50 

### was

− 3.192 M for OPP in Runs 1 and 2; RBA values were not determined. However, OPP is classified as equivocal in the ER assay because the OPP binding curves yielded steep Hill slopes (

− 2.262

to

− 4.588), indicating that the data did not fit the four-parameter, non-linear regression model for analysis of one-site binding.

The estimated mean log IC 50 

### was

− 8.943 M for 17 β -estradiol and

− 5.454 M for the weak positive control. The mean RBA was 0.032% for th e weak positive control. All performance criteria were met.

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

 Only a single saturation binding run was performed instead of three runs as recommended in the test guideline.

 The protein concentration ( μ g/tube) used in the a ssay was not reported.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 1 of 78 Data Requirement: EPA DP Barcode 064103

OECD Data Point 229

EPA MRID 48672606

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Ortho-Phenylphenol Purity: 99.9%

Common name O-Phenylphenol

Chemical name: IUPAC

CAS name:

CAS No.: 90-43-7
Synonyms: Preventol O Extra; OPP; (1,1’-Biphenyl)-2-ol

EPA PC Code: 064103

Primary Reviewer: Joan Gaidos Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 08/08/2012

Secondary Reviewer: Teri S. Myers Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 10/15/2012

Primary and Final Additional Reviewer: Robin Sternberg Signature:

Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 10/26/2012, 05/27/2015

Additional Reviewer: Elyssa Arnold Signature:

Biologist, USEPA/OCSPP/OPP/EFED/ERB2 Date: 02/07/2014

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126
Date: 2015.06.02 11:15:42 -04'00'Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 2 of 78 CITATION: Lehman C.M., J.A. Fiting, K.L. Hutchinson, D. W. Louch, N.A. Malowinski, L.G. McFadden and J.

Thomas. 2012. Ortho-Phenylphenol: The Fish Shor t-term Reproduction Assay Using the Fathead

Minnow, Pimephales promelas . Unpublished study performed by Toxicology and Environmental

Research and Consulting, Midland, Michigan. Lab Study No.: 111032. Study sponsored by OPP

EDSP Consortium, Dow Chemical Company, Midland, Michigan and LANXESS Corporation,

Pittsburgh, Pennsylvania. Study completed January 04, 2012.

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

screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with
the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within

the assay, where available, and redundancy across the battery. Thus, the results of each individual assay

should not be considered in isolation but rather should be considered in the context of other assays in the
battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has
the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay
results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

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

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

nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 3 of 78 EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of ortho-phenylphenol (OPP) 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 OPP at nominal concentrations of 0 (negative
control), 0.02, 0.20, and 2.0 mg a.i./L; time-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

25.5 o 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). OPP 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.
OPP 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 OPP treatment. There were no other significant effects on
endpoints ( e.g. , nuptial tubercle score, gonado-somatic index (GSI), or gonadal staging). There were noData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 4 of 78 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 concentrationresponse relationship. Plasma testosterone and 17 β -estradiol levels were not measured.

The study met all performance and validity criteria with the following exceptions. Spawning did not occur at

least every 4 days in three of the four negative control replicates, and the fecundity in the negative control

was 13.9 eggs/female/day/replicate which was less than the guideline criterion of ≥ 15

eggs/female/day/replicate. 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. The coefficient of
variation (CV) for measured test concentrations ranged from 28 to 60%, exceeding the guideline criterion of

<20%. Analytical verification of the test solutions at Day 0 to 21 yielded recoveries ranging from 23.6 to 115%

of nominal concentrations with a pattern of decline in the higher treatment level due to biodegradation of the

test material. Therefore, flow rates were periodically adjusted in an attempt to maintain nominal concentrations.

These deviations did not impact the interpretation of the study.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay

(OCSPP Guideline 890.1350).

Results Synopsis

Test Organism age at test initiation: 5.5 months

Mean body weight at test initiation: Male 2.0 g; female 1.4 g

Mean length at test initiation: Not reported

Test Type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 5 of 78Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Ortho-Phenylphenol.Treatment(mg a.i./L)[TWAmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.0158NoNoNoNoNoNoNANANoNoNANANANA0.0755NoNoNoNoNoNoNANANoNoNANANANA0.876Yes3Yes3NoNoNoNoNANANoYesNANANANAAbbreviations:E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.3Although not significantly different (p>0.05) from the negative control, these endpoints were markedly decreased compared tothe negative control. Therefore, thedecreases were considered biologically significant.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 6 of 78 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350:
“Fish Short-Term Reproductive Assay” and OECD 229 (2009). The

following deviations were noted:

1. In the negative control, spawning did not occur at l east every 4 days in three of the four replicates.

The fecundity in the negative control was 13.9 eggs/female/day/replicate and did not meet the

guideline criterion of ≥ 15 eggs/female/day/replicate. Two of the four control replicates were below

the minimum value at 7.3 and 9.2 eggs/female/day/replicate.

2. Analytical recoveries dropped over the course of the exposure period due to biodegradation of the test

material; therefore, flow rates were periodically adjusted in an attempt to maintain nominal

concentrations.

3. Analytical verification of the test solutions on Days 0, 4, 7, 8, 12, 14, 19 and 21 yielded recoveries

ranging from 23.6 to 115% of nominal concentrations with a pattern of decline in the higher treatment

levels. The coefficient of variation for measured concentrations ranged from 28 to 60%, exceeding the

guideline criterion of <20%.

4. The physico-chemical properties of the test material were not reported.

5. The total organic carbon, unionized ammonia and residual chlorine in the test water were not reported.

Guideline performance criteria establish maximum levels for these values; and it is unclear if the

maximum recommendations were exceeded.

These deviations do not impact the interpretation of this study.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims

statements were provided. This study was conducted in accordance with GLP
Standards as published by the U.S. EPA (40 CFR Parts 160), and OECD
Principles of GLP [ENV/MC/CHEM(98)17].

A. TEST MATERIAL: Ortho-Phenylphenol (OPP); CAS# 90-43-7Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 7 of 78 Description: White solid; Recertification date February 22, 2013; solubility 700 mg/L at

20 q C; Kow = 3.18

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

vapor pressure of test compound, expiration date.

Lot No./Batch No. : CHHYDD0051

Purity: 99.9%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 4, 7, 8, 9, 12, 14, 19
and 21 yielded daily average recoveries (mean of n=4 replicates per test
concentration) of 50.5% (Day 9) to 115% (Day 0) of nominal for the lowest
test concentration (nominal 0.02 mg/L), 29.7% (Day 4) to 79.1% (Day 0)
of nominal for the mid test concentration (nominal 0.2 mg/L), and 23.6%
(Day 21) to 115% (Day 0) of nominal for the high test concentration (nominal

2.0 mg/L). Stock solution fortified with OPP at concentrations of 3.0, 30.0,

and 300 mg a.i./L yielded recoveries of 17.2 to 101%. There was a pattern

of decline during the study period, especially at the higher test concentrations.

The study authors attributed this decline to biodegradation which has been

documented previously at concentrations similar to those used in this study.

These data indicate the test material was not stable under the test conditions.

Storage Conditions of

Test Chemicals: Storage conditions were not reported.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 8 of 78B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?YesAquatic Research Organisms, Inc., Hampton,New Hampshire. EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameasdefinitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:19 days EPA recommends a minimum two-week acclimation period. Note that the acclim ation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 9 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortality during the last 2weeks prior to preexposure was 3.0%.Mortality during the 7days prior to preexposure period was1.5%.Fish did not receive anytreatment for diseaseduring acclimation period.Fish with behavioralabnormalities or clinicalsigns were not used inthe study. EPA recommends that mortality during the 7 days prior to the pre -exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality

is between 5 - 10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation

period, EPA recommends that the fish not be

used.Type of food:Thawed frozen brineshrimpAccording to the study authors, measuredconcentrations of pesticides, organics,metals, and other inorganics were belowlevels of concern in a representative sampleof the frozen brine shrimp used for fish feed. EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 10 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsSource of food:Brine Shrimp Direct,Ogden, UtahFrequency of feeding:2 times/dayDetails on feeding:At least twice daily at arate sufficient to promoteactive reproduction andmaintain body condition;1.5-2.5 mL per replicateduring the pre-exposurephase; 2.5-3.5 mL perreplicate (or pro-rated toaccount for mortality)during the exposurephaseThe amount of frozen shrimp provided wasmeasured and an equal proportion pernumber of surviving fish was given to eachtank. Food was withheld from the fish for atleast 12 hours prior to test termination to aidin histological processingof the fish.Uneaten food and fecal material wereremoved from the test vessels at least threetimes per week by siphoning.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:5.5 monthsHatched on February 22, 2011 EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 11 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at testinitiation (if determined):2.0 gBased on 10 males used to stockaquaria for pre-exposure phase. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):Not reportedIndividual weights within ± 20% of themean weight of the sub sampled fish ofthe same sex.Mean weight of females at testinitiation (if determined):1.4 gBased on 10 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):Not reportedIndividual weights within ± 20% of themeanweight of the sub sampled fish ofthe same sex.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:15 days EPA recommends a minimum of 14 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 12 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:24 EPA recommends that additional tanks set up at the beginnin g of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:>15 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 13 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns during preexposure:> two times in theimmediately preceding7 daysSpawning occurred at least every 3days. EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:NoneC. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a

flow - through system. As noted in the Corrections and Clarifications

document 1 , the use of a static renewal system is not recommended for this assay.1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 14 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Continuous-flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:45 ± 4.5 mL/minute (2.7L/hr) to provide completevolume turnovers ca. 2.7hrs(equivalent to 6 volumereplacements/day)L/hr calculated by the reviewer based on 45 mL/minute.Because analytical recoveries dropped over the course of theequilibrium and exposure periods due to the biodegradation of thetest material, flow rates were periodically adjusted in an attemptto maintain nominal concentrations. These changes were asfollows. On the last day of equilibrium (testDay -1), flow ratefrom delivery tubing was increased to 90 ± 9 mL/minute andstock injection rate was doubled to 3.0 mL/min. On exposureDay 5, flow rate from delivery tubing was reduced to 45 ± 4.5mL/min for all concentration levels and stock injection rate wasreduced to 1.5 mL/min in low concentration (where analyticalrecoveries were acceptable); stock injection rate remained at 3.0mL/min in the mid- and high levels. On exposure Day 8, the useof the mid and low level stocks was discontinued due to observedbiodegradation in these solutions; the high-concentration stocksolution (300 mg/L) was then usedfor all treatment levelsat aninjection rate of 0.015 mL/min (low), 0.3 mL/min (mid), and 3 Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges

per day.2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 15 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsmL/min (high). On exposure Day 11, the injection rate to the lowtreatment level was increased to 0.03 mL/min.Details on toxicant mixing forflow-through systems:For each treatment level, aperistaltic pump continuouslydelivered a concentrated testsubstance stock solution at adesignated rate (1.5mL/min) from aconcentration-specific stockvessel to a stainless steelmixing chamber where itwas mixed with dilutionwater.The test solution was gravity-fed from the mixing chamberand split equally to replicate test vessels via a Teflonmanifold with Teflon delivery tubing.The flow rates of the stock solutions and of the diluents(via test solution flow to each replicate vessel) weretargeted not to vary more than 20% throughout theexposure; adjustments were made as necessary tomaintain or re-establish flow rates within this range. Recommended toxicant mixing for

flow - through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A

de monstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.Aeration?No EPA recommends aeration if dissolved oxygen reaches < 4.9 mg/L (<60% saturation).Source of dilution water:Upper Saginaw Bay of LakeHuronoff Whitestone PointPrior to use, dilution water was limed and flocculated withferric chloride, sand-filtered, pH-adjusted with gaseousCO2, carbon-filtered and UV-irradiated. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, andData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 16 of 78ParameterValue(s)Details or RemarksGuideline Recommendations other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?YesAccording to the study authors, measured concentrationsof pesticides, organics, metals, and other inorganics werebelow levels of concern.Test vessel type/materials:Glass aquaria sealedtogether withsiliconeadhesive EPA and OECD recommend that

water -contact portions of the system not compromise the study

(e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:39 cm long x 20 cm wide x25 cm high; solution depth13 cm EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x

20 cm).Fill volume:10 L EPA recommends 10 L solution

per tank.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 17 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsSpawning substrate material:10 cm diameter PVC cutlengthwise in half (arches)and into9 cm sectionsandpositioned in a screened,lipped stainless steel (ca. 13cm long) collection tray.3 spawning substrates per tank EPA recommends that each tank contain three semi -circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size:PVC cut in half (arches) andinto 9 cm sections.Each substrate was placed on screened stainlesscollection tray.Additional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.6 25.5 25.11Continuously in onetest vessel; weekly ineach test vessel EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH7.27.47.31Weekly, in each testvessel EPA recommends pH 6.5 to 9.0.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 18 of 78ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsDissolved oxygen (mg/L)6.4 8.1 7.31Weekly, in each testvessel EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)28 48 381Weekly, from sampleof a control and thehighest exposure levelsolution EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3) 60 66 641Weekly, from sampleof a control and thehighest exposure levelsolutionTotal organic carbon (mg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that residual chlorine in dilution water be < 10 P g/L.Conductivity (Pmhos/cm) 159 198 1751Weekly, from sampleof a control and thehighest exposure levelsolution General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly andData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 19 of 78ParameterMinimumMaximumMeanMeasurement IntervalGuideline Recommendations that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?No EPA recommends conducting a range-finder if

96 -hour LC

50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?NA EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 20 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies:NALife stage:NA EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:NA EPA recommends a 96-hour exposure.Additional details:NA EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration -response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96 -hr LC

50, may be selected as the highest exposur e concentration in the 21- day

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 21 of 78Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive

test be 21 days.Method for selecting the highesttest concentration in the definitivetest:The acute toxicity 96-hrLC50value for OPP withfathead minnows wasreported to be between5.9 and 6.3 mg a.i./L.The highest test concentration was selectedas 1/3 of 6.0 mg/L, equal to 2.0 mg/L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Holcombe et al., 1984;Geiger et al., 1985Separation of test concentrations:Step down factor of 0.1from highest testconcentration0 (control), 0.02, 0.20,and 2.0 mg a.i./L EPA suggests that a concentration separation of between 0.33 (orthree-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:3 and a control: 0(control), 0.02, 0.20, and2.0 mg a.i./L EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 22 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Time-Weighted AverageLimit of quantification (LOQ):0.00495 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:0, 4, 5, 7, 8, 11, 12, 14,19, 20 and 21daysAlso, Day 9 for 0.02 mg a.i./L replicate testvessels It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks dur ing pre- exposure. Tanks are then ranked according to pre -exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 23 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration (ifapplicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series on TestingData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 24 of 78ParameterValue(s)Details or RemarksGuideline RecommendationsWas a positive control used?NoPositive control (if applicable):NAPositive control concentration(s) (ifapplicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:721-885 luxCool white fluorescent bulbs EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:NoneAppearance of the test solution was notdescribedand Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 25 of 78Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Phosmet.Treatment IDNominalConcentration(mg a.i./L)Time-WeightedAverageConcentration(mg a.i./L)Mean CV (%)Details or Remarks1Guideline RecommendationsNegative Control0<LOQNA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Treatment 10.020.015828Reviewer-calculated meanmeasured: 0.0162 ± 0.0045Reviewer-calculated TWA: 0.0165Treatment 20.200.075541Reviewer-calculated meanmeasured: 0.0785 ± 0.0785Reviewer-calculated TWA: 0.0755Treatment 32.00.87660Reviewer-calculated meanmeasured: 0.9276 ± 0.5532Reviewer-calculated TWA: 0.8767Abbreviations:CVCoefficient of variation.LOQ=0.00495 mg a.i./L1The reviewer-calculated time-weighted averages were slightlydifferent that those of the study author, likely due to roundingdifferences.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 26 of 78 E. Observations

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

length, tubercle score, gonadal staging and histopathology, and plasma vitellogenin were evaluated.

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics,
and other clinical signs occur at least daily. At test termination (Day 21), additional observations include body

weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma

sex steroids (testosterone and 17 β -estradiol, if measured). Gonado-somatic index (GSI) is calculated using
a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at

test termination.

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other

abnormal behavior.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 27 of 78 I. RESULTS AND DISCUSSION

A. Results

Overall mean survival values were 100, 100, 100, and 71% in the negative control and TWA 0.0158,

0.0755, and 0.876 mg a.i./L treatment levels, respectively. However, the study author reported that six of

seven fish deaths occurred in a single tank over the course of 2 days and therefore the deaths are possibly

tank-related rather than treatment-related. Since the acute toxicity 96-hr LC 50 value for OPP with fathead

minnows is between 5.9 and 6.3 mg a.i./L, very little mortality at the high concentration of 0.875 mg

a.i./L would be expected.

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

Treatment

(mg a.i./L)

[TWA measured] Males Females

n 1 # Surviving % Survival n 1 # Surviving % Survival

Negative Control

(<LOQ) 8 8 100 16 16 100

0.0158 8 8 100 16 16 100

0.0755 8 8 100 16 16 100

0.876 8 6 75 16 11 69

LOQ=0.00495 mg a.i./L

1 Total number of fish at test initiationData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 28 of 78 The average weight of male fathead minnows (n=48) used to stock aquaria for the pre-exposure phase of
the study was 2.0 ± 0.2 g (CV=12.1%), while the average weight of the females (n=96) was 1.4 ± 0.2 g
(CV=13.0%). Based on six fish per replicate vessel (2 males and 4 females) and the minimum targeted
test solution flow rate to each vessel (45 mL/min), the resulting loading rate was approximately 0.1 g

fish/L of solution passing through the test vessel in a 24-hour period.

Mean male body weight values were 3.00, 2.48, 2.35, and 2.54 g, and female body weight values were

1.58, 1.47, 1.51 and 1.51 g in the negative control and TWA 0.0158, 0.0755 and 0.876 mg a.i./L treatment
levels, respectively (Table 10). Mean male body length values were 50.2, 47.8, 47.0, and 48.9 mm, and

female body length values were 41.6, 40.6, 40.4 and 41.0 mm in the negative control and TWA 0.0158,

0.0755 and 0.876 mg a.i./L treatment levels, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 29 of 78Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA measured]Body WeightLengthMalesFemalesMalesFemalesn1Mean(g)±SDn1Mean(g)±SDn1Mean(mm)±SDn1Mean(mm)±SDNegative Control(<LOQ)43.000.3414 1.580.176450.21.69441.61.680.015842.480.41641.470.061447.83.01440.60.7970.075542.350.49941.510.207447.02.33440.42.190.87632.540.52931.510.065348.91.62341.00.261Abbreviations:SDStandard deviation.LOQ=0.00495 mg a.i./L1Number of replicate tanksData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 30 of 78 At the end of the 15 day pre-exposure period, fecundity measurements for the previous seven days across
all replicate vessels with surviving fish (n=23) ranged from 0.0-59.3 eggs/female/reproductive day with
an average fecundity (± standard deviation) of 18.3 ± 10.8 eggs/female/reproductive day. Fecundity

measurements for the previous seven days across all pre-exposure phase replicate vessels carried over for
use in the exposure phase of the study (n=16) ranged from 15.1-59.3 eggs/female/reproductive day with

an average fecundity of 22.0 ± 10.5 eggs/female/reproductive day.

Mean fecundity values were 13.9, 13.9, 21.0 and 7.3, and fertilization success was 97.2, 98.0, 98.9 and

66.0% in the negative control and TWA 0.0158, 0.0755 and 0.876 mg a.i./L treatment levels, respectively
(Table 11). For fecundity, the variance in the 0.876 mg a.i./L treatment group was nearly as high as the
mean (7.3 ± 6.38). The fecundity in the negative control was 13.9 eggs/female/reproductive day/replicate

which did not meet the guideline criterion of ≥ 15 eggs/female/reproductive day/replicate. In the negative

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

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Fecundity 1 Fertilization Success 2

Mean ±SD Mean (%) ±SD

Negative Control (<LOQ) 13.9 6.59 97.2 3.40

0.0158 13.9 1.35 98.0 2.04

0.0755 21.0 8.90 98.9 0.83

0.876 7.3 6.83 66.0 57.13

LOQ=0.00495 mg a.i./L

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 31 of 78 Median male tubercle scores were 14.5, 15.5, 19.3 and 17.0 in the negative control and TWA 0.0158,
0.0755, and 0.876 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were

found to have tubercles.

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

n 1 Median Tubercle Score 2 n 1 Median Tubercle Score

Negative Control (<LOQ) 4 15 4 0

0.0158 4 16 4 0

0.0755 4 19 4 0

0.876 3 17 3 0

LOQ=0.00495 mg a.i./L

1 Number of replicate tanks

2 Mean tubercle scores: 16, 15, 20, and 17 for the negative control and TWA 0.0158, 0.0755, and 0.876 mg a.i./L

treatment levels, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 32 of 78 Mean male GSI was 1.30, 1.19, 1.31 and 1.09, and mean female GSI was 15.73, 13.46, 14.11 and 13.06

in the negative control and TWA 0.0158, 0.0755, and 0.876 mg a.i./L treatment levels, respectively (Table

13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

n Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative Control (<LOQ) 4 1.30 0.297 4 15.73 4.439

0.0158 4 1.19 0.224 4 13.46 2.478

0.0755 4 1.31 0.173 4 14.11 3.969

0.876 3 1.09 0.207 3 13.06 2.108

LOQ=0.00495 mg a.i./L

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 33 of 78 Median male gonadal stage was 2, 2, 2 and 2, and median female gonadal stage was 3, 3, 3 and 3 in
the negative control and TWA 0.0158, 0.0755 and 0.876 mg a.i./L treatment levels, respectively (Table

14).

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Males Females

n Median Stage 1 n Median Stage 2

Negative Control (<LOQ) 8 2 16 3

0.0158 8 2 16 3

0.0755 8 2 16 3

0.876 6 2 11 3

LOQ=0.00495 mg a.i./L

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

One male fish had moderately increased proportions of spermatogonia and a single small developing oocyte
in one testis in the 0.876 mg a.i./L treatment group compared to the control (Table 15). This finding in

male fish is considered incidental and unrelated to treatment due to isolated occurrences and other

treatment-related findings. In female fish, there was an increase in the incidence and severity of oocytes
atresia in treated fish compared to controls (Table 17); however, the effects lacked a concentration-response

relationship. The study author found no treatment-related histopathological observations in ovaries or testes

at any test concentration and interpreted findings to be background spontaneous changes not associated

with exposure to OPP due to lack of concentration-response relationships.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 34 of 78 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA

measured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Increased

Testicular

Degeneration Duct

Mineralization Seminiferous

tubule

mineralization

n Incidence n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 8 8 8 8 NA NA 8 8 8 8

1 8 0 8 0 NA NA 8 0 8 0

2 8 0 8 0 NA NA 8 0 8 0

3 8 0 8 0 NA NA 8 0 8 0

4 8 0 8 0 NA NA 8 0 8 0

0.0158 0 8 8 8 8 NA NA 8 8 8 7

1 8 0 8 0 NA NA 8 0 8 1

2 8 0 8 0 NA NA 8 0 8 0

3 8 0 8 0 NA NA 8 0 8 0

4 8 0 8 0 NA NA 8 0 8 0

0.0755 0 8 8 8 8 NA NA 8 7 8 8

1 8 0 8 0 NA NA 8 1 8 0

2 8 0 8 0 NA NA 8 0 8 0

3 8 0 8 0 NA NA 8 0 8 0

4 8 0 8 0 NA NA 8 0 8 0

0.876 0 6 7 6 7 NA NA 6 7 6 8

1 6 0 6 1 NA NA 6 0 6 0

2 6 0 6 0 NA NA 6 1 6 0

3 6 1 6 0 NA NA 6 0 6 0

4 6 0 6 0 NA NA 6 0 6 0

LOQ=0.00495 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 35 of 78Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)0NANANANANANANANA861NANANANANANANANA822NANANANANANANANA803NANANANANANANANA804NANANANANANANANA800.01580NANANANANANANANA881NANANANANANANANA802NANANANANANANANA803NANANANANANANANA804NANANANANANANANA800.07550NANANANANANANANA861NANANANANANANANA812NANANANANANANANA813NANANANANANANANA804NANANANANANANANA80Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 36 of 78Treatment(mg a.i./L)[TWA measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.8760NANANANANANANANA651NANANANANANANANA612NANANANANANANANA603NANANANANANANANA604NANANANANANANANA60Abbreviation:NANot applicableLOQ=0.00495 mg a.i./L1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 37 of 78 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA

measured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation Aggregates of

macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Negative Control

(<LOQ) 0 16 1 NA NA NA NA 16 10

1 16 8 NA NA NA NA 16 6

2 16 4 NA NA NA NA 16 0

3 16 0 NA NA NA NA 16 0

4 16 3 NA NA NA NA 16 0

0.0158 0 16 2 NA NA NA NA 16 13

1 16 4 NA NA NA NA 16 3

2 16 3 NA NA NA NA 16 0

3 16 0 NA NA NA NA 16 0

4 16 7 NA NA NA NA 16 0

0.0755 0 16 3 NA NA NA NA 16 12

1 16 1 NA NA NA NA 16 4

2 16 4 NA NA NA NA 16 0

3 16 3 NA NA NA NA 16 0

4 16 5 NA NA NA NA 16 0

0.876 0 11 0 NA NA NA NA 11 6

1 11 5 NA NA NA NA 11 5

2 11 1 NA NA NA NA 11 0

3 11 1 NA NA NA NA 11 0

4 11 4 NA NA NA NA 11 0

Abbreviation: NA Not applicable

LOQ=0.00495 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 38 of 78 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales

promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Additional Diagnostic Observations 1

Severity Subacute to Chronic/

Interstitium/ M ultifocal

Inflammation Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative Control

(<LOQ) 0 16 13 NA NA 16 13 NA NA

1 16 3 NA NA 16 3 NA NA

2 16 0 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

0.0158 0 16 13 NA NA 16 12 NA NA

1 16 3 NA NA 16 4 NA NA

2 16 0 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

0.0755 0 16 13 NA NA 16 14 NA NA

1 16 3 NA NA 16 2 NA NA

2 16 0 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

0.876 0 11 10 NA NA 11 9 NA NA

1 11 3 NA NA 11 2 NA NA

2 11 0 NA NA 11 0 NA NA

3 11 0 NA NA 11 0 NA NA

4 11 0 NA NA 11 0 NA NA

Abbreviation: NA Not applicable

LOQ=0.00495 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 39 of 78 Mean male VTG was 452, 1220, 866 and 561 ng/mL, and female VTG was 66.3x10 6 , 71.7x10 6 , 60.8x10 6

and 42.1x10 6 ng/mL in the negative control and TWA 0.01 58, 0.0755 and 0.876 mg a.i./L treatment
levels, respectively (Table 19).

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA measured] Plasma Vitellogenin (VTG)

Males Females

N Mean

(ng/mL plasma) ±SD N Mean

(ng/mL plasma) ±SD

Negative Control (<LOQ) 4 452 174 4 66.3 x 10 6 22.9 x 10 6

0.0158 4 1220 1170 4 71.7 x 10 6 18.6 x 10 6

0.0755 4 866 765 4 60.8 x 10 6 29.6 x 10 6

0.876 4 528 188 4 42.1 x 10 6 5.50 x 10 6

Abbreviations: SD Standard deviation.

LOQ=0.00495 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 40 of 78Plasma testosterone and plasma 17β-estradiol in male and females were not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[TWA measured]Plasma Testosterone (T)Plasma 17β-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control (<LOQ)NANANANANANANANANANANANA0.0158NANANANANANANANANANANANA0.0755NANANANANANANANANANANANA0.876NANANANANANANANANANANANAAbbreviations:NANot applicable.SDStandard deviation.LOQ=0.00495 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 41 of 78There were no notable observations with regards to behavior, coloration/banding, changes in ovipositor appearance or size of dorsal nape pad, with theexception of one lethargic male in the TWA 0.876 mg a.i./L treatment level (Table 21).Table 21: Secondary Sex Characteristicsand Clinical Signs in Fathead Minnow ( Pimephales promelas)Treatment(mg a.i./L)[TWA measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceNegative Control (<LOQ)None8NANone16NA0.0158None8NANone16NA0.0755None8NANone16NA0.876Lethargy61None11NAAbbreviation:NANot applicableLOQ=0.00495 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 42 of 78 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility, fecundity, and VTG. Data

were gender specific and analyzed in comparison to the control.

Descriptive statistics (mean, standard deviation, etc. ) were determined for each endpoint. Significant effects

were detected for p<0.05 with the exception of Shapiro-Wilk’s Test, which was based on p<0.01 (statistical

program used was not reported). Survival data were analyzed using a Cochran-Armitage Linear Trend Test
(with Yate’s Continuity Correction). Male and female length and weight, GSI, fecundity, and VTG, were

analyzed using ANOVA and a one-tailed Dunnett’s test. Fertility was analyzed by Mann-Whitney-Wilcoxon
U test (with Bonferroni-Holm adjustment), and male tubercle score was analyzed by Multi-Quantal

Jonckheere-Terpstra Test. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Levene’s Tests

to test for normality and homogeneity of variances, respectively. If normality and homogeneity tests passed
(p>0.01), a parametric analysis was performed. If non-normality or unequal variance were indicated
(p<0.01) even after transformation, a non-parametric analysis was performed on the ranks of the data.

These methods appear to be consistent with the methods recommended in the guideline.

Adult survival (combined sexes) at the 0.876 mg a.i./L treatment level (71%) was significantly different
compared to the controls (100%; p<0.05; Cochran-Armitage Trend test with Yate’s Continuity Correction;

Table 9). There was no statistically significant difference in fertilization success or fecundity at any treatment
compared to the control (Table 11). However, for fecundity, the variance in the 0.876 mg a.i./L treatment
group was nearly as high as the mean (7.3 ± 6.38), therefore it was difficult to discern statistical differences
(p>0.05; Dunnett’s test). Also, because of non-normality and no acceptable normalizing transformations,

fertility in the 0.876 mg a.i./L treatment group was analyzed using Mann-Whitney-Wilcoxon U test with

Bonferroni-Holm adjustment and no si gnificance was found (p=1 .000). Finally, the fecundity in the negative

control, was 13.9 eggs/female/reproductive day/replicate and did not meet the Guideline validity criterion

minimum of ≥ 15 eggs/female/reproductive day/replicate. Ther e were no significant differences between

treated groups and control for any other endpoint.

Although not analyzed statistically, the study author found no treatment-related histopathological observations

in ovaries or testes at any test concentration compared to controls and interpreted findings to be backgroundData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 43 of 78spontaneous changes not associated with exposure to ortho-phenylphenol due to lack of concentrationresponse relationships (Tables 15-18). There were no differences in secondary sex characteristics or clinical
signs for any treatment group compared to the controls (Table 21).

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via Toxstat

3.5. Male and female body weight and length, male mean vitellogenin (VTG), male and female gonadal
somatic index (GSI), male tubercle score, fertility and fecundity data were not consistent with a monotonic

concentration-response. All data were tested for normality using Shapiro-Wilks test and for homogeneity of
variance using Levene’s test. Data which met the assumptions of normality and homogeneity of variance

were then analyzed using the parametric Dunnett’s test. Data which did not meet the parametric assumptions

were analyzed using the non-parametric Mann-Whitney U test.

Female body weight and length, and fertility data were neither monotonic nor parametric and were analyzed

using Mann-Whitney. Female VTG exhibited monot onic trends and was analyzed using the non-parametric

Jonckheere-Terpstra test, as recommended by the OCSPP 890.1350 guideline. None of the surviving

females were found to have tubercles. Unless otherwise indicated, effects were considered statistically

significant at p<0.05. These analyses were conducted using SAS ® (SAS Institute, Cary, NC; version 8.1).

Both the study authors and reviewer relied on the time-weighted mean concentrations to discuss effects in

this study.

Conclusions:

Survival was significantly reduced (p<0.05) to 71% of the control at the 0.876 mg a.i./L treatment level
(Table 9). Female VTG (Table 13) was significantly reduced (p<0.05) to 36.5% of control at the 0.876

mg a.i./L treatment level. There were no other significant effects on endpoints. Although not analyzed

statistically, there were no treatment-related histopathological observations, secondary sex characteristics or

clinical signs for any treatment group compared to the controls.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 44 of 78Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Ortho-Phenylphenol.Treatment(mg a.i./L)[TWA measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p% Diff.pNegative Control (<LOQ)15NA0NANo0NANANANANA0.0158160.977-8.430.848No170.450.364NANANANA0.0755190.7660.771.000No91.660.775NANANANA0.876170.998-16.220.526No16.620.998NANANANAStatistical TestDunnett’s3Dunnett’sNADunnett’sNANAAbbreviations:Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.00495 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Mean tubercle scores: 16, 15, 20, and 17 for the negative control and TWA 0.0158, 0.0755, and 0.876 mg a.i./L treatment levels, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 45 of 78Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Ortho-Phenylphenol.Treatment(mg a.i./L)[TWA measured]FecundityFert. SuccessTubercle ScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.pMedianp% Diff.pEffect?(Yes/No)% Diff.p%Diff.p% Diff.pNegative Control(<LOQ)0 NA0 NA 0NA0 NANo 0 NANANANANA0.0158-0.361.0000.771.0000NA-14.430.695No8.100.718NANANANA0.075550.720.3281.700.7800NA-10.250.854No-8.370.330NANANANA0.876-47.480.434-32.101.0000NA-16.960.643No-36.520.040NANANANAStatistical TestDunnett’sMann-WhitneyNADunnett’sNAJonckheereNANAAbbreviations:Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.00495 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 46 of 78 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with OrthoPhenylphenol.

Treatment

(mg a.i./L)

[TWA measured] Body Weight Length

Males Females Males Females

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

Negative Control (<LOQ) 0 NA 0 NA 0 NA 0 NA

0.0158 -17.33 0.282 -6.66 0.343 -4.83 0.353 -2.35 0.678

0.0755 -21.50 0.153 -4.12 0.889 -6.32 0.177 -2.83 0.346

0.876 -15.22 0.432 -4.49 0.615 -2.57 0.807 -1.22 1.000

Statistical Test Dunnett’s Mann-Whitney Dunnett’s Mann-Whitney

Abbreviations: Diff. Difference. NA Not applicable.

LOQ=0.00495 mg a.i./L

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

2 Unless otherwise specified, effects are considered statistically significant at p<0.05.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 47 of 78 E. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER. The

validity and performance were met with the following exceptions. In the negative control, spawning did not

occur at least every 4 days in three of the four replicates. The fecundity in the negative control was 13.9

eggs/female/day/replicate which was less than the guideline criterion of ≥ 15 eggs/female/day/replicate.

Specifically, two of the four negative control replicates were below the minimum value at 7.3 and 9.2

eggs/female/reproductive day/replicate. The coefficient of variation (CV) for measured test concentrations

ranged from 28 to 60%, exceeding the guideline criterion of <20%. Analytical verification of the test

solutions at Days 0, 4, 7, 8, 12, 14, 19 and 21 yielded recoveries ranging from 23.6 to 115% of nominal

concentrations with a pattern of decline in the higher treatment level due to biodegradation of the test

material. Therefore, flow rates were periodically adjusted in an attempt to maintain nominal concentrations.

These deviations do not impact the interpretation of this study.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in agreement regarding effects on survival; the reviewer
also detected a significant reduction (p<0.05) in female VTG. Both the study author’s and reviewer’s
analysis detected statistically significant reductions (p<0.05) in survival at the 0.876 mg a.i./L treatment
level, using either the study author’s analysis method (Cochran-Armitage Trend test) or the reviewer’s
analysis method (Fisher’s Exact test). Also, the reviewer found a significant reduction (36.5%) in female
VTG at the 0.876 mg a.i./L treatment level (p<0.05) following the OCSPP 890.1350 flowchart (using the

results of Jonckheere-Terpstra test). The reviewer’s conclusions based on the OCSPP 890.1350 flowchart

are presented in the Executive Summary and Conclusions sections of this DER.

The method recoveries of OPP in stock solution fortified at 3.0, 30.0 and 300 mg a.i./L were 41.1 ± 112%
(n=4), 85.3 ± 13.0% (n=7), and 92.4 ± 4.32% (n=18) using a Phenomenex Gemini 3 P C6-Phenyl

column.

The study author reported that analytical recoveries dropped over t he course of the eq uilibrium and exposure

periods due to biodegradation of the test material; therefore, flow rates were periodically adjusted in anData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 48 of 78 attempt to maintain nominal concentrations. On the last day of equilibrium (-1 day), the flow rate from the

delivery tubing was increased to 90 ± 9 mL/min and stock injection rate doubled to 3.0 mL/min. On

exposure Day 5, the delivery tubing flow rate was reduced to 45 ± 4.5 mL/min for all concentration levels
and stock injection reduced to 1.5 mL/min only in the low concentration (where recoveries were acceptable).

On exposure Day 8, the use of mid and low treatment level stock solutions were discontinued due to
biodegradation and the high level stock solution (300 mg/L) was used for all treatment levels at an injection
rate of 0.015 mL/min (low concentration), 0.3 mL/min (mid concentration) and 3 mL/min (high

concentration). On exposure Day 11, the injection rate to the low level treatment was increased to 0.03

mL/min.

For mortality, the study author reported that six of seven fish deaths occurred in a single tank over the

course of 2 days and therefore the deaths are possibly tank-related rather than treatment-related. Since

the acute toxicity 96-hr LC 50 value for OPP with fathead minnows is between 5.9 and 6.3 mg a.i./L, very

little mortality at the high concentration of 0.875 mg a.i./L would be expected. The study author analyzed

the data without including the six fish from the affected tank and the effect of treatment on mortality is no
longer significant (p=0.1374).

Because high variability in the endpoint makes male VTG results difficult to interpret, this endpoint is

presented graphically in the boxplot below.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 49 of 78Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 50 of 78 III. REFERENCES

Birk, K. H., Dixon, T. S., Harfmann, R., and Johnson, R. (2011). Characterization of Preventol O Extra
(ort/zo-phenylphenol) Lot # CHHYDD0051 for use as a Test Substance, in a Toxicology Study. ML AL2011 -000666. Report of The Dow Chemical Company, Midland, Michigan.

Bentley, R. E. (1976). Acute toxicity of Dowicide CO antimicrobial to bluegill (Lepomis macrochirus) and
rainbow trout (Salmo gairdneri). Bionomics EG&G Environmental Consultants, Wareham, Massachusetts.

Unpublished Report Sponsored by The Dow Chemical Company, Midland, Michigan.

Geiger, D. L., Northcott, C. E., Call, D. J., and Brooke, L. T. (1985). Acute toxicities of organic chemicals

to fathead minnows (Pimephales promelas), Vol. 2. Center for Lake Superior Environmental Studies,

University of Wisconsin-Superior, Wisconsin; 326 pp.

Gonsior, S. J. and Tryska, T. J. (1997). Evaluat ion of the biodegradability of o-Ph enylphenol. R eport of

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

Gonsior, S. J., Rhinehart, W. L., Spence, M. W., and Bailey, R. E. (1984). Biodegradation of oPhenylphenol in river water and activated sludge. /. Agric. Food Chem. 32(3), 593-596.

Grim, K. C., Wolfe, M., Hawkins, W., Johnson, R and Wolf, J. (2007) Intersex in Japanese medaka
(Oryzias latipes) used as negative controls in toxicologic bioassays: a review of 54 cases from 41 studies.

Environmental Toxicology and Chemistry 26: 1636-1643.

Holcombe, G. W., Phipps, G. L., Knuth, M. L., and Felhaber, T. (1984). The acute toxicity of selected

substituted phenols, benzenes and benzoic acid esters to fathead minnows, Pimephales promelas.

Environmental Pollution Series A 35, 367-381.

Hollander, M. and D. A. Wolfe. (1973). Nonparametric Statistical Methods. John Wiley & Sons, Inc. New

York. Pp 120-123.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 51 of 78 OECD (2009). Organisation for Economic Cooperation and Development (2009). OECD Guideline for

Testing of Chemicals: Fish Short Term Reproduction Assay. OECD 229. Adopted 7 September 2009.

USEPA (2009). Endocrine Disrupter Screening Program Test Guidelines, OCSPP 890.1350: Fish ShortTerm Reproduction Assay. EPA 740-C-09-007, October 2009.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 52 of 78APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpointMonotonic?Parametric?EDSPEFEDCommentsFemale body weightNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale body lengthNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale VTGYesYesJonckheere:Dose 3 p=0.040William’s:n.s. p>0.05EDSP and EFED conclusions differ, 36.5% significantreduction at the 0.876 mga.i./L level, according toOCSPP 890.1350 flowchart guidance.Male VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale GSINoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale GSINoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale tuberclescoreNANANANANo scoreData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-PhenylphenolEPA MRID Number 48672606Page 53 of 78EndpointMonotonic?Parametric?EDSPEFEDCommentsMale tubercle scoreNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFecundityNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFertilityNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05EDSP and EFED same conclusions, no effectF testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANAData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 54 of 78 Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 8 0 8

0.0158 8 0 8

----------------------------------------------------------------------------

TOTAL 16 0 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 8. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 8 0 8

0.0755 8 0 8

----------------------------------------------------------------------------

TOTAL 16 0 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 8. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 55 of 78 Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 8 0 8

0.876 6 2 8

----------------------------------------------------------------------------

TOTAL 14 2 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 6. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 8 0
1 0.0158 8 0
2 0.0755 8 0
3 0.876 8 2

----------------------------------------------------------------------------

Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 16 0 16

0.0158 16 0 16

----------------------------------------------------------------------------

TOTAL 32 0 32

============================================================================

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 16. Since b is greater than 11.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 56 of 78 Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 16 0 16

0.0755 16 0 16

----------------------------------------------------------------------------

TOTAL 32 0 32

============================================================================

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 16. Since b is greater than 11.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 16 0 16

0.876 11 5 16

----------------------------------------------------------------------------

TOTAL 27 5 32

============================================================================

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 11. Since b is less than or equal to 11.0 there is a significant difference
between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------- NUMBER NUMBER SIG
GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 16 0
1 0.0158 16 0
2 0.0755 16 0
3 0.876 16 5 *

----------------------------------------------------------------------------Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 57 of 78 Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

0.0158 24 0 24

----------------------------------------------------------------------------

TOTAL 48 0 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24. Since b is greater than 19.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

0.0755 24 0 24

----------------------------------------------------------------------------

TOTAL 48 0 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24. Since b is greater than 19.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 58 of 78 Fisher's Exact Test

============================================================================ NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

0.876 17 7 24

----------------------------------------------------------------------------

TOTAL 41 7 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 17. Since b is less than or equal to 19.0 there is a significant difference
between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 24 0
1 0.0158 24 0
2 0.0755 24 0
3 0.876 24 7 *

----------------------------------------------------------------------------Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 59 of 78 test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.956 0.618 3.743 0.045 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.58 0.17 0.09 11.09 1.30, 1.86 Dose1 4 1.47 0.06 0.03 4.20 1.37, 1.57 Dose2 4 1.51 0.21 0.10 13.63 1.18, 1.84 Dose3 3 1.51 0.07 0.04 4.32 1.35, 1.67

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.59 1.36 1.77 . .
Dose1 1.50 1.38 1.51 93.34 6.66
Dose2 1.55 1.26 1.70 95.88 4.12
Dose3 1.51 1.44 1.57 95.51 4.49

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.35 0.789

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.58 . 1.58 . . . . . .
Dose1 1.47 0.640 1.50 0.271 . . . . .
Dose2 1.51 0.870 1.50 0.289 0.980 . . . .
Dose3 1.51 0.868 1.50 0.322 0.990 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.32 0.723

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1.59 . .
Dose1 1.50 0.343 0.123
Dose2 1.55 0.889 0.279
Dose3 1.51 0.615 0.359

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 60 of 78 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.35 0.789

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.58 . -1.52 . . . . . .
Dose1 -1.47 0.640 -1.52 0.794 . . . . .
Dose2 -1.51 0.870 -1.52 0.825 0.980 . . . .
Dose3 -1.51 0.868 -1.52 0.830 0.990 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.32 0.723

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1.59 . .
Dose1 -1.50 0.343 0.877
Dose2 -1.55 0.889 0.721
Dose3 -1.51 0.615 0.641

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.884 0.054 0.624 0.614 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.00 0.34 0.17 11.32 2.46, 3.54 Dose1 4 2.48 0.41 0.21 16.67 1.82, 3.14 Dose2 4 2.36 0.50 0.25 21.23 1.56, 3.15 Dose3 3 2.54 0.53 0.31 20.78 1.23, 3.86

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 2.93 2.70 3.45 . .
Dose1 2.55 1.92 2.91 82.67 17.33
Dose2 2.41 1.79 2.81 78.50 21.50Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 61 of 78 Dose3 2.68 1.96 2.99 84.78 15.22

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.60 0.245

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.00 . 3.00 . . . . . .
Dose1 2.48 0.282 2.48 0.074 . . . . .
Dose2 2.36 0.153 2.44 0.062 0.978 . . . .
Dose3 2.54 0.432 2.44 0.080 0.998 0.943 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.05 0.256

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 2.93 . .
Dose1 2.55 0.156 0.042
Dose2 2.41 0.235 0.039
Dose3 2.68 0.262 0.091

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.60 0.245

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -3.00 . -2.60 . . . . . .
Dose1 -2.48 0.282 -2.60 0.938 . . . . .
Dose2 -2.36 0.153 -2.60 0.953 0.978 . . . .
Dose3 -2.54 0.432 -2.60 0.952 0.998 0.943 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-valueData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 62 of 78 3 4.05 0.256

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -2.93 . .
Dose1 -2.55 0.156 0.958
Dose2 -2.41 0.235 0.961
Dose3 -2.68 0.262 0.909

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.969 0.841 5.063 0.019 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 41.58 1.70 0.85 4.08 38.88, 44.27 Dose1 4 40.60 0.80 0.40 1.98 39.32, 41.88 Dose2 4 40.40 2.20 1.10 5.45 36.90, 43.90 Dose3 3 41.07 0.25 0.15 0.61 40.44, 41.69

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 41.35 40.10 43.50 . .
Dose1 40.75 39.60 41.30 97.65 2.35
Dose2 40.90 37.50 42.30 97.17 2.83
Dose3 41.10 40.80 41.30 98.78 1.22

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.47 0.706

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 41.58 . 41.58 . . . . . .
Dose1 40.60 0.701 40.65 0.244 . . . . .
Dose2 40.40 0.582 40.65 0.261 0.998 . . . .
Dose3 41.07 0.947 40.65 0.293 0.977 0.937 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groupsData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 63 of 78 Degrees of Freedom TestStat P-value
3 0.85 0.836

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 41.35 . .
Dose1 40.75 0.678 0.282
Dose2 40.90 0.346 0.232
Dose3 41.10 1.000 0.359

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.47 0.706

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -41.58 . -40.86 . . . . . .
Dose1 -40.60 0.701 -40.86 0.822 . . . . .
Dose2 -40.40 0.582 -40.86 0.852 0.998 . . . .
Dose3 -41.07 0.947 -41.07 0.803 0.977 0.937 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.85 0.836

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -41.35 . .
Dose1 -40.75 0.678 0.718
Dose2 -40.90 0.346 0.768
Dose3 -41.10 1.000 0.641

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes ConclusionData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 64 of 78 Test Stat P-value Test Stat P-value
0.969 0.846 0.642 0.604 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 50.23 1.68 0.84 3.34 47.56, 52.89 Dose1 4 47.80 3.02 1.51 6.32 42.99, 52.61 Dose2 4 47.05 2.33 1.17 4.95 43.34, 50.76 Dose3 3 48.93 1.64 0.95 3.36 44.85, 53.02

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 50.55 47.90 51.90 . .
Dose1 48.30 43.70 50.90 95.17 4.83
Dose2 46.95 44.90 49.40 93.68 6.32
Dose3 48.30 47.70 50.80 97.43 2.57

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.45 0.280

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 50.23 . 50.23 . . . . . .
Dose1 47.80 0.353 47.84 0.099 . . . . .
Dose2 47.05 0.177 47.84 0.105 0.965 . . . .
Dose3 48.93 0.807 47.84 0.129 0.914 0.709 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.93 0.403

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 50.55 . .
Dose1 48.30 0.346 0.124
Dose2 46.95 0.156 0.054
Dose3 48.30 0.615 0.129

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.45 0.280

Dunnett - testing each trt mean signif. different than controlData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 65 of 78 Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -50.23 . -48.36 . . . . . .
Dose1 -47.80 0.353 -48.36 0.921 . . . . .
Dose2 -47.05 0.177 -48.36 0.940 0.965 . . . .
Dose3 -48.93 0.807 -48.93 0.883 0.914 0.709 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.93 0.403

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -50.55 . .
Dose1 -48.30 0.346 0.876
Dose2 -46.95 0.156 0.946
Dose3 -48.30 0.615 0.871

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.885 0.056 1.943 0.181 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 66325000 22842705 11421352 34.44 29977159,1.0267E8 Dose1 4 71700000 18628115 9314057 25.98 42058512,1.0134E8 Dose2 4 60775000 29537702 14768851 48.60 13773925,1.0778E8 Dose3 3 42100000 5500000 3175426 13.06 28437243,55762757

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 65300000 41500000 93200000 . .
Dose1 63650000 60200000 99300000 108.10 -8.10
Dose2 46900000 44300000 1.05E8 91.63 8.37
Dose3 42100000 36600000 47600000 63.48 36.52

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.14 0.376Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 66 of 78 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 66325000 . 69012500 . . . . . .
Dose171700000 0.972 69012500 0.654 . . . . .
Dose260775000 0.970 60775000 0.460 0.893 . . . .
Dose342100000 0.379 42100000 0.114 0.337 0.688 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 5.19 0.158

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 65300000 . .
Dose1 63650000 0.678 0.718
Dose2 46900000 0.889 0.330
Dose3 42100000 0.262 0.040

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences)
Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.14 0.376

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -6.633E7 . -6.143E7 . . . . . .
Dose1 -7.17E7 0.972 -6.143E7 0.709 . . . . .
Dose2-6.078E7 0.970 -6.143E7 0.743 0.893 . . . .
Dose3 -4.21E7 0.379 -6.143E7 0.753 0.337 0.688 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 5.19 0.158

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -6.53E7 . .
Dose1 -6.365E7 0.678 0.282Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 67 of 78 Dose2 -4.69E7 0.889 0.670
Dose3 -4.21E7 0.262 0.960

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.957 0.642 3.039 0.075 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 452.75 173.93 86.97 38.42 175.98, 729.52 Dose1 4 1224.48 1166.91 583.45 95.30 -632.33, 3081.28 Dose2 4 867.75 766.03 383.02 88.28 -351.18, 2086.68 Dose3 3 528.00 188.80 109.00 35.76 59.00, 997.00

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 464.00 251.00 632.00 . .
Dose1 1051.50 4.90 2790.00 270.45 -170.45
Dose2 903.50 64.00 1600.00 191.66 -91.66
Dose3 558.00 326.00 700.00 116.62 -16.62

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.88 0.482

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 452.75 . 848.33 . . . . . .
Dose1 1224.48 0.364 848.33 0.845 . . . . .
Dose2 867.75 0.775 848.33 0.872 0.902 . . . .
Dose3 528.00 0.998 528.00 0.692 0.620 0.929 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.30 0.730

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 464.00 . .Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 68 of 78 Dose1 1051.50 0.346 0.876
Dose2 903.50 0.889 0.768
Dose3 558.00 0.733 0.601

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.88 0.482

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -452.75 . -452.75 . . . . . .
Dose1-1224.48 0.364 -904.81 0.242 . . . . .
Dose2 -867.75 0.775 -904.81 0.259 0.902 . . . .
Dose3 -528.00 0.998 -904.81 0.291 0.620 0.929 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.30 0.730

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -464.00 . .
Dose1 -1051.50 0.346 0.124
Dose2 -903.50 0.889 0.232
Dose3 -558.00 0.733 0.399

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.939 0.374 1.417 0.290 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 15.73 4.44 2.22 28.21 8.67, 22.79 Dose1 4 13.46 2.48 1.24 18.44 9.51, 17.41Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 69 of 78 Dose2 4 14.12 3.97 1.98 28.12 7.80, 20.43 Dose3 3 13.06 2.11 1.22 16.15 7.82, 18.30

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 14.49 12.08 21.85 . .
Dose1 13.46 11.24 15.68 85.57 14.43
Dose2 14.25 9.91 18.05 89.75 10.25
Dose3 12.32 11.42 15.44 83.04 16.96

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.42 0.740

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 15.73 . 15.73 . . . . . .
Dose1 13.46 0.695 13.79 0.267 . . . . .
Dose2 14.12 0.854 13.79 0.286 0.993 . . . .
Dose3 13.06 0.643 13.06 0.223 0.999 0.978 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.74 0.628

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 14.49 . .
Dose1 13.46 0.346 0.124
Dose2 14.25 0.678 0.279
Dose3 12.32 0.411 0.205

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.42 0.740

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -15.73 . -14.16 . . . . . .
Dose1 -13.46 0.695 -14.16 0.813 . . . . .Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 70 of 78 Dose2 -14.12 0.854 -14.16 0.843 0.993 . . . .
Dose3 -13.06 0.643 -14.16 0.847 0.999 0.978 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 1.74 0.628

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -14.49 . .
Dose1 -13.46 0.346 0.876
Dose2 -14.25 0.678 0.721
Dose3 -12.32 0.411 0.795

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.906 0.116 2.067 0.163 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.31 0.30 0.15 23.16 0.82, 1.79 Dose1 4 1.20 0.23 0.11 19.06 0.83, 1.56 Dose2 4 1.32 0.17 0.09 12.99 1.04, 1.59 Dose3 3 1.09 0.20 0.12 18.66 0.59, 1.60

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.31 1.01 1.59 . .
Dose1 1.17 0.97 1.48 91.57 8.43
Dose2 1.26 1.18 1.56 100.77 -0.77
Dose3 1.14 0.87 1.27 83.78 16.22

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.68 0.581

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.31 . 1.31 . . . . . .Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 71 of 78 Dose1 1.20 0.848 1.26 0.455 . . . . .
Dose2 1.32 1.000 1.26 0.484 0.885 . . . .
Dose3 1.09 0.526 1.09 0.171 0.939 0.615 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.19 0.533

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1.31 . .
Dose1 1.17 0.494 0.193
Dose2 1.26 0.889 0.558
Dose3 1.14 0.615 0.252

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.68 0.581

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.31 . -1.24 . . . . . .
Dose1 -1.20 0.848 -1.24 0.745 . . . . .
Dose2 -1.32 1.000 -1.24 0.778 0.885 . . . .
Dose3 -1.09 0.526 -1.24 0.786 0.939 0.615 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.19 0.533

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1.31 . .
Dose1 -1.17 0.494 0.807
Dose2 -1.26 0.889 0.442
Dose3 -1.14 0.615 0.748

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenolData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 72 of 78 ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.00 0.00 0.00 . .
Dose1 0.00 0.00 0.00 . .
Dose2 0.00 0.00 0.00 . .
Dose3 0.00 0.00 0.00 . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
. . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . .
Dose1 0.00 . . . . . . . .
Dose2 0.00 . . . . . . . .
Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.00 . .
Dose1 0.00 1.000 .
Dose2 0.00 1.000 .
Dose3 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1
Jonckheere Dose1Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 73 of 78 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
. . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . .
Dose1 0.00 . . . . . . . .
Dose2 0.00 . . . . . . . .
Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.00 . .
Dose1 0.00 1.000 .
Dose2 0.00 1.000 .
Dose3 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1
Jonckheere Dose1

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.971 0.875 1.850 0.197 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 16.25 4.63 2.31 28.48 8.89, 23.61 Dose1 4 14.75 3.48 1.74 23.57 9.22, 20.28 Dose2 4 20.00 8.03 4.02 40.16 7.22, 32.78 Dose3 3 17.00 9.50 5.48 55.88 -6.60, 40.60

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 14.50 13.00 23.00 . .
Dose1 15.50 10.00 18.00 90.77 9.23Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 74 of 78 Dose2 19.25 12.50 29.00 123.08 -23.08
Dose3 17.00 7.50 26.50 104.62 -4.62

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.45 0.721

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 16.25 . 17.00 . . . . . .
Dose1 14.75 0.977 17.00 0.650 . . . . .
Dose2 20.00 0.766 17.00 0.685 0.679 . . . .
Dose3 17.00 0.998 17.00 0.699 0.969 0.931 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.55 0.907

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 14.50 . .
Dose1 15.50 1.000 0.500
Dose2 19.25 0.678 0.721
Dose3 17.00 0.866 0.696

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 0.45 0.721

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -16.25 . -15.50 . . . . . .
Dose1 -14.75 0.977 -15.50 0.650 . . . . .
Dose2 -20.00 0.766 -18.71 0.386 0.679 . . . .
Dose3 -17.00 0.998 -18.71 0.417 0.969 0.931 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groupsData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 75 of 78 Degrees of Freedom TestStat P-value
3 0.55 0.907

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -14.50 . .
Dose1 -15.50 1.000 0.500
Dose2 -19.25 0.678 0.279
Dose3 -17.00 0.866 0.304

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.983 0.986 3.160 0.068 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 13.90 6.59 3.29 47.38 3.42, 24.38 Dose1 4 13.85 1.35 0.67 9.73 11.71, 15.99 Dose2 4 20.95 8.90 4.45 42.47 6.79, 35.11 Dose3 3 7.30 6.38 3.68 87.38 -8.55, 23.15

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 14.10 7.30 20.10 . .
Dose1 14.35 11.90 14.80 99.64 0.36
Dose2 21.50 9.80 31.00 150.72 -50.72
Dose3 10.10 0.00 11.80 52.52 47.48

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 2.62 0.103

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 13.90 . 16.23 . . . . . .
Dose1 13.85 1.000 16.23 0.776 . . . . .
Dose2 20.95 0.328 16.23 0.809 0.437 . . . .
Dose3 7.30 0.434 7.30 0.134 0.562 0.073 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all testsData Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 76 of 78 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.68 0.197

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 14.10 . .
Dose1 14.35 1.000 0.500
Dose2 21.50 0.235 0.906
Dose3 10.10 0.615 0.379

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 2.62 0.103

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -13.90 . -13.88 . . . . . .
Dose1 -13.85 1.000 -13.88 0.586 . . . . .
Dose2 -20.95 0.328 -15.10 0.502 0.437 . . . .
Dose3 -7.30 0.434 -15.10 0.527 0.562 0.073 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.68 0.197

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -14.10 . .
Dose1 -14.35 1.000 0.500
Dose2 -21.50 0.235 0.094
Dose3 -10.10 0.615 0.621

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - O phenyl phenol ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 77 of 78 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.663 <.001 21.414 <.001 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 97.20 3.40 1.70 3.49 91.80, 102.60 Dose1 4 97.95 2.04 1.02 2.08 94.70, 101.20 Dose2 4 98.85 0.83 0.42 0.84 97.52, 100.18 Dose3 3 66.00 57.16 33.00 86.61 -76.00, 208.00

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 98.35 92.40 99.70 . .
Dose1 98.00 95.90 99.90 100.77 -0.77
Dose2 98.95 97.90 99.60 101.70 -1.70
Dose3 98.20 0.00 99.80 67.90 32.10

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.37 0.302

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 97.20 . 98.00 . . . . . .
Dose1 97.95 1.000 98.00 0.603 . . . . .
Dose2 98.85 0.999 98.00 0.637 1.000 . . . .
Dose3 66.00 0.277 66.00 0.079 0.364 0.342 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.32 0.956

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 98.35 . .
Dose1 98.00 1.000 0.558
Dose2 98.95 0.780 0.697
Dose3 98.20 1.000 0.562

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 11 1.37 0.302Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol

EPA MRID Number 48672606

Page 78 of 78 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -97.20 . -91.60 . . . . . .
Dose1 -97.95 1.000 -91.60 0.712 . . . . .
Dose2 -98.85 0.999 -91.60 0.746 1.000 . . . .
Dose3 -66.00 0.277 -91.60 0.756 0.364 0.342 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.32 0.956

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -98.35 . .
Dose1 -98.00 1.000 0.442
Dose2 -98.95 0.780 0.303
Dose3 -98.20 1.000 0.438

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

# DATA EVALUATION RECORD

ORTHO-PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672602)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Kelly Luck, M.S. Date: 05/07/2012

Secondary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 05/14/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/24/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/24/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).ORTHO-PHENYLPHENOL/ 064103



### Primary Reviewer: Ayaad Assaad, D.V

. M., Ph.D.


### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division In vivo Hershberger Assay (2012) I Page 1 of 16
OCSPP 890.1400/ OECD 441



### Signature:
Date: Signature:
Date: 

# ()**/j ~,;~** 

# ~~15**~I/** l 

# It < Template version i0/2011



### DATA EVALUATION RECORD

STUDY TYPE: In Vivo Hershberger Assay (Rat) ; OCSPP 890.1400; OECD 441



### PC CODE: 064103 

### DP BARCODE: D398785



### TXR#: 0055304 

### CAS#: 90-43-7



### TEST MATERIAL (PURITY): Ortho-phenylphenol (99.9% a.i.)



### SYNONYMS: OPP; Prevento! 

### 0 Extra ; (1

, 1 ' -Biphenyl)-2-ol



### CITATION: Marty

, M. S

. , Marshall

, V. A., and Jeong, Y. 

### C. (2012). Ortho-phenylphenol: Hershberger assay in castrated male Crl:CD(SD) rats. Toxicology &


### Environmental Research and Consulting

, Dow Chemical Company, Midland

, MI. Laboratory Project Study ID: 111084

, January 5, 2012. MRID 48672602. Unpublished.



### SPONSOR: OPP E DSP Consortium: Dow Microbial Control

, The Dow Chemical Company,
Midland

, MI ; and LANX E SS Corporation

, Materials Protection Products

, 111
RIDC Park West Drive

, Pittsburgh

, PA



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48672602) screening for androgenic activity, ortho-phenylphenol (OPP; 99.9% a.i.

, Lot # CHHYDD0051) in com oil was administered dail y 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 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

, OPP (99.9% a.i.

, batch/lot # CHHYDD0051) in

com 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 dail y dose of reference androgen TP at 0.4 mg / kg / day by s.c. injection. The anti-androgenic positi v e 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 negati v e control.

For both component s of the assay , body weights and food consumption were determined daily.
The animals were dosed for 10 consecutive da y s and terminated approximately 24 hours after the
final dose admini s tration

. At necropsy , the five androgen-dependent tissues

, adrenal glands

, kidney s, and liver were collected and weighed.In vivo Hershberger Assay (201 2) / Page 2 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### All animals survived until sc heduled termination. One anim al in the high dose group was observed to have mottled lungs at necropsy, indicati ng a gavage error; therefore, the results for this animal were excluded from the study. Th ere were no treatment-related clinical signs in animals dosed at ≤ 250 mg/kg/day in either assay. In bot h assays, the majority of high-dose animals (6/7 androgen assay; 5/7 in anti-andr ogen assay) had some or all of the following observations: clear or red peri oral soiling, perineal soiling (u rine 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 assa y, 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 wei ghts 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 gain s 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 trea tment-related effects onweights of the accessory sex organs/tissues, adrenal glands, kidneys, or liver for any OPPtreatment group. Animals in the TP group had increased (p<0.05) accessory sex organ weightsas 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.

In the anti-androgen assay, body weights and body we ight gains in the OPP treatment groups and positive control group were comparable to the nega tive control over the duration of the study. In the 1,000 mg/kg/day group, food consumption was d ecreased (p<0.05) by 19% during Days 4-7. Food consumption in the 50 and 250 mg/kg/day groups and the positiv e 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 th e weights of the seminal vesicles, LABC, Cowper’s glands, glans penis, adrenal glands, ki dneys, or liver for any OPP-trea tment 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 Cowpe r’s gland; and 18% in glans penis. Adrenal glands, kidney, and liv er 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. A ll %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 assa y (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%).

Statis tically significant changes were not seen in two or more of the five androgen sensitive tissue weights. OPP was negative for androgenici ty and anti-androgenicity in the Hershberger

assay.In vivo Hershberger Assay (201 2) / Page 3 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### The assay satisfies the EDSP Tier 1 Test Order require ments for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.In vivo Hershberger Assay (201 2) / Page 4 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, Dow Chemical Company

Location: Midland, MI Study Director: V. A. Marshall Other Personnel: M. S. Marty (Lead Scientist) and YC. Jeong (D.V.M.) Study Period: May 9, 2011 to January 5, 2012



### 2. Test Substance: Ortho-phenylphenol (OPP) Description: Molecular weight 170.2 g/mol Source: Lanxess Deutschland GmbH (Leverkusen, Germany) Lot #: CHHYDD0051 (expiration date not reported) Purity: 99.9% Stability: Stable in corn oil for 15 days at room temperature CAS #: 90-43-7 Structure: OH



### 3. Reference Androgen: Testosterone propionate (TP) Supplier: Sigma Aldrich (St. Louis, MO) Lot #: 048K1328 (expiration date not reported) Purity: ≥ 98% CAS #: 57-85-2



### 4. Reference Anti-androgen: Flutamide (FT) Supplier: Sigma Aldrich (St. Louis, MO) Lot #: 099K1112 (expiration date not reported) Purity: >99% CAS #: 13311-84-7



### 5. Solvent/Vehicle Control: Corn oil Supplier: Sigma Aldrich (St. Louis, MO) Lot/Batch #: Not provided Rationale (if other than water): Selected due to solubility properties of the test substance Final concentration: Not applicableIn vivo Hershberger Assay (201 2) / Page 5 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### 6. Test Animals: Species: Rat (castrated males only) Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: Post-natal day (PND) 55/ 197.1 – 308.8 g Source: Charles River (Portage, MI) Housing: Rats were housed 2-3 per cage in stainl ess steel cages with wire mesh floors suspended above absorbent paper. Diet: LabDiet Certified Rodent Di et #5002 (PMI Nutrition In ternational, St. Louis, MO) , ad libitum Phytoestrogen content was not reported
Water: Municipal drinking water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22  3 oC 40-70% 12-15/hr (average) 12 hrs light/ 12 hrs dark Acclimation period: Rats were castrated at the supplier and received at the facility 4-5 days postcastration; rats were then allowed a 5- to 6-day acclimation period at the facility, such that dosing was in itiated 10 days post-castration



### B. STUDY DESIGN

1. In life dates: Start: June 18, 2011 End: June 29, 2011

2. Study Design: In a Hershberger Assay conducted to screen for pot ential androgenic activity, OPP was administered daily via oral gavage to castrated male rats. A positive control group received the vehicle plus (TP) by s.c. injection. Positive androgenic activity is defined as a significant increase in two or mo re target organ weights compared to the vehicle control. To screen for the pote ntial anti-androgenic ac tivity, OPP was also administered daily via oral gavage to castrated male rats in conjuncti on with a daily dose of TP (0.4 mg/kg/day) by s.c. injection. The pos itive control group rece ived flutamide (FT) plus TP. Anti-androgenic activity is indicated by a statistical ly significant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP-only control group. For both assays, the an imals were dosed for 10 consecutive days and necropsied approximately 24 hours after th e final dose administration for organ weight

measurements.

3. Study Schedule: Male rats were castrated on PND 45 at the supplier and were received at the test facility 4 or 5 days later, at PND 49 or 50. The animals were acclimated at the facility for 5-6 days prior to initiation of dosing. The dose administration period was from PND 55 through 64. Rats were euthanized on PND 65 approximately 24 hours after the last dose and necropsied for organ weight measurements.

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1 using a computer program designed to increase the probability of uniform group mean weights and standard devi ations at the start of the study. Statistical analysis indicated that th ere were no significant differe nces in group means at study initiation. However, for two of the dosing groups (50 and 1000 mg/kg/day groups in the androgen agonist assay), the weight for one anim al in the group was greater than +20% of the group mean (animal #4296 weighed 306.9 g, with a group mean weight of 242.0 g; and animal #4311 weighed 308.8 g, with a group mean weight of 245.7 g).In vivo Hershberger Assay (201 2) / Page 6 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (negative control) 0 7 Low (OPP) 50 7 Mid (OPP) 250 7 High (OPP) 1000 7 Testosterone propionate (TP), positive control b 0.4 7 Anti-Androgen Assay Low (OPP + TP) 50 + 0.4 7 Mid (OPP + TP) 250 + 0.4 7 High (OPP + TP) 1000 + 0.4 7 Flutamide + TP, positive control 3 + 0.4 7 a Data were obtained from Text Table 1 on page 17 of the study report. b This dose group served as the positive control for the androgen agonist assay and the negative control for the anti-androgen assay.



### 5. Dose Selection Rationale: The high dose level was selected based on the results from a preliminary probe study 1 

### in which four Crl:CD(SD) rats/sex/dose were dosed at 0, 250, 500, 700, or 900 mg/kg/day by oral gavage for 15 da ys. For males in the highest dose group, there was a 5-7% decrease in body weight throughout most of the dose period; body weight gains were 10-26% lower in this group across the intervals evaluated. The body weight gain differential between the high-dose males and the control group d ecreased as the study progressed (at the end of the study, gains were 10% less than controls). Feed consumption was decreased by 6-12% on study days 1-6 in high-dose males, but this decrease was ≤ 4% of control values thereafter. There were no ma rked effects on clinical chemistry parameters and electrolytes. Relative liver weights were increased in males in the 700 and 900 mg/kg/day groups (11-16%). The high dose level of 1000 mg/kg/day selected for the current study was expected to produce some syst emic toxicity but was not expected to cause a greater than 10% decrease in terminal body weight compared to the control group. The lower dose levels were selected to provide dose response data for any treatment-related effects that may have been observe d among the high-dose group rats.

6. (a) Dose Preparation: Dose formulations were prepar ed once at the beginning of the study based on the established stability of the test substance in corn oil. Homogeneity and achieved concentration were tested prior to st udy initiation in each of the dose formulations by sampling the top, middle, and bottom portions. The stability of OPP in corn oil during ambient temperature storage was tested at concentrations of 0. 25, 2.50, and 250 mg/mL.

( b) Dose Analysis:

Results

Homogeneity (%CV): 0.6-4.9%

1 Marty, M. S. and Marshall, V. A. (In progress). Or thophenylphenol: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxico logy & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.In vivo Hershberger Assay (201 2) / Page 7 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### Stability(% of Day 1): 99.7-101.5% after 8 days and 104.2-106.1% after 15 days storage at ambient temperature

Concentration (% of nominal): 91.0-95.5%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage administration: Test formulations were administ ered to the animals daily via oral gavage (4 mL/kg bw) for 10 days. TP was give n via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gava ge at 4 mL/kg bw. Dose volum es were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Body weights, body weight gains, absolu te organ weights, and feed consumption were evaluated using Bartlett’s test for equal ity of variances. Based on the outcome of the Bartlett’s test (p=0.01), a pa rametric or non-parametric an alysis of variance (ANOVA) was performed. If the ANOVA was significant, analys is by the one-sided Dunnett’s test or the Wilcoxon Rank-Sum test with Bonferroni’s co rrection was performe d. Significance was designated at p ≤ 0.05. The statistical analyses were considered adequate.

Statistical outliers were iden tified using a sequential me thod (p=0.02) and excluded if justified by sound scientific reasons.

C. METHODS

1. Clinical Examinations: During dosing, cage-side checks fo r mortality and morbidity were conducted twice daily and hand-held examinati ons for clinical signs of toxicity were conducted once daily.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout the dosing period, and at termination. St atistical analyses of body wei ghts were performed on Days 1, 4, 7, and 11.

3. Food Consumption: Food consumption was measured and statistically analyzed for the following intervals: Days 1-4, Days 4-7, and Days 7-11. Valu es were measured for each cage and reported as group mean da ily food consumption (g/animal/day).

4. Serum Hormone Measurements (Optional): At study termination, animals, except those dosed with FT, were anesthetized with isoflurane/oxygen and blood was collected via cardiac puncture for potential future serum hormone analyses; however, serum hormone analyses were not condu cted for this study.

5. Dissection and Measurement of Tissue and Organ Weights: On PND 65 (approximately 24 hours after final administration of the test substance), all animals were checked for preputial separation, anesthetized using isof lurane/oxygen (all animals except those dosed with FT) for blood collection, euthanized by deca pitation, and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesicles, LABC,In vivo Hershberger Assay (201 2) / Page 8 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### Cowper’s gland, and glans penis) were ex cised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standa rd operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Additionally, th e liver, kidneys, and adrenals were weighed. Tissues except liver were preserved in neutral, phosphate-bu ffered 10% formalin. Three sections of the liver we re preserved in neutral, phosphate-buffered 10% formalin for possible future histological ev aluation and a portion of the remainder was quick frozen in liquid nitrogen for possible future evaluation of liver enzyme induction.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination.

2. Clinical signs of toxicity: There were no treatment-related clinical signs in animals dosed

at ≤ 250 mg/kg/day in either assay.

The incidences of clinical observations in the 1,000 mg/kg/day group in the androgen agonist assay are presented in Table 2. At the high dose, six of seven animals had some or all of the following observations: clear or red perioral soiling, perineal soiling (urine and/or feces), decreased activity, noisy respirati on, and soft feces. The incidence of these observations decreased as the study progressed. In addition, one animal was noted as cold to touch (Day 2) and anot her had red urine (Day 7).

TABLE 2. Incidence of Clinical Observations in th e 1000 mg/kg/day Group in the Androgen Agonist Assay a

Observation Study Day Interval Days 1-4 Days 4-7 Days 7-11
# Observed # Examined # Observed # Examined # Observed # Examined
Perioral (clear) soiling 4 7 2 7 1 7 Perineal (urine/feces) soiling 3 7 2 7 0 7

Perioral (red) soiling 2 7 0 7 0 7
Decreased activity 2 7 2 7 0 7 Noisy respiration 2 7 1 7 2 7
Soft feces 1 7 0 7 0 7
Cold to touch 1 7 0 7 0 7
Red urine 0 7 1 7 0 7 a Data were obtained from Text Table 3 on page 26 of the study report.



### The incidences of clinical observations in the 1,000 mg/kg/day group in the anti-androgen assay are presented in Table 3. At the high dose, five animals had some or all of the following observations: clear or red perioral soiling, perineal soiling (urine and/or feces), decreased activity, and noisy respiration. Many of these observations were noted in the later portion of the study (Days 7-11). In addition, on e animal had slow respiration (Days 9 and 10) and perinasal soiling (Day 11).In vivo Hershberger Assay (201 2) / Page 9 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### One animal in the 1,000 mg/kg/day group (Ani mal # 4346) had clinical signs including decreased activity, noisy, labored, and/or slow respiration, sneezing, a nd perioral/perineal soiling in the initial (Days 1-4) and final (Days 7-11) portions of the study. At necropsy, this animal was observed to have mottled lungs , indicating a gavage error. The results for this animal were excluded from the study.

TABLE 3. Incidence of Clinical Observations in th e 1000 mg/kg/day Group in the Anti-Androgen Assay a

Observation Study Day Interval Days 1-4 Days 4-7 Days 7-11
# Observed # Examined # Observed # Examined # Observed # Examined
Perioral (clear) soiling 0 6 0 6 2 6
Perioral (red) soiling 1 6 1 6 0 6 Perineal (urine/feces) soiling 1 6 1 6 1 6

Decreased activity 1 6 0 6 1 6 Noisy respiration 1 6 1 6 1 6
Slow respiration 0 6 0 6 1 6
Perinasal (red) soiling 0 6 0 6 1 6 a Data were obtained from Text Table 4 on page 27 of the study report.



### B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are presen ted in Table 4. Body we ights 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 st udy were similar to controls. Animals in this group lost body weight over Days 1-7 but gained weight in the later portion of the study, such that mean overall body weight gain (Days 1-11) was not significantly different from the controls. Body weights and body weight gains in the 50 and 250 mg/kg/day groups were comparable to controls throughout the duration of the st udy. 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%).

There was inter-animal variability in respons e to OPP treatment. Three animals in the 1,000 mg/kg/day group gained <60% of the mean body weight gain in the control group; two of these animals had decreased terminal body weights ( ↓ 16% and 18%) relative to the control group. One animal, which had the highest body weight on Day 1, lost weight over the dosing period. The remaining three animal s gained 67-114% of the mean control body weight gains over the dosing peri od and had terminal body weights ≥ 92% of the control

group.In vivo Hershberger Assay (201 2) / Page 10 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 4. Selected Group Mean Body Weights and Cumulati ve Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) OPP (50) OPP (250) OPP (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 7 241.1 24.3 7 243.8 27.7 7 242.0 34.0 7 243.8 23.7 7 245.7 32.6
4 7 259.2 24.1 7 270.1 33.8 7 259.2 29.5 7 263.8 24.5 7 239.4 35.6
7 7 274.2 26.4 7 293.4 34.1 7 276.4 29.9 7 282.1 25.1 7 232.1* ( ↓ 15) 34.0

11 7 296.5 30.8 7 319.0 40.9 7 302.9 36.4 7 311.3 29.8 7 278.3 28.0

B ody Weight Gain (Days 1-4) 7 18.1 6.8 7 26.3* ( ↑ 45) 6.7 7 17.1 5.8 7 20.0 5.6 7

− 6.3 34.5

B ody Weight Gain (Days 1-7) 7 33.1 13.0 7 49.6* ( ↑ 50) 10.4 7 34.4 10.7 7 38.3 14.5 7

− 13.6 44.9

B ody Weight Gain (Days 1-11) 7 55.4 14.5 7 75.2* ( ↑ 36) 16.7 7 60.8 11.7 7 67.5 20.0 7 32.7 25.9

a Data were obtained from Table 4 on pages 41 and 42 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### Selected body weight and body weight gain data for the anti-androgen assay are presented in Table 5. Body weights and body weight gains in the OPP treatmen t groups and positive control group were comparable to the negative control over the durat ion of the study.

As for the androgen agonist assay, there was inter-animal variability in response to OPP treatment. Two animals in the 1,000 mg/kg/ day group had decreased terminal body weights ( ↓ 16% and 52%) relative to the control group; th ese animals gained <52% of the mean body weight gain in the control group. The remaini ng four animals gained 80-111% of the mean control body weight gains over the dosi ng period and had terminal body weights ≥ 91% of the control group.In vivo Hershberger Assay (201 2) / Page 11 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 5. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Anti-Androgen Assay a

Study Day # Dose (mg/kg/day)
Vehicle Control + TP (0.4) Positive Control TP + FT (0.4 + 3) OPP + TP (50 + 0.4) OPP + TP (250 + 0.4) OPP + TP (1000 + 0.4)
N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 7 243.8 27.7 7 243.0 23.2 7 246.4 23.7 7 243.4 24.7 6 241.9 30.2
4 7 270.1 33.8 7 269.2 26.5 7 273.8 22.4 7 267.6 25.1 6 259.1 26.8
7 7 293.4 34.1 7 288.6 25.0 7 299.9 16.6 7 290.8 26.2 6 272.8 41.3
11 7 319.0 40.9 7 319.4 30.9 7 338.4 23.3 7 323.9 31.2 6 296.4 51.8

B ody Weight Gain (Days 1-4) 7 26.3 6.7 7 26.2 4.3 7 27.4 2.6 7 24.2 5.0 6 17.2 16.2

B ody Weight Gain (Days 1-7) 7 49.6 10.4 7 45.6 6.9 7 53.5 11.9 7 47.4 9.1 6 30.9 21.9

B ody Weight Gain (Days 1-11) 7 75.2 16.7 7 76.4 10.2 7 92.0 10.8 7 80.5 11.5 6 54.5 25.4

a Data were obtained from Table 4 on pages 43 and 44 of the study report. N Number of animals in the group SD Standard Deviation



### C. FOOD CONSUMPTION: Food consumption data for the androgen agonist assay are presented in Table 6. In the 1,000 mg/kg/ day group, food consumption was decreased (p<0.05) by 42% during Days 4-7. Food cons umption was increased (p<0.05) in the 50 mg/kg/day group on Days 1-4 ( ↑ 6%) and in the positive control group on Days 1-4 ( ↑ 14%), Days 4-7 ( ↑ 14%), and Days 7-11 ( ↑ 12%).

TABLE 6. Food Consumption (g/animal/d ay) in the Androgen Agonist Assay a

Study Day

Interval Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) OPP (50) OPP (250) OPP (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
1-4 7 17.2 0.8 7 19.6* ( ↑ 14) 0.9 7 18.3* ( ↑ 6) 0.3 4 18.5 0.3 7 11.3 5.0

4-7 7 18.5 0.8 7 21.0* ( ↑ 14) 0.6 7 19.1 1.1 7 20.0 1.4 7 10.7* ( ↓ 42) 3.4

7-11 7 18.5 0.6 7 20.8* ( ↑ 12) 1.3 7 19.7 0.7 7 20.4 2.2 7 18.2 1.8

a Data were obtained from Table 5 on pages 45 and 46 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### Food consumption data for the anti-androgen as say are presented in Table 7. In the 1,000 mg/kg/day group, food consumption was decr eased (p<0.05) by 19% during Days 4-7. Food consumption in the 50 and 250 mg/kg/day groups and the positiv e control group was similar to controls over th e duration of the study.In vivo Hershberger Assay (201 2) / Page 12 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### In general, increases or decreases in f ood consumption corresponded with increased or decreased body weight gains in both assays.

TABLE 7. Food Consumption (g/anima l/day) in the Anti-Androgen Assay a

Study Day

Interval Dose (mg/kg/day)
Vehicle Control + TP (0.4) Positive Control TP + FT (0.4 + 3) OPP + TP (50 + 0.4) OPP + TP (250 + 0.4) OPP + TP (1000 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1-4 7 19.6 0.9 7 19.5 0.9 7 18.9 0.6 7 18.1 0.5 6 15.6 3.8
4-7 7 21.0 0.6 7 20.7 1.0 7 22.1 1.8 7 20.5 1.4 6 17.1* ( ↓ 19) 1.9

7-11 7 20.8 1.3 7 21.1 1.1 7 23.1 0.9 7 21.7 0.4 6 17.3 2.8 a Data were obtained from Table 5 on pages 47 and 48 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone measurements were not conducted.

E. ORGAN WEIGHTS: Weights for the accessory sex or gans, adrenal glands, kidneys, and liver from the androgen agonist assay are presented in Table 8. There were no treatment related effects in accessory sex organs, ad renal glands, kidneys, or liver for any OPP treatment group. Animals in the TP group had increased (p<0.05) accessory sex organ weights as follows: 1760% in seminal vesicl es; 1117% in ventral pr ostate; 226% in 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.

One animal in the vehicle control group had en larged Cowper's glands (0.0148 g) compared to the group mean weight for th is tissue (0.0050 g). This tissue weight was identified as a statistical outlier, alt hough other tissue weights from this animal were similar to control values. Because this outlier va lue increased variability for Co wper’s glands in the vehicle control group, reducing the statis tical power to identify a treatment-related effect, statistical comparisons for Cowper's glands were carried ou t with or without this animal. In either case, there was no significant treatment-r elated effect on Cowper's gland.

Percent CVs for the vehicle control and th e 1,000 mg/kg/day treatment groups were compared to the performance criteria in the Gu idelines. All %CV values were less than the maximum recommended values, with the exception of the %CV for Cowper’s glands for the control group (CV = 88%; maximum recommended CV = 55%).In vivo Hershberger Assay (201 2) / Page 13 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 8. Accessory Sex Organ We ights from Androgen Agonist Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) OPP (50) OPP (250) OPP (1000)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Seminal vesicles (mg) 7 38.2 8.2 21 7 710.4* ( ↑ 1760) 183.9 26 7 36.9 15.4 42 7 35.7 6.6 18 7 37.5 8.6 23

Ventral prostate (mg) 7 18.4 4.3 23 7 223.9* ( ↑ 1117) 39.1 17 7 15.6 2.7 17 7 16.4 3.1 19 7 17.7 3.4 19

LABC (mg) 7 191.1 37.4 20 7 622.3* ( ↑ 226) 92.3 15 7 188.9 63.0 33 7 188.3 29.7 16 7 159.2 22.8 14

Cowper’s glands (mg) 7 5.0 4.4 88 7 45.3* ( ↑ 806) 7.9 17 7 4.7 2.1 45 7 4.2 1.9 45 7 3.0 0.7 23

Glans penis (mg) 7 54.6 6.5 12 7 91.1* ( ↑ 67) 5.8 6 7 56.0 7.2 13 7 53.5 9.7 18 7 52.0 7.1 14

Adrenal glands (mg) 7 55.0 6.9 13 7 56.6 10.9 19 7 58.9 13.1 22 7 62.3 14.5 23 7 58.8 14.0 24

Kidneys (g) 7 1.934 0.178 9 7 2.128 0.312 15 7 1.975 0.239 12 7 2.069 0.257 12 7 2.173 0.449 21 Liver (g) 7 11.3 1.3 12 7 12.3 2.0 16 7 12.3 2.0 16 7 12.9 2.1 16 7 12.9 1.5 12 a Data were obtained from Table 6 on pages 49 and 50 of the stud y report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation; calculated by the reviewers * Significantly different from controls at p<0.05



### Weights for the accessory sex organs, adrenal glands, kidneys, and liver from the antiandrogen assay are presented in Table 9. At 1,000 mg/kg/day, the weights for ventral prostate were decreased (p<0.05) by 28% co mpared to the controls. There were no treatment-related effects on the weights of th e seminal vesicles, LABC, Cowper’s glands, glans penis, adrenal glands, kidneys, or liver for any OPP treatment group. Animals dosed with TP + FT (positive control) had decrea sed (p<0.05) accessory sex organ weights as follows: 83% in seminal vesicles; 77% in vent ral prostate; 46% in LABC; 61% in Cowper’s gland; and 18% in glans penis. Adrenal gl ands, kidney, and liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and th e 1,000 mg/kg/day treatment groups were compared to the performance criteria in the Gu idelines. All %CV values were less than the maximum recommended values, with the exceptio n of the %CV for LABC for the high dose group (CV = 22%; maximum recommended CV = 20%).In vivo Hershberger Assay (201 2) / Page 14 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 9. Accessory Sex Organ Weight s from Anti-Androgen Agonist Assay in Sprague Dawley or Wistar Rats a

Organ Dose (mg/kg/day)
Vehicle Control + TP (0.4) Positive Control TP + FT (0.4 + 3) OPP + TP (50 + 0.4) OPP + TP (250 + 0.4) OPP + TP (1000 + 0.4)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Seminal vesicles (mg) 7 710.4 183.9 26 7 121.1* ( ↓ 83) 55.5 46 7 588.9 116.0 20 7 603.1 138.4 23 6 627.4 110.6 18

Ventral prostate (mg) 7 223.9 39.1 17 7 51.3* ( ↓ 77) 10.8 21 7 200.8 35.4 18 7 208.0 33.4 16 6 161.1* ( ↓ 28) 30.5 19

LABC (mg) 7 622.3 92.3 15 7 336.9* ( ↓ 46) 42.0 12 7 603.4 72.2 12 7 589.3 93.4 16 6 596.7 130.8 22

Cowper’s glands (mg) 7 45.3 7.9 17 7 17.7* ( ↓ 61) 4.8 27 7 43.5 7.9 18 7 40.5 9.3 23 6 40.6 6.8 17

Glans penis (mg) 7 91.1 5.8 6 7 75.0* ( ↓ 18) 9.9 13 7 93.2 7.3 8 7 86.0 8.3 10 6 81.2 8.5 10

Adrenal glands (mg) 7 56.6 10.9 19 7 56.4 12.1 21 7 52.1 8.9 17 7 50.4 8.5 17 6 46.7 10.1 22

Kidneys (g) 7 2.128 0.312 15 7 2.086 0.221 11 7 2.163 0.137 6 7 2.162 0.181 8 6 2.232 0.263 12 Liver (g) 7 12.3 2.0 16 7 12.9 1.4 11 7 13.8 1.6 12 7 13.4 1.1 8 6 14.3 3.0 21 a Data were obtained from Table 6 on pages 51 and 52 of the stud y report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation; calculated by the reviewers * Significantly different from controls at p<0.05



### F. MACRO- AND HISTOPATHOLOGY : All animals in both the androgen and antiandrogen assays achieved preputial separation by study termination. Two animals, one each in the 1,000 mg/kg/day group (with or without TP ), were found to have thickening of the nonglandular mucosa of the stomach, which is reportedly consistent with previous observation of gastric irritati on following gavage exposure to OPP. In addition, one highdose animal in the anti-androgen assay had mottled lungs at necropsy, indicating a gavage error; the results for this animal were excl uded from the study. There were no other gross pathological findings.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There was no mortality in any study animals; however, there was notable inter-animal variabi lity in responses to high-dose OPP treatment with some animals exhibiting significant clinical signs and marked effects on body weight/body weight gains. Treatment-relate d clinical signs in the 1000 mg/kg/day OPP group included decreased activity, noisy or sl ow respiration, perior al/perinasal/perineal soiling, soft feces, red urine, and cold to touch. High-dose animals had significantly decreased body weight gains (2 8-41%) in both the androgeni c and anti-androgenic portion of the assay, resulting in 6- 7% decreases in terminal body weights. Decreases in feed consumption corresponded with decreases in body weight gains. All animals underwent preputial separation prior to the study termination. At n ecropsy, two high-dose animals had thickened glandular stomach mucosa, which was attributed to OPP treatment. There were no treatment-related increases in androgen-se nsitive tissue (AST) weights in OPP-treatedIn vivo Hershberger Assay (201 2) / Page 15 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### animals in the androgenic portion of assay. In the anti-androgenic portion of the assay, high-dose animals had a significant decrease in ventral prostate weight, but there were nosignificant effects of OPP treatment on any other AST weights. There were no treatmentrelated findings at doses ≤ 250 mg/kg/day OPP. The positive and negative control compounds produced the expected responses. Ov erall, this assay met the CV performance criteria outlined in the corres ponding test guidelines. Based on the lack of statistically significant, treatment-related changes in two AST organ weights, OPP at doses ≤ 1000 mg/kg/day (assay limit dose) was deemed nega tive for both androgenic and anti-androgenic activity in the Hershberger assay.

B. AGENCY COMMENTS: 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; the results for this animal were ex cluded from the study. There were no treatmentrelated clinical signs in animals dosed at ≤ 250 mg/kg/day in either assay. In the 1,000 mg/kg/day group in the androgen agonist assay, si x of seven animals 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. The incidence of these observations decreased as the study progresse d. In the anti-androgen assa y, five animals in the 1,000 mg/kg/day group had some or all of the followi ng observations: cl ear or red perioral soiling, perineal soiling (urine and/or feces), decreased activity, and noisy respiration. Many of these observations were noted in th e later portion of the study (Days 7-11). 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 a ssay, body weights were decrease d (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 consump tion in this group was decreased (p<0.05) by 42% during Days 4-7. Body we ights 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 accesso ry sex organs, adrenal glands, kidneys, or liver for any OPP treatment group. Animals in the positive control group had the expected increases in accessory sex organ weights; weight s of adrenal glands, kidney, and liver were comparable to the vehicle control.

In the anti-androgen assay, body weights and body weight gains in the OPP 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 ventral prostate were decreased (p<0.05) by 28% compared to the controls. There were no treatment-relate d effects on the weights of seminal vesicles, LABC, Cowper’s glands, glans penis, adre nal glands, kidneys, orliver for any OPPtreatment group. Animals dosed with TP + FT (positive control) had the expected decreasesIn vivo Hershberger Assay (201 2) / Page 16 of 16 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441



### in accessory sex organ weights; weights of adrenal glands, kidney, and liver were comparable to the vehicle control.

Percent CVs for the vehicle control and th e 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, w ith 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 antiandrogen assay (CV = 22%; maximum recommended CV = 20%).

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

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

 For two of the dosing groups (50 and 1000 mg /kg/day groups in the androgen agonist assay), the initial weight for one animal in the group was greater than +20% of the

group mean.

# DATA EVALUATION RECORD

O -PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-72-2012 (MRID 48686601)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 6/22/2012

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/09/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/11/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/11/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).0-PHENYLPHENOL (OPP) I 064103



### Primary Reviewer: Patience Brown, Ph.D. 

### Office of Science Coordination and Policy Secondary Reviewer: Elizabeth Mendez, Ph.D. 

### Health Effects Division, Office of Pesticide Programs Female Pubertal Assay (2012) I Page I of 16
OCSPP 890.1450 I OECD None



### DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450



### PC CODE: 064103 

### DP BARCODE: D401755



### TXR#: 0055304 

### CAS No.: 90-43-7



### TEST MATERIAL (PURITY: 0-Phenylphenol (OPP, 99.9% purity)



### SYNONYMS: (1, 

### l '-Biphenyl)-2-ol; ortho-phenylphenol; 2-phenylphenol; Prevental 

### 0 Extra



### CITATION: Marty, M.S., Zablotny, C.L., Sura, R

. (2012). Ortho-Phenylphenol: Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Female Cr! : CD(SD) Rats

. Toxicology and Environmenta l Research and Consulting

, The
Dow Chemical Company, Midland

, MI. Laboratory Project Study ID No.:
111087 ; April 13, 2012. MRID 4868660 I. 

### Unpublished.



### SPONSOR: OPP EDSP Consortium, Dow Microbial Control

, The Dow Chemical Company, Midland, MI and LANXESS Corporation

, Materials Protection
Products

, 111 RIDC Park West Drive, Pittsburgh

, PA

.



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48686601), 15 Sprague Dawley Crl:CD(SD) rats / dose group were treated daily via oral gavage with 0-phenylphenol (OPP; 99.9% purity, Lot No. CHHYDD0051) in corn oil at doses ofO (vehicle), 50

, 250, or 900 mg / kg / day from post-natal day (PND) 22 to 42

. Anima l s 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

, th y roxine (T4) and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay or chemiluminescent immunoassay. Liver

, pituitary

, adrenals, and uro ge nital organ weights were recorded

, and microscopic examinations

were performed on the ovaries, uterus

, thyroid

, and kidneys

.

All animals survived until sc heduled termination

, with the exception of one animal in the highdose gro up ( death due to gavage error). Additionally, no treatment-related clinical signs of toxicity were observed in animals for any dose groups.Female Pubertal Assay (2012) / Page 2 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### 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 OPP. Initial a nd terminal body weights, body weight gains, and body weight at attain ment of VO were similar across all dose groups. However, the 900 mg/kg/day group weighed 8% less than cont rol animals at PND 35. The 900 mg/kg/day group gained 10% to 13% less th an control between PND 22-35, but these decreases were not statistically (NS) different from control animal s. 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 en tire 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 incr ease (p<0.05) of 9% was noted in relative liver weight. Relative pituitary weights were decreased (p<0.0 5) by 10%. There were no dose-related effects on the weights of kidneys, ova ries, 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 c ontrol from 93.3% to 64.3%, and the percent regularly cycling was de creased (p<0.05) from 86.7% to 28.6%. However, the estrous cycle status at necropsy (d iestrus, proestrus, es trus, not cycling) was generally similar across all groups.

Serum urea nitrogen (BUN) and phosphorus leve ls 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 tr eatment-related kidney effects. Significantly increased alanine aminotransferase activity (ALT; p<0.05; 102%) was also considered treatment-related.

OPP was not associated with any signifi cant treatment-related changes in thyroid histopathology. At 900 mg/kg/day, increased inci dences 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 anim als, 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 anim als vs. 0/15 controls).

The doses tested were adequa te based on the increased serum ALT, BUN, phosphorus, renal lesions, and effects on estr ous cyclicity seen at th e high dose (900 mg/kg/day).

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Female Pubertal Assay (2012) / Page 3 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology and Environmental Research and Consulting

Location: Midland, MI. Study Director: C.L. Zablotny
Other Personnel: M.S. Marty, Lead Scientist R. Sura, Pathologist Study Period: August 12, 2011 to April 13, 2012



### 2. Test Substance: O -Phenylphenol (OPP)
Description: Not reported (CoA not provided)
Source: Lanxess Deutschland GmbH , Leverkusen, Germany
Lot #: CHHYDD0051, exp. not reported Purity: 99.9% purity Stability: Reported to be stable in corn oil for ≥ 15 days (0.25–250 mg/mL)
CAS #: 90-43-7
Structure: OH



### 3. Vehicle: Corn oil

4. Test Animals:
Species: Rat (females only)
Strain: Sprague Dawley Crl:CD(SD) Age/Mean Weight at Study Initiation: PND 22/ 48.0-48.3 g
Source: Charles River Laboratorie s, Inc., Portage, MI Housing: 2 animals/cage in plastic solid bottom cages w ith heat-treated, aspen wood shavings (Harlan Laboratories, Inc., Indianapolis, IN)
Diet: Teklad Diet #2016 (Harlan Laboratories, Inc.), ad libitum Total genistein equiva lents <325 μg/g diet
Water: Deionized water, ad libitum
Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3°C
40–70%
12–15 times/hr 12-hr light/dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 19, 2011 End: September 9, 2011

2. Mating: Time-mated pregnant nulliparous dams were received from the supplier on Gestation Day 7–10 and allowed to deliver their pups naturally. Litters with more than 10 pups were standardized by culling on PND 4 to 10 pups per litter wi th equal numbers of males and females whenever possible. 3. Animal Assignment: Offspring were weaned on P ND 21 and weighed. Animals were assigned to dose groups such that the mean body weights (and variances) were similarFemale Pubertal Assay (2012) / Page 4 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### among the test groups noted in Table 1, and the assignment of littermates to the same treatment group was avoided. Because study animals were housed two females per cage, and the number of animals per treatment was 15, four extra females from the lactation phase (1/group) were housed with the single female in each group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females
Control 0 15
Low 50 15
Mid 250 15 High 900 15 a Data were obtained from page 19 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from two previously performed studies. In the Hershberger assay 1 

### , OPP was administered by oral gavage at dose concentrations of 0, 50, 250, a nd 1000 mg/kg/day to castrated male CD rats that were 55–64 days old. The 1000 mg/kg/day dose resulted in decr eases in terminal body weights of 6-10% in the androgenic and an ti-androgenic portions of the assay, and decreased body weights gains of 41% in both phases. No effects on body weights/gains or feed consumption was observed in the 250 mg/kg/day animals.

In a 15-day dose range finding toxicity (p robe) study conducted in-house (Marty and Marshall, in progress), four rats /sex/dose were administered either corn oil (0) or OPP in corn oil at dose concentrations of 250, 500, 700 or 900 mg/kg/day. Body weights and body weight gains were decreased (5–7% and 10–26%, respectively) in the 900 mg/kg/day males, associated with decreased feed consum ption. There were no si gnificant changes in body weight, body weight gain, or feed consumption in males at ≤ 700 mg/kg/day and all treated females. Increases in absolute and relative liver weights in male and female rats administered 900 and 700 mg/kg/day, and increa sed absolute kidney weights in females administered 900 mg/kg/day were also observed.

Based on these studies, 900 mg/kg/day was se lected as the high dose was considered sufficient.

5. Dose Preparation and Analysis: Dose formulations were prepared periodically by mixing appropriate amounts of test substance with corn oil at Toxicology and EnvironmentalResearch and Consulting; samples from the top, middle and bottom of the low- and highdose formulations were analyzed for homogene ity and concentration analyses. Duplicate samples from the control and mid-dose formulations were taken from random locations. Stability analysis was conducted by Toxi cology and Environmental Research and Consulting 1 

### in a separate study (data not provided).

Results of Dose Analysis

Homogeneity (% R.S.D.): 1.6 to 6.0%

1 Marty et al. (2012). Orthophenylphenol: Hershberger A ssay in Castrated Male Crl:CD(SD) Rats. Toxicology and Environmental Research and Consulting, The Dow Chem ical Company, Midland, MI. Unpublished (MRID # 48672602).Female Pubertal Assay (2012) / Page 5 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### Stability: Reported to be stable for ≥ 15 days

Concentration (% nominal): 99.6 to 103.8%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 through PND 42, in a volume of 4 mL/kg of bo dy weight. Dose volumes were adjusted based on the individual daily body weights.

7. Statistics: Mean values were presented with st andard deviations and coefficient of variation. The following st atistical scheme was used:

Classification Procedure Initial body weight Body weight gain Clinical chemistry Mean cycle length Age at vaginal opening Body weight at vaginal opening Age at first estrus Organ weights (except uterus) Organ-to-body weight ratios (kidney, adrenal and pituitary weights) Serum hormones A Bartlett’s test was conducted to check for homogeneity of variance (p=0.01).

Two-way analysis of variance (ANOVA) with block and treatment as main effects was performed.

When significant dose effects were determined in the two-way ANOVA at p = 0.05, then individual dose groups were compared to controls with Dunnett's test at p = 0.05 (exper iment-wise error).

Uterine weight Liver-to-body weight ratio A Bartlett’s test was conducted to check for homogeneity of variance (p=0.01). A nonparametric analysis (Kruskal-Wallis test) was run on these data. When the Kruskal-Wallis test was significant at alpha=0.05, the Wilcoxon ranksum test was performed with a Bonferroni-Holm adjustment for multiple comparisons to the control. Age and body weight at vaginal opening Organ weights (thyroid and ovary) A Bartlett’s test was conducted to check for homogeneity of variance (p=0.01).

Analysis of covariance (ANCOVA) was conducted with body weight on PND 22 as the covariate.

When the dose effect was si gnificant at p = 0.05, least square means with a Dunnett’s correction was used to determine differences (experiment wise p = 0.05).

When the ANCOVA was not statistically significant, a Dunnett’s test was performed. Cycling Status Percent cycling regularly A Chi-square test was performed, and when significant, it was followed by pairwise comparisons to the control group via a Fi sher's exact test with a Bonferroni-Holm adjustment. Specific histopathologic observations Pair-wise Chi-square test with Yates' continuity correction (alpha = 0.05, two-sided)Female Pubertal Assay (2012) / Page 6 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### Statistical outliers were identified by a sequent ial test (p = 0.02). Outliers and questionable data points (i.e., values due to technica l errors) were excluded only for documented scientifically sound reasons. Hi stopathology results were not s ubjected to outlier tests. The statistical analyses were considered adequate.

C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined at least twice daily for mortality, moribundity, availability of food/wate r, and clinical abnormalities. Clinical, hand-held examinations were conducted daily post-dose.

2. Body Weight: Animals were weighed prior to a llocation to dose groups (PND 21) and daily prior to dosing. Body weights determin ed prior to dosing on PND 42 were used as the terminal body weights. Body weights we re statistically an alyzed on PND 21, 22, 25, 28, 32, 35, and 42; body weight gains were statis tically analyzed beginning with the PND 22-25, followed by subsequent intervals, in addition to the entire dosing period (PND 22- 42).

3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset of VO. Age and weight on the day of comple tion of VO were recorded. Examination for the onset of puberty ceased with acquisiti on or on PND 42 if VO was not yet acquired (PND 43 used for calculation of mean age of puberty onset).

4. Estrous Cyclicity: Beginning on the day of VO through scheduled termination, daily vaginal smears were obtained, evaluated, and cla ssified as diestrus, pr oestrus, or estrus. The overall cycling pattern for each female was characterized as regular, irregular, or not cycling, where regular cycling was defined as recurring 4 to 5 day cycles and irregular cycling was periods of estrus longer than 2 days and/or di estrus longer than 3 days. Animals were considered acyclic if there were ≥ 3 consecutive days of estrus or
≥ 5 consecutive days of diestrus. The age at fi rst vaginal estrus was recorded. Mean age at first estrus, mean cycle length, % cycling, % regularly cycling, and estrus stage at termination were presented.

5. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the afternoon of PND 41. All surviving animals were anesthetized with 5% isoflurane approximately 2 hours after the final dose (starting at approximately 0800 hours) on PND 42. Blood was collected immediately into serum separation tubes after puncture of the inferior vena cava (or cardiac puncture). Blood samples for hormone analysis were kept refrigerated or maintained on wet i ce until centrifugati on, and stored at

− 80°C until subsequent hormone evaluations. Animals were euthanized by exsanguination, followed by decapitation.

Groups were counter-balanced during necr opsy, and necropsies were completed by 1300 hours. Animals were euthanized by exsanguination followed by decapitation. A complete necropsy was performed on all animals. Anim als found dead were necropsied on the same day. Similar necropsy procedures were followed for these animals to examine gross lesions; however, blood was not collected, te rminal body and organ weights were not recorded, and organs were not examined histopathologically.Female Pubertal Assay (2012) / Page 7 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### a. Hormone Analysis: Samples were sent on dry ice to WIL Research Laboratories, LLC (Ashland, OH) for analysis. TSH concentr ations were analyzed by radioimmunoassay (RIA), and a chemiluminescent immunoassay was used for total T 4



### .
b. Clinical Chemistry: The following CHECKED (X) pa rameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol

X Potassium X Globulins (calculated)

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALP/also ALK) X Total protein

Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin/globulin ratio (calculated) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on Guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Ovaries, without oviducts (paire d, without oviducts)*+ XX Thyroid* + XX Uterus, imbibed, w ith oviducts and cervix a *+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary*
a With and without fluid * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on Guideline 890.1450



### All organs, except the thyroid/tr achea and pituitary, were weighe d prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed again after each uterine horn wa s cut three times to allow removal of the fluid in the lumen (blotted weight).

All tissues were fixed in 10% phosphate-bu ffered formalin prior to embedding. After fixation, the thyroid was dissect ed from the trachea and weig hed. All collected tissues were routinely processed into paraffin bloc ks, sectioned, stained with hematoxylin and eosin, and examined microscopically.Female Pubertal Assay (2012) / Page 8 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### Two sections of each of the two lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5- point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA. Other histopathologi cal findings were graded as very slight, slight, moderate, or severe.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination, with the exception of one animal in the high-dose group. Hemorrhages in the lungs and nasal cavity of this animal were consistent with death due to a gava ge error or aspiration of test material due to a dosing error. This death was not attributed to OPP treatment.

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clini cal signs of toxicity were observed in animals for any dose groups.

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, and age and weight at attainment of VO are presented in Table 2.

Mean body weights were similar across all dose groups on PND 21 (45.5–45.6 g) with CVs that were ≤ 10.5%. There were no effects on age at at tainment of VO for any dose of OPP. The mean age at VO for control was PND 33.5 (unadjusted and adjusted), and the mean age at VO for OPP-treated animals ranged fr om PND 33.7–34.0 (unadjuste d and adjusted). Initial and terminal body weights, body weight gains, and body weight at attainment of VO were similar across all dose groups. Howe ver, 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 decrease s were not statistically 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 d ecreased (3%) over the entire dosing period (PND 22-42).

The mean age at VO, body weight at VO, wean ling weight, and final body weight in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1450); however, th e % CV for final body weight was outside the acceptable range, as follows: 10.6% CV for final body weight (maximum 8.93%).Female Pubertal Assay (2012) / Page 9 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) Initial body weight (PND 22; g) U 15 48.07 4.85 10.1 15 48.3 3.19 6.62 15 48.3 5.3 11.0 15 48.0 5.22 10.9 Body weight at VO (g) U 15 105.5 14.6 13.8 15 105.5 16.4 15.5 15 106.7 14.3 13.4 14 98.1 10.6 10.8
A 15 105.7 3.19 NA 15 105.3 3.19 NA 15 106.5 3.19 NA 14 98.4 3.30 NA Final body weight (PND 42; g) U 15 152.0 16.2 10.6 15 150.8 14.6 9.7 15 153.1 16.2 10.6 14 148.7 17.2 11.6
A 15 152.3 2.93 NA 15 150.5 2.93 NA 15 152.8 2.93 NA 14 149.1 3.04 NA Final body weight (% of control) U NA NA NA NA 15 99.2 9.6 9.7 15 100.7 10. 7 10.6 14 97.8 11.3 11.6
A NA NA NA NA NR NR NR NR NR NR NR NR NR NR NR NR Body weight gain (final – initial; g) U 15 104.0 12.5 12.0 15 102.5 13.2 12.8 15 104.8 12.3 11.7 14 100.7 14.5 14.37
A NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR Age at VO (PND) U 15 33.5 1.5 4.5 15 33.8 2.1 6. 3 15 34 2 5.8 14 33.7 2.4 7.2
A 15 33.5 0.52 NA 15 33.8 0.52 NA 15 34 0.52 NA 13 33.7 0.54 NA Proportion unopened (#/N) 0/15 NA 0/15 NA 0/15 NA 0/14 NA

a Data were obtained from Table 7 on page 54 of the study report. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined CV Coefficient of Variation NA Not applicable NR Not reported * Significantly different fro m controls at p<0.05.



### D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 3. Relative liver weight was increased (p< 0.05) at 900 mg/kg/day ( ↑ 9%), but unadjusted and adjusted liver weights were not significantly affected in the high-dose animals. Adjusted (but not unadjusted) and relative pituitary weight s were decreased (p<0.05) 12% and 10%, respectively, in the 900 mg/kg/day group. Adju sted adrenal weight was decreased (p<0.05) in the 900 mg/kg/day animals ( ↓ 13%), but unadjusted and relativ e adrenal weights were not significantly affected. There were no dose-related effects on the weights of kidneys, ovaries, uterus, or thyroid.

The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1450) with the following exceptions: (i) mean adrenals weight (31.9 mg; acceptable range 38.34-48.84 g) and (ii) CV for liver weight (13.14%; maximum 13.13%).Female Pubertal Assay (2012) / Page 10 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%)

Liver (g) U 15 7.18 0.94 13.14 15 6.92 0.93 13.45 15 7.23 1.04 14.41 14 7.69 1.34 17.37
A 15 7.19 0.223 NA 15 6.90 0.223 NA 15 7.21 0.223 NA 14 7.71 0.230 NA

R 15 4.71 0.25 5.22 15 4.58 0. 28 6.18 15 4.71 0.23 4.94 14 5.15* ( ↑ 9) 0.50 9.68

Kidneys (g) U 15 1.23 0.13 10.59 15 1.21 0.15 12.17 15 1.20 0.15 12.10 14 1.22 0.15 12.00
A 15 1.23 0.029 NA 15 1.20 0.029 NA 15 1.20 0.029 NA 14 1.22 0.030 NA
R 15 0.81 0.04 4.97 15 0.80 0.05 6.78 15 0.78 0.05 6.96 14 0.82 0.09 11.33 Pituitary (mg) U 15 7.8 1.1 13.5 15 7.6 0.9 12. 4 15 7.6 1.3 17.5 14 6.8 1.5 22

A 15 7.8 0.27 NA 15 7.6 0. 27 NA 15 7.6 0.27 NA 14 6.9* ( ↓ 12) 0.28 NA

R 15 5.1 0.5 9.1 15 5.1 0.5 9.5 15 5.0 0.6 12 14 4.6* ( ↓ 10) 0.7 15.0

Adrenals (mg) U 15 31.9 5.2 16.4 15 30.1 4.8 16 15 32.1 5.6 17.5 14 27.7 5.1 18.6

A 15 31.9 1.17 NA 15 30.1 1. 17 NA 15 32.0 1.17 NA 14 27.8* ( ↓ 13) 1.22 NA

R 15 21.0 2.6 12.5 15 20.1 3.5 17.6 15 21.0 2.9 13.8 14 18.6 2.7 14.5
Ovaries (mg) U 15 59.3 13.1 22.1 15 57.9 11.0 19 15 59.1 14.1 23.8 14 51.1 14.5 28.3
A 15 59.4 3.22 NA 15 57.8 3.22 NA 15 59.0 3.22 NA 14 51.2 3.34 NA Uterus, wet (mg) U 15 319.1 130.6 40.9 15 251.7 58.8 23.4 15 281.1 110.3 39.2 14 279.9 145.1 51.8
A 15 318.9 29.92 NA 15 252.0 29.92 NA 15 281.5 29.93 NA 14 279.5 30.98 NA Uterus, blotted (mg) U 15 275.9 74.0 26.8 15 239.2 52.8 22.1 15 257.0 77.9 30.3 14 240.0 79.7 33.2
A 15 276.0 18.67 NA 15 239.1 18.67 NA 15 256.9 18.67 NA 14 240.2 19.32 NA Thyroid, fixed (mg) U 15 8.5 1.2 14.6 15 8.7 1.1 12.2 15 9.2 1.6 16.9 14 9.5 1.1 11.5
A 15 8.5 0.33 NA 15 8.7 0.34 NA 15 9.2 0.33 NA 14 9.5 0.34 NA a Data were obtained from Table 10 on pages 57 of the study re port. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined NA Not applicable CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### E. ESTROUS CYCLICITY: Estrous cycle data are presen ted in Table 4. There were no effects of treatment on mean age at first estr us or mean cycle length. At 900 mg/kg/day, the percent cycling was decreased (not signi ficant) from 93.3% to 64.3%, and the percent regularly cycling was decreased (p<0.05) fr om 86.7% to 28.6%, with considerable variability in the high-dose animals (five female s with irregular cycli ng and five that were not cycling). The cycle status at necropsy (diestrus, proestrus, estrus, not cycling) was generally similar across all groups.Female Pubertal Assay (2012) / Page 11 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 4. Estrous Cyclicity in Female Rats Treated with OPP a

Treatment

Groups N Mean Age at First Estrus (PND) N Mean Cycle Length (days) N Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling

Vehicle 15 34.3 14 4.7 15 93.3 86.7 10 1 4 0 50 mg/kg/day 15 34.4 12 4.7 15 93.3 73.3 12 0 3 0 250 mg/kg/day 15 35.1 13 5.2 15 86.7 80 8 0 7 0 900 mg/kg/day 10 33.0 8 5.7 14 64.3 28.6* 12 0 2 0
a Data were obtained from Table 8 on pages 55 of the study report. N Number of animals * Significantly different from controls; p<0.05.



### F. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry concentrations are presented in Ta ble 5. No dose-depende nt treatment effects were observed on serum T 4 

### or TSH concentrations after exposure to OPP.

The following significant increases (p<0.05) were observed in clinical chemistry parameters at 900 mg/kg/day: (i) ALT; ↑ 102%); (ii) sodium ( ↑ 1%); (iii) blood urea nitrogen ( ↑ 23%); and (iv) phosphorus ( ↑ 14%). There were no dose-related effects on the other measured clinical chemistry parameters. Historical control va lues were not provided for either the hormone or cl inical chemistry parameters.

The results for serum T 4 

### and TSH in the control group were within the acceptable range of the performance criteria provide d in the Guideline (890.1450).Female Pubertal Assay (2012) / Page 12 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mea n SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Hormones
Serum T 4 , Total (μg/dL) 15 4.13 0.67 16.3 15 3.99 0.71 17.8 15 3.74 0.87 23.2 14 3.65 0.77 21.1

Serum TSH (ng/mL) 15 3.09 1.80 59.1 15 2.14 1.16 54.2 15 3.03 2.10 69.5 14 3.86 2.20 57.4

Clinical Chemistry
Creatinine (μmol/L) 15 0.13 0.05 36.14 15 0.11 0.04 31.05 15 0.13 0.05 36.60 14 0.12 0.04 35.07
Serum urea nitrogen (mg/dL) 15 13 2 14 15 13 2 18 15 15 2 16 14 16* ( ↑ 23) 2 15
Alkaline phosphatase (U/L) 15 310 54 18 15 281 45 16 15 296 66 22 14 275 48 18 ALT (U/L) 15 57 4 8 15 57 11 19 15 63 12 19 14 115* ( ↑ 102) 40 34 AST (U/L) 15 107 18 17 15 108 20 19 15 109 20 19 14 115 23 20 GGT (U/L) 15 1.5 0.0 0.0 15 1.5 0.0 0.0 15 1.5 0.0 0.0 14 1.5 0.0 0.0
Albumin (g/dL) 15 4.2 0.2 5.0 15 4.2 0.2 4.5 15 4.2 0.2 4.3 14 4.1 0.3 8.2
Albumin/ globulin ratio 15 3.4 0.4 11.8 15 3.3 0. 5 13.8 15 3.2 0.4 12.1 14 3.3 0.3 9.6
Cholesterol (mg/dL) 15 92 18 20 15 83 13 16 15 87 12 14 14 91 12 13
Globulin (g/dL) 15 1.3 0.1 9.9 15 1.3 0.2 12 15 1.3 0.2 11.9 14 1.3 0.2 15.5
Total bilirubin (mg/dL) 15 0.05 0.00 0.00 15 0.05 0.00 0.00 15 0.05 0.00 0.00 14 0.05 0.01 24.9 Total protein (g/dL) 15 5.5 0.3 4.6 15 5.4 0.2 4 15 5.5 0.2 4.3 14 5.4 0.5 9.4
Sodium (mmol/L) 15 143 1 1 15 143 1 1 15 142 1 1 14 144* ( ↑ 1) 2 1
Potassium (mmol/L) 15 4.7 0.3 6.2 15 4.6 0.4 8.1 15 4.6 0.2 5.2 14 4.6 0.3 5.8
Chloride (mmol/L) 15 101 1 1 15 101 2 2 15 101 1 1 14 101 3 3
Calcium (mg/dL) 15 11.4 0.3 2.7 15 11.3 0.3 2.4 15 11.4 0.3 2.8 14 11.2 0.3 3.1 Phosphorus (mg/dL) 15 8.5 0.7 8.5 15 8. 5 0.5 5.5 15 9 0.8 8.8 14 9.7* ( ↑ 14) 0.9 9.2 a Data were obtained from Table 9 on pages 56 of the study re port. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### G. GROSS PATHOLOGY: No treatment-related effects were observed on gross pathology.

H. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 6. OPP was not associated with any significant treatmentrelated changes in thyroid histopathology.Female Pubertal Assay (2012) / Page 13 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 6. Thyroid Gland Follicular Cell Height and Colloid Area a

Treatment Groups Parameter Evaluated
Colloid Area Follicular Cell Height

Grade b Incidence Grade Incidence
Observed Examined Observed Examined

Vehicle Control 1 0 15 1 10 15
2 0 15 2 5 15
3 0 15 3 0 15
4 5 15 4 0 15
5 10 15 5 0 15

OPP (900 mg/kg/day) 1 0 14 1 12 14
2 0 14 2 2 14
3 0 14 3 0 14
4 2 14 4 0 14
5 12 14 5 0 14 a Data were obtained from Table 12 on page 63 of the study report. b Thyroid histopathology is graded 1–5. Follicular cell height: 1 = lowest, 5 = highest; and Colloid area: 1 = most colloid, 5 = least colloid.



### The incidence of histopathological findings of the ovaries, uterus and kidneys are presented in Table 7. At 900 mg/kg/day, increased inci dences of the following histological lesions were noted in the kidneys: (i) multifocal re nal tubular dilatation (12/14 treated animals, very slight and slight severity vs. 4/15 cont rols, very slight severity); (ii) multifocal collecting duct epithelium hypertrophy (3/14 trea ted 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 renal tubular dilation was charact erized by the presence of multiple ectatic tubules (predominantly proximal convoluted tubules) in the cortex and medulla that were lined by flattened to cuboidal epithelial cells that often contained proteinaceous material mixed with sloughed cellular debris. In a few animals, the dilated tubules were accompanied by small numbers of lymphocytes and de generated tubules. Other re nal lesions in the treatment groups occurred in single anim als or not dose-dependently.

Evaluation of the uteri and ovaries were perf ormed with knowledge of the estrous stage; there were no significant treatment-related fi ndings. Two rats administered 900 mg/kg/day OPP had a very slight decrease in the size of the uterus, and one of these also had a juvenile appearance of the ovary. These effects on the ov ary and the uterus were interpreted to be secondary to treatment-related decreases on th e body weight of these two animals. There were no treatment-related hi stopathological findings noted in animals in the 50 and 250 mg/kg/day groups.Female Pubertal Assay (2012) / Page 14 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None

TABLE 7. Incidence of Histopathological Le sions of the Ovaries, Uterus and Kidney a

Findings Dose Level (mg/kg/day) Vehicle Control OPP (50) OPP (250) OPP (900)

# Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined

Ovaries
Within Normal Limits 15 15 15 15 15 15 13 14 Juvenile appearance (very slight) 0 15 0 15 0 15 1 14

Uterus
Within Normal Limits 15 15 15 15 15 15 12 14 Decreased size (slight) 0 15 0 15 0 15 2 14 Kidney
Within Normal Limits 6 15 2 15 1 15 0 14 Casts; proteinaceous; collecting duct; unilateral; multifocal (slight) 0 15 0 15 0 15 1 14

Degeneration; tubule; focal (very slight) 5 15 3 15 3 15 0 14

Degeneration; tubule; multifocal (very slight) 4 15 9 15 9 15 11 14

Degeneration;, tubule; multifocal (slight) 0 15 0 15 0 15 1 14

Dilatation; pelvis; unilateral 0 15 1 15 0 15 0 14

Dilatation; tubule; focal (very slight) 1 15 3 15 3 15 2 14

Dilatation; tubule; multifocal (very slight) 1 15 0 15 1 15 7 14

Dilatation; tubule; multifocal (slight) 0 15 0 15 0 15 2 14

Fibrosis; corticomedullary junction; interstitium; focal (very slight) 0 15 1 15 0 15 0 14

Fibrosis; corticomedullary junction; interstitium; multifocal (very slight) 1 15 0 15 0 15 0 14

Fibrosis; glomerulus; multifocal (very slight) 0 15 1 15 0 15 0 14

Hyperplasia; epithelium; papilla; unilateral; multifocal, slight 0 15 0 15 0 15 1 14

Hypertrophy; collecting duct; multifocal (very slight) 0 15 0 15 0 15 3 14

Infarct; cortex; focal (very slight) 1 15 0 15 0 15 1 14

Necrosis; with accompanying inflammation; tubule; focal (slight) 0 15 0 15 0 15 2 14

a Data were obtained from Table 13 on pages 64–65 of the study report.Female Pubertal Assay (2012) / Page 15 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Overall, systemic toxicity was confirmed for the high-dose group (900 mg/kg/day; based on decr eased body weight gain as well as liver weight increase and histopathol ogical changes in the kidneys). Based on the lack of toxicologically meaningful, treatment-relate d effects on puberty onset, estrous cycle, endocrine-sensitive organ weights, serum T 4 

### and TSH levels and uterine, ovarian, and thyroid histopathology, there was no evidence of endocrine activity for OPP in the female pubertal assay at doses ≤ 900 mg/kg/day.

B. AGENCY COMMENTS: All animals survived until sc heduled termination, with the exception of one animal in the high-dose group (died due to gavage error). Additionally, no treatment-related clinical signs of toxicity were observed in animals for any dose groups.

Mean body weights were similar across all do se groups on PND 21. There were no effects on age at attainment of VO for any dose of OPP. The unadjusted and adjusted mean ages at VO for control and for OPP-treated animals were not statistically different. Initial and terminal body weights, body weight gains, and body weight at attainment of VO were similar across all dose groups.

At 900 mg/kg/day, modest decreases (p<0.05) were noted in adjusted pituitary ( ↓ 12%) and adrenal ( ↓ 13%) weights. A minor incr ease (p<0.05) of 9% was noted in relative liver weight, and relative pituitary weights were decreased (p<0.05) by 10%. There were no dose-related effects on the weights of kidneys, ovaries, uter us, or thyroid.

There were no effects of treatment on mean age at first estrus or mean cycle length. In the high-dose animals, the percent cycling was decreased (NS) compared to the controls from 93.3% to 64.3%, and the percen t regularly cycling was decreased (p<0.05) from 86.7% to 28.6%. The cycle status at necropsy (diestrus, proestrus, estrus, not cycling) was generally similar across all groups.

Serum urea nitrogen, ALT, and phosphorus leve ls were increased (p<0.05) in the 900 mg/kg/day dose group.

OPP was not associated with any signifi cant treatment-related changes in thyroid histopathology. At 900 mg/kg/day, increased incidences of the following histological lesions were noted in the kidney: (i) very slight and slight multifocal renal tubular dilatation; (ii) very slight mu ltifocal collecting duct epithelium hypertrophy; (iii) very slight to slight multifocal renal tubule dilatation; and (iv) slight focal renal tubular necrosis, with accompanying inflammation. The renal tubular dilation was characterized by the presence of multiple ectatic tubules (predominantly proxi mal convoluted tubules) in the cortex and medulla that were lined by flattened to c uboidal epithelial cells that often contained proteinaceous material mixed with sloughed cellul ar debris. In a few animals, the dilated tubules were accompanied by small numbers of lymphocytes and degenerated tubules. Other renal lesions in the treatment groups occurred in single animals and were not considered dose-dependent.Female Pubertal Assay (2012) / Page 16 of 16 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1450 / OECD None



### Thyroid function was not altered at any dose of OPP. The only evidence of an effect on the sex tissues were changes in the percent cyc ling and percent regularly cycling. However, these parameters are variable and may be influenced by body weight. At PND 35, the 900 mg/kg/day group weighed 8% less than controls.

Increased serum ALT, BUN, phosphorus, rena l lesions, and effects on estrous cyclicity may indicate adverse effects at 900 mg/kg/day in this study.

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

 Total genistein-equivalent content of the laboratory diet (Teklad Diet #2016) was reported as <325 μg/g; Guideline stat es that the content should be ≤ 300 μg/g.

 Control CV for final body weight (10.63%) wa s greater than the performance criteria maximum of 8.93%.

 Control mean adrenal weight (31.9 mg) was below the performance criteria acceptable range of 38.34-48.84 mg

# DATA EVALUATION RECORD

O -PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-72-2012 (MRID 48686602)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 6/22/2012

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/09/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/11/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/11/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).0-PHENYLPHENOL (OPP) I 064103



### Primary Reviewer: Patience Browne, Ph.D.


### Office of Science Coordination and Policy Secondary Reviewer: John Liccione, Ph.D.

# Signature~.**~~~~~=~~** Health Effects Division, Office of Pesticide Programs Date:**--t6~'f'Tt'-L- =-2-** **--**



### DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500



### PC CODE: 064103 

### DP BARCODE: D401755



### TXR#: 0055304 

### CAS No.: 90-43- 7



### TEST MATERIAL (PURITY: 0-Phenylphenol (OPP

, 99.9% purity)



### SYNONYMS: (1

, 1 ' -Biphenyl)-2-ol; ortho-phenylphenol ; 2-phenylphenol; Prevental 

### 0 Extra



### CITATION: Marty

, M

. S

. , Andrus, A.K., Sura

, R. (2012). Ortho-Phenylphenol: Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Male Crl: CD(SD) Rats

. Toxicology and Environmental Research and Consulting

, The
Dow Chemical Company

, Midland, MI. Laboratory Project Study ID No.:
111088 ; April 13

, 2012. MRID 48686602

. Unpublished

.



### SPONSOR: OPP EDSP Consortium

, Dow Microbial Control, The Dow Chemical Company

, Midland, MI and LANXESS Corporation

, Materials Protection Products

, 111
RIDC Park West Drive

, Pittsburgh, PA.



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48686602), 16 Sprague Dawley Crl:CD(SD) rats / dose group were treated daily via oral gavage with 0-phenylphenol (OPP ; 99

. 9 % purity

, Lot No. CHHYDD005 I) in corn oil at doses of 0 (vehicle)

, 50, 250, or 900 m g / 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 OPP-dose group) was considered to be gavage

e rror. The cause of death of the fourth animal (high-dose group) was not determined

, but was
not attributed to OPP treatment. Additionally

, no treatment-related clinical signs of toxicity were observed in animals for any dose groups.Male Pubertal Assay (2012) / Page 2 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### Adjusted terminal body weights were decrease d 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 ga ins 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 administratio n of OPP included increased relative liver weight at 250 mg/kg/day ( ↑ 8%), and adjusted and re lative 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 wei ghts were decreased by 11% at ≤ 250 mg/kg/day and by 16% at 900 mg/kg/day. However, the most relevant measur es 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 ventra l 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 we re observed in three andr ogen sensitive tissues, these effects were considered treatment-relate d. There were no dose-related effects on the weights of kidneys, dorsolateral pros tate, right testis, or thyroid.

No treatment-related effects were observed on serum testosterone or TSH concentrations. Serum
T 4 

### 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 th e thyroid. At 900 mg/kg/day, an apparent treatment-related effect was observe d on serum urea nitrogen concentrations, which corroborated the renal histological evidence. Significantly incr eased alanine aminotransferase (ALT) activity ( ↑ 95%) was considered treatment-relate d, 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 OPP dose. However, in the kidneys, there were in creased incidences of renal tubular dilatation (combined focal and multifocal, very slight a nd 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 incide nces of renal tubular degeneration were similar in high-dose and contro l 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 overa ll 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.Male Pubertal Assay (2012) / Page 3 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2012) / Page 4 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology and Environmental Research and Consulting

Location: Midland, MI. Study Director: A.K. Andrus
Other Personnel: M.S. Marty, Lead Scientist R. Sura, Pathologist Study Period: August 12, 2011 to April 13, 2012



### 2. Test Substance: O -Phenylphenol (OPP)
Description: Not reported (CoA not provided)
Source: Lanxess Deutschland GmbH , Leverkusen, Germany
Lot #: CHHYDD0051, exp. not reported Purity: 99.9% purity Stability: Reported to be stable in corn oil for ≥ 15 days (0.25–250 mg/mL)
CAS #: 90-43-7
Structure: OH



### 3. Vehicle: Corn oil

4. Test Animals:
Species: Rat (males only)
Strain: Sprague Dawley Crl:CD(SD) Age/Weight at Study Initiation: PND 23/ 37.4 – 64.9 g
Source: Charles River Laboratorie s, Inc., Portage, MI Housing: 2 animals/cage in plastic solid bottom cages w ith heat-treated, aspen wood shavings (Harlan Laboratories, Inc., Indianapolis, IN)
Diet: Teklad Diet #2016 (Harlan Laboratories, Inc.), ad libitum Total genistein equiva lents <325 μg/g diet
Water: Deionized water, ad libitum
Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22±1°C (with a permissible excursion of ±3)
40–70%
12–15 times/hr 12-hr light/dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 20, 2011 End: September 20, 2011

2. Mating: Time-mated pregnant nulliparous dams were received from the supplier on Gestation Day 7-10 and allowed to deliver their pups naturally. Litters with more than 10 pups were standardized by culling on PND 4 to 10 pups per litter wi th equal numbers of males and females whenever possible. 3. Animal Assignment: Animals were weaned on PND 21 and weighed. Animals were assigned to dose groups such that the mean body weights (and variances) were similarMale Pubertal Assay (2012) / Page 5 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### among the test groups noted in Table 1, and the assignment of littermates to the same treatment group was avoided.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males
Control 0 16
Low 50 16
Mid 250 16 High 900 16 a Data were obtained from page 18 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from two previously performed studies. In the Hershberger assay 1 

### , OPP was administered by oral gavage at dose concentrations of 0, 50, 250, a nd 1000 mg/kg/day to castrated male CD rats that were 55-64 days old. The 1000 mg/kg/da y dose resulted in decr eases in terminal body weights of 6-10% in the androgenic and an ti-androgenic portions of the assay, and decreased body weights gains (Days 1-11) of 41% in both phases. No effects on body weights/gains or feed consumption was observed in the 250 mg/kg/day animals.

In a 15-day dose range finding toxicity (p robe) study conducted in-house (Marty and Marshall, in progress), four rats /sex/dose were administered either corn oil (0) or OPP in corn oil at dose concentrations of 250, 500, 700 or 900 mg/kg/day. Body weights and body weight gains (Days 3-15) were decreased (5-7% and 10-26%, resp ectively) in the 900 mg/kg/day males, associated with decreased feed consumption. There were no significant changes in body weight, body weight gai n, or feed consumption in males at ≤ 700 mg/kg/day and all treated females. Increases in absolute and relative liver weights in male and female rats administered 900 and 700 mg/kg/day, and in creased absolute kidney weights in females administered 900 mg/kg/day were also observed.

Based on these studies and the guideline requirements of dosing immature rats (PND 23) for a period of 31/32 days, 900 mg/kg/da y was selected as the high dose.

5. Dose Preparation and Analysis: Dose formulations were prepared periodically by mixing appropriate amounts of test substance with corn oil at Toxicology and EnvironmentalResearch and Consulting; samples from the top, middle and bottom of the low- and highdose formulations were analyzed for homogene ity and concentration analyses. Duplicate samples from the control and mid-dose formulations were taken from random locations. Stability analysis was conducted by Toxi cology and Environmental Research and Consulting 1 

### in a separate study (data not provided).

Results of Dose Analysis

Homogeneity (% R.S.D.): 1.6 to 6.0%

1 Marty et al. (2012). Orthophenylphenol: Hershberger A ssay in Castrated Male Crl:CD(SD) Rats. Toxicology and Environmental Research and Consulting, The Dow Chem ical Company, Midland, MI. Unpublished (MRID 48672602).Male Pubertal Assay (2012) / Page 6 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### Stability: Stable for ≥ 15 days

Concentration (% nominal): 99.6 to 103.8%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: All doses were administered once daily by gavage, from PND 23 through PND 53, in a volume of 4 mL/kg of bo dy weight. Dose volumes were adjusted based on the individual daily body weights.

7. Statistics: Mean values were presented with st andard deviations and coefficient of variation. The following st atistical scheme was used:

Classification Procedure Body weight Body weight gain Clinical chemistries* Age at PPS Body weight at PPS Organ weights Organ-to-body weight ratios (liver, kidney, adrenal and pituitary weights) Serum hormones A Bartlett’s test was conducted to check for homogeneity of variance (p=0.01).

Transform and retest when necessary.

Two-way analysis of variance (ANOVA) with block and treatment as main effects was performed.

When significant dose effects were determined in the two-way ANOVA at p = 0.05, then individual dose groups were compared to controls with Dunnett's test at p = 0.05 (exper iment-wise error). Age and body weight at PPS Organ weights A Bartlett’s test was conducted to check for homogeneity of variance (p=0.01).

Analysis of covariance (ANCOVA) was conducted with body weight on PND 23 as the covariate.

When the dose effect was si gnificant at p = 0.05, least square means with a Dunnett’s correction was used to determine differences (experiment wise p = 0.05).

When the ANCOVA was not statistically significant, a Dunnett’s test was performed. Specific histopathologic observations Pair-wise Chi-square test with Yates' continuity correction (alpha = 0.05, two-sided)



### * The Bartlett’s test was significant for the raw and transf ormed alanine aminotransfera se data. A non-parametric Kruskal Wallis test (p = 0.05), follo wed by a Wilcoxon rank-sum test with Bonferroni-Holm adjustment for dose groups compared to control.



### Statistical outliers were identified by a sequent ial test (p = 0.02). Outliers and questionable data points (i.e., values due to technica l errors) were excluded only for documented scientifically sound reasons. Hi stopathology results were not s ubjected to outlier tests. The statistical analyses were considered adequate.Male Pubertal Assay (2012) / Page 7 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined at least twice daily for mortality, moribundity, availability of food/wate r, and clinical abnormalities. Clinical, hand-held examinations were conducted daily post-dose.

2. Body Weight: Animals were weighed prior to a llocation to dose groups (PND 21) and daily prior to dosing. Body weights determined prior to dosing on PND 53 were used as the terminal body weights. Body weights were statistically analyzed on PND 21, 23, 26, 30, 37, 42, 45, and 53; body weight gains were statistically analyzed beginning with the PND 23-26 interval, followed by subsequent intervals, in addition to the entir e dosing period (PND 23- 53).

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily at approximately the same time each day for onset of PPS. Age and weight on the day of completion of PPS were recorded. PPS was reco rded as: progression not initiated, partial separation, a persistent thread of tissue between the glans and prepuce, or complete PPS. Examination for the onset of puberty ceased wi th acquisition or on PND 53 if PPS was not yet acquired (PND 54 used for calculation of mean age of puberty onset).

4. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the afternoon of PND 52. All surviving animals were anesthetized with 5% isoflurane approximately 2 hours after the final dose (starting at approximately 0800 hours) on PND 53. Blood was collected immediately into serum separation tubes after puncture of the inferior vena cava (or cardiac puncture). Bl ood samples for hormone analysis were kept refrigerated or maintained on wet ice until centrifugation, and stored at

− 80°C until subsequent hormone evaluations . Animals were euthanized by exsanguination, followed by decapitation.

Groups were counter-balanced during necr opsy, and necropsies were completed by 1300 hours. A complete necropsy was performed on all animals. Animals found dead were necropsied on that day during the normal work week or, if found dead after routine working hours, on weekends, or holidays, were refrigerated until necropsy on the next scheduled workday. When appropriate, similar necropsy procedures were followed for these animals to examine gross lesions; however, blood wa s not collected, terminal body and organ weights were not recorded, and organs were not examined histopathologically.

a. Hormone Analysis: Samples were sent on dry ice to WIL Research Laboratories, LLC (Ashland, OH) for analysis. TSH concentr ations were analyzed by radioimmunoassay (RIA), and chemiluminescent immunoassays were used for total T 4 

### and testosterone.

b. Clinical Chemistry: The following CHECKED (X) pa rameters were examined.Male Pubertal Assay (2012) / Page 8 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol

X Potassium X Globulins

X Sodium Glucose

ENZYMES X Total bilirubin
X Alkaline phosphatase (ALP/also ALK) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in ma le rats based on Guideline 890.1500.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid* + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glands (w ith and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + * Weights required base d on guideline 890.1500 + Histopathological examination re quired based on Guideline 890.1500



### All organs except the thyroid/tr achea were weighed prior to fixation. The paired kidneys and adrenals were weighed together. The se minal vesicle with coagulating glands was weighed. After each side was cut sagitally to allow removal of the fluid in the lumen (blotted weight), the semina l vesicle was weighed again.

The gross lesions, adrenals, dorsolateral pros tate, kidneys, LABC, liver, pituitary, seminal vesicle with coagulating glands , thyroid, and ventral prostate were fixed in 10% phosphatebuffered formalin prior to embedding. After fixation, the thyroid was dissected from the trachea and weighed. All collected tissues were routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and examined microscopically.

Two sections of each of the two lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA.Male Pubertal Assay (2012) / Page 9 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### The testes and epididymides were placed in Bouin’s fixative for 24–72 hours, rinsed with 50% ethanol, and stored in 70% ethanol prior to embedment in paraffin. For the qualitative assessment of the spermatogenesis stages, a cros s-section from the center of the testes was embedded in paraffin, and stained with PAS/hematoxylin. The histopathological examination of the testes include d a qualitative assessment of th e stages of spermatogenesis; a qualitative assessment of the relationships between spermatagonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the semi niferous tubules; and identification of any degenera tive changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Examination of the intact epid idymis included the caput, corpus, and cauda, accomplished by evaluation of a longitudinal sect ion, and was conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium.

Other histopathological findings were graded as very slight, slight, moderate, or severe.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination, with the exception of four animals. One animal in each of the thre e OPP dose groups apparently died from either from a gavage error or aspiration of test materi al due to a dosing error. The cause of death for a fourth animal, in the 900 mg/kg/day gr oup, was undetermined, but none of the deaths were attributed to OPP treatment.

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clini cal signs of toxicity were observed in animals for any dose groups. On e control animal had dermatitis (PND 52-53), one animal in the 50 mg/kg/day group had an abrasion (PND 32-40), and one animal in the 900 mg/kg/day group had red, perinasal soiling, perineal soiling, and noisy respiration on PND 46-47, but continued to gain weight unt il scheduled necropsy. These observations were considered isolated, transient occu rrences, and not related to treatment.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age of attainment of PPS and weight at day of PPS are presented in Table 2. Data for one animal (animal #8004) in the vehicle contro l group were excluded from analysis due to excessively low initial body weight (21.2 g co mpared to group mean weight of 47.7 g on PND 21).

The control group gained more weight than animals assigned to the treated groups by PND 23; the difference in mean body weight wa s significant (p<0.05) only in the low-dose group on PND 23 ( ↓ 11%), although a minor decrease ( ↓ 7%; NS) was also noted in the 900 mg/kg/day group. This difference in body weights continued through PND 30, with no difference in body weight gain, but wa s resolved at la ter time-points.

Unadjusted terminal (PND 53) body weights were decreased (p<0.05) by 12% in the 900 mg/kg/day group, and adjusted terminal body weights were decr eased by 8% (NS). Males in the 900 mg/kg/day group only ga ined 205.7 g during PND 23-53 (p<0.05; ↓ 13%)Male Pubertal Assay (2012) / Page 10 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### compared with 235.4 g in the control animals during this interval. Body weights and body weight gains were not affected at doses of ≤ 250 mg/kg/day.

The age of attainment of PPS, adjusted for body weight on PND 23, was delayed by 1.6 days (NS) at 900 mg/kg/day (45.1 days) compar ed to control (43.5 days). The unadjusted age at attainment of PPS was delayed (p<0.05) by 2.1 days in the 900 mg/kg/day group (45.2 days) compared to control (43.1 days). There were no effects on age at attainment of PPS for the 50 and 250 mg/kg/day dose groups.

The mean age at PPS, body weight at PPS, wean ling weight, and final body weight in the control group were within the acceptable range of the performance criteria provided in the Guideline; however, the % CV for weight at PPS and final body wei ght were outside the acceptable range, as follows: 8.36% CV for we ight at PPS (maximum 7.57%) and 8.2% CV for final body weight (maximum 7.47%).

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) Initial body weight (PND 23; g) U 15 56.8 3.68 6.48 16 50.5* ( ↓ 11) 6.27 12.4 16 54.8 5.37 9.79 16 52.6 ( ↓ 7) 7.55 14.4

Body weight at PPS (g) U 15 206.0 17.2 8.36 15 204.0 17.8 8.73 15 201.2 14.2 7.06 14 203.7 20.0 9.79

A 15 201.8 4.23 NA 15 208.9 4.28 NA 15 199.4 4.09 NA 14 204.9 4.22 NA

Final body weight (PND 53; g) U 15 292.2 24.0 8.20 15 280.5 24. 5 8.72 15 285.1 26.2 9.20 14 258.4* ( ↓ 12) 22.9 8.84

A 15 283.9 5.24 NA 15 290.0 5. 30 NA 15 281.7 5.07 NA 14 260.7 ( ↓ 8) 5.22 NA

Final body weight (% of control) U NA NA NA NA 15 96.0 8.40 8.70 15 97.6 9.00 9.20 14 88.4 7.80 8.80

A NA NA NA NA NR NR NR NR NR NR NR NR NR NR NR NR

Body weight gain (final – initial; g) U 15 235.4 22.0 9.35 15 230.4 22. 0 9.54 15 230.2 22.7 9.87 14 205.7* ( ↓ 13) 18.5 9.01

A NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR

Age at PPS (PND) U 15 43.1 1.50 3.40 15 43.9 1. 90 4.40 15 43.2 2.30 5.30 14 45.2* ( ↑ 5) 1.90 4.30

A 15 43.5 0.49 NA 15 43.5 0.50 NA 15 43.4 0.48 NA 14 45.1 0.49 NA Proportion unseparated (#/N) 0/15 0/15 0/15 0/14

a Data were obtained from Table 7 on page 56 of the study report. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 N Number of animals examined CV Coefficient of Variation NA Not applicable NR Not reported * Significantly different fro m controls at p<0.05.Male Pubertal Assay (2012) / Page 11 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 3. Relative liver weight was increased by 8% (p<0.05) at 250 mg/ kg/day. Adjusted and relative liver weights were increased (p<0.05) at 900 mg/kg/day by 10% and 21%, respectiv ely, compared to controls. There was a dose-dependent decr ease in pituitary weights, though only the unadjusted and adjusted pituitary weights of the 900 mg/kg/day gr oup were significantly decreased (p<0.05;15% and 10%, re spectively) . A dose-depende nt decrease in unadjusted adrenal weights was significan t all treatment levels (p<0.05 ); 11% at 50 and 250 mg/kg/day, and by 16% at 900 mg/kg/day).

Weights of androgen-dependent organs were significantly de creased in the high treatment group. At 900 mg/kg/day, the unadjusted weight s of the seminal vesicle + coagulating glands (with and without flui d) were decreased (p<0.05) by 23% and 18%, respectively. Ventral prostate and LABC wei ghts (unadjusted and adjusted) were also decreased (p<0.05) by 17-20% and 16-18%, respectively. Unadju sted weights for the left and right epididymides were decreased (p<0.05) 10% and 9%, respectively, and the unadjusted and adjusted weights for the left testis were d ecreased (p<0.05) 9% and 7%, respectively. There were no significant decreases between contro l and treatment mean weights of kidneys, dorsolateral prostate, righ t testis, or thyroid.

The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1500) with the following exceptions: (i) mean kidney weight (2.01 g; acceptable range 2.242-3.050 g); (ii) mean thyroid weight (12.4 mg; acceptable range 14-26 mg); and (iii) CV for seminal vesicles (21.2%; maximum 21.06%).

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%)

Liver (g) U 15 13.2 1.36 10.27 15 12.8 1 .33 10.4 15 13.9 1.43 10.26 14 14.1 1.32 9.4

A 15 12.8 0.33 NA 15 13.2 0. 33 NA 15 13.8 0.32 NA 14 14.2* ( ↑ 10) 0.33 NA

R 15 4.51 0.19 4.25 15 4.54 0.18 4.07 15 4.87* ( ↑ 8) 0.24 4.86 14 5.45* ( ↑ 21) 0.27 4.92

Kidneys (g) U 15 2.01 0.2 9.82 15 1.96 0.22 11.35 15 2.05 0.24 11.55 14 1.9 0.19 10.3
A 15 1.96 0.05 NA 15 2.01 0.05 NA 15 2.03 0.05 NA 14 1.91 0.05 NA
R 15 0.69 0.04 6.47 15 0.7 0.07 9.73 15 0.72 0.06 7.94 14 0.73 0.05 6.5 Pituitary (mg) U 15 9.9 1.3 12.8 15 9.7 1.2 12.6 15 9.1 1 10.7 14 8.4* ( ↓ 15) 1.4 17

A 15 9.6 0.3 NA 15 10 0. 3 NA 15 9 0.29 NA 14 8.5* ( ↓ 10) 0.3 NA

R 15 3.4 0.4 10.7 15 3.4 0.3 8.6 15 3.2 0.2 6.7 14 3.2 0.4 13.7
Adrenals (mg) U 15 44.6 6.6 14.7 15 39.6* ( ↓ 11) 5.2 13 15 39.6* ( ↓ 11) 4.6 11.6 14 37.5* ( ↓ 16) 8.4 22.5

A 15 42.8 1.48 NA 15 41.7 1.5 NA 15 38.9 1.43 NA 14 38 1.48 NA
R 15 15.3 1.9 12.1 15 14.2 1.7 12 15 13.9 1 6.9 14 14.4 2.6 17.8
Seminal vesicles + U 15 480.8 102.1 21.2 15 422 104. 7 24.8 15 455.8 100.1 22 14 370.0* ( ↓ 23) 120.1 32.4Male Pubertal Assay (2012) / Page 12 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) coagulating glands, with fluid (mg) A 15 463.7 27.7 NA 15 441.9 28. 1 NA 15 448.6 26.81 NA 14 374.8 ( ↓ 19) 27.6 NA

Seminal vesicles + coagulating glands, without fluid (mg) U 15 237.3 44.5 18.8 15 215.3 50.4 23.4 15 230.3 48.3 21 14 193.3* ( ↓ 18) 51.1 26.4

A 15 228.7 12.5 NA 15 225.3 12. 6 NA 15 226.7 12.08 NA 14 195.7 ( ↓ 14) 12.4 NA

Ventral prostate (mg) U 15 219.8 36.4 16.5 15 193 44. 8 23.2 15 219 43.6 19.9 14 175.3* ( ↓ 20) 33.6 19.2

A 15 212.9 10.3 NA 15 201 10. 4 NA 15 216.1 9.97 NA 14 177.3* ( ↓ 17) 10.3 NA

Dorsolateral prostate (mg) U 15 177.2 34.3 19.3 15 162.8 29.5 18. 1 15 183.5 37 20.2 14 150.7 39.8 26.4

A 15 172 9.2 NA 15 168.9 9.33 NA 15 181.3 8.91 NA 14 152.1 9.18 NA

LABC (mg) U 15 511.5 43.8 8.6 15 491.6 85. 8 17.4 15 520.9 79.2 15.2 14 418* ( ↓ 18) 74.9 17.9

A 15 501.2 19.0 NA 15 503.6 19. 3 NA 15 516.5 18.4 NA 14 420.9* ( ↓ 16) 19.0 NA

Epididymis, left (mg) U 15 205.2 22.2 10.8 15 200 20. 4 10.2 15 202 24.9 12.3 14 185.4* ( ↓ 10) 23.7 12.8

A 15 199.6 5.53 NA 15 206.6 5.6 NA 15 199.6 5.36 NA 14 187 5.52 NA Epididymis, right (mg) U 15 208.7 21.7 10.4 15 201.4 19. 3 9.6 15 211.7 27.7 13.1 14 190.4* ( ↓ 9) 22.2 11.7

A 15 201.4 5.07 NA 15 209.9 5.14 NA 15 208.6 4.91 NA 14 192.4 5.06 NA Testis, left (mg) U 15 1423 106.8 7.5 15 1379 98.4 7.1 15 1436 111 7.7 14 1294* ( ↓ 9) 113.7 8.8

A 15 1394 25.5 NA 15 1412 25. 8 NA 15 1424 24.6 NA 14 1302* ( ↓ 7) 25.4 NA

Testis, right (mg) U 15 1428 118.7 8.3 15 1390 81 5.8 15 1441 115.3 8 14 1342 172.5 12.9
A 15 1401 31.2 NA 15 1421 31.6 NA 15 1430 30.2 NA 14 1350 31.1 NA Thyroid, fixed (mg) U 15 12.4 1.5 11.9 15 12.3 2.3 18. 5 15 13.3 1.9 14.6 14 12.8 1.8 14

A 15 12.3 0.51 NA 15 12.5 0.52 NA 15 13.2 0.49 NA 14 12.8 0.51 NA a Data were obtained from Table 9 on pages 59–61 of the study re port. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined NA Not applicable CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry concentrations are presented in Table 4. Although no dose-dependent treatment effects were observed on serum te stosterone or TSH, serum T 4 

### concentrations were significantly decreased (p<0.05) 14-23% at all OPP dose levels.

The following significant changes (p<0.05) were observed at 900 mg/kg/day in serum clinical chemistry parameters: (i) urea nitroge n levels were increased by 27%; (ii) ALT was increased by 95%; (iii) AST was increased by approximately 32%; and (iv) GGT wasMale Pubertal Assay (2012) / Page 13 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### decreased by 40%. GGT was also decrease d (p<0.05) by 40% at 250 mg/kg/day. There were no significant differences between contro l and treatment means on the other measured clinical chemistry parameters.

The results for serum T 4 

### , TSH, and testosterone in th e control group were within the acceptable range of the performance criteria provided in the Guideline (890.1500).Male Pubertal Assay (2012) / Page 14 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control OPP (50 mg/kg/day) OPP (250 mg/kg/day) OPP (900 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones
Serum T 4 , Total (μg/dL) 15 5.64 0.80 14.1 15 4.82* ( ↓ 14) 0.62 12.9 15 4.33* ( ↓ 23) 0.52 12.1 14 4.38* ( ↓ 22) 0.71 16.1
Serum TSH (ng/mL) 15 7.72 3.34 43.2 15 8.37 3.64 43.5 15 7.67 2.07 27.0 14 9.10 2.73 30.0
Serum

testosterone (ng/mL) 15 2.89 1.71 59.1 15 3.81 2.58 67.6 15 4.06 2.87 70.7 14 2.13 1.69 79.7 Clinical Chemistry
Creatinine (μmol/L) 15 0.1 0 36.1 15 0.1 0 31 15 0.1 0 34.5 14 0.1 0.1 46.7
Serum urea nitrogen (mg/dL) 15 15 2 12 15 14 3 18 15 16 2 15 14 19* ( ↑ 27) 3 16
Alkaline phosphatase (U/L) 15 346 56 16 15 338 64 19 15 325 51 16 14 302 57 19

ALT (U/L) 15 61 13 22 15 56 12 21 15 63 13 21 14 119* ( ↑ 95) 32 27

AST (U/L) 15 91 24 26 15 93 24 25 15 91 17 19 14 120* ( ↑ 32) 36 30

GGT (U/L) 15 2.5 1.5 59.4 15 2.2 1.2 56.6 15 1.5* ( ↓ 40) 0 0 14 1.5* ( ↓ 40) 0 0 Albumin (g/dL) 15 3.7 0.2 5.5 15 3.8 0.2 4.9 15 3.7 0.3 9.3 14 3.9 0.2 4.1 Albumin/ globulin ratio 15 1.9 0.4 19.4 15 2 0.2 11. 2 15 1.9 0.4 21.1 14 2.2 0.3 15.2
Cholesterol (mg/dL) 15 96 13 14 15 93 18 20 15 89 12 13 14 91 17 19 Globulin (g/dL) 15 2 0.3 14.3 15 1.9 0.2 8 15 2 0.3 15.4 14 1.8 0.3 13.8
Total bilirubin (mg/dL) 15 0.07 0.03 50.5 15 0.06 0.03 51. 4 15 0.05 0 0 14 0.07 0.06 90.9 Total protein (g/dL) 15 5.7 0.2 3.3 15 5.7 0.2 3.4 15 5.7 0.2 3.9 14 5.7 0.3 5 Sodium (mmol/L) 15 143 2 1 15 142 2 1 15 142 2 1 14 144 2 1
Potassium (mmol/L) 15 5.5 0.4 7.1 15 5.4 0.4 7.5 15 5.4 0.4 6.7 14 5.3 0.4 7.2 Chloride (mmol/L) 15 99 1 1 15 99 1 1 15 99 1 1 14 98 2 2 Calcium (mg/dL) 15 11.3 0.3 2.9 15 11.2 0.3 2.7 15 11.2 0.4 3.5 14 11.1 0.4 3.2 Phosphorus (mg/dL) 15 8.9 0.6 7.2 15 8.5 0.6 6.6 15 8.7 0.6 7.4 14 8.8 0.9 9.8 a Data were obtained from Table 8 on pages 57 and 58 of the st udy report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### F. GROSS PATHOLOGY: No treatment-related effects we re observed on gross pathology.

G. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 5. OPP was not associated with any significant treatmentrelated changes in thyroid histopathology.Male Pubertal Assay (2012) / Page 15 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

TABLE 5. Thyroid Gland Follicular Cell Height and Colloid Area a

Treatment Groups Parameter Evaluated
Colloid Area Follicular Cell Height

Grade b Incidence Grade b Incidence
Observed Examined Observed Examined

Vehicle Control 1 0 15 1 5 15
2 0 15 2 7 15
3 3 15 3 3 15
4 7 15 4 0 15
5 5 15 5 0 15

OPP (900 mg/kg/day) 1 0 14 1 9 14
2 0 14 2 3 14
3 2 14 3 2 14
4 3 14 4 0 14
5 9 14 5 0 14 a Data were obtained from Table 11 on page 67 of the study report. b Thyroid histopathology is graded 1–5. Fo llicular cell height: 1 = lowe st, 5 = highest; and Colloid area: 1 = most colloid, 5 = least colloid.



### The incidence of histopathological findings of the testes, epididymides and kidneys are presented in Table 6. No treatment-related findi ngs were observed in the right testes or right epididymides at any OPP dose.

At 900 mg/kg/day, an increased incidence of renal tubular dilatati on (combined focal and multifocal, very slight and slight severity) was noted (12/14 animals) compared to controls (5/15 animals). An increased incidence of collecting duct epithelium hypertrophy (combined focal and multifocal, very slight seve rity) was also observed (5/14) compared to controls (0/15). Alt hough the incidence of renal tubular degeneration was similar, the severity was increased in high-dose animals; the incidence of slight multifocal tubular degeneration was 3/14 compared to 0/15 in controls. The renal tubular dilation was characterized by the presence of multiple ectatic tubules (predominantly proximal convoluted tubules) in the cort ex and medulla that were li ned by flattened to cuboidal epithelial cells, and often cont ained proteinaceous material mixed with sloughed cellular debris. The dilated tubules were occas ionally accompanied by lymphocytes and degenerated tubules. Other renal lesions in the treatment groups were similar to controls or occurred in single animals.

Immature and decreased spermatic elements we re noted in the right epididymis; however, these effects were not dose-dependent and ar e consistent with the juvenile age of the animals. There were no treatment-related histopathologic effects in the right testis or right epididymis.Male Pubertal Assay (2012) / Page 16 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

TABLE 6. Incidence of Histopathological Lesi ons of the Testes, Epididymides and Kidney a

Findings Dose Level (mg/kg/day) Vehicle Control OPP (50) OPP (250) OPP (900)

# Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined

Testes
Within Normal Limits 15 15 NA NA NA NA 14 14 Epididymides
Within Normal Limits 3 15 0 15 0 15 0 14 Decreased spermatic elements, very slight 7 15 6 15 5 15 4 14

Decreased spermatic elements, slight 1 15 4 15 1 15 5 14

Immature spermatic elements, very slight 5 15 6 15 5 15 4 14

Immature spermatic elements, slight 4 15 7 15 9 15 7 14

Immature spermatic elements, moderate 1 15 2 15 1 15 3 14

Kidney
Within Normal Limits 0 15 2 15 2 15 0 14 Aggregates of mononuclear cells; interstitium, multifocal, very slight 5 15 3 15 0 15 2 14

Cyst, cortex, focal, very slight 2 15 0 15 0 15 0 14

Degeneration, tubule, focal, very slight 2 15 6 15 7 15 1 14

Degeneration, tubule, multifocal, very slight 12 15 5 15 4 15 9 14

Degeneration, tubule, multifocal, slight 0 15 0 15 0 15 3 14

Dilatation, pelvis, unilateral, slight 1 15 0 15 1 15 1 14

Dilatation, t ubule, focal, very slight 2 15 2 15 4 15 1 14

Dilatation, t ubule, focal, slight 1 15 0 15 0 15 0 14

Dilatation, tubule, multifocal, very slight 1 15 0 15 2 15 10 14

Dilatation, tubule, multifocal, slight 0 15 0 15 0 15 1 14

Fibrosis, interstitium, focal, very slight 0 15 0 15 1 15 0 14

Hyperplasia, epithelium, papilla; unilateral, multifocal, very slight 0 15 0 15 0 15 1 14

Hyperplasia, epithelium, papilla; bilateral, multifocal, very slight 0 15 0 15 0 15 1 14

Hypertrophy, collecting duct, epithelium, focal, very slight 0 15 1 15 0 15 2 14

Hypertrophy, collecting duct, epithelium, multifocal, very slight 0 15 0 15 0 15 3 14

a Data were obtained from Table 12 on pages 68–69 of the study report.Male Pubertal Assay (2012) / Page 17 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Overall, systemic toxicity was confirmed for the high-dose group (900 mg/kg/day; based on decreased body weight as well as liver weight increases, altered clinical chemistry paramete rs and histopathological changes in the kidneys) with some effect extending to the mid-dose gr oup (250 mg/kg/day; incr eased relative liver weights). Thyroid function was not altered at any dose of OPP. The small delay in preputial separation and decreased accessory sex tissue and reproductive or gan weights were attributed to decreased terminal body weight s in the high-dose animals. Thus, there was no evidence of direct effects on endocrine function in the male pubertal assay at doses up to and including 900 mg/kg/day OPP, the hi ghest dose level tested.

B. AGENCY COMMENTS: All animals survived until sc heduled termination, with the exception of four animals, but none of the deat hs were attributable to OPP treatment. Additionally, no treatment-related clinical signs of toxicity were observed in animals for any dose groups.

Terminal body weights were significantly decr eased in the 900 mg/kg/day group due to a treatment-related effect on body weight gain over the duration of th e study. Body weights and body weight gains were not significantly affected at OPP dose concentrations of
≤ 250 mg/kg/day. The adjusted age of attainme nt of PPS was delayed, but not significantly, compared to the control (45.1 vs. 43.5 days) in the 900 mg/kg/day gr oup. The significant delay in the unadjusted age of attainment of PPS may have been related to the observed decreased terminal body weights and body weight gain.
Organ weight changes (p<0.05) after administra tion of OPP included in creased relative liver weight at 250 mg/kg/day, and adjusted and relative liver weights at 900 mg/kg/day. There was a dose-dependent decrease in pituitary we ights, though only the una djusted and adjusted pituitary weights were significantly decrease d (p<0.05) 15% and 10%, respectively in the 900 mg/kg/day group. A dose-dependent decr ease in unadjusted adrenal weights was significant all treatment levels (decreased (p <0.05) by 11% at 50 and 250 mg/kg/day, and by 16% at 900 mg/kg/day).Mi nor but significant increases (p<0. 05) were noted in the adjusted liver and adrenal weights at 900 mg/kg/day. .

Additionally, decreased (p<0.05) organ weights at 900 mg/kg/day included: unadjusted weights of seminal vesicles + coagulating gla nds with and without fluid; unadjusted and adjusted ventral prostate a nd LABC weights; unadjusted we ights for the left and right epididymides; and unadjusted and adjusted weight s for the left testis. The adjusted organ weights were decreased (p<0.05) in the ventral prostate by 17%, in the LABC by 16%, and in the testis by 7%. As adjusted weight decrea ses were observed in three androgen sensitive tissues, these effects were considered treatment-related. There were no statistically significant differences between mean contro l and treatment organ weights of kidneys, dorsolateral prostate, righ t testis, or thyroid.

Although no dose-dependent treatment effects we re observed on serum testosterone or TSH concentrations, serum T 4 

### concentrations were decreased (p <0.05) at all dose levels by 15- 23% (p<0.05). No gross or histological lesions of the thyroid were observed.Male Pubertal Assay (2012) / Page 18 of 18 O -PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None



### At 900 mg/kg/day, an apparent treatment-related effect was observed on serum urea nitrogen concentrations, which corroborated observed renal histopathology. .

No treatment-related histological findings were observed in the right test es or right epididymides at any OPP dose. However in the kidneys, an increased incidence of renal tubular dilatation (combined focal and multifocal, very slight a nd slight severity) was noted in animals at 900 mg/kg/day compared to contro ls. An increased incidence of collecting duct epithelium hypertrophy (combined focal and multif ocal, very slight severity) was also observed at 900 mg/kg/day compared to contro ls. Although the incidence of renal tubular degeneration was similar in the high-dose and control, the seve rity was increased at the high dose.

The high dose of 900 mg/kg/day was adequate based on a decreased adjusted terminal body weight of 8%, decreased overall body weight gain of 13%, decreased androgen-sensitive organ weights of 7-17%, increa sed serum ALT of 95%, and in creased incidences and/or severity of renal lesions.

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

 CV for weight at PPS was 8.36% (maximum 7.57%);

 CV for final body weight was 8.2% (maximum 7.47%);

 Mean kidney weight was 2.01 g ( acceptable rang e 2.242-3.050 g);

 Mean thyroid weight was 12.4 mg (acceptable range 14-26 mg)

# DATA EVALUATION RECORD

ORTHO-PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672601)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Michelle Sharpe-Kass, M.S. Date: 5/14/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/24/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/24/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).ORTHO-PHENYLPHENOL/064103



### Primary Reviewer: Ayaad Assaad, D

. V

. M

. , Ph.D

. 

### Health Effects Division Secondary Reviewer: John Liccione, Ph.D. 

### Health Effects Division Steroidogenesis Assay (2012) I Page 1 of 13 OCSPP 890.1550/ OECD None



### Signature:**---F==:J---=-~~~F'--7~** 

### Date:**--T--:=-t'----±"7'-7~.-+-:t:---** 

### S ign a tu re:--.**~:::::i~.z~~::....~-** 

### D ate:

__ ____...;;L-..,,.._.,<-L--1--



### DA TA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells) ; OCSPP 890.1550



### PC CODE: 064103 

### DP BARCODE: D398785



### TXR#: 0055304 

### CAS No.: 90-43-7



### TEST MATERIAL (PURITY): Ortho-Phenylphenol (99.9% a.i

. )



### SYNONYMS: (1

, 

### 1 '-Biphenyl)-2-ol ; OPP ; Prevento! 

### 0 Extra



### CITATION: LeBaron

, M.J.

, Kan, H.L.

, Perala

, A.W. (2012). Evaluation ofOrthoPhenylphenol (OPP) in the Jn Vitro 

### Steroidogenesis Assay. Toxicology &


### Environmental Research and Consulting, Midland

, Ml. Laboratory Report No .: 111014

, Jan 4

, 2012. MRID 48672601. Unpublished.



### SPONSOR: OPP EDSP Consortium

, The Dow Chemical Company

, Midland

, MI and
LANXESS Corporation

, 111 RIDC Park West Drive

, Pittsburgh

, PA



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48672601 )

, H295R cells
cultured in vitro 

### in 24-well plates were incubated with ortho-phenylphenol (OPP, 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 concurrentl y 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.

No precipitation was observed at OPP concentration up to 100 μM, and OPP was not cytotoxic

to the H295R cells. Cell viability for the QC and test plates was 2: 95.4% for OPP and the
reference standards.

Four independent test runs of the steroidogenesis assay with OPP were conducted, but the fourth

was not statistically evaluated as three acceptable independent assays for OPP 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 recommendedSteroidogenesis Assay (2012) / Page 2 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### 40 pg/mL level. However, basal estradiol produc tion was still >26 pg/mL which is >2.5-fold the minimum detection limit (MDL), as per Guideline recommendation. Forskolin at 10 μ M induced testosterone and estr adiol production as required ( ≥ 2-fold and ≥ 7.5-fold, respectively), and 1 μ M prochloraz inhibited testosterone production by >50% as required; however, inhibition of estradiol production (40%) was below the required 50%.

The intra- and inter-plate vari ability (%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 OPP in Run 3 where the intraplate CV was 39.9%, due to in creased responsiveness.

Conflicting results were observed for testosterone production, with a statistically significant 30% increase at 10 μM OPP and a 30% decrease at 100 μM OPP. Statistically significant increases in estradiol production were obs erved at 10 μM OPP (2.6-fold) and 100 μM OPP (4.0-fold). These effects on estradiol were reproducible a nd concentration-dependent in all three runs.

Based on the hormone responses in the three in dependent runs, OPP treatment resulted in statistically significant and repr oducible increases in estradio l and testosterone production; however, the alterations in testoste rone production were inconsistent in direction of the response.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Steroidogenesis Assay (2012) / Page 3 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting

Location: Midland, MI Study Director: Kan, H.L
Other Personnel: LeBaron, M.J, Lead Scientist Perala, A.W., Analytical Chemist Gollapudi, B.B., Technical Reviewer Study Period: March 9, 2011 - January 4, 2012



### 2. Test Substance: Ortho-Phenylphenol (OPP)
Description: Technical, solid Lot #: (Expiration Date) CHHYDD0051 (not reported) Purity: 99.9 % Solubility (in Solvent): Ethanol(30 mM), ca. 0.5-0.6 g/L in water @ 20°C Volatility: ca. 0.0007 mbar (@ 20°C) Stability: 2 year shelf life Storage conditions: Ambient
CAS #: 90-43-7 Molecular weight: 170.2
Structure: OH



### 3. Positive Control (Inducer): Forskolin
Description: White powder, 410.50 MW
Source: Sigma, St. Louis, MO (Cat # not reported) Lot #: (Expiration Date): Lot # 097K50653 (Exp date not reported) Purity: 99% Solubility (in Solvent): DMSO, up to 0.01 M Storage conditions: Ambient
CAS #: 66575-29-9



### 4. Positive Control (Inhibitor): Prochloraz
Description: Off-White Powder, MW 376.67
Source: Sigma, St. Louis, MO (Cat # not reported) Lot #: (Expiration Date) SZE6220X (Exp date not reported) Purity: 99.1% Solubility (in Solvent): DMSO, up to 0.01 M Storage conditions: Ambient
CAS #: 67747-09-5Steroidogenesis Assay (2012) / Page 4 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO)
Description: Not reported
Source: Sigma-Aldrich, St. Louis, MO (Cat # 276855) Lot #: (Expiration Date) 16496CPV and 68996LMV (Exp date not reported) Purity: ≥ 99.9% Storage conditions: Ambient (room temperature)
CAS #: 67-68-5
Justification for choice of solvent: Not reported

Final concentration: (% volume in assay) 0.1%



### 6. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture
Description: Not reported
Source: Sigma, St. Louis, MO (Cat # D-6434) Lot #: (Expiration Date) RNBB 6106 (not reported)
Sodium bicarbonate: Not reported
Nu-Serum: BD-Biosciences (Cat # 355100), Lot # 355100; tested for background hormone concentrations by performing laboratory ITS+ premix: BD-Biosciences (Cat # 354352), Lot # 05245



### 7. Test Cells: H295R human adrenocortical carcinom a cells (ATCC, Manassas, VA, CLR2128; lot # not reported). Cells were maintained in DMEM/Ham-F12, including ITS+Premix, and 2.5% Nu-Serum, in a 5% CO 2 

### incubator at 37±2oC. The background hormone concentrations present in the Nu-S erum were 974 pg/mL for testosterone and 681 pg/mL for estradiol. It was stated that ce lls were cultured following the specific protocol described in the USEPA Endocri ne Disruptor Screening Program Guideline. Cells from frozen stocks were thawed and placed in tissu e culture flasks and allowed to recover from frozen by culturing for at least four additional passages prior to testing. Cells were plated into wells of 24-well culture plates at a density of approximately 2 to 3 × 10 5 

### cells/mL. Plates were incubated in a 5% CO 2 

### atmosphere at 37±2oC for approximately 24 hours prior to exposure to the test chemi cal or the positive controls.

The following performance criteria were met (indicated by an “x”): x Cell passage identifier. Cell Passage #: 7.5-8.5 x Cells frozen down at passage 5 x Frozen cells cultured for 4 additional passages x Total number of passages does not exceed 10



### B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Hormone interference te sts were not conducted because testosterone and estradiol concentr ations were determined by LC/APPI-MS/MS. Hormone cross-reactivity also was not determined.

b. Hormone Extraction: Briefly, standards and samples were vortexed with methylene chloride containing both testoste rone and estradiol internal st andards, and the organic phase were transferred to clean vials. The extrac ts were evaporated to dryness, and the finalSteroidogenesis Assay (2012) / Page 5 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### residue in each vial was deri vatized with dansyl chloride in sodium bicarbonate buffer (100 mM, pH 10.5).

c. Laboratory Proficiency Test: The report stated that laborat ory proficiency assays were performed to optimize and validate the H295R steroidogenesis assay. These non-GLP, unpublished experimental results with positive control chemicals demonstrated laboratory proficiency and validation a nd are reported separately.

2. Test Solutions: Stock solutions were prepared in DMSO and diluted in the final treatment medium. A total of seven con centrations were tested along with a DMSO vehicle control. None of the test and QC solution prep methods are explicitly stated in the study report, but analytical testing of actual OPP concentrations in the test solutions indicated that the target concentrations were achieved.

3. Cell Plating and Preincubation: It was stated that H295R (ATCC CLR-2128) cells were cultured following the specific protocol de scribed in the USEPA Endocrine Disruptor Screening Program Guideline. Cells from frozen stock were thawed a nd cultured for at least four additional passages. The cells were then seeded in 24-well plates using 1 mL of cells at a concentration of 200,000 to 300,000 cells/mL, yi elding approximately 50-60% confluency at 24 hours. The seeded plates were incubated for 24 hours at 37°C in a 5% CO 2 

### atmosphere.

4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed and re placed (1 mL). The cells were then exposed to the OPP or solvent vehicle alon e (DMSO) in triplicate according to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H 295R Cells to OPP (Final Concentrations in μM) a

1 2 3 4 5 6

A DMSO DMSO DMSO 0.1 0.1 0.1
B 100 100 100 0.01 0.01 0.01
C 10 10 10 0.001 0.001 0.001
D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 18 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



### A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner with 1 μL of either forskolin or prochloraz according to the schematic presented in Table 2.Steroidogenesis Assay (2012) / Page 6 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Po sitive Controls (Final Concentrations in μM) a

1 2 3 4 5 6

A Blank b Blank Blank Blank + MeOH c Blank + MeOH Blank + MeOH

B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH
C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochlor az 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Proc hloraz 1μM Prochloraz 1μM Prochloraz 1μM a Data were obtained from page 17 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well. b Blank wells received medium only. c MeOH = 70% methanol was added to these wells for 30 mi nutes at room temperature following medium removal.



### Following dosing, the plates were incubated for 48 hours under the conditions previously described. The medium from each well was re moved, split into two 0.45-mL aliquots, and frozen at

− 80°C until hormone measurements.

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined using the CellTiter 96 Aq ueous 

### One Solution Cell Proliferation Assay Kit (Promega, Madison WI, Cat# G3580), immediately after removal of the culture medi um after the 48-hour in cubation. This is a colorimetric modified MTT test using a te trazolium compound [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide] and an electron coupling agent. The quantity of formazan product, measured by the amount of 4 90 nM absorbance is directly proportional to the number of viable cells in the culture.

6. Hormone Measurement System: Concentrations of testoste rone and estradiol in the supplemented medium were determined by reverse-phase LC/APPI-MS/MS using an Applied Biosystems Sciex API 4000 tandem mass spectrometer equipped with an Agilent HP 1100 HPLC system.

The following performance criteria were met (indicated by an “x”):

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable fo r two concentrations of testosterone and estradio l (mean measured amount from triplicate samples within 30% of nominal concentration) . The report indicated that internal spike-in controls to measure extraction efficiency was used, but no results presented. NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) X Solvent control within 75% range be low maximum response on standard curve NA Test compound tested for interf erence with meas urement system



### C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations (SD) for testosterone and estradiol concentrations were calculated for each concentration of the reference chemicals and the SC, as well as the blank and background wells, on the QC plates and for each concentration of OPP and SC on the test chemical plate. Relative changes in hormone production were calculated w ith the following equation:

Relative change = (hormone concentrati on in each well) ÷ (mean SC hormone concentration)

Concentrations that exhibited ≥ 20% cytotoxicity, or where pr ecipitation was observed, were omitted from further analysis.

Prior to conducting statistical analyses, the assumptions of normality and variance homogeneity were evaluated. Homogeneity of va riance were evaluated by Bartlett's test andSteroidogenesis Assay (2012) / Page 7 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### normality by Shapiro-Wilk's test at alpha = 0.0 1. If the data were not homogeneous or normally distributed, then the data were tr ansformed to approximate homogeneity or a normal distribution. If the da ta were homogeneous and appr oximately normally distributed, differences between chemical treatments and SC were analyzed using parametric analysis of variance followed by Dunnett's test, if significan t. If the data were not homogeneous or normally distributed, a non-parametric test was used (Kruskal Wallis) and if significant, was followed by the Wilcoxon rank sum test with a Bonferroni-Holm correction. Differences were considered significant at p ≤ 0.05.

II. RESULTS

A. TEST COMPOUND: The hormone concentrations after exposure to OPP, and the folddifference change relative to the SC and the mean (±SD) for the three assay runs are presented in Table 3 and shown graphically in Figures 1 and 2. Precipitation was not observed at any tested OPP concentration

The %CV within each plate for the SC on the test chemical plates (calculated by the reviewers) ranged from 2.48-6.63% for testosterone and 3.78-10.1% for estradiol, and the between-plate %CVs for the test plates were 4.62% and 14.7% for testosterone and estradiol, respectively. These %CVs are within the Guideline limit of ≤ 30%.

A statistically significant slight increase (1.3-fold) in test osterone production was observed at 10 μM OPP, and a statistically significan t slight decrease (0.7-fo ld) was observed at 100 μM OPP; these effects on testosterone were inconsistent in direction. A statistically significant increase in estradiol production was observed at 10 μM OPP (2.6-fold) and 100 μM OPP (4.0-fold). These effects on es tradiol were reproduc ible, concentrationdependent and statistically significant in all three runs of the assay.Steroidogenesis Assay (2012) / Page 8 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with OPP for 48 Hours. a

Nominal Concentration (μM) Trial 1 Trial 2 Trial 3 Tria l 1 Trial 2 Trial 3 Mean ± SD b Statistical Significance

Testosterone (pg/mL) Fold Difference
DMSO 778.0 719.0 781.3 1.0 1.0 1.0 1.0 — —
0.0001 808.7 764.3 659.7 1.0 1.1 0.8 1.0 0.15 None
0.001 749.3 726.7 771.0 1.0 1.0 1.0 1.0 0.0 None
0.01 784.3 708.3 745.0 1.0 1.0 1.0 1.0 0.0 None
0.1 776.0 716.0 792.7 1.0 1.0 1.0 1.0 0.0 None

1 868.0 720.3 798.3 1.1 1.0 1.0 1.0 0.06 None
10 1,046.7 910.7 976.3 1.3 1.3 1.2 1.3 0.06 All 3 trials

100 556.3 523.7 525.0 0.7 0.7 0.7 0.7 0.0 All 3 trials Estradiol (pg/mL) Fold Difference
DMSO 28.5 34.2 38.3 1.0 1.0 1.0 1.0 — —
0.0001 27.1 37.8 39.6 1.0 1.1 1.0 1.0 0.06 None
0.001 28.3 36.9 43.6 1.0 1.1 1.1 1.1 0.06 None
0.01 30.6 41.3 45.0 1.1 1.2 1.2 1.2 0.07 None
0.1 28.3 37.5 44.7 1.0 1.1 1.2 1.1 0.10 None

1 31.4 38.8 44.4 1.1 1.1 1.2 1.1 0.06 None
10 82.2 93.7 84.7 2.9 2.7 2.2 2.6 0.36 All 3 trials

100 131.3 132.7 135.3 4.6 3.9 3.5 4.0 0.56 All 3 trials a Data were obtained from page 32 of the study report. b Calculated by the reviewers.Steroidogenesis Assay (2012) / Page 9 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Produc tion Relative to OPP Concentration.

* Significantly different from the solvent control at p ≤ 0.05.Steroidogenesis Assay (2012) / Page 10 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### FIGURE 2. Change in Estradiol Producti on Relative to OPP Concentration.

* Significantly different from the solvent control at p ≤ 0.05.Steroidogenesis Assay (2012) / Page 11 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### B. CYTOTOXICITY: No evidence of cytotoxicity was present from the MTT cell viability assay conducted on the QC and OP P test plates (Table 4). Me thanol exposure reduced cell viability to 28-35%.

TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or OPP for 48 Hours. a

Compound Concen. (μM) Cell Viability – Run 1 Cell Viabilit y – Run 2 Cell Viability – Run 4
Mean SD Mean SD Mean SD

DMSO NA 100.0 NR 100.0 NR 100.0 NR
Forskolin 1 115.5 NR 133.1 NR 116.4 NR
Forskolin 10 111.6 NR 120.1 NR 110.5 NR
Prochloraz 0.1 105.7 NR 121.0 NR 103.6 NR
Prochloraz 1 92.5 NR 99.8 NR 107.3 NR Media only NA 96.0 NR 98.4 NR 95.4 NR
Blank + MeOH NA 27.8 NR 32.1 NR 28.3 NR
SC + MeOH NA 29.1 NR 34.6 NR 29.1 NR

DMSO NA 100.0 NR 100.0 NR 100.0 NR
OPP 0.0001 106.1 NR 110.9 NR 105.8 NR
OPP 0.001 113.1 NR 118.0 NR 111.7 NR
OPP 0.01 104.2 NR 113.6 NR 96.9 NR
OPP 0.1 103.2 NR 104.8 NR 111.0 NR
OPP 1 110.7 NR 122.1 NR 105.6 NR
OPP 10 99.0 NR 122.7 NR 111.5 NR
OPP 100 113.1 NR 128.5 NR 122.5 NR a Data were obtained from page 51 of the study report. NA Not applicable NR Not reported SC Solvent control



### C. QC PLATE: The hormone concentrations after exposure to OPP, SC, blank and background samples, as well as the fold-differe nce change relative to SC (individual and mean ± SD) for the three assay runs, are presen ted in Table 5. The minimum basal hormone production levels for testosterone (500 pg/mL ) were met in both blank and SC wells for testosterone. The minimum basal hormone pr oduction levels for es tradiol (26.3-38.3 pg/ml) were below the recommended concentration of 40 pg/mL. Although the basal estradiol production was <40 pg/mL, basal production was st ill >26 pg/mL, which is >2.5-fold the MDL as per guideline recommendation. Testoste rone and estradiol in the presence of 10

μ M forskolin met induction requirements ( ≥ 2-fold and ≥ 7.5-fold, respectively). Inhibition of testosterone production >50%, as required, in the presence of 1 μM prochloraz; however only 40% inhibition of estradiol was achieve d in each run. Because all other guideline requirements for basal hormone concentrations , and hormone induction and inhibition, were met for testosterone and estradiol, these results were deemed acceptable.

The intra- and inter-plate variability (%CV) was reported to be 0.5-24.4% for the testosterone and estr adiol assays, with the exception of 0.01 μM OPP in Run 3 where the intraplate CV was 39.9%, due to increased responsiveness in one assay. Calculation of the variability by the reviewers confirme d the within- and between-plate %CVs.Steroidogenesis Assay (2012) / Page 12 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

TABLE 5. Mean ± Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours. a

Compound Conc. (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Testosterone (pg/mL) Fold Difference

Blank NA 1041 936 1040 — — — — —

DMSO NA 778 719 781 1.0 1.0 1.0 1.0 —
Forskolin 1 1377 1217 1310 1.8 1.7 1.7 1.7 0.06
Forskolin 10 2007 1757 1733 2.6 2.4 2.2 2.4 0.20
Prochloraz 0.1 515 514 534 0.7 0.7 0.7 0.7 0.0
Prochloraz 1 250 233 218 0.3 0.3 0.3 0.3 0.0 Estradiol (pg/mL) Fold Difference
Blank NA 26.3 34.8 37.2 — — — — —

DMSO NA 28.5 34.2 38. 3 1.0 1.0 1.0 1.0 —
Forskolin 1 348 366 492 12.2 10.7 12.8 11.9 1.08
Forskolin 10 610 626 780 21.4 18.3 20.4 20.0 1.58
Prochloraz 0.1 26.4 29.7 28. 6 0.9 0.9 0.7 0.8 0.12
Prochloraz 1 17.6 19.5 21. 9 0.6 0.6 0.6 0.6 0.0 a Data were obtained from page 33 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the combined hormone responses in each of three independent H295R steroidogene sis assays, it was determined that OPP treatment resulted in a statistic ally significant increase in es tradiol (E2) production at the highest two concentrations tested, including the assa y limit concentration (10 -4 

### M), while no effects on estradiol production we re seen at lower concentrati ons. There were very slight (<1.5-fold), divergent alterations in te stosterone production at the highest two concentrations of OPP. The slight increase in testosterone production did not meet the 1.5- fold cut-off criterion established in the valida tion program for this assay, and hence was not considered to be biologically re levant. Thus, the results of this in vitro steroidogenesis assay indicate that, under the conditions of this study, OPP increased estradiol production at the highest two concentrations tested.

B. AGENCY COMMENTS: Precipitation of OPP was not observed at any concentration tested ( ≤ 100 μ M), and OPP was not cytotoxic to th e H295R cells. Cell viability was
≥ 95.4% for OPP and the reference compounds on the QC and/or test plates.

Four independent test runs of the steroidoge nesis assay with OPP were conducted, but the fourth was not evaluated statistically as th ree acceptable independent assays for OPP had been completed. The minimum basal hormone production levels for estradiol were below the recommended concentration of 40 pg/mL; however, basal estradiol production >26 pg/mL, which is 2.5-fold greater than the MD L as per guideline reco mmendation. Induction of testosterone and estradiol production by 10 μ M forskolin met guideline requirements ( ≥ 2- fold and ≥ 7.5-fold, respectively). Inhibition of testosterone produc tion by 1 μM prochloraz met guideline requirements, but inhibition of estrad iol production was <50% in each run.

The intra- and inter-plate variability (%CV) was reported to be 0.5-24.4% for the testosterone and estradiol, w ith the exception of 0.01 μM OPP in Run 3 where the intraplate CV was 39.9%, due to increased responsiveness in one assay. Calculation of the variabilitySteroidogenesis Assay (2012) / Page 13 of 13 ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None



### by the reviewers confirmed that the within- and between-plate % CVs were identical to those calculated for the test plates because th e SC data from the QC plates was used to replace the SC data from the test plates due to test material vol atility and apparent contamination of the SC wells by the test material.

A statistically significant slight increase (1.3-fold) in test osterone production was observed at 10 μM OPP, and a statistically significan t slight decrease (0.7-fo ld) was observed at 100 μM OPP; these effects on testosterone were inconsistent in direction. A statistically significant increase in estradiol production was observed at 10 μM OPP (2.6-fold) and 100 μM OPP (4.0-fold). These effects on es tradiol were reproduc ible, concentrationdependent and statistically significant in all three runs of the assay.

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

 No Certificates of Analysis for the test mate rial or reference chemicals were included in the study report.

 The specificity, accuracy and recovery for testosterone and estr adiol by the LC/APPIMS/MS method were not provided.

 Minimum basal production levels for estr adiol in the SC wells were not met.

 Inhibition of estradio l production (40%) by 1 μ M prochloraz was less than the guideline specified 50% inhibition.

# DATA EVALUATION RECORD

ORTHO-PHENYLPHENOL (OPP)

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-42-2012 (MRID 48672605)

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

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Kelly Luck, M.S. Date: 5/07/2012

Secondary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 5/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/24/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/24/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).ORTHO-PHENYLPHENOL/ 064103 In vivo Uterotrophic Assay (2012) I Page I of8
OCSPP 890.1600/ OECD 440



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D 

### Signature:

# A~**· ~J**  Health Effects Division Date:**~** 

### _ Secondary Reviewer: _J_e_ss_R_ow_la_n_d

_________ 

### Signature: 

## de:: ">-O**~** 

### Health Effects Division Date:

__**b~/~!t'-'l~l_)-=---** Template version 09/2011



### DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat) ; OCSPP 890.1600; OECD 440



### PC CODE: 064103 

### DP BARCODE: D398785



### TXR#: 0055304 

### CAS#: 90-43-7



### TEST MATERIAL (PURITY): Ortho-phenylphenol (99.9% a.i.)



### SYNONYMS: OPP; Prevento! 

### 0 Extra ; 

### (1

, l ' -Biphenyl)-2-ol



### CITATION: Marty, M. S.

, Zablotny

, C. L.

, Passage, J. 

### K., and Jeong, YC. (2012). Orthophenylphenol (OPP): uterotrophic assay in the immature female Crl:CD(SD)

rat. Toxicology & 

### Environmental Research and Consulting, Dow Chemical Company

, Midland, MI. Laboratory Project Study ID: 111086, January 5

, 2012

. MRID 48672605. Unpublished.



### SPONSOR: OPP EDSP Consortium : Dow Microbial Control

, The Dow Chemical Company

, Midland, MI; and LANXESS Corporation, Materials Protection Products

, 111
RlDC Park West Drive

, Pittsburgh, PA



### TEST ORDER#: CON-064103-27



### EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48672605) conducted to screen for potential estrogenic activity

, ortho-phenylphenol (OPP ; 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 l 7a-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 OPP dosed animals. Overall body weight gains

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 OPP 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.In vivo Uterotrophic Assay (2012) / Page 2 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### No statistically significant changes were seen in uterine weight in this as say. OPP is negative in the uterotrophic assay.

The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance and Quality Assurance statements were provided.In vivo Uterotrophic Assay (2012) / Page 3 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, Dow Chemical Company

Location: Midland, MI Study Director: C. L. Zablotny
Other Personnel: M. S. Marty (Lead Scientist), J. K. Passa ge (Study Director in Training), and YC. Jeong (Toxicologist) Study Period: June 9, 2011 to January 5, 2012



### 2. Test Substance: Ortho-phenylphenol (OPP) Description: Molecular weight 170.2 g/mol
Source: Lanxess Deutschland GmbH (Leverkusen, Germany)
Lot #: CHHYDD0051 (expiration date not reported) Purity: 99.9% Stability: Stable in corn oil for 15 days at room temperature
CAS #: 90-43-7 Structure: OH



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich
Lot #: 090m1241v (expiration date not reported) Purity: Not reported
CAS #: 57-63-6



### 4. Solvent/Vehicle Control: Corn oil Supplier: Sigma Aldrich (St. Louis, MO)
Lot/Batch #: Not reported Rationale (if other than water): Selected due to solubility properties of the test substance
Final concentration: Not applicable



### 5. Test Animals: Species: Rat (immature females only)
Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: PND 19/ 42.4 – 52.7 g females
Source: Charles River Laboratories, Inc. (Portage, MI); immature rats (PND 11) were received with lactating dams. Pups were weaned PND 18 (1 day prior to dosing). Housing: Weanlings were housed 6/cage in solid bottom cages with paper pulp bedding.
Diet: LabDiet Certified Rodent Di et #5002 (PMI Nutrition In ternational, St. Louis, MO) , ad libitum (total genistein equiva lents <325 μg/g diet)
Water: Municipal water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22  3 oC 40-70% 12-15/hr (average) 12 hrs light/ 12 hrs dark Acclimation period: 8 days (7 days housed with thei r dam plus 1 day after weaning )



### B. STUDY DESIGN

1. In Life Dates: Start: June 17, 2011 End: June 20, 2011In vivo Uterotrophic Assay (2012) / Page 4 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### 2. Study Design: Following an initial 8-day acclimation period, immature, intact female rats were administered the test substance from PND 19-21. Rats were euthanized (PND 22) approximately 24 hours after the last dose and ne cropsied for uterine weight measurements. The pups were housed with their dam fo r 7 days prior to weaning on PND 18.

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1 using a computer program designed to increase the probability of uniform group mean weights and standard devi ations at the start of the study. Statistical analysis indicated that th ere were no significant differe nces in group means at study initiation. Furthermore, the body we ight of each animal was within  20% of the overall

mean.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay
Vehicle Control 0 6
OPP Low 50 6
OPP Mid 250 6 OPP High 1000 6
17 α -ethynyl estradiol (EE), Reference Estrogen 10 μg/kg/day 6 a Data were obtained from Text Table 1 on page 14 of the study report.



### 4. Dose Selection Rationale: The high dose level was selected based on the results from a preliminary probe study 1 

### in which four Crl:CD(SD) rats/sex/dose were dosed at 0, 250, 500, 700, or 900 mg/kg/day by oral gavage for 15 days. For females, body weight, body weight gain, and feed consumption in all dose groups were similar to controls. There were no marked effects on clinical chemistry paramete rs and electrolytes. Females in the 900 mg/kg/day group had increased absolu te and relative liver weights ( ↑ 11% and 16%, respectively) and increased absolute kidney weights ( ↑ 8%); increases in absolute and relative liver weights were also seen in fe males in the 700 mg/kg/day group. The high dose level was expected to cause a significant decrease in body we ight gain over the dosing period in the current study. The lower dose leve ls were selected to provide dose response data for any estrogenic effects that may have been observed among the high-dose group rats.

5. Dose Preparation and Analysis: Dose formulations were prepared once at the beginning of the study based on the establishe d stability of the test substan ce in corn oil. Homogeneity and achieved concentration were tested prio r to study initiation in each of the dose formulations by sampling the top, middle, and bo ttom portions. The stability of OPP in corn oil was tested in conjunction with the Hershberger study (see DER for MRID 48672602; concurrently reviewed).

Results of Dose Analysis

1 Marty, M. S. and Marshall, V. A. (In progress). Or thophenylphenol: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxico logy & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.In vivo Uterotrophic Assay (2012) / Page 5 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### Homogeneity (% R.S.D.): 0.3-0.8%

Stability (% of Day 1): 104.2-106.1% at concentrations of 0.25-250 mg/mL for up to 15 days at ambient temperature

Concentration (% of nominal): 97.0-100.3%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Animals were administered th e test formulation, the positive control (EE), or the vehicle control daily via or al gavage for three consecutive days in a dose volume of 4 mL/kg body weight. Dose volum es were adjusted daily based on the concurrent body weight measurement.

7. Statistics: Body weights and gains were analyzed by a forced parametric test. For uterine weights (blotted and wet), a Bartlett’s test was conducted to check for homogeneity of variance. Based on the outcome of the Bartle tt’s test (p=0.01), we t uterine weights for vehicle control versus positive control were l og transformed prior to further analysis. An analysis of covariance (ANCOVA) was perfor med with dose in the model and terminal body weight as the covariate. Statistical anal yses were conducted for data from the vehicle controls versus OPP treatment groups and da ta from the vehicle controls versus the EEtreated group. If the dose effect was significant (p<0.05), one -sided least squared means with Dunnett’s correction were used to determine differences, since the least square means were adjusted for the covariant. An additional analysis of variance (ANOVA) was run separately on relative uterine weights. Th e incidence of vaginal opening was reported but not statistically analyzed. The statisti cal analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality, morbidity and clinical signs of toxicity were conducted twice daily, except fo r Day 1 when the check was conducted once. Detailed physical examinations were perf ormed prior to initia tion of dosing and at termination.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout the dosing period, and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine Weight: On PND 22 (approximately 24 hours after final administration of the test substance), all animals were checked for precocious vaginal opening, anesthetized by carbon dioxide inhalation, euthanized by cervical dislocation, and subjected to a gross necropsy. Dissection of the uterus was performed according to the Guidelines (OCSPP 890.1600). The “wet” uterus (i.e., containing the luminal fluid) was weighed. If a leak of fl uid was noted, this observation was recorded and the weight excluded from statistical analysis. Subsequently, each uterine horn was “nicked”In vivo Uterotrophic Assay (2012) / Page 6 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### three times, covered with saline-moistened abso rbent paper, and blotted by gently placing a 500 g weight on the paper-covered uterus to remove the luminal fluid while preventing desiccation. The blotted ut erus was then weighed.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived unt il scheduled termination.

2. Clinical Signs of Toxicity: No treatment-related clinical si gns of toxicity were observed in animals for any dose groups. No precocious vaginal opening was observed in any rats on the day of termination.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and overall body weight gain data are presented in Table 2. Overall body weight gains were decreased (p<0.05) by 25% in the 1,000 mg/kg/day group compared to controls. 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.

TABLE 2. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control OPP (50) OPP (250) OPP (1000) Reference Estrogen
EE (10 μg/kg/day)
N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 46.1 3.1 6 46.9 3.5 6 46.4 2.9 6 46.8 3.9 6 46.1 3.4
2 6 49.8 3.5 6 49.5 3.2 6 49.3 2.5 6 48.3 4.7 6 48.6 3.6
3 6 54.4 3.8 6 54.6 3.1 6 54.8 3.0 6 52.0 5.4 6 53.1 3.9
4 6 59.2 3.9 6 59.1 2.7 6 60.1 3.2 6 56.6 6.0 6 58.2 4.4 Body Weight Gain (1 - 4) 6 13.0 0.8 6 12.2 2.0 6 13.7 1.5 6 9.8* ( ↓ 25) 2.7 6 12.1 2.0

a Data were obtained from Table 3 on page 28 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY

1. Uterine Weights: Uterine weight data are presented in Table 3. Uterine weights in the OPP treatment groups were comparable to the vehicle controls. Absolute wet and blottedIn vivo Uterotrophic Assay (2012) / Page 7 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### uterus weights for the positive control (EE) group were increased (p<0.05) by 897% and 432%, respectively. The increased uterine we ights were within the expected range.

The mean blotted uterine weight for the vehicle control group was 0.046% of the mean vehicle control terminal body weight, which meets the Guideline requirement (<0.09%).

TABLE 3. Uterine Weights from Estrogen Agonist Assay in Sprague Dawley Rats a

Parameter Dose (mg/kg/day)

Vehicle Control OPP (50) OPP (250) OPP (1000) Reference Estrogen EE (10 μ g /k g /da y )

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 6 59.2 3.9 6 59.1 2.7 6 60.1 3.2 6 56.6 6.0 6 58.2 4.4 Wet, absolute (mg) 5 27.9 5.6 6 28.5 3.1 6 29.7 5.0 6 30.1 4.0 6 278.3* ( ↑ 897) 56.2

Wet, relative (%) b 5 0.048 0.009 6 0.048 0.005 6 0.049 0.006 5 0.052 0.009 6 0.483 0.115 Blotted, absolute (mg) 6 27.1 4.9 6 27.3 2.9 6 28. 1 4.7 6 28.1 3.7 6 144.2* ( ↑ 432) 18.4

Blotted, relative (%) b 6 0.046 0.007 6 0.046 0.004 6 0.047 0.006 6 0.050 0.008 6 0.249 0.039 a Data for terminal BW and absolute weights were obtained from Table 4 on page 29 of the study report. Percent differences from controls are included in parentheses. b Relative weights were calculated by the reviewers, usi ng the individual terminal BW and uterine weights from Appendix Table 2 on pages 34 and 35 of the study report. BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There were no animal mortalities or treatmentrelated clinical observations in this st udy. There were no significant effects on body weights in any OPP-treatment group and no signi ficant differences in body weight gains at OPP doses ≤ 250 mg/kg/day. At 1000 mg/kg bw/day, body weight gains were significantly decreased at all test intervals with an overall 25% decrease in body weight gains from days 1-4. There were no treatment-rela ted increases in mean uterine weights (wet or blotted) in any OPP treatment group. In the positive contro l group, significant increases in wet and blotted uterine weights were observed without any significant changes in body weights or body weight gains. None of the animals in th is study exhibited precoc ious vaginal opening. Uterine weights from vehicle-treated controls met the performance cr iteria outlined in the applicable test guidelines, indicating acceptable assay sens itivity. Overall, under the conditions of this study, there was no indication of estrogenicity in immature female rats at doses ≤ 1000 mg/kg bw/day OPP, the limit dose level in the uter otrophic assay.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any OPP treatment groups, and no precocious vaginal opening was observed in the treated i mmature females. Overall body weight gainsIn vivo Uterotrophic Assay (2012) / Page 8 of 8 ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440



### were decreased (p<0.05) by 25% in the 1,000 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 st udy. Uterine weights in th e OPP treatment groups were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increa sed (p<0.05) by 897% and 432%, respectively, as expected. No statistically significant changes were seen in uterine weight in this assay. OPP is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None