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 Effects Division (7509P) 

THROUGH: Jess Rowland ~-v-51--­ Deputy Director 

Health Effects Division 

TO: Jolene Trujillo 

Biologist/Chemical Review Manager 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7505P) 

I. ACTION REQUESTED 

Ver.Apr. 20 I 0 

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

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays 

submitted in response to the agency's Test Order for o-phenylphenol: Test Order# CON064103-27. 

Page I of2 

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Page 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 other scientifically relevant information (OSRI)) 

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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'

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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.

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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). 

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Results Synopsis:

Test organism NF stage at test initiation: 51 

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

Test type: Flow-through 

Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects1,2 in the Amphibian 

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

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross 

and 

Histopathology 

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

0.0175 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). 

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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 CaCO3) exceeded the recommended range of 40-48 

mg/L as CaCO3. 

 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, US 

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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 reported 

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B. Test Organism

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: African clawed frog EPA recommends African clawed frog 

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a list 

of all of the necessary protocol deviations to 

accommodate this species is recommended 

for inclusion in the study report. The guideline 

recommends that the performance criteria 

used to support the reliability of the test be 

identified.

Species scientific name: Xenopus laevisSpecies strain (if stated): Not reported 

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

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as in-house stock? 

Yes Breeding pairs, from which tadpoles were 

obtained, were acquired from Xenopus

Express (Brooksville, Florida). 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Not specified

Acclimation period for parental frogs (if applicable): 

Not specified

Details on parental feeding: Not specified Details on parental health: Not specified 

Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? Not specified EPA and OECD recommend that the best 2 – 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of spawns evaluated (if applicable): 

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 per spawn:

NA 

NF stage at test initiation 51 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 days 

post-fertilization (dpf)

Mean total length at test initiation (if reported): 

Not reported 

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

Not reported 

Was the optional size selection method used? 

No 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on larval selection: Not described No details were provided on selection of 

individual tadpoles used in the test.Adult males and females were injected with human chorionic gonadotropin the day before the desired breeding and placed in 10-gallon glass aquaria. Adults 

were removed after breeding and egg masses were distributed over several hatching tanks such that the embryos were not overcrowded. After approximately 4 days, tadpoles were free swimming and were transferred to rearing vessels (10 tadpoles/L). Tadpoles were reared until NF stage 51. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Loading 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 

demonstrate equal performance to Sera 

Micron® .

Source of food: Sera North America, 

Inc., Montgomeryville, Pennsylvania

Iodide concentration in diet (if known):

50 μg/g 

Frequency of feeding: 2 times/day EPA recommends that feeding occur at least 

twice per day.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: 5 days post hatch to NF 

stage 51: 30 mg/animal/dayDuring exposure, increased from 30 mg/animal/day (days 0-4) to 80 mg/animal/day (days 15-21) 

Days 

mg Sera 

micron/Tank 

0-4 30 

5-7 40 

8-10 50 

11-14 70 

15-21 80 

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.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Continuous flow Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

32 ± 3 mL/min This flow-rate provided a complete volume 

turnover approximately every 2.7 hours. Day 

14 analytical recoveries had dropped substantially from day 7 values. Therefore, in an attempt to increase recovery rates, the flow of stock solutions to the test chambers 

was increased by ~70% on day 16. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems (if applicable):

For each treatment level, a continuous-flow syringe 

pump continuously delivered a concentrated test substance stock solution at a designated rate from a stock vessel to a stainless steel mixing chamber where it mixed with dilution water; no test substance stock was added 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 system is recommended; 4) The 

recommended flow splitting accuracy is 

within 10%.

Renewal period for static renewal (if applicable):

NA If static renewal is used, EPA recommends 

24-hr renewal; renewal period is 

recommended not to exceed 72 hours.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? 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 water 

was Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. Prior to use 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

thyroid gland (e.g., fluoride, chlorate, 

perchlorate). OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

in the laboratory, the water was sand-filtered, 

pH-adjusted with gaseous 

CO2, carbon-filtered, and 

UV-irradiated. 

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

Yes, all below levels of concern

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 μg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide (e.g. 

in Sera Micron).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel type/materials: Glass sealed together 

with clear silicone adhesive

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 cm Fill volume: ~5.2 LAdditional details on exposure system: 

NA 

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

Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 64 78 71 

Weekly in the control 

and highest 

treatment group 

replicates 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.0 7.6 7.3 

Weekly in all 

replicate 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 8.2 Weekly in all 

replicate 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 22.8 22.4 

Weekly in all 

replicate test vessels 

and continuously in 

one control vessel 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

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Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

Iodide Not reported 

EPA recommends aquatic iodide range 0.5 

– 10 μg/L (supplemental iodide should not 

exceed 2 μg/L).

Ammonia Not 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 the 

Fluoride Not detectable Perchlorate Not detectable Chlorate Not detectable Alkalinity (mg/L as CaCO3)Conductivity (μmhos/cm) 

36 

159 

48 

226 

41 

181 

Weekly in the control 

and highest 

treatment group 

replicates 

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Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

beginning and end of the test.

1 Not reported by study author; reviewer calculated 

 D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the range-finder? 

No toxicity data were available to Xenopus

and the reported 

solubility was 700 mg/L at 20°C. A maximum 

concentration of 25 mg a.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 toxicity (e.g., <10% 

mortality), whichever concentration is lowest.

Species: Xenopus laevis 

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Life stage: Not specified Test duration: 4 daysAdditional details: A non-GLP, 96-hr, 

static renewal test was conducted in general accordance with ASTM methods. Nominal concentrations were 0 (negative control), 1.6, 3.1, 6.3, 12.5, and 25 mg a.i./L. After 96 hours, complete mortality was observed in the two highest treatment groups, yielding an LC50 of 8.87 

mg a.i./L. 

Dividing the LC50 by 3 yielded 

approximately 3.0 mg a.i./L, which was set as the highest concentration in the definitive AMA. 

Behavioral abnormalities (e.g., laying on 

the bottom, floating near the top) were observed in the 1.3 and 3.1 mg a.i./L treatment groups, an additional low concentration was included to ensure that 

an adequate number of treatments did not exhibit overt toxicological effects.

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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

Range-finder; 1/3 of the LC50

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., <10% 

mortality), whichever concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations: 0.2 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Time-weighted average 

Limit 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 reported Frequency of measurement: Days 0, 7, 14, 17, and 

21

It is recommended that test item concentration 

be measured in one tank at each treatment 

level at test initiation and every week 

thereafter.

Number of replicates in control: 4 EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in solvent control (if applicable):

NA EPA and OECD recommend the use of a 

concurrent solvent control when a solubilizing 

agent is used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 (20/rep) 

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

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any 

other adverse effects and that concentration 

not be greater than 0.1 ml/L4.

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

Aquatic Toxicology, 76, pp.69–92. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a positive control used? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

cool white fluorescent bulbs EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water’s surface: 0.759-0.893 klux measured weekly EPA recommends light intensity 0.6 – 2 Klux 

(at water’s surface).

Additional details: NA 

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

on Testing and Assessment. No. 23. Paris, France. 

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Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Ortho-Phenylphenol. 

Treatment ID

Nominal 

Concentration 

(mg a.i./L)

Time-Weighted 

Average 

Measured 

Concentrationa

(mg a.i./L) 

Mean CV1 (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA Per the study report, the 

decline in concentrations was likely due to increased 

biomatter and enhanced biodegradation throughout the test. 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient of 

variation (CV) ≤20%.

Treatment 1 0.024 0.0175 42.7 Treatment 2 0.12 0.0673 68.8 Treatment 3 0.60 0.352 72.2 Treatment 4 3.0 1.92 57.1 

Abbreviations: CV Coefficient of variation. 1 As reported by study author 

 E. Observations

Biological Endpoints: Day 7- NF stage, hind-limb length (HLL), snout-vent length (SVL), HLL normalized by SVL, wet weight 

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 Day 21- NF stage, HLL, SVL, normalized HLL, wet weight, histopathology  Daily- mortality and sub-lethal effects 

Were 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 (Days 7 and 21); 

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

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

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II. RESULTS AND DISCUSSION 

A. Results

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 71 Day 21 

n2 Mortality # Mortality % n2 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 replicate 

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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 

n1 Median 

Stage2 # Asynchronous n1 Median Stage2 # Asynchronous 

Negative Control 4 54.5 0 4 59.53 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 author 

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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 212

n3 Mean4

(mm) 

±SD4 HLL: 

SVL1 n3 Mean4(mm) 

±SD4 HLL: 

SVL1

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-calculated 

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Day 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 ranged 

from 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 Weight1

Day 7 Day 212 Day 7 Day 212

n3

Mean

(mm) 

±SD n3

Mean

(mm) 

±SD n3

Mean

(g) 

±SD n3

Mean

(g) 

±SD

Negative Control 4 19.2 1.12 4 30.5 0.213 4 0.632 0.077 4 2.30 0.113 

0.0175 4 18.7 0.924 4 30.3 0.285 4 0.594 0.068 4 2.20 0.103 

0.0673 4 19.2 0.360 4 30.0 1.29 4 0.629 0.035 4 2.17 0.190 

0.352 4 19.1 0.516 4 30.3 0.842 4 0.614 0.050 4 2.30 0.117 

1.92 4 18.4 0.262 4 29.4 1.16 4 0.563 0.050 4 2.06 0.231 

Abbreviations: SD Standard deviation. 1 Also referred to as “wet weight” in the test guideline. 2 Results based on non-late stage tadpoles (NF < 61) only. 3 number of replicate tanks 

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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. 

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

Treatment

(mg a.i./L)

[TWAmeasured]

Diagnostic Observations1

Severity

Thyroid Gland 

Hypertrophy 

Thyroid Gland 

Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 20 20 20 20 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.

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

0.0175 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

0.0673 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

0.352 0 NA NA NA NA NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

1.92 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

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

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All 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 resting on 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 Signs1

Type n2 Incidence 

Negative Control No effects 80 -- 

0.0175 No effects 80 -- 

0.0673 Resting on surface 80 1 (1.3%) 

0.352 No effects 80 -- 

1.92 Resting on bottom 79 1 (1.3%) 

1 Note that asynchronous development (unable to stage) is reported previously in Table. 2 Total number of tadpoles per replicate 

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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 20th to the 80th 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 significant 

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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. 

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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. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Ortho-Phenylphenol (Non-Late Stage). 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

Median p Median p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 NA 59.5 NA NA NA NA NA NA NA NA NA No 

0.0175 54 0.62 59.5 1.0 -4.09 0.79 2.01 0.81 NA NA NA NA No 

0.0673 54 0.22 59 0.62 0.18 1.0 -0.29 0.67 NA NA NA NA No 

0.352 54 0.62 59.5 1.0 -4.26 0.76 -2.01 0.35 NA NA NA NA No 

1.92 54 0.22 59 0.18 -7.28 0.35 -7.54 0.04 NA NA NA NA No 

Statistical Test Mann-Whitney Mann-Whitney Dunnett’s Jonckheere NA NA NA 

Abbreviations: Diff. Difference. NA Not applicable.

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

by the expert opinion of a board-certified pathologist. 

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

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Table 17: Growth Endpoints1,2 in the AMA with Ortho-Phenylphenol (Non-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 -- -- -- -- -- -- -- -- 

0.0175 -2.60 0.73 -0.70 0.19 -6.84 0.66 -4.39 0.78 

0.0673 0.00 1.0 -1.76 0.33 -0.47 1.0 -5.74 0.60 

0.352 -0.39 1.0 -0.84 0.32 -2.61 0.98 0.08 1.0 

1.92 -4.30 0.34 -3.76 0.06 -10.84 0.30 -10.3 0.16 

Statistical Test Dunnett’s Jonckheere Dunnett’s Dunnett’s 

Abbreviations: Diff. Difference. 

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

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

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Table 17: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Ortho-Phenylphenol (Late Stage). 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

Median p Median p 

% 

Diff. 

p % Diff. p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control NA NA NA NA NA NA -- -- NA NA NA NA No 

0.0175 NA NA NA NA NA NA -3.54 0.73 NA NA NA NA No 

0.0673 NA NA NA NA NA NA 4.73 0.52 NA NA NA NA No 

0.352 NA NA NA NA NA NA -0.44 1.0 NA NA NA NA No 

1.92 NA NA NA NA NA NA 6.51 0.32 NA NA NA NA No 

Statistical Test NA NA NA Dunnett’s NA NA NA 

Abbreviations: Diff. Difference. NA Not applicable.

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

by the expert opinion of a board-certified pathologist. 

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

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Table 19: Growth Endpoints1,2 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. 

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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. 

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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. 

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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-value 

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 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) 

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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 control 

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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) ) 

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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-value 

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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=0.01 

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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 0.413 

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 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 Conclusion 

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 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 0.072 

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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 TESTS 

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

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) 

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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.Interval 

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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 tests 

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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, 32.06 

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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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 0.425 

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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, 18.52 

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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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 trend 

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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 -2.01 

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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-values 

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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 0.42 

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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 Dose5 

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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 26.97 

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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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 . . . . . 

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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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-test 

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 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 . . 

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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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 0.059 

Page 73 of 281
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 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 tests 

Page 74 of 281
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 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 LOEC 

Page 75 of 281
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 EPA MRID Number 48916901 (revised report); 48672607 (original)

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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 0.126 

Page 76 of 281
Data 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) 

Page 77 of 281
DATA EVALUATION RECORD 

O-PHENYLPHENOL (OPP) 

Study Type: OCSPP 890.1150, Androgen Receptor 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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 78 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 79 of 281
0-PHENYLPHENOL/ 064103 

Androgen Receptor Binding (Rat Prostate Cytosol) (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 in 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. 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 androgen receptor (AR) binding assay (MRID 48672603), 

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

competitive binding experiment, which measured the binding of a single concentration of 

[3H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10- 10 

to 10-3 M) of o-phenylphenol (OPP, 99.9% purity, Lot #CHHYDD0051). Ethanol was used as a 

vehicle at a final assay concentration of <3%. A total of three runs were performed, and each run 

included dexamethasone as a weak positive control, and R 1881 as the ligand reference standard. 

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

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

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

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

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

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

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

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

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

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

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

Page 80 of 281
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 13 

O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

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

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

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

In the competitive binding experiment, OPP reduced the mean specific binding of the 

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

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

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

concentrations ≤10−5

 M (92.8-103.7% binding). Specific [3H]-R1881 binding was reduced to 

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

The estimated mean log IC50 was −3.749 M for 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 these numbers is high due to the small variation. 

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

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

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

Binding Assay.

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

Assay (OCSPP 890.1150). 

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

Assurance statements were provided. 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.

Page 81 of 281
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 provided 

Page 82 of 281
Androgen 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 (Wilmington, MA). Male Sprague Dawley rats 

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

approximately 24 hours later. The ventral prostate 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-salt TEDG buffer [0.01 M Tris buffer, 10% 

glycerol, 1 mM sodium molybdate, 1.5 mM EDTA] and 1 mM phenylmethylsulfonyl 

fluoride (PMSF) with dithiothreitol (DTT)] at pH 7.4 to the ventral prostate tissues at 10 

mL/g of tissue. The tissues were minced, homogenized on ice, and centrifuged for 30 min at 

30,000 × g at 4°C. The supernatant was collected, 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 the Pierce BCA method 

(Thermo Scientific Pierce Research Lab, Rockford, IL) compatible with DTT in the TEDG 

buffer.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-R1881 was performed to demonstrate that 

the AR was present in reasonable concentrations and had the appropriate affinity for the 

R1881 ligand. The conditions for the saturation binding experiment are summarized in 

Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand (as serial dilutions) 0.25-10 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 (less than 25-35% of the radiolabeled R1881). 

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

H]-R1881 was not 

adjusted for decay over time, and serial dilutions 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-specific binding, solutions of non-labeled R1881 

Page 83 of 281
Androgen 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 concentrations that were 100-fold greater than 

each respective radiolabeled concentration, resulting in final concentrations in cytosol of 25, 

50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, the radiation found in 

7.5, 15, 21, 30, or 45 μL of 10 nM [3

H]-R1881 and 7.5, 15, or 30 μL of 100 nM [3H]-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 concentration was determined to be 1.97 mg 

protein/assay tube, which resulted in the binding of 8.6-9.0% of the total radioactivity 

added. As indicated in the guideline for acceptable assay performance the receptor 

concentration bound less than 25 to 35% of the radiolabeled R1881. Cytosolic protein used 

in this assay was thawed fresh for this experiment at ~4°C and maintained at ~4°C during 

the binding assay. Each run contained three concurrent replicates at each concentration, 

resulting in the 72 samples depicted in Table 2. 

 

Table 2. Saturation Binding Experiment Runa,b

Total Binding Non-Specific Binding Radioligand alone 

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

[

3

H]-R1881 

Final conc. (nM) 

[

3

H]-R1881 

Final conc. (nM) 

R1881 

Final conc. (nM) 

[

3

H]-R1881 

Initial conc. (nM) 

[

3

H]-R1881 

(μL) 

0.25 0.25 25 10 7.5 

0.50 0.50 50 10 15 

0.70 0.70 70 10 21 

1.0 1.0 100 10 30 

1.5 1.5 150 10 45 

2.5 2.5 250 100 7.5 

5.0 5.0 500 100 15 

10 10 1000 100 30 

a Data were obtained from page 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 triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. Samples were dried, 

and 300 μl of prostate cytosol were added. 

d Tubes 25-48 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate 

cytosol were added. 

e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [3

H]-R1881 or 7.5, 15, 21, or 30 μL of 100 

nM [3

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

Following addition of triamcinolone 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 maintained 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 of 

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Androgen 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 binding profiles. Final determination of 

acceptable AR binding assay performance was 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 single 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 visually. No precipitation was noted. 

Dilutions of the test substance, 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 three 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 calculations) were run the day before each analysis run (the 

day of preparation of sample tubes). 

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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) Concentrations in Competitive Binding Assay a b

Solvent Control Reference standard Weak 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 triplicate 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 cytosol 

were 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 Tubes 6-8 and 75-77 that contained 30 μL 

of non-radiolabeled R1881 (used to evaluate non-specific binding). Six tubes analyzed the day prior 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 overnight 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 [3H]-R1881. 

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

(IC50) were calculated with nonlinear regression analysis by using 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 nonlinear 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 least 25% but less than 50%), or the 

Hill slope for the curve falls outside the range for the weak positive control (−0.6 to −1.4). 

Non-Binder: The average lowest portion of curves across runs is greater than 75% activity 

(i.e. less than 25% displacement of radioligand), or the data do not fit the model. 

Untestable: If the test compound is not soluble above 1×10−6

 M and the binding curve does 

not cross 50%, the chemical is judged to be untestable. 

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O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constants

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

by the competitor

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

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding data were not 

provided in the study report; however, a summary data for the two saturation binding 

runs were provided in the study profile (MRID 48618303) and the information is 

provided in Table 5. The dissociation constant (Kd) for [3H]-R1881 was 0.4641, and the 

estimated Bmax was 0.6392 nM for the batch of prostate cytosol used to evaluate 

competitive binding. The mean and individual Kd 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 (R2

 = 0.9871-0.9937) and the small variation among the 

two runs. 

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

Cytosola

Parameter Run 1 Run 2 Run 3 Mean b

R2 (unweighted) 0.994 0.987 ND 0.987-0.994 

Bmax (nM) 0.062 0.066 ND 0.064 

Bmax (fmol/100 μg protein) 3.146 3.343 ND 3.245 

Kd (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 R2

 is reported and the mean is reported for the other parameters. 

R2

 Goodness of fit for curve calculated for specific binding 

ND not determined

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

H]-R1881 to 

the androgen receptor for the three independent runs. The specific binding reached a 

plateau in each run, and non-specific binding was less than 20% of total binding. Figure 

2 contains the Scatchard plots that illustrate the binding of [3

H]-R1881 to the androgen 

receptor. 

Page 87 of 281
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 13 

O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

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

Binding Experiment.

FIGURE 2. Scatchard Plots of the Binding of [3H]-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-

Page 88 of 281
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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 IC50 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 control. Confidence in these numbers is high 

due to the small variation. No precipitation of the test compound was reported. The solvent 

control responses indicated no drift in the study assay. Repeat runs were unnecessary. 

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

Parameter Run 1 Run 2 Run 3 Mean ± SEb

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 IC50 (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 

IC50 (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 RBAb 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 R2

 is reported and the mean ± SE is reported for the other parameters. Log RBA and SE 

values are reviewer-calculated, as well as mean values 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 affinity

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O-PHENYLPHENOL/ 064103 OCSPP 890.1150/ OECD None

FIGURE 3. Percentage R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, and OPP (Run 1).

FIGURE 4. Percentage R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, and OPP (Run 2). 

FIGURE 5. Percentage R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, and OPP (Run 3).

Page 90 of 281
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 evaluated 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 Kd (nM) 0.685 to 1.57 NR X 

Non-specific binding (%) ≤10.0 7.09 to 7.96c

 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 include 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 approximately 2 log units). Examination across the 

Page 91 of 281
Androgen 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 reference radiolabeled androgen 

(R1881) at the highest two concentrations tested, including 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 (dexamethasone), 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 prostate 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 the test guideline. The dissociation constant (Kd) 

for [3H]-R1881 was 0.4641, and the estimated Bmax 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 concentration-dependent 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 6.3-7.4%. 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 IC50 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 reported. The solvent control 

responses indicated no drift in the study assay. All performance 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 performed instead of three runs as recommended 

in the test guideline. 

 The specific activity of the [3

H]-R1881 stock solution was not checked prior to use on 

each assay day. 

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

Page 92 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 93 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 94 of 281
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. 

Unpublished. 

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 10-10 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 (10-10 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% and 

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

background controls was acceptable. 

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

nmol·mg-protein−1

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

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

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

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

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

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

−1.17. 

For 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 recommended limit of 15%. The data for OPP were 

modeled for all runs, and the goodness of fit (r2) values were 0.87-0.98. The best fit Hill slope 

and log IC50 values for OPP across the three independent runs did not differ significantly. 

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

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

OPP. The average log IC50 and Hill slope for OPP were −3.97 M and −0.99, respectively. 

Based on the data from the average response curve, OPP is classified as an Inhibitor of 

aromatase activity in this assay. 

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

890.1200). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

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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; ([3H]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/min 

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B. METHODS

1. Assay Components and Preparations: A mixture of non-labeled and radiolabeled 

[

3

H]ASDN was prepared such that the final concentration of ASDN in the assay was 

approximately 2 μM, and the amount of tritium added to each incubation tube yielded a 

predicted radioactivity of 1.0 μCi/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 prepared 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 achieved by the addition of 20 μL 

of the dilution for a final assay volume of 2 mL. 

Human recombinant microsomes were purchased from Gentest (Woburn, MA), and stored 

at approximately –80oC for an unspecified time period. Microsomes were portioned into 

individual vials based on the protein concentration of the 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 

[3H]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 protein concentration was determined on 

each day the aromatase assay was run. The details of the protein determinations were not 

reported, except that the analyses were conducted in accordance with SOP No. TOX-BMT192.05 (Quantitation of Proteins). Microsomes were diluted with 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 determined to be 0.080 nmol·mg-protein−1

·min−1, which is slightly 

below the minimum recommended aromatase 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, 

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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 tubes 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 3H2O 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 inhibitor 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 replicates within each sample type and concentration was 

<15%. 

(2) Demonstration of Proficiency of New Technician for Conducting Assay (when 

applicable): The positive control data for slope, top and bottom percent met the criteria as 

listed in section (i) of OCSPP 890.1200. 

TABLE 2. Performance Criteria for the Positive Controla

Parameter Lower Limit Criteria Upper Limit Criteria 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 IC50 (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. 

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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 Activity with Test Chemical(s): The response of aromatase 

activity to the presence of eight concentrations of a test chemical 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 chemical 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 componentsb plus solvent vehicle N/A 

Bkgd Activity Control 4 Same as above without NADPH N/A 

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

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

4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10−6.5

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

4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10−7.5

4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10−8

4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10−9

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

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

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

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

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

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

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

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

OPP Conc 8c 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, propylene glycol, microsomal protein, [3H]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 included in the report (Table 5). 

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TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

DPM/mL for each portion of extracted aqueous incubation mixture X 

Average DPM/mL for each aqueous portion (after extraction) X 

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol·mg protein−1·min−1 X 

Average aromatase activity in the full activity control tubes X 

Percentage of control activity remaining in the presence of various inhibitor concentrations 

a Data were obtained from pages 43-45 of the study report. 

DPM Disintegrations per minute

2. Statistical Methods: Statistical analyses and graphical 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 IC50 values across 

three independent runs were compared based on a one-way random effects analysis of 

variance, treating runs as random effects. 

The response curve was fitted by nonlinear regression 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 concentration or 4-OH ASDN 

concentration. Each run was plotted with the data’s best fit curve. 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 categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter nonlinear 

regression model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is 

between 50% and 75% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

Non-inhibitor 

Data do not fit model --- 

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near −1. Unusually steep curves 

may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than −2.0, the result is classified as 

equivocal. 

b If the test compound was not soluble above 10−6 M and the inhibition curve does not cross 50%, the chemical is typically 

determined to be untestable in the aromatase assay. 

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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 average full activity, with the exception of the 3rd

replicate in Run 2 (89.0%) and 3rd replicate in Run 3 (83.1%). Activity in the background 

controls ranged from 2.38% to 7.88% and averaged 5.7% of the full control activity. The 

response of the full activity controls and background controls was acceptable. 

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 positive control (expressed as % full control 

activity) for each concentration tested across all 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 within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration. 

TABLE 7. Effect of OPP on Aromatase Activity (as percent of control) 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 Variance 

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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 each concentration tested across all three runs 

is presented in Table 7 (above), along with the overall standard deviation and %CV. 

Inhibition response curves for OPP from each run are shown in Figure 2, and the average 

inhibition response curve across all runs is shown in Figure 3. 

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

FIGURE3. Mean Inhibition Response Curve for OPP. 

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The effect of OPP on the inhibition of aromatase activity is presented in Table 8. The 

estimated log IC50 ranged from -4.0 to -3.9 log M for OPP and averaged -3.97 M compared 

to the positive control 4-OH ASDN, which averaged -7.1 M. The average slope of the 

concentration response curve was -0.99 for OPP 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 Aromatase Activity (as Percent of Control) From Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SEM %CV 

Log IC50 (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. Mean, 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 curve and the criteria listed above in Table 7, 

the results support the conclusion that OPP inhibits 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% inhibition 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 average full activity, with the exception of the 3rd

replicate in Run 2 (89.0%) and 3rd replicate in Run 3 (83.1%). Activity in the background 

controls ranged from 2.38% to 7.88% and averaged 5.7% of the full control activity. The 

response of the full activity controls and background controls was acceptable. 

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

nmol·mg-protein−1

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

nmol·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 IC50, and coefficient of variation for replicates of each concentration, with the 

exceptions of the overall %CV for the highest three concentrations (51.2, 36.2, and 129%) 

exceeded the recommended limit of 15%. 

Page 105 of 281
Aromatase (Human Recombinant) Assay (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 recommended limit of 15%. The data for OPP were 

modeled for all runs, and the goodness of fit (r2) values were 0.87-0.98. For test material 

curves, the best fit Hill slope and log IC50 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 IC50 and Hill slope for OPP were −3.97 M and −0.99, 

respectively. 

For 4-OH ASDN, the estimated log IC50 averaged −7.1 M and the Hill slope was −1.17. 

Confidence in the mean log IC50 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 curve, 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 substance in the stock solution was not reported. 

Page 106 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 107 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 108 of 281
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-phenylphenol (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- 10 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 (Bmax) 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. 

Page 109 of 281
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O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

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

positive control. All performance criteria were met. 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 binding curve exhibited steep Hill slopes (−2.262 to 

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

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

RBA value was not determined. 

Based on the results from the three runs, OPP is classified as Equivocal in the Estrogen Receptor 

Binding Assay. 

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

Assay (OCSPP 890.1250). 

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

Assurance statements were provided. All phases of the study were conducted in compliance 

with FIFRA GLP Standards except that concentration checks for the control substances used in 

the study were not performed. 

Page 110 of 281
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: 17β-estradiol 

 Supplier: Sigma, St. Louis, MO 

 Catalog #: E8875 

 Lot #: 098K1372 

 Purity: 100% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-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 #: 68-22-4 

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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 purchased from Charles River (Wilmington, MA). 

The uteri were weighed, minced and homogenized in ice-cold TEDG (Tris, EDTA, DTT, 

glycerol) buffer + PMSF (phenylmethylsulfonyl fluoride), then centrifuged for 10 min at 

2500 × g at 4oC. Supernatant was transferred and centrifuged for 60 minutes at 105,000 × g, 

discarding the resulting pellets. Protein concentration of the cytosol was determined to be 

3.573 mg/mL using a protein kit compatible with DTT in the TEDG buffer (Pierce BCA 

method; Thermo Scientific Pierce Research Lab). Cytosol was divided into aliquots 

(volume not reported) that 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 [3H]-17β-estradiol was reportedly performed to 

demonstrate that the ER was present in reasonable 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 mMb 

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 guideline; however, all other values 

including ligand depletion met the performance criteria. 

 

 

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

H]-17β-estradiol was not 

adjusted for decay over time. Serial dilutions in TEDG + PMSF buffer were prepared to 

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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 17β-estradiol were prepared in a similar manner to achieve concentrations that 

were 100-fold greater than each respective radiolabeled concentration to result in final 

concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. For each batch of cytosol, the 

optimal protein concentration was determined by testing serial amounts of protein per tube, 

using 0.03 nM radiolabeled estradiol. The optimal protein concentration was determined to 

be 0.1191 mg protein/assay tube, which resulted in the binding of 40.77% of the total 

radioactivity added. This value was slightly higher than the suggested range in the guideline, 

however all other values met the performance criteria. Cytosolic 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 replicates at each concentration, resulting in the 

72 samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Runa

Total binding b Non-specific binding c Radioligand alone d

Assay Components Tubes 1-24 Tubes 25-48 Tubes 49-72 

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17β-estradiol (8 serial 

dilutions, 100x each respective labeled 

concentration) f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate 

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]-17β-estradiol bound to ER 

c Non-specific binding = [3

H]-17β-estradiol and 100-fold greater non-labeled bound to ER 

d Total [3

H]-17β-estradiol alone for dpm determination at each concentration 

e Final concentrations of [3

H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. 

f Final concentrations of non-labeled 17β-estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM. 

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

competitive binding experiment is included in Table 3. 

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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 specific activity of the stock solution [3H]-17β-estradiol 

was not adjusted for decay over time, and diluted in TEDG + PMSF buffer to achieve a final 

concentration of 1 nM. Uterine cytosol was used at a 1:2 dilution to achieve 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 17β-estradiol) were prepared to 

achieve the concentrations shown in Table 4. Each assay consisted of three independent runs 

on three different days. Each run included duplicate blanks, and triplicate sets of solvent 

blank and 0.1 μM estradiol (non-specific binding, NSB) at the beginning and end of each run, 

along with triplicate tubes of the reference standard, the weak positive control, negative 

control, and OPP at each concentration, resulting in a total of 104 samples per run. In 

addition, duplicate blanks followed by six replicates of [3H]-17β-estradiol only (for total 

binding calculations) were run the day before each analysis run (the day of preparation of 

sample tubes). 

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

OPP 

Positive control Negative control Reference Chemical 

19-Norethindrone Octyltriethoxysilane Non-labeled 

17β-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, positive control, negative control, solvent control, or 

non-labeled 17β-estradiol; 390 μL of TEDG + PMSF buffer with [3

H]-17β-estradiol; and 100 μL of uterine 

cytosol (with ER), for 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) slurry 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 [3H]-17β-estradiol, followed by vortexing, and 

centrifugation for 10 min at 1000 × g. Aliquots of supernatant were radioassayed by liquid 

scintillation counting. The temperature was maintained at 4-8 oC throughout 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 conduct non-linear regression analyses and to fit a 

curve (for 17β-estradiol, the positive control, and the test substance) to the Hill equation 

formula which incorporated log IC50 as a parameter to be estimated. For parameters reported 

from the competitive binding experiment (log IC50 and %RBA), means were calculated for 

each run and for the composite three runs.

1. Definitions 

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

three runs. Each run was first individually classified as follows: 

Interactive = lowest point on the fitted curve within the range of the data is less than 50% 

(i.e., >50% of the radiolabeled estradiol has been displaced from the ER). 

Not interactive = there are usable data points at or above 10-6 M and either the lowest point 

on the fitted response curve within the range of the data is above 75% (i.e., 

<25% of the radiolabeled estradiol has been displaced from the ER) or a 

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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 fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

The values for each run were then averaged across runs and the chemical classified using the 

following ranges: 

Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

b. Descriptors for receptor binding: 

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

concentration of active receptor sites 

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

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

from the receptor 

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

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: 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-phenylphenol. For the 

prepared rat uterine cytosol, the Kd for [3H]-17β-estradiol was 0.1032 nM. The Bmax was 

0.07097 nM, which fell within the expected range of 0.03 to 1.5 nM. The data produced a 

linear Scatchard plot. Confidence in these numbers is high for the run due to the goodness 

of fit (R2

 =0.9617). 

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TABLE 5. Saturation Binding Experiment of [3H]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosol a

Parameter Run 1b Run 2 Run 3 Mean ± SE 

R2 (unweighted) 0.9617 ND ND NA 

Bmax (nM) 0.07097 ND ND NA 

Bmax (fmol/100 μg protein) 59.28 ND ND NA 

Kd (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. 

R2 Goodness of fit for curve calculated for specific binding. 

ND Not determined 

NA Not applicable 

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

Binding Experiment. 

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O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

FIGURE 2. Scatchard Plot of the binding of [3H]-17β-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 (r2, IC50, log IC50, and RBA) are presented in Table 6. 

OPP exhibited >50% displacement of radiolabeled estradiol at concentrations of 10-3 M in 

Runs 1 and 2; however, it was classified as “equivocal” due to failure to fit the 4-parameter 

nonlinear regression model for analysis of one-site binding. There were no apparent 

alterations in radiolabeled 17β-estradiol binding at OPP 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 IC50 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 IC50 was 

−8.943 M for 17β-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%). Confidence in these numbers is high due to 

the small variation. Additional runs were unnecessary. 

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TABLE 6. Competitive Binding Assay of O-phenylphenol with Estrogen Receptor from Rat Uterine 

Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

r

2

 (unweighted) 17β-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 IC50 (M) 17β-estradiol −8.963 −8.939 −8.929 −8.943 ± 0.010 

 19-norethindrone −5.475 −5.442 −5.445 −5.454 ± 0.011 

 OPP ~-3.018c ~-3.365 c NA NA 

IC50 (M) 17β-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 expressed 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: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of 

concentrations tested (“missing”, i.e., not included in calculation of mean). 

c Mean of three runs expressed to the tenths place 

d Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5 

FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Unlabeled 

E2, 19-Norethindrone, Octyltriethoxysilane or OPP, Run 1.

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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 Estrogen Receptor in the Presence of Unlabeled 

E2, 19-Norethindrone, Octyltriethoxysilane or OPP, Run 2.

FIGURE 5. Percentage E2 Bound to the Estrogen 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.

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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 competitive binding assay functioned 

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

TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge residual SD ≤2.35 −0.1540 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 (Log10(M)−1) −1.1 to −0.7 −0.9981 to −0.9491 X 

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge 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 (Log10(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]-17β-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-norethindrone. Acceptance criteria were only 

defined for norethynodrel, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control. 

c Reviewer-calculated. The minimum specific binding of [3

H]-17β-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 17β-estradiol displaced [3H]-17β-estradiol in a manner consistent with one-site 

binding, as indicated by a descent from 91.41-92.79% to 10.11-10.73% binding over 

approximately an 81-fold increase in concentration 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 concentration of the estradiol 

standard 99.34-100.03%. Examination across the 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 determined that ortho-phenylphenol 

(OPP) had no appreciable effect in the binding of the reference estrogen (17β-estradiol) at 

any concentration up to 10−4

 M. At the guideline limit concentration (10−3 M) a reproducible 

decrease in 17β-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 the 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 14 of 14 

O-PHENYLPHENOL/ 064103 OCSPP 890.1250/ OECD None

model, consistent with a false positive response in this assay. The results of the in vitro 

estrogen receptor binding assay using rat uterine cytosol indicate 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 (Bmax) of 59.28 fmol/100 μg protein with the dissociation 

constant (Kd) of 0.1032 nM. Nonspecific binding as a percent of total binding was 1.7-8.6% 

across the concentration range. In the competitive binding experiment, 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). The mean estimated log IC50 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 IC50 was −8.943 M for 17β-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. 

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 a single saturation binding run was performed instead of three runs as 

recommended in the test guideline. 

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

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Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol 

EPA MRID Number 48672606 

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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'

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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 Short-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.

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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 

25.5o

C and a pH of 7.2 to 7.4. 

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

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

control and all other treatment groups. Six of seven deaths occurred in a single tank over two days, potentially 

indicating a tank-related issue (the high treatment level was well below any acute toxicity level). 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 no 

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Data Evaluation Record on the Fish Short-Term Reproduction Assay with Ortho-Phenylphenol 

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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 17β-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-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Ortho-Phenylphenol. 

Treatment 

(mg a.i./L)

[TWA 

measured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M F M F 

M F M F M F M F

0.0158 No No No No No No NA NA No No NA NA NA NA 

0.0755 No No No No No No NA NA No No NA NA NA NA 

0.876 Yes3 Yes3 No No No No NA NA No Yes NA NA NA NA 

Abbreviations: E2 17β-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. T Testosterone. VTG Vitellogenin. 1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. 2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavily 

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

3 Although not significantly different (p>0.05) from the negative control, these endpoints were markedly decreased compared to the negative control. Therefore, the 

decreases were considered biologically significant. 

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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 least 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# 90-43-7

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Description: White solid; Recertification date February 22, 2013; solubility 700 mg/L at 

20qC; 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. 

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 B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes Aquatic Research Organisms, Inc., Hampton, 

New Hampshire. 

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test? 

Yes EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test. 

Acclimation period: 19 days EPA recommends a minimum two-week 

acclimation period. Note that the acclimation 

period is different from the subsequent, in situ 

pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortality during the last 2 

weeks prior to preexposure was 3.0%.Mortality during the 7 days prior to preexposure period was 1.5%. Fish did not receive any treatment for disease during acclimation period. Fish with behavioral abnormalities or clinical signs were not used in the 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 brine 

shrimp

According to the study authors, measured concentrations of pesticides, organics, metals, and other inorganics were below levels of concern in a representative sample of 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of food: Brine Shrimp Direct, 

Ogden, Utah

Frequency of feeding: 2 times/day Details on feeding: At least twice daily at a 

rate sufficient to promote active reproduction and maintain body condition; 1.5-2.5 mL per replicate

during the pre-exposure 

phase; 2.5-3.5 mL per 

replicate (or pro-rated to 

account for mortality) during the exposure 

phase 

The amount of frozen shrimp provided was measured and an equal proportion per number of surviving fish was given to each tank. Food was withheld from the fish for at least 12 hours prior to test termination to aid in histological processing of the fish. Uneaten food and fecal material were removed from the test vessels at least three times per week by siphoning.

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 5.5 months Hatched on February 22, 2011 EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

2.0 g Based on 10 males used to stock 

aquaria 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 (if determined): 

Not reported Individual weights within ± 20% of the 

mean weight of the sub sampled fish of the same sex. 

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

1.4 g Based on 10 females used to stock 

aquaria for pre-exposure phase. 

Range of individual weights (females) at test initiation (if determined): 

Not reported Individual weights within ± 20% of the 

mean weight of the sub sampled fish of 

the same sex. 

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

Not reported 

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

Not reported 

Duration of pre-exposure phase: 15 days EPA recommends a minimum of 14 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical to the definitive test?

Yes EPA recommends that pre-exposure 

conditions, including temperature, 

photoperiod, feeding, etc., be identical to 

definitive test conditions. 

Number of pre-exposure tanks: 24 EPA recommends that additional tanks set 

up at the beginning of pre-exposure will 

ensure that sufficient replicates with the 

correct sex ratio are available for the 

definitive test. 

Number of males per tank: 2 Number of females per tank: 4 Pre-exposure fecundity: >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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of spawns during preexposure: > two times in the immediately preceding 

7 days 

Spawning occurred at least every 3 days.

EPA recommends that spawning occur at 

least twice in the 7 days prior to the 

definitive test. 

Details on pre-exposure: None 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a 

flow-through system. As noted in 

the Corrections and Clarifications 

document1 , the use of a static 

renewal system is not 

recommended for this assay.

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

Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. 

(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system:

Continuous-flow diluter Intermittent flow proportional 

diluters or continuous flow serial 

diluters are recommended.2

Flow-through rate: 45 ± 4.5 mL/minute (2.7 

L/hr) to provide complete volume turnovers ca. 2.7 hrs 

(equivalent to 6 volume replacements/ day) 

L/hr calculated by the reviewer based on 45 mL/minute. Because analytical recoveries dropped over the course of the equilibrium and exposure periods due to the biodegradation of the test material, flow rates were periodically adjusted in an attempt to maintain nominal concentrations. These changes were as follows. On the last day of equilibrium (test Day -1), flow rate from delivery tubing was increased to 90 ± 9 mL/minute and stock injection rate was doubled to 3.0 mL/min. On exposure Day 5, flow rate from delivery tubing was reduced to 45 ± 4.5 mL/min for all concentration levels and stock injection rate was reduced to 1.5 mL/min in low concentration (where analytical recoveries were acceptable); stock injection rate remained at 3.0 mL/min in the mid- and high levels. On exposure Day 8, the use 

of the mid and low level stocks was discontinued due to observed biodegradation in these solutions; the high-concentration stock 

solution (300 mg/L) was then used for all treatment levels at an 

injection 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.

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

mL/min (high). On exposure Day 11, the injection rate to the low treatment level was increased to 0.03 mL/min. 

Details on toxicant mixing for flow-through systems: 

For each treatment level, a peristaltic pump continuously delivered a concentrated test substance stock solution at a designated rate (1.5 mL/min) from a concentration-specific stock 

vessel to a stainless steel mixing chamber where it was mixed with dilution water. 

The test solution was gravity-fed from the mixing chamber and split equally to replicate test vessels via a Teflon manifold with Teflon delivery tubing.The flow rates of the stock solutions and of the diluents (via test solution flow to each replicate vessel) were targeted not to vary more than 20% throughout the exposure; adjustments were made as necessary to maintain 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 

demonstration that the test 

solution is completely mixed 

before introduced into the test 

system is recommended; 4) The 

recommended flow splitting 

accuracy is within 10%.

Aeration? No EPA recommends aeration if 

dissolved oxygen reaches 

<4.9 

mg/L (<60% saturation).

Source of dilution water: Upper Saginaw Bay of Lake 

Huron off Whitestone Point 

Prior to use, dilution water was limed and flocculated with ferric chloride, sand-filtered, pH-adjusted with gaseous CO2, 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, and 

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Parameter Value(s) Details or Remarks Guideline 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 for pesticides, heavy metals, and other contaminants? 

Yes According to the study authors, measured concentrations 

of pesticides, organics, metals, and other inorganics were below levels of concern. 

Test vessel type/materials: Glass aquaria sealed 

together with silicone adhesive

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 x 

25 cm high; solution depth 13 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Spawning substrate material: 10 cm diameter PVC cut 

lengthwise in half (arches) and into 9 cm sections and 

positioned in a screened, 

lipped stainless steel (ca. 13 cm 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) and 

into 9 cm sections. 

Each substrate was placed on screened stainless collection tray. 

Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 24.6 25.5 25.1 1

Continuously in one test vessel; weekly in each test vessel 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials should not exceed 1oC.

pH 7.2 7.4 7.3 1 Weekly, in each test 

vessel 

EPA recommends pH 6.5 to 9.0.

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Dissolved oxygen (mg/L) 6.4 8.1 7.3 1 Weekly, in each test 

vessel 

EPA recommends dissolved oxygen (DO) 

>4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3) 28 48 38 1

Weekly, from sample of a control and the highest exposure level solution 

EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 60 66 64 1

Weekly, from sample of a control and the highest exposure level solution 

Total organic carbon (mg/L) Not reported Not reported Not reported Not reported EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

Unionized ammonia (Pg/L) Not reported Not reported Not reported Not reported

EPA recommends that unionized ammonia in 

the dilution water be <1 

Pg/L. 

Residual chlorine (Pg/L) Not reported Not reported Not reported Not reported

EPA recommends that residual chlorine in 

dilution water be < 10Pg/L. 

Conductivity (Pmhos/cm) 159 198 175 1

Weekly, from sample of a control and the highest exposure level solution 

General recommendations for frequency of 

measurements: EPA recommends that 

temperature, pH, and dissolved oxygen be 

measured in all test tanks at least weekly and 

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Parameter Minimum Maximum Mean Measurement Interval Guideline 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. 

1 Means 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 Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No EPA recommends conducting a range-finder if 

96-hour LC50 data for the fathead minnow are 

unavailable. 

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

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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Species: NA Life 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 LC50, may be selected as 

the highest exposure concentration in the 21-day 

test.

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

test be 21 days. 

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

The acute toxicity 96-hr LC50 value for OPP with fathead minnows was reported to be between 5.9 and 6.3 mg a.i./L.

The highest test concentration was selected as 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 LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Holcombe et al., 1984; Geiger et al., 1985 

Separation of test concentrations: Step down factor of 0.1 

from highest test concentration0 (control), 0.02, 0.20, 

and 2.0 mg a.i./L 

EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable1 . 

Number of test concentrations: 3 and a control: 0 

(control), 0.02, 0.20, and 2.0 mg a.i./L 

EPA recommends a minimum of 3 

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Time-Weighted Average 

Limit 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 reported Frequency of measurement: 0, 4, 5, 7, 8, 11, 12, 14, 

19, 20 and 21 days 

Also, Day 9 for 0.02 mg a.i./L replicate test vessels

It is recommended that test item concentration 

be measured prior to the addition of fish in all 

tanks and at least weekly thereafter in two 

replicates per treatment level.

Was the randomized complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure 

fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive 

test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls. 

Number of replicates in control: 4 EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in solvent control (if applicable):

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is used. 

EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any other 

adverse effects and that concentration not be 

greater than 0.1 ml/L4

.

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

Toxicology, 76, pp.69–92. 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a positive control used? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

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

Light intensity at water’s surface: 721-885 lux Cool white fluorescent bulbs EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

Additional details: None Appearance of the test solution was not 

described 

and Assessment. No. 23. Paris, France. 

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

Treatment ID 

Nominal 

Concentration

(mg a.i./L) 

Time-Weighted 

Average 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks1 Guideline Recommendations 

Negative Control 0 <LOQ NA EPA recommends that test item 

concentrations be maintained at a 

coefficient of variation (CV) ≤20%. Treatment 1 0.02 0.0158 28 

Reviewer-calculated meanmeasured: 0.0162 ± 0.0045 

Reviewer-calculated TWA: 0.0165 

Treatment 2 0.20 0.0755 41 

Reviewer-calculated meanmeasured: 0.0785 ± 0.0785 

Reviewer-calculated TWA: 0.0755 

Treatment 3 2.0 0.876 60 

Reviewer-calculated meanmeasured: 0.9276 ± 0.5532 

Reviewer-calculated TWA: 0.8767 

Abbreviations: CV Coefficient of variation. LOQ=0.00495 mg a.i./L 1 The reviewer-calculated time-weighted averages were slightly different that those of the study author, likely due to rounding differences.

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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 17β-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. 

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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 LC50 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 

n1 # Surviving % Survival n1 # 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 initiation 

 

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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. 

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

Treatment 

(mg a.i./L) 

[TWA measured]

Body Weight Length 

Males Females Males Females 

n1

Mean

(g) 

±SD n1

Mean

(g) 

±SD n1

Mean

(mm) 

±SD n1

Mean

(mm) 

±SD

Negative Control 

(<LOQ) 4 3.00 0.341 4 1.58 0.176 4 50.2 1.69 4 41.6 1.68

0.0158 4 2.48 0.416 4 1.47 0.061 4 47.8 3.01 4 40.6 0.797 

0.0755 4 2.35 0.499 4 1.51 0.207 4 47.0 2.33 4 40.4 2.19 

0.876 3 2.54 0.529 3 1.51 0.065 3 48.9 1.62 3 41.0 0.261 

Abbreviations: SD Standard deviation. 

LOQ=0.00495 mg a.i./L

1 Number of replicate tanks 

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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]

Fecundity1 Fertilization Success2

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. 

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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 

n1 Median Tubercle Score2 n1 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. 

 

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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 GSI1

(%) 

±SD n Mean GSI1

(%) 

±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. 

 

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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 Stage1 n Median Stage2

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. 

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

Treatment 

(mg a.i./L) 

[TWA 

measured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Duct 

Mineralization

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. 

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

Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 NA NA NA NA NA NA NA NA 8 6 

1 NA NA NA NA NA NA NA NA 8 2 

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

0.0158 0 NA NA NA NA NA NA NA NA 8 8 

1 NA NA NA NA NA NA NA NA 8 0 

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

0.0755 0 NA NA NA NA NA NA NA NA 8 6 

1 NA NA NA NA NA NA NA NA 8 1 

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

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Treatment 

(mg a.i./L) 

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.876 0 NA NA NA NA NA NA NA NA 6 5 

1 NA NA NA NA NA NA NA NA 6 1 

2 NA NA NA NA NA NA NA NA 6 0 3 NA NA NA NA NA NA NA NA 6 0 4 NA NA NA NA NA NA NA NA 6 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. 

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

Treatment 

(mg a.i./L) 

[TWA 

measured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Aggregates of 

macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

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. 

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

promelas).

Treatment

(mg a.i./L)

[TWA measured] 

Additional Diagnostic Observations1

Severity 

Subacute to Chronic/ 

Interstitium/ Multifocal 

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. 

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Mean male VTG was 452, 1220, 866 and 561 ng/mL, and female VTG was 66.3x106, 71.7x106, 60.8x106 

and 42.1x106

 ng/mL in the negative control and TWA 0.0158, 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 106 22.9 x 106

0.0158 4 1220 1170 4 71.7 x 106 18.6 x 106

0.0755 4 866 765 4 60.8 x 106 29.6 x 106

0.876 4 528 188 4 42.1 x 106 5.50 x 106

Abbreviations: SD Standard deviation. 

LOQ=0.00495 mg a.i./L 

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Plasma testosterone and plasma 17

β-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 17

β-estradiol (E2) 

Males Females Males Females 

n

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD

Negative Control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

0.0158 NA NA NA NA NA NA NA NA NA NA NA NA 

0.0755 NA NA NA NA NA NA NA NA NA NA NA NA 

0.876 NA NA NA NA NA NA NA NA NA NA NA NA 

Abbreviations: NA Not applicable. SD Standard deviation. 

LOQ=0.00495 mg a.i./L 

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There were no notable observations with regards to behavior, coloration/banding, changes in ovipositor appearance or size of dorsal nape pad, with the exception of one lethargic male in the TWA 0.876 mg a.i./L treatment level (Table 21). 

Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow (Pimephales promelas)

Treatment 

(mg a.i./L) 

[TWA measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Negative Control (<LOQ) None 8 NA None 16 NA 

0.0158 None 8 NA None 16 NA 

0.0755 None 8 NA None 16 NA 

0.876 Lethargy 6 1 None 11 NA 

Abbreviation: NA Not applicable 

LOQ=0.00495 mg a.i./L 

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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 significance 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. There 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 background 

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spontaneous 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 monotonic 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. 

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

Treatment 

(mg a.i./L) 

[TWA measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control (<LOQ) 15 NA 0 NA No 0 NA NA NA NA NA 

0.0158 16 0.977 -8.43 0.848 No 170.45 0.364 NA NA NA NA 

0.0755 19 0.766 0.77 1.000 No 91.66 0.775 NA NA NA NA 

0.876 17 0.998 -16.22 0.526 No 16.62 0.998 NA NA NA NA 

Statistical Test Dunnett’s3 Dunnett’s NA Dunnett’s NA NA 

Abbreviations: Diff. Difference. E2 17β-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. LOQ=0.00495 mg a.i./L

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 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. 

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

Treatment 

(mg a.i./L) 

[TWA measured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2

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

Effect? 

(Yes/No) 

% Diff. p

% 

Diff. p % Diff. p

Negative Control 

(<LOQ) 

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

0.0158 -0.36 1.000 0.77 1.000 0 NA -14.43 0.695 No 8.10 0.718 NA NA NA NA 

0.0755 50.72 0.328 1.70 0.780 0 NA -10.25 0.854 No -8.37 0.330 NA NA NA NA 

0.876 -47.48 0.434 -32.10 1.000 0 NA -16.96 0.643 No -36.52 0.040 NA NA NA NA 

Statistical Test Dunnett’s Mann-Whitney NA Dunnett’s NA Jonckheere NA NA 

Abbreviations: Diff. Difference. E2 17β-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. 

LOQ=0.00495 mg a.i./L

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

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

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

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Table 24: Growth Endpoints1,2 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. 

 

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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 3P C6-Phenyl 

column. 

The study author reported that analytical recoveries dropped over the course of the equilibrium and exposure 

periods due to biodegradation of the test material; therefore, flow rates were periodically adjusted in an 

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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 LC50 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. 

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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). 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 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. 

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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. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification table 

Endpoint Monotonic? Parametric? EDSP EFED Comments Female body weight No No Mann-Whitney: 

n.s. p>0.05 

Mann-Whitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female body length No No Mann-Whitney: 

n.s. p>0.05 

Mann-Whitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG Yes Yes Jonckheere: 

Dose 3 p=0.040 

William’s: n.s. p>0.05

EDSP and EFED conclusions differ, 36.5% significant reduction at the 0.876 mg a.i./L level, according to OCSPP 890.1350 flowchart guidance. 

Male VTG No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female GSI No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male GSI No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female tubercle score 

NA NA NA NA No score 

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Endpoint Monotonic? Parametric? EDSP EFED Comments Male tubercle score No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Fecundity No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Fertility No No Mann-Whitney: 

n.s. p>0.05 

Mann-Whitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

F testosterone NA NA NA NA NA M testosterone NA NA NA NA NA F estradiol NA NA NA NA NA M estradiol NA NA NA NA NA 

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 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. 

 

 

 

 

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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. 

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 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 * 

 ---------------------------------------------------------------------------- 

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 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. 

 

 

 

 

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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 * 

 ---------------------------------------------------------------------------- 

 

 

 

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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) 

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

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 21.50 

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 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-value 

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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 groups 

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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 Conclusion 

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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 control 

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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 0.376 

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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 0.282 

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 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 . . 

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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, 17.41 

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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 . . . . . 

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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 . . . . . . 

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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 phenol 

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Data 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 Dose1 

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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 9.23 

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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 groups 

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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 tests 

Page 197 of 281
Data 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. 

Page 198 of 281
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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 0.302 

Page 199 of 281
Data 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) 

Page 200 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 201 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 202 of 281
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 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 Hershberger Assay (MRID 48672602) screening for 

androgenic activity, ortho-phenylphenol (OPP; 99.9% a.i. , 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 (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 daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The 

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

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

negative control. 

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

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

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

kidneys, and liver were collected and weighed. 

Page 203 of 281
In vivo Hershberger Assay (2012) / Page 2 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

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

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

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

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

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

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

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

assays) was found to have thickening of the nonglandular mucosa of the stomach. 

In the androgen agonist assay, body weights were decreased (p<0.05) by 15% on Day 7 in the 

1,000 mg/kg/day group compared to controls; however, body weights at the end of the study 

were similar to controls. Food consumption in this group was decreased (p<0.05) by 42% during 

Days 4-7. Body weights and body weight gains in the 50 and 250 mg/kg/day groups were 

comparable to controls throughout the duration of the study; food consumption was increased 

(p<0.05) in the 50 mg/kg/day group on Days 1-4 (↑6%). In the positive control group, 

cumulative body weight gains were increased (p<0.05) on Days 1-4 (↑45%), Days 1-7 (↑50%), 

and Days 1-11 (↑36%); food consumption was increased (p<0.05) in this group on Days 1-4 

(↑14%), Days 4-7 (↑14%), and Days 7-11 (↑12%). There were no treatment-related effects on 

weights of the accessory sex organs/tissues, adrenal glands, kidneys, or liver for any OPPtreatment group. Animals in the TP group had increased (p<0.05) accessory sex organ weights 

as follows: 1760% in seminal vesicles; 1117% in ventral prostate; 226% in levator anibulbocavernosus (LABC); 806% in Cowper’s gland; and 67% in glans penis. Adrenal glands, 

kidney, and liver weights in the positive control group were comparable to the vehicle control. 

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 the 

ventral prostate were decreased (p<0.05) by 28% compared to the controls. However, there were 

no treatment related effects on the weights of the seminal vesicles, LABC, Cowper’s glands, 

glans penis, adrenal glands, kidneys, or liver for any OPP-treatment group. Animals dosed with 

TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as follows: 83% 

in seminal vesicles; 77% in ventral prostate; 46% in LABC; 61% in Cowper’s gland; and 18% in 

glans penis. Adrenal glands, kidney, and liver weights in the positive control group were 

comparable to the vehicle control. 

Percent CVs for the vehicle control and the 1,000 mg/kg/day treatment groups for both assays 

were compared to the performance criteria in the guidelines. All %CV values were less than the 

maximum recommended values, with the exception of the %CV for Cowper’s glands for the 

control group in the androgen agonist assay (CV = 88%; maximum recommended CV = 55%) 

and the %CV for LABC for the high dose group in the anti-androgen assay (CV = 22%; 

maximum recommended CV = 20%). 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. OPP was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Page 204 of 281
In vivo Hershberger Assay (2012) / Page 3 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

Assurance statements were provided. 

Page 205 of 281
In vivo Hershberger Assay (2012) / 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 applicable 

Page 206 of 281
In vivo Hershberger Assay (2012) / 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 stainless steel cages with wire mesh floors 

suspended above absorbent paper. 

 Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, 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 initiated 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 potential 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 more target organ weights compared to the 

vehicle control. To screen for the potential anti-androgenic activity, OPP was also 

administered daily via oral gavage to castrated male rats in conjunction with a daily dose of 

TP (0.4 mg/kg/day) by s.c. injection. The positive control group received flutamide (FT) 

plus TP. Anti-androgenic activity is indicated by a statistically significant decrease in two 

or more target organ weights of the treated groups (test substance + TP) compared to the 

TP-only control group. For both assays, the animals were dosed for 10 consecutive days 

and necropsied approximately 24 hours after the 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 deviations at the start of the study. Statistical 

analysis indicated that there were no significant differences 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 animal 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). 

Page 207 of 281
In vivo Hershberger Assay (2012) / Page 6 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

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 controlb 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 study1 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 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 decreased 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 marked 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 systemic 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 observed among the high-dose group rats. 

6. (a) Dose Preparation: Dose formulations were prepared 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 study 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). Orthophenylphenol: Hershberger, Uterotrophic, and Pubertal 

Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow 

Chemical Company, Midland, Michigan. 

Page 208 of 281
In vivo Hershberger Assay (2012) / 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 adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

7. Dosage administration: Test formulations were administered to the animals daily via oral 

gavage (4 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT 

was administered via oral gavage at 4 mL/kg bw. Dose volumes were adjusted daily based 

on the concurrent body weight measurement. 

8. Statistics: Body weights, body weight gains, absolute organ weights, and feed consumption 

were evaluated using Bartlett’s test for equality of variances. Based on the outcome of the 

Bartlett’s test (p=0.01), a parametric or non-parametric analysis of variance (ANOVA) was 

performed. If the ANOVA was significant, analysis by the one-sided Dunnett’s test or the 

Wilcoxon Rank-Sum test with Bonferroni’s correction was performed. Significance was 

designated at p≤0.05. The statistical analyses were considered adequate. 

Statistical outliers were identified using a sequential method (p=0.02) and excluded if 

justified by sound scientific reasons. 

C. METHODS

1. Clinical Examinations: During dosing, cage-side checks for mortality and morbidity were 

conducted twice daily and hand-held examinations for clinical signs of toxicity were 

conducted once daily. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and at termination. Statistical analyses of body weights 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. Values were measured for each 

cage and reported as group mean daily 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 conducted 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 isoflurane/oxygen (all animals except those dosed 

with FT) for blood collection, euthanized by decapitation, and subjected to a gross necropsy. 

The five mandatory androgen-dependent organs (ventral prostate, seminal vesicles, LABC, 

Page 209 of 281
In vivo Hershberger Assay (2012) / Page 8 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and 

weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. 

EPA Guideline (OCSPP 890.1400). Additionally, the liver, kidneys, and adrenals were 

weighed. Tissues except liver were preserved in neutral, phosphate-buffered 10% formalin. 

Three sections of the liver were preserved in neutral, phosphate-buffered 10% formalin for 

possible future histological evaluation 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 respiration, 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 another had red urine (Day 7). 

TABLE 2. Incidence of Clinical Observations in the 1000 mg/kg/day Group in the Androgen Agonist 

Assaya

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, one animal had slow respiration (Days 9 and 

10) and perinasal soiling (Day 11). 

Page 210 of 281
In vivo Hershberger Assay (2012) / Page 9 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

One animal in the 1,000 mg/kg/day group (Animal # 4346) had clinical signs including 

decreased activity, noisy, labored, and/or slow respiration, sneezing, and 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 the 1000 mg/kg/day Group in the Anti-Androgen Assaya

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 presented in Table 4. Body weights were decreased 

(p<0.05) by 15% on Day 7 in the 1,000 mg/kg/day group compared to controls; however, 

body weights at the end of the study were similar to controls. 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 study. 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 response 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 animals gained 67-114% of the mean control body 

weight gains over the dosing period and had terminal body weights ≥92% of the control 

group. 

Page 211 of 281
In vivo Hershberger Assay (2012) / Page 10 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 4. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist 

Assaya

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

Body 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

Body 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

Body 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 treatment groups and positive 

control group were comparable to the negative control over the duration 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; these animals gained <52% of the mean body 

weight gain in the control group. The remaining four animals gained 80-111% of the mean 

control body weight gains over the dosing period and had terminal body weights ≥91% of 

the control group. 

Page 212 of 281
In vivo Hershberger Assay (2012) / Page 11 of 16 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 5. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen 

Assaya

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

Body 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

Body 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

Body 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 consumption 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/day) in the Androgen Agonist Assaya

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 assay are presented in Table 7. 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. 

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ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

In general, increases or decreases in food consumption corresponded with increased or 

decreased body weight gains in both assays. 

TABLE 7. Food Consumption (g/animal/day) in the Anti-Androgen Assaya

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 CONCENTRATIONS (Optional): Serum hormone measurements 

were not conducted. 

E. ORGAN WEIGHTS: Weights for the accessory sex organs, 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, adrenal 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 vesicles; 1117% in ventral prostate; 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 enlarged Cowper's glands (0.0148 g) compared 

to the group mean weight for this tissue (0.0050 g). This tissue weight was identified as a 

statistical outlier, although other tissue weights from this animal were similar to control 

values. Because this outlier value increased variability for Cowper’s glands in the vehicle 

control group, reducing the statistical power to identify a treatment-related effect, statistical 

comparisons for Cowper's glands were carried out with or without this animal. In either 

case, there was no significant treatment-related effect on Cowper's gland. 

Percent CVs for the vehicle control and the 1,000 mg/kg/day treatment groups were 

compared to the performance criteria in the Guidelines. All %CV values were less than the 

maximum recommended values, with the exception of the %CV for Cowper’s glands for the 

control group (CV = 88%; maximum recommended CV = 55%). 

Page 214 of 281
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ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 8. Accessory Sex Organ Weights from Androgen Agonist Assay in Sprague Dawley Ratsa

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 study 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% compared to the controls. There were no 

treatment-related effects on the weights of the seminal vesicles, LABC, Cowper’s glands, 

glans penis, adrenal glands, kidneys, or liver for any OPP treatment group. Animals dosed 

with TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as 

follows: 83% in seminal vesicles; 77% in ventral prostate; 46% in LABC; 61% in Cowper’s 

gland; and 18% in glans penis. Adrenal glands, kidney, and liver weights in the positive 

control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 1,000 mg/kg/day treatment groups were 

compared to the performance criteria in the Guidelines. All %CV values were less than the 

maximum recommended values, with the exception of the %CV for LABC for the high dose 

group (CV = 22%; maximum recommended CV = 20%). 

Page 215 of 281
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ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1400/ OECD 441

TABLE 9. Accessory Sex Organ Weights from Anti-Androgen Agonist Assay in Sprague Dawley or Wistar Ratsa

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 study 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 irritation 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 excluded 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 variability in responses to high-dose OPP treatment 

with some animals exhibiting significant clinical signs and marked effects on body 

weight/body weight gains. Treatment-related clinical signs in the 1000 mg/kg/day OPP 

group included decreased activity, noisy or slow respiration, perioral/perinasal/perineal 

soiling, soft feces, red urine, and cold to touch. High-dose animals had significantly 

decreased body weight gains (28-41%) in both the androgenic 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 necropsy, two high-dose animals had 

thickened glandular stomach mucosa, which was attributed to OPP treatment. There were 

no treatment-related increases in androgen-sensitive tissue (AST) weights in OPP-treated 

Page 216 of 281
In vivo Hershberger Assay (2012) / 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 no 

significant 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. Overall, this assay met the CV performance 

criteria outlined in the corresponding 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 negative 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 excluded 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, 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 respiration, and soft feces. The incidence of these observations 

decreased as the study progressed. In the anti-androgen assay, five animals in the 1,000 

mg/kg/day group 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). At 

necropsy, one animal in each high dose group (both assays) was found to have thickening of 

the nonglandular mucosa of the stomach. 

In the androgen agonist assay, body weights were decreased (p<0.05) by 15% on Day 7 in 

the 1,000 mg/kg/day group compared to controls; however, body weights at the end of the 

study were similar to controls. Food consumption in this group was decreased (p<0.05) by 

42% during Days 4-7. Body weights and body weight gains in the 50 and 250 mg/kg/day 

groups were comparable to controls throughout the duration of the study; food consumption 

was increased (p<0.05) in the 50 mg/kg/day group on Days 1-4 (↑6%). In the positive 

control group, cumulative body weight gains were increased (p<0.05) on Days 1-4 (↑45%), 

Days 1-7 (↑50%), and Days 1-11 (↑36%); food consumption was increased (p<0.05) in this 

group on Days 1-4 (↑14%), Days 4-7 (↑14%), and Days 7-11 (↑12%). There were no 

treatment-related effects on weights of accessory 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; weights 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-related effects on the weights of seminal vesicles, 

LABC, Cowper’s glands, glans penis, adrenal glands, kidneys, or liver for any OPPtreatment group. Animals dosed with TP + FT (positive control) had the expected decreases 

Page 217 of 281
In vivo Hershberger Assay (2012) / 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 the 1,000 mg/kg/day treatment groups for both 

assays were compared to the performance criteria in the guidelines. All %CV values were 

less than the maximum recommended values, with the exception of the %CV for Cowper’s 

glands for the control group in the androgen agonist assay (CV = 88%; maximum 

recommended CV = 55%) and the %CV for LABC for the high dose group in the 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 androgenicity and anti-androgenicity in the 

Hershberger assay. 

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered 

to have had an adverse impact on the results, interpretation or conclusions of this study: 

 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. 

Page 218 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 219 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 220 of 281
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 Environmental 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. Animals were examined for vaginal 

opening (VO) daily beginning on PND 22, and the body weight at day of attainment was 

recorded. Following sacrifice on PND 42, thyroxine (T4) and thyroid stimulating hormone 

(TSH) levels were analyzed by radioimmunoassay or chemiluminescent immunoassay. Liver, 

pituitary, adrenals, and urogenital organ weights were recorded, and microscopic examinations 

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

All animals survived until scheduled termination, with the exception of one animal in the highdose group (death due to gavage error). Additionally, no treatment-related clinical signs of 

toxicity were observed in animals for any dose groups. 

Page 221 of 281
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 and terminal body weights, body weight 

gains, and body weight at attainment of VO were similar across all dose groups. However, the 

900 mg/kg/day group weighed 8% less than control animals at PND 35. The 900 mg/kg/day 

group gained 10% to 13% less than control between PND 22-35, but these decreases were not 

statistically (NS) different from control animals. High-dose rats gained more weight than 

control animals during the PND 35-42 interval, such that body weight gains were only slightly 

decreased (3.2%, NS) over the entire dosing period (PND 22-42). 

At 900 mg/kg/day, modest decreases (p<0.05) were noted in adjusted pituitary (↓12%) and 

adrenal (↓13%) weights, and a minor increase (p<0.05) of 9% was noted in relative liver weight. 

Relative pituitary weights were decreased (p<0.05) by 10%. There were no dose-related effects 

on the weights of kidneys, ovaries, uterus, or thyroid. 

There were no effects of treatment on mean age at first estrus or mean cycle length. In the highdose animals, the percent cycling was decreased (NS) relative to the control from 93.3% to 

64.3%, and the percent regularly cycling was decreased (p<0.05) from 86.7% to 28.6%. 

However, the estrous cycle status at necropsy (diestrus, proestrus, estrus, not cycling) was 

generally similar across all groups.

Serum urea nitrogen (BUN) and phosphorus levels were increased (p<0.05) in the 900 

mg/kg/day dose group (↑23.1 and ↑14.1%, respectively). These increases were considered 

treatment-related, and were associated with treatment-related kidney effects. Significantly 

increased alanine aminotransferase activity (ALT; p<0.05; 102%) was also considered 

treatment-related. 

OPP was not associated with any significant treatment-related changes in thyroid 

histopathology. At 900 mg/kg/day, increased incidences of the following histological lesions 

were noted in the kidney: (i) multifocal renal tubular dilatation (12/14 treated animals, very 

slight and slight severity vs. 4/15 controls, very slight severity); (ii) multifocal collecting duct 

epithelium hypertrophy (3/14 treated animals, very slight severity vs. 0/15 controls); (iii) 

multifocal renal tubule dilatation (9/14 treated animals, very slight to slight severity vs. 1/15 

controls, very slight severity; and (iv) slight focal renal tubular necrosis, with accompanying 

inflammation (2/14 treated animals vs. 0/15 controls). 

The doses tested were adequate based on the increased serum ALT, BUN, phosphorus, renal 

lesions, and effects on estrous cyclicity seen at the high dose (900 mg/kg/day). 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

Assurance statements were provided. 

Page 222 of 281
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 Laboratories, Inc., Portage, MI 

 Housing: 2 animals/cage in plastic solid bottom cages with heat-treated, aspen wood shavings (Harlan 

Laboratories, Inc., Indianapolis, IN) 

 Diet: Teklad Diet #2016 (Harlan Laboratories, Inc.), ad libitum

Total genistein equivalents <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 with equal numbers of 

males and females whenever possible. 

3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were 

assigned to dose groups such that the mean body weights (and variances) were similar 

Page 223 of 281
Female 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 Designa

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 assay1, OPP was administered by oral 

gavage at dose concentrations of 0, 50, 250, and 1000 mg/kg/day to castrated male CD rats 

that were 55–64 days old. The 1000 mg/kg/day dose resulted in decreases in terminal body 

weights of 6-10% in the androgenic and anti-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 (probe) 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 consumption. There were no significant 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 increased absolute kidney weights in females 

administered 900 mg/kg/day were also observed. 

Based on these studies, 900 mg/kg/day was selected 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 Environmental 

Research and Consulting; samples from the top, middle and bottom of the low- and highdose formulations were analyzed for homogeneity and concentration analyses. Duplicate 

samples from the control and mid-dose formulations were taken from random locations. 

Stability analysis was conducted by Toxicology and Environmental Research and 

Consulting1 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 Assay in Castrated Male Crl:CD(SD) Rats. Toxicology and 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Unpublished (MRID # 

48672602). 

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Stability: Reported to be stable for ≥15 days 

Concentration (% nominal): 99.6 to 103.8% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 

through PND 42, in a volume of 4 mL/kg of body weight. Dose volumes were adjusted 

based on the individual daily body weights.

7. Statistics: Mean values were presented with standard deviations and coefficient of 

variation. The following statistical 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 (experiment-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 significant 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 Fisher'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)

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Statistical outliers were identified by a sequential test (p = 0.02). Outliers and questionable 

data points (i.e., values due to technical errors) were excluded only for documented 

scientifically sound reasons. Histopathology results were not subjected to outlier tests. 

The statistical analyses were considered adequate. 

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined at least twice daily for 

mortality, moribundity, availability of food/water, and clinical abnormalities. Clinical, 

hand-held examinations were conducted daily post-dose. 

2. Body Weight: Animals were weighed prior to allocation to dose groups (PND 21) and 

daily prior to dosing. Body weights determined prior to dosing on PND 42 were used as 

the terminal body weights. Body weights were statistically analyzed on PND 21, 22, 25, 

28, 32, 35, and 42; body weight gains were statistically 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 completion of VO were recorded. Examination for 

the onset of puberty ceased with acquisition 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 classified as diestrus, proestrus, 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 diestrus 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 first 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 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 necropsy, and necropsies were completed by 1300 

hours. Animals were euthanized by exsanguination followed by decapitation. A complete 

necropsy was performed on all animals. Animals 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, terminal body and organ weights were not 

recorded, and organs were not examined histopathologically. 

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a. Hormone Analysis: Samples were sent on dry ice to WIL Research Laboratories, LLC 

(Ashland, OH) for analysis. TSH concentrations were analyzed by radioimmunoassay 

(RIA), and a chemiluminescent immunoassay was used for total T4. 

b. Clinical Chemistry: The following CHECKED (X) parameters 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin/globulin ratio (calculated) 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on Guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination. 

 UROGENITAL OTHER 

XX Ovaries, without oviducts (paired, without oviducts)*+ XX Thyroid*+

XX Uterus, imbibed, with oviducts and cervixa*+ X Liver* 

XX Kidneys (paired)*+ X Adrenals (paired)* 

 X Pituitary* 

a With and without fluid 

* Weights required based on guideline 890.1450 

+ Histopathological examination required based on Guideline 890.1450 

All organs, except the thyroid/trachea and pituitary, were weighed prior to fixation. Paired 

organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed 

again after each uterine horn was cut three times to allow removal of the fluid in the lumen 

(blotted weight). 

All tissues were fixed in 10% phosphate-buffered 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. 

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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 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 

one animal in the high-dose group. Hemorrhages in the lungs and nasal cavity of this 

animal were consistent with death due to a gavage 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 clinical 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 attainment 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 from PND 33.7–34.0 (unadjusted and adjusted). 

Initial and terminal body weights, body weight gains, and body weight at attainment of VO 

were similar across all dose groups. However, the 900 mg/kg/day group weighed 8% less 

than control animals at PND 35. The 900 mg/kg/day group gained 10% to 13% less than 

control between PND 22-35, but these decreases were not statistically different from 

control animals. High-dose rats gained more weight than control animals during the PND 

35-42 interval, such that body weight gains were only slightly decreased (3%) over the 

entire dosing period (PND 22-42). 

The mean age at VO, body weight at VO, weanling 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, the % CV for final body weight was outside the acceptable 

range, as follows: 10.6% CV for final body weight (maximum 8.93%). 

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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/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

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 standard 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 from controls at p<0.05. 

D. ORGAN WEIGHTS: Organ weights at necropsy are presented 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 weights were decreased (p<0.05) 12% and 10%, 

respectively, in the 900 mg/kg/day group. Adjusted adrenal weight was decreased (p<0.05) 

in the 900 mg/kg/day animals (↓13%), but unadjusted and relative 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%). 

 

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TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control OPP 

(50 mg/kg/day) 

OPP 

(250 mg/kg/day) 

OPP 

(900 mg/kg/day) 

N Mean 

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

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 report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses. 

b Standard deviation (SD) reported for unadjusted values and standard 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 from controls at p<0.05. 

E. ESTROUS CYCLICITY: Estrous cycle data are presented in Table 4. There were no 

effects of treatment on mean age at first estrus or mean cycle length. At 900 mg/kg/day, 

the percent cycling was decreased (not significant) from 93.3% to 64.3%, and the percent 

regularly cycling was decreased (p<0.05) from 86.7% to 28.6%, with considerable 

variability in the high-dose animals (five females with irregular cycling and five that were 

not cycling). The cycle status at necropsy (diestrus, proestrus, estrus, not cycling) was 

generally similar across all groups.

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TABLE 4. Estrous Cyclicity in Female Rats Treated with OPPa

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 Table 5. No dose-dependent treatment effects 

were observed on serum T4 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 values were not provided 

for either the hormone or clinical chemistry parameters. 

The results for serum T4 and TSH in the control group were within the acceptable range of 

the performance criteria provided in the Guideline (890.1450). 

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TABLE 5. Hormone Levels and Clinical Chemistrya

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 T4, 

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 report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05. 

G. GROSS PATHOLOGY: No treatment-related effects were observed on gross pathology. 

H. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 6. OPP was not associated with any significant treatmentrelated changes in thyroid histopathology. 

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TABLE 6. Thyroid Gland Follicular Cell Height and Colloid Area a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb 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 incidences of the following histological lesions 

were noted in the kidneys: (i) multifocal renal tubular dilatation (12/14 treated animals, 

very slight and slight severity vs. 4/15 controls, very slight severity); (ii) multifocal 

collecting duct epithelium hypertrophy (3/14 treated animals, very slight severity vs. 0/15 

controls); (iii) multifocal renal tubule dilatation (9/14 treated animals, very slight to slight 

severity vs. 1/15 controls, very slight severity; and (iv) slight focal renal tubular necrosis, 

with accompanying inflammation (2/14 treated animals vs. 0/15 controls). The renal 

tubular dilation was characterized 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 degenerated tubules. Other renal lesions in the treatment 

groups occurred in single animals or not dose-dependently. 

Evaluation of the uteri and ovaries were performed with knowledge of the estrous stage; 

there were no significant treatment-related findings. 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 ovary and the uterus were interpreted to be 

secondary to treatment-related decreases on the body weight of these two animals. There 

were no treatment-related histopathological findings noted in animals in the 50 and 

250 mg/kg/day groups. 

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TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidneya

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. 

Page 234 of 281
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 decreased body weight gain as well as liver 

weight increase and histopathological changes in the kidneys). Based on the lack of 

toxicologically meaningful, treatment-related effects on puberty onset, estrous cycle, 

endocrine-sensitive organ weights, serum T4 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 scheduled 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 dose 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 increase (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, uterus, 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 percent 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 levels were increased (p<0.05) in the 900 

mg/kg/day dose group. 

OPP was not associated with any significant treatment-related changes in thyroid 

histopathology. At 900 mg/kg/day, increased incidences of the following histological 

lesions were noted in the kidney: (i) very slight and slight multifocal renal tubular 

dilatation; (ii) very slight multifocal 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 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 degenerated tubules. 

Other renal lesions in the treatment groups occurred in single animals and were not 

considered dose-dependent. 

Page 235 of 281
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 cycling 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, renal 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, interpretation or conclusions of 

this study: 

 Total genistein-equivalent content of the laboratory diet (Teklad Diet #2016) was 

reported as <325 μg/g; Guideline states that the content should be ≤300 μg/g. 

 Control CV for final body weight (10.63%) was 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 

Page 236 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 237 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 238 of 281
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 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial 

separation (PPS) daily beginning on PND 30, and the body weight at day of attainment was 

recorded. Following sacrifice on PND 53 , total serum testosterone, thyroxine (T4), and thyroid 

stimulating hormone (TSH) levels were analyzed by radioimmunoassay or chemiluminescent 

immunoassay. Liver, pituitary, adrenal and urogenital organ weights were recorded, and 

microscopic examinations were performed on the testes, epididymides, thyroid, and kidneys. 

All animals survived until scheduled termination, with the exception of four animals. The cause 

of death for three of these animals (one in each OPP-dose group) was considered to be gavage 

error. The cause of death of the fourth animal (high-dose group) was not determined, but was 

not attributed to OPP treatment. Additionally, no treatment-related clinical signs of toxicity were 

observed in animals for any dose groups. 

Page 239 of 281
Male Pubertal Assay (2012) / Page 2 of 18 

O-PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

Adjusted terminal body weights were decreased by 8% in the 900 mg/kg/day group, due to a 

treatment-related effect on body weight gain (↓13%; p<0.05) over the duration of the study. 

Body weights and body weight gains were not affected at ≤250 mg/kg/day. The adjusted age of 

attainment of PPS was delayed by 1.6 days (not significant; NS) in the 900 mg/kg/day group 

compared to the control (45.1 vs. 43.5 days). Delayed PPS appeared to be related to the 

decreased terminal body weight and body weight gain observed in the high-dose group. 

Significant (p<0.05) organ weight changes after administration of OPP included increased 

relative liver weight at 250 mg/kg/day (↑8%), and adjusted and relative liver weights at 

900 mg/kg/day (↑10% and ↑21%, respectively). Compared to the control, unadjusted and 

adjusted pituitary weights were decreased by 15% and 10%, respectively, in the 900 mg/kg/day 

group, and unadjusted adrenal weights were decreased by 11% at ≤250 mg/kg/day and by 16% at 

900 mg/kg/day. However, the most relevant measures are the adjusted weights, and only minor 

increases (p<0.05) were noted in the liver and adrenal at 900 mg/kg/day. 

Additionally, decreased (p<0.05) organ weights at 900 mg/kg/day included: unadjusted weights 

of seminal vesicles + coagulating glands with and without fluid (↓23% and ↓18%, respectively); 

unadjusted and adjusted ventral prostate and LABC weights (↓17-20% and ↓16-18%, 

respectively); unadjusted weights for the left and right epididymides (↓10% and ↓9%, 

respectively); and unadjusted and adjusted weights for the left testis (↓9% and ↓7%, 

respectively). As adjusted weight decreases were observed in three androgen sensitive tissues, 

these effects were considered treatment-related. There were no dose-related effects on the 

weights of kidneys, dorsolateral prostate, right testis, or thyroid. 

No treatment-related effects were observed on serum testosterone or TSH concentrations. Serum 

T4 concentrations were decreased (p<0.05) at all dose levels by 15-23%, but without clear dosedependence. There were no gross or histological lesions of the thyroid. At 900 mg/kg/day, an 

apparent treatment-related effect was observed on serum urea nitrogen concentrations, which 

corroborated the renal histological evidence. Significantly increased alanine aminotransferase 

(ALT) activity (↑95%) was considered treatment-related, but the 32% increase in aspartate 

aminotransferase activity (AST) was a slight response and not adverse. The 40% decreases 

(p<0.05) in gamma glutamyl transferase (GGT) activities at 250 and 900 mg/kg/day are 

considered slight and were not dose-dependent. 

No treatment-related findings were observed in the right testes or right epididymides at any OPP 

dose. However, in the kidneys, there were increased incidences of renal tubular dilatation 

(combined focal and multifocal, very slight and slight severity) at 900 mg/kg/day (12/14 

animals) compared to controls (5/15 animals). Increased incidences of collecting duct 

epithelium hypertrophy (combined focal and multifocal, very slight severity) were also observed 

at 900 mg/kg/day (5/14) compared to controls (0/15). Although the incidences of renal tubular 

degeneration were similar in high-dose and control animals, the severity was increased at the 

high dose; the incidence of slight multifocal tubular degeneration was 3/14 at 900 mg/kg/day 

compared to 0/15 in controls. 

The high dose of 900 mg/kg/day was sufficient based on the 8% decrease in the adjusted 

terminal body weight, the 13% decrease overall body weight gain, the 7-17% decreases in 

androgen-sensitive organ weights, the 94% increase in serum ALT, and increased incidences 

and/or severity of renal lesions. 

Page 240 of 281
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 Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

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

Assurance statements were provided. 

Page 241 of 281
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 Laboratories, Inc., Portage, MI 

 Housing: 2 animals/cage in plastic solid bottom cages with heat-treated, aspen wood shavings (Harlan 

Laboratories, Inc., Indianapolis, IN) 

 Diet: Teklad Diet #2016 (Harlan Laboratories, Inc.), ad libitum

Total genistein equivalents <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 with 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 similar 

Page 242 of 281
Male 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 Designa

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 assay1, OPP was administered by oral 

gavage at dose concentrations of 0, 50, 250, and 1000 mg/kg/day to castrated male CD rats 

that were 55-64 days old. The 1000 mg/kg/day dose resulted in decreases in terminal body 

weights of 6-10% in the androgenic and anti-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 (probe) 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%, respectively) in the 900 

mg/kg/day males, associated with decreased feed consumption. There were no significant 

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 increased 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/day 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 Environmental 

Research and Consulting; samples from the top, middle and bottom of the low- and highdose formulations were analyzed for homogeneity and concentration analyses. Duplicate 

samples from the control and mid-dose formulations were taken from random locations. 

Stability analysis was conducted by Toxicology and Environmental Research and 

Consulting1 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 Assay in Castrated Male Crl:CD(SD) Rats. Toxicology and 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Unpublished (MRID 

48672602). 

Page 243 of 281
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 adequate 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 body weight. Dose volumes were adjusted 

based on the individual daily body weights.

7. Statistics: Mean values were presented with standard deviations and coefficient of 

variation. The following statistical 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 (experiment-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 significant 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 transformed alanine aminotransferase data. A non-parametric 

Kruskal Wallis test (p = 0.05), followed by a Wilcoxon rank-sum test with Bonferroni-Holm adjustment for dose 

groups compared to control. 

Statistical outliers were identified by a sequential test (p = 0.02). Outliers and questionable 

data points (i.e., values due to technical errors) were excluded only for documented 

scientifically sound reasons. Histopathology results were not subjected to outlier tests. 

The statistical analyses were considered adequate. 

Page 244 of 281
Male Pubertal Assay (2012) / Page 7 of 18 

O-PHENYLPHENOL (OPP) / 064103 OCSPP 890.1500 / OECD None

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined at least twice daily for 

mortality, moribundity, availability of food/water, and clinical abnormalities. Clinical, 

hand-held examinations were conducted daily post-dose. 

2. Body Weight: Animals were weighed prior to allocation 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 entire 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 recorded 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 with 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). Blood 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 necropsy, 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 was 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 concentrations were analyzed by radioimmunoassay 

(RIA), and chemiluminescent immunoassays were used for total T4 and testosterone. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

Page 245 of 281
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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in male rats based on Guideline 890.1500. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected 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 (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 

X Levator ani-bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

* Weights required based on guideline 890.1500 

+ Histopathological examination required based on Guideline 890.1500 

All organs except the thyroid/trachea were weighed prior to fixation. The paired kidneys 

and adrenals were weighed together. The seminal vesicle with coagulating glands was 

weighed. After each side was cut sagitally to allow removal of the fluid in the lumen 

(blotted weight), the seminal vesicle was weighed again. 

The gross lesions, adrenals, dorsolateral prostate, 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. 

Page 246 of 281
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 cross-section from the center of the testes was 

embedded in paraffin, and stained with PAS/hematoxylin. The histopathological 

examination of the testes included a qualitative assessment of the stages of spermatogenesis; 

a qualitative assessment of the relationships between spermatagonia, spermatocytes, 

spermatids, and spermatozoa seen in cross sections of the seminiferous tubules; and 

identification of any degenerative changes (e.g., vacuolation of the germinal epithelium, a 

preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and 

fibrosis). Examination of the intact epididymis included the caput, corpus, and cauda, 

accomplished by evaluation of a longitudinal section, 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 three OPP dose groups apparently died from either 

from a gavage error or aspiration of test material due to a dosing error. The cause of death 

for a fourth animal, in the 900 mg/kg/day group, was undetermined, but none of the deaths 

were attributed to OPP treatment. 

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clinical signs of toxicity were 

observed in animals for any dose groups. One 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 until scheduled necropsy. These observations 

were considered isolated, transient occurrences, 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 control group were excluded from analysis due to 

excessively low initial body weight (21.2 g compared 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 was 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 was resolved at later 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 decreased by 8% (NS). 

Males in the 900 mg/kg/day group only gained 205.7 g during PND 23-53 (p<0.05; ↓13%) 

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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) compared 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, weanling 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 weight were outside the 

acceptable range, as follows: 8.36% CV for weight 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/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

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 standard 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 from controls at p<0.05. 

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D. ORGAN WEIGHTS: Organ weights at necropsy are presented 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%, respectively, compared to 

controls. There was a dose-dependent decrease in pituitary weights, though only the 

unadjusted and adjusted pituitary weights of the 900 mg/kg/day group were significantly 

decreased (p<0.05;15% and 10%, respectively) . A dose-dependent decrease in unadjusted 

adrenal weights was significant 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 decreased in the high treatment 

group. At 900 mg/kg/day, the unadjusted weights of the seminal vesicle + coagulating 

glands (with and without fluid) were decreased (p<0.05) by 23% and 18%, respectively. 

Ventral prostate and LABC weights (unadjusted and adjusted) were also decreased (p<0.05) 

by 17-20% and 16-18%, respectively. Unadjusted 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 decreased (p<0.05) 9% and 7%, respectively. There 

were no significant decreases between control and treatment mean weights of kidneys, 

dorsolateral prostate, right 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 Necropsya

Organ 

Vehicle Control OPP 

(50 mg/kg/day) 

OPP 

(250 mg/kg/day) 

OPP 

(900 mg/kg/day) 

N Mean 

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

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 32.4

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TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control OPP 

(50 mg/kg/day) 

OPP 

(250 mg/kg/day) 

OPP 

(900 mg/kg/day) 

N Mean 

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean 

SD/ 

SEb

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 report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses. 

b Standard deviation (SD) reported for unadjusted values and standard 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 from 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 testosterone or TSH, serum T4 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 nitrogen levels were increased by 27%; (ii) ALT was 

increased by 95%; (iii) AST was increased by approximately 32%; and (iv) GGT was 

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decreased by 40%. GGT was also decreased (p<0.05) by 40% at 250 mg/kg/day. There 

were no significant differences between control and treatment means on the other measured 

clinical chemistry parameters. 

The results for serum T4, TSH, and testosterone in the control group were within the 

acceptable range of the performance criteria provided in the Guideline (890.1500). 

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TABLE 4. Hormone Levels and Clinical Chemistrya

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 T4, 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 study report. Percent differences from controls, calculated by 

the reviewers, are included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05. 

F. GROSS PATHOLOGY: No treatment-related effects were observed on gross pathology. 

G. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 5. OPP was not associated with any significant treatmentrelated changes in thyroid histopathology. 

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TABLE 5. Thyroid Gland Follicular Cell Height and Colloid Area a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb 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. Follicular cell height: 1 = lowest, 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 findings were observed in the right testes or right 

epididymides at any OPP dose. 

At 900 mg/kg/day, an increased incidence of renal tubular dilatation (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 severity) was also observed (5/14) compared to 

controls (0/15). Although 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 cortex and medulla that were lined by flattened to cuboidal 

epithelial cells, and often contained proteinaceous material mixed with sloughed cellular 

debris. The dilated tubules were occasionally 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 were noted in the right epididymis; however, 

these effects were not dose-dependent and are consistent with the juvenile age of the 

animals. There were no treatment-related histopathologic effects in the right testis or right 

epididymis. 

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TABLE 6. Incidence of Histopathological Lesions of the Testes, Epididymides and Kidneya

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, tubule, focal, 

very slight 2 15 2 15 4 15 1 14 

Dilatation, tubule, 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. 

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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 parameters and histopathological changes in the kidneys) 

with some effect extending to the mid-dose group (250 mg/kg/day; increased 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 organ weights were attributed 

to decreased terminal body weights 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 highest dose level tested. 

B. AGENCY COMMENTS: All animals survived until scheduled termination, with the 

exception of four animals, but none of the deaths 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 decreased in the 900 mg/kg/day group due to a 

treatment-related effect on body weight gain over the duration of the study. Body weights 

and body weight gains were not significantly affected at OPP dose concentrations of 

≤250 mg/kg/day. The adjusted age of attainment of PPS was delayed, but not significantly, 

compared to the control (45.1 vs. 43.5 days) in the 900 mg/kg/day group. 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 administration of OPP included increased 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 weights, though only the unadjusted and adjusted 

pituitary weights were significantly decreased (p<0.05) 15% and 10%, respectively in the 

900 mg/kg/day group. A dose-dependent decrease 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).Minor 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 glands with and without fluid; unadjusted and 

adjusted ventral prostate and LABC weights; unadjusted weights for the left and right 

epididymides; and unadjusted and adjusted weights 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 decreases were observed in three androgen sensitive 

tissues, these effects were considered treatment-related. There were no statistically 

significant differences between mean control and treatment organ weights of kidneys, 

dorsolateral prostate, right testis, or thyroid. 

Although no dose-dependent treatment effects were observed on serum testosterone or TSH 

concentrations, serum T4 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. 

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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 testes or right 

epididymides at any OPP dose. However in the kidneys, an increased incidence of renal 

tubular dilatation (combined focal and multifocal, very slight and slight severity) was noted 

in animals at 900 mg/kg/day compared to controls. An increased incidence of collecting duct 

epithelium hypertrophy (combined focal and multifocal, very slight severity) was also 

observed at 900 mg/kg/day compared to controls. Although the incidence of renal tubular 

degeneration was similar in the high-dose and control, the severity 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%, increased serum ALT of 95%, and increased 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 range 2.242-3.050 g); 

 Mean thyroid weight was 12.4 mg (acceptable range 14-26 mg) 

Page 256 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 257 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 258 of 281
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 concurrently with each independent run of a test chemical plate to demonstrate that 

the assay responded properly to positive control agents at two concentration levels. The positive 

controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol 

and testosterone production. 

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 recommended 

Page 259 of 281
Steroidogenesis Assay (2012) / Page 2 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

40 pg/mL level. However, basal estradiol production was still >26 pg/mL which is >2.5-fold the 

minimum detection limit (MDL), as per Guideline recommendation. Forskolin at 10 μM 

induced testosterone and estradiol production as required (≥2-fold and ≥7.5-fold, respectively), 

and 1 μM prochloraz inhibited testosterone production by >50% as required; however, inhibition 

of estradiol production (40%) was below the required 50%. 

The intra- and inter-plate variability (%CV) was reported to be 0.5-24.4% for the testosterone 

and estradiol in the test runs, with the exception of 0.01 μM OPP in Run 3 where the intraplate 

CV was 39.9%, due to increased responsiveness. 

Conflicting results were observed for testosterone production, with a statistically significant 30% 

increase at 10 μM OPP and a 30% decrease at 100 μM OPP. Statistically significant increases 

in estradiol production were observed at 10 μM OPP (2.6-fold) and 100 μM OPP (4.0-fold). 

These effects on estradiol were reproducible and concentration-dependent in all three runs. 

Based on the hormone responses in the three independent runs, OPP treatment resulted in 

statistically significant and reproducible increases in estradiol and testosterone production; 

however, the alterations in testosterone production were inconsistent in direction of the response. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

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

Assurance statements were provided. 

 

Page 260 of 281
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 #: 67747-09-5 

Page 261 of 281
Steroidogenesis 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 carcinoma 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% CO2 incubator at 37±2oC. The background 

hormone concentrations present in the Nu-Serum were 974 pg/mL for testosterone and 681 

pg/mL for estradiol. It was stated that cells were cultured following the specific protocol 

described in the USEPA Endocrine Disruptor Screening Program Guideline. Cells from 

frozen stocks were thawed and placed in tissue 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 × 105 cells/mL. 

Plates were incubated in a 5% CO2 atmosphere at 37±2oC for approximately 24 hours prior 

to exposure to the test chemical or the positive controls.

The 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 tests were not conducted 

because testosterone and estradiol concentrations 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 testosterone and estradiol internal standards, and the organic phase 

were transferred to clean vials. The extracts were evaporated to dryness, and the final 

Page 262 of 281
Steroidogenesis Assay (2012) / Page 5 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

residue in each vial was derivatized with dansyl chloride in sodium bicarbonate buffer 

(100 mM, pH 10.5). 

c. Laboratory Proficiency Test: The report stated that laboratory 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 and are reported separately.

2. Test Solutions: Stock solutions were prepared in DMSO and diluted in the final treatment 

medium. A total of seven concentrations 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 described in the USEPA Endocrine Disruptor 

Screening Program Guideline. Cells from frozen stock were thawed and 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, yielding approximately 50-60% confluency 

at 24 hours. The seeded plates were incubated for 24 hours at 37°C in a 5% CO2

atmosphere. 

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and replaced (1 mL). The cells were then 

exposed to the OPP or solvent vehicle alone (DMSO) in triplicate according to the 

schematic presented in Table 1. 

TABLE 1. Dosing Schematic for the Exposure of H295R 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 total 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 response to forskolin (an inducer of testosterone 

and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol 

production). The QC plate was prepared and dosed in the same manner with 1 μL of either 

forskolin or prochloraz according to the schematic presented in Table 2. 

Page 263 of 281
Steroidogenesis Assay (2012) / Page 6 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM)a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Blank + MeOHc Blank + MeOH Blank + MeOH 

B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH 

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM 

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM 

a Data were obtained from page 17 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

b Blank wells received medium only. 

c MeOH = 70% methanol was added to these wells for 30 minutes 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 removed, 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 

Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison WI, Cat# G3580), 

immediately after removal of the culture medium after the 48-hour incubation. This is a 

colorimetric modified MTT test using a tetrazolium 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 490 nM absorbance is directly proportional to 

the number of viable cells in the culture. 

6. Hormone Measurement System: Concentrations of testosterone 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 for two concentrations of testosterone and estradiol (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 below maximum response on standard curve 

NA Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations (SD) for testosterone 

and estradiol concentrations were calculated for each concentration of the reference 

chemicals and 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 with the following equation:

Relative change = (hormone concentration in each well) ÷ (mean SC hormone 

concentration) 

Concentrations that exhibited ≥20% cytotoxicity, or where precipitation was observed, were 

omitted from further analysis. 

Prior to conducting statistical analyses, the assumptions of normality and variance 

homogeneity were evaluated. Homogeneity of variance were evaluated by Bartlett's test and 

Page 264 of 281
Steroidogenesis Assay (2012) / Page 7 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

normality by Shapiro-Wilk's test at alpha = 0.01. If the data were not homogeneous or 

normally distributed, then the data were transformed to approximate homogeneity or a 

normal distribution. If the data were homogeneous and approximately normally distributed, 

differences between chemical treatments and SC were analyzed using parametric analysis of 

variance followed by Dunnett's test, if significant. 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 testosterone production was observed 

at 10 μM OPP, and a statistically significant slight decrease (0.7-fold) 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 estradiol were reproducible, concentrationdependent and statistically significant in all three runs of the assay. 

Page 265 of 281
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 Trial 1 Trial 2 Trial 3 Mean ± SDb 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. 

 

Page 266 of 281
Steroidogenesis Assay (2012) / Page 9 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to OPP Concentration. 

* Significantly different from the solvent control at p≤0.05. 

Page 267 of 281
Steroidogenesis Assay (2012) / Page 10 of 13 

ORTHO-PHENYLPHENOL/064103 OCSPP 890.1550/ OECD None

FIGURE 2. Change in Estradiol Production Relative to OPP Concentration. 

* Significantly different from the solvent control at p≤0.05. 

Page 268 of 281
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 OPP test plates (Table 4). Methanol 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 Viability – 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-difference change relative to SC (individual and 

mean ± SD) for the three assay runs, are presented 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 production levels for estradiol (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 still >26 pg/mL, which is >2.5-fold the 

MDL as per guideline recommendation. Testosterone and estradiol in the presence of 10 

μM 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 achieved 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 estradiol 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 confirmed the within- and between-plate %CVs. 

Page 269 of 281
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 steroidogenesis assays, it was determined that OPP 

treatment resulted in a statistically significant increase in estradiol (E2) production at the 

highest two concentrations tested, including the assay limit concentration (10-4 M), while no 

effects on estradiol production were seen at lower concentrations. There were very slight 

(<1.5-fold), divergent alterations in testosterone 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 validation program for this assay, and hence was not 

considered to be biologically relevant. 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 μM), and OPP was not cytotoxic to the 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 steroidogenesis assay with OPP were conducted, but the 

fourth was not evaluated statistically as three 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 MDL as per guideline recommendation. Induction 

of testosterone and estradiol production by 10 μM forskolin met guideline requirements (≥2-

fold and ≥7.5-fold, respectively). Inhibition of testosterone production by 1 μM prochloraz 

met guideline requirements, but inhibition of estradiol 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, 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 

Page 270 of 281
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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 the SC data from the QC plates was used to 

replace the SC data from the test plates due to test material volatility and apparent 

contamination of the SC wells by the test material. 

A statistically significant slight increase (1.3-fold) in testosterone production was observed 

at 10 μM OPP, and a statistically significant slight decrease (0.7-fold) 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 estradiol were reproducible, 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 material or reference chemicals were included in 

the study report. 

 The specificity, accuracy and recovery for testosterone and estradiol by the LC/APPIMS/MS method were not provided. 

 Minimum basal production levels for estradiol in the SC wells were not met. 

 Inhibition of estradiol production (40%) by 1 μM prochloraz was less than the guideline 

specified 50% inhibition. 

Page 271 of 281
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 272 of 281
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

assay, where available, and redundancy across the battery. Thus, the results of each 

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 273 of 281
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. 

Page 274 of 281
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 assay. OPP is negative in 

the uterotrophic assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

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

Assurance statements were provided. 

 

Page 275 of 281
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. Passage (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: 17α-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 Diet #5002 (PMI Nutrition International, St. Louis, 

MO), ad libitum (total genistein equivalents <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 their dam plus 1 day after weaning ) 

B. STUDY DESIGN 

1. In Life Dates: Start: June 17, 2011 End: June 20, 2011 

Page 276 of 281
In 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 necropsied for uterine weight measurements. 

The pups were housed with their dam for 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 deviations at the start of the study. Statistical 

analysis indicated that there were no significant differences in group means at study 

initiation. Furthermore, the body weight of each animal was within 20% of the overall 

mean. 

TABLE 1. Study Designa

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 

17α-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 study1 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 parameters and electrolytes. Females in the 900 

mg/kg/day group had increased absolute 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 females in the 700 mg/kg/day group. The high dose 

level was expected to cause a significant decrease in body weight gain over the dosing 

period in the current study. The lower dose levels 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 established stability of the test substance in corn oil. Homogeneity 

and achieved concentration were tested prior to study initiation in each of the dose 

formulations by sampling the top, middle, and bottom 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). Orthophenylphenol: Hershberger, Uterotrophic, and Pubertal 

Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow 

Chemical Company, Midland, Michigan. 

Page 277 of 281
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 adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Animals were administered the test formulation, the positive 

control (EE), or the vehicle control daily via oral gavage for three consecutive days in a dose 

volume of 4 mL/kg body weight. Dose volumes 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 Bartlett’s test (p=0.01), wet uterine weights for 

vehicle control versus positive control were log transformed prior to further analysis. An 

analysis of covariance (ANCOVA) was performed with dose in the model and terminal 

body weight as the covariate. Statistical analyses were conducted for data from the vehicle 

controls versus OPP treatment groups and data 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. The incidence of vaginal opening was reported but 

not statistically analyzed. The statistical 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 for Day 1 when the check was conducted once. 

Detailed physical examinations were performed prior to initiation of dosing and at 

termination. 

2. Body Weight: Animals were weighed at randomization, daily throughout 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 fluid was noted, this observation was recorded and 

the weight excluded from statistical analysis. Subsequently, each uterine horn was “nicked” 

Page 278 of 281
In vivo Uterotrophic Assay (2012) / Page 6 of 8 

ORTHO-PHENYLPHENOL/ 064103 OCSPP 890.1600/ OECD 440

three times, covered with saline-moistened absorbent 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 uterus was then weighed. 

5. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: No treatment-related clinical signs 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 blotted 

Page 279 of 281
In 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 weights 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 Ratsa

Parameter 

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 

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, using 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 study. There were no significant effects on body 

weights in any OPP-treatment group and no significant 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-related increases in mean uterine weights (wet or blotted) in 

any OPP treatment group. In the positive control 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 this study exhibited precocious vaginal opening. 

Uterine weights from vehicle-treated controls met the performance criteria outlined in the 

applicable test guidelines, indicating acceptable assay sensitivity. 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 uterotrophic 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 immature females. Overall body weight gains 

Page 280 of 281
In 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 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. 

No statistically significant changes were seen in uterine weight in this assay. OPP is negative 

in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

Page 281 of 281