UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: 6/30/2015 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: EPTC: Data Evaluation Records (DERs) for EDSP Tier 1 Assays 

PC Code: 041401 

Decision No.: 459078, 466072 

Petition No.: NA 

DP Barcode: D397177, D402841 

Registration No.: NA 

Regulatory Action: NA 

Risk Assessment Type: NA 

TXRNo.: 0051762 

MRID No.: See Table 

FROM: Greg Akerman, Ph.D. 

Immediate Office 

Health Effects Division (7509P) 

Case No.: NA 

CAS No.: 759-94-4 

40 CFR: NA 

THROUGH: Jess Rowland de-~~ 

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 EPTC: Test Order # EDSP-041401-64. 

Page I of2 

Page 1 of 310
Page 2 of 2 

II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for EPTC.

III. MRID Table 

Chemical: EPTC PC Code: 041401 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48827601 

890.1150 Androgen Receptor Binding (Rat Prostate) 48616403, 48843501 

890.1200 Aromatase Assay (Human Recombinant) 48616402 

890.1250 Estrogen Receptor Binding 48616404, 48843501 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48616405 

890.1350 Fish Short-Term Reproduction 48827602 

890.1400 Hershberger (Rat) 48616406 

890.1450 Female Pubertal (Rat) 48827603 

890.1500 Male Pubertal (Rat) 48827603 

890.1550 Steroidogenesis (Human Cell Line – H295R) 48816401 

890.1600 Uterotrophic (Rat) 48616407 

Page 2 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 1 of 80

DER Template Version: 22 September 2011 

Data Requirement: EPA DP Barcode 402838 

OECD Data Point 231 

EPA MRID 48827601

EPA Guideline 890.1100 

 Amphibian Metamorphosis Assay (Frog) 

Test Material: EPTC Purity (%): >99.0% 

Common Name EPTC 

Chemical Name IUPAC Not reported 

 CAS Name Not reported 

CAS No. 759-94-4 

Synonyms Eptam® Technical (EPTC); S-Ethyl dipropylthiocarbamate

EPA PC Code 041401

Primary Reviewer: John Marton Date: 9/24/12 

Staff Scientist, Cambridge Environmental, Inc. 

Secondary Reviewer(s): Teri S. Myers Date: 9/27/12

Senior Scientist, Cambridge Environmental, Inc. 

Reviewer: Frank T. Farruggia, Ph.D. Date: 3/22/13 

USEPA/OPP/EFED/ERB3 

Reviewer: Robin Sternberg Date: 11/1/13

Wildlife Biologist, USEPA/OPP/EFED/ERB1 

Additional Reviewer: Amy Blankinship Date: 6/5/15 

USEPA/OPP/EFED/ERB6 

FRANK 

FARRUGGIA

Digitally signed by FRANK FARRUGGIA 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=FRANK 

FARRUGGIA, dnQualifier=0000048865 

Date: 2015.06.11 08:18:40 -04'00'

Digitally signed by ROBIN STERNBERG 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=ROBIN 

STERNBERG, dnQualifier=0000039126 

Date: 2015.06.11 11:19:25 -04'00'

AMY 

BLANKINS

HIP

Digitally signed by AMY 

BLANKINSHIP 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, 

dnQualifier=0000040917 

Date: 2015.06.15 17:33:27 

-04'00'

Page 3 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 2 of 80

DER Template Version: 22 September 2011 

Date Evaluation Completed: 6/5/15

CITATION: Lee, Michael R. 2012. EPTC- Amphibian Metamorphosis Assay with African Clawed Frog (Xenopus 

laevis) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 231. Unpublished study 

performed by Smithers Viscient, Wareham, Massachusetts, 02571-1037. Laboratory report number 

12791.6155. Study sponsored by Gowan Company, Yuma, Arizona. Study completed May 4, 2012. 

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

assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, 

or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to 

identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across 

the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered 

in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine 

if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay 

results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document). 

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

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

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

Page 4 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 3 of 80

DER Template Version: 22 September 2011 

EXECUTIVE SUMMARY

The 21-day assay of EPTC (>99.0% purity) on amphibian metamorphosis of the African clawed frog (Xenopus 

laevis) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage 

(NF) 51 (80 tadpoles per control or treatment, 20/replicate) were exposed to nominal concentrations of 0 

(negative control), 0.024, 0.12, 0.60, and 3.0 mg a.i./L. Mean-measured concentrations were <0.0063 (<LOQ; 

control), 0.019, 0.10, 0.50, and 2.6 mg a.i./L. The test system was maintained at 21 to 23°C and a pH of 7.0

to 8.2. 

There were no treatment-related effects on survival of tadpoles during the 21-day test: survival was 100% in the 

negative control and 98, 99, 100, and 96% in the mean-measured low, mid-low, mid-high, and high treatment 

groups. At test termination, spinal curvature was observed in 33% of tadpoles in the negative control and 41, 44, 

28, and 0% in the mean-measured low, mid-low, mid-high, and high treatment groups. The incidence of spinal 

curvature was not considered to be treatment-related. 

At the three highest test concentrations, day 7 snout-vent length (SVL) was significantly reduced (p<0.05) by 

6.7, 7.8, and 15.2%, respectively, and day 7 body weight was significantly reduced (p<0.05) by 16.1, 22.2, and 

41.9%, respectively, compared to the negative control. At the three highest test concentrations, day 21 body weight 

was significantly reduced (p<0.05) by 5.9, 12.0, and 22.9%, respectively, compared to the negative control. Day 

21 SVL was significantly reduced (p<0.05) at all treatment concentrations compared to the negative control 

(range: 2.2-8.5%). 

EPTC significantly decreased (p<0.05) day 21 normalized hind-limb length (HLL) by 17.5% at the high treatment 

level compared to the negative control; no inhibitions (p>0.05) were observed in Day 7 normalized HLL. There 

was no significant acceleration or delay of median NF developmental stage at any treatment level throughout the 

exposure period. No asynchronous development was observed. 

Mild follicular cell hyperplasia and hypertrophy were observed in the negative control and all treatment groups, 

and one to two individuals with increased follicular lumen area (mild) were observed in each of the negative control 

Page 5 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 4 of 80

DER Template Version: 22 September 2011 

and mid-low and high treatment groups. However, these histopathology findings were not considered to be 

treatment-related. 

The study met all of the performance and validity criteria. 

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

Guideline 890.1100). 

Results Synopsis:

Test organism NF stage at test initiation: NF 51 

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

Test type: flow-through 

Page 6 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 5 of 80

DER Template Version: 22 September 2011 

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

Metamorphosis Assay (AMA) with EPTC. 

Treatment 

(mg a.i./L) 

[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 

0.019 No No No No No No No 

0.10 No No No No No No No 

0.50 No No No No No No No 

2.6 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). 

I. MATERIALS AND METHODS 

 Guideline Followed: This study was conducted following guidelines outlined in the U.S. Environmental 

Protection Agency’s Endocrine Disruptor Screening Program Test Guidelines 

890.1100, Amphibian Metamorphosis (Frog), and OECD Guideline for Testing 

of Chemicals No. 231, Amphibian Metamorphosis Assay. The following 

deviations from OCSPP 890.1100 were noted: 

1. The flow rate resulted in a 90% replacement time in the test vessels of approximately 8 hours. OCSPP 

guideline 890.1100 recommends complete volume turnover every 2.7 hours. The flow-through rate 

of 32.5 mg/min as calculated by the reviewer is greater than recommended (i.e., 25 mL/min). 

Page 7 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 6 of 80

DER Template Version: 22 September 2011 

2. Results from the periodic screening analysis of the dilution water were not provided. 

 These deviations do not impact the acceptability of the study. 

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

were provided. This study was conducted in compliance with all pertinent OECD 

(OECD, 1998) and U.S. EPA (40 CFR, Part 160 and 40 CFR, Part 792) Good 

Laboratory Practice regulations with the following exception: routine food and 

water contaminant screening analyses were conducted using standard U.S. 

EPA procedures by GeoLabs, Inc., Braintree, Massacusetts. 

A. Test Material EPTC 

Description: Liquid

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

vapor pressure of test compound, expiration date.

Lot No./Batch No. : P1105096 (Lot No.) 

Purity: >99.9% 

Impurities: Not reported 

Stability of Compound: Stable. Mean-measured concentrations over the 21-day exposure period 

yielded recoveries of 78-87% of nominal with coefficients of variation of 3.5 

to 6.8%.

Storage Conditions of 

Test Chemicals: Stored at room temperature in a dark, ventilated cabinet 

Page 8 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 7 of 80

DER Template Version: 22 September 2011 

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 specified 

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). 

Page 9 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 

8 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as in-house stock? 

Yes The brood stock was originally obtained 

from Nasco, Fort Atkinson, Wisconsin and was maintained as in-house breeders for approximately one year prior to use for generating tadpoles used in this exposure.

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable): 

At least one year

Details on parental feeding: Not specified During the pre-exposure period, tadpoles 

were fed twice daily increasing from 5 to 30 mg/tadpole. 

Page 10 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 

9 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on parental health: The brood stock did not 

show any signs of disease, sickness, injuries, or abnormalities form the day of receipt to the day of pre-exposure initiation. 

Page 11 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 10 of 80

DER Template Version: 22 September 2011 

Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? Yes EPA and OECD recommend that the best 2 – 

3 individual spawns, with a minimum of 1500 

larvae/spawn, be evaluated to identify the 

best single spawn, and that the larvae selected 

for testing originate from the best single spawn 

(i.e., the spawns are not co-mixed)

Number of spawns evaluated (if applicable): 

3, one from each of three breeding pairs 

From hatch to feeding stage (approximately 6 days post-fertilization), the quality of each of the three spawns was quantitatively assessed; criteria were embryo survival and the quantity of embryos. 

Number of eggs sampled per spawn: 

Not specified Each spawn produced at least 1,500 eggs 

Once all tadpoles were at feeding stage (day 6 post-fertilization), larvae from the highest quality spawn were transferred to nine, 10-L rearing tanks. All transferred tadpoles originated from a single spawn. 

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: 12 days post-fertilization 

(dpf) 

Page 12 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 11 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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 

Details on larval selection: Healthy tadpoles at NF 

stage 51 were selected. No further details were provided. 

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: Xenopus Express 

tadpole food 

Representative samples of the food source were periodically analyzed for the 

EPA recommends Sera Micron® throughout 

pre-exposure (after NF stage 45/46) and 

Page 13 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 12 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected in the food samples at concentrations considered toxic to the test organisms. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern, in agreement with ASTM (2005). 

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: Xenopus Express, 

Brooksville, Florida

Iodide concentration in diet (if known): 

Not reported 

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

twice per day.

Page 14 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 13 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: Feeding regime followed 

guideline recommendations. 

It is recommended that food rations during the 

pre-exposure period be increased along with 

larval growth to approximately 30 

mg/larva/day by test initiation. EPA and 

OECD recommend that food rations increase 

from 30 mg/larva/day at test initiation (Study 

Day 0-4) to 80 mg/larva/day in the last week 

of the test (Study Day 15-21).

Page 15 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 14 of 80

DER Template Version: 22 September 2011 

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.

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

Intermittent-flow proportional diluter 

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable): 

32.5 mL/min (calculated by reviewer) 

The flow rate provided approximately 7.2 volume additions every 24-hours, with a 90% replacement time of ~8 hours. (6.5 L*7.2*1000 mL/L)/(24 hr * 60 min/hr)

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. 

Page 16 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 15 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Test material was delivered to a mixing chamber using a syringe pump and a gas-tight syringe. The mixing chamber was positioned over a magnetic stir plate which aided in mixing the stock solution with the dilution water. The solution in the mixing chamber constituted the highest nominal test concentration (3.0 mg a.i./L) and was subsequently proportionally diluted (20%) to provide the remaining nominal exposure concentrations (0.60, 0.12, and 0.024 mg a.i./L). 

Flow-splitting cells were employed to equally distribute the solutions to the four replicate vessels for each concentration or control group. Flow-splitting accuracy of the diluter cells was within ±5% of the nominal value. 

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.

Page 17 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 16 of 80

DER Template Version: 22 September 2011 

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.

Page 18 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 17 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: FETAX solution was used as 

dilution water. Laboratory well water was fortified according to the FETAX formulation in “Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)” (ASTM, 2004). The laboratory well water used to prepare FETAX solution originated from an on-site 100-meter bedrock well. The water was pumped to a concrete reservoir where it was supplemented on demand with de-chlorinated Town of Wareham well water. The water was aerated prior to use to ensure proper equilibration of dissolved gases. 

Solutions were prepared in 3,400-L batches and mixed for approximately 48 hours prior to use to ensure all salts were properly dissolved and that dissolved gases were at equilibrium with the atmosphere. 

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.

Page 19 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 18 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes. No pesticides, PCBs, or toxic metals were detected at concentrations considered toxic in any of the water samples analyzed, in agreement with ASTM (2005) standard practices. 

Iodide supplementation in water? 

None reported If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 

սg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

Test vessel type/materials: Glass 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).

Page 20 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 19 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel size: 30 x 15 x 20 cm 2.5 gallon exposure aquaria 

14.5-cm high side drain 

Fill volume: ~6.5 LAdditional details on exposure system: 

The exposure system was constructed of glass, silicone sealant, stainless steel, and Teflon®. All test chambers were brushed and siphoned daily to remove detritus and uneaten food during the test. Calibration of the diluter system was conducted prior to exposure initiation and confirmed at test termination (Day 21) by measuring delivery volumes of toxicant and dilution water. 

The function of the diluter was monitored daily and a visual check was performed twice daily. In addition, weekly analysis of exposure solutions for test substance concentration was also used to verify proper operation of the diluter system. 

Page 21 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 20 of 80

DER Template Version: 22 September 2011 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Hardness (mg/L as CaCO3) 160 180 1701

Measured in replicate A of the control, low, and high test concentrations on Day 0 and in alternating replicates weekly. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.01 8.16 7.74 

Measured in all test vessels on Day 0 and daily in alternating replicates. Temperature was continuously monitored in replicate A of the control 

EPA recommends pH 7.5 ± 1, 

inter-replicate and inter-treatment 

differentials should not exceed 0.5.

Dissolved oxygen (mg/L) 3.97 9.23 6.97 

EPA recommends dissolved oxygen 

(DO) >3.5 mg/L (>40% air 

saturation). OECD recommends DO 

concentration >3.5 mg/L (>40% air 

saturation).

Temperature (°C) 21.4 22.7 21.9 

EPA recommends temperature 

22±1oC; inter-replicate and 

inter-treatment differentials should 

not exceed 0.5oC.

Page 22 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 21 of 80

DER Template Version: 22 September 2011 

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Iodide (սg/L) NR NR 7.8 

Well water (January 2012) EPA recommends aquatic iodide 

range 0.5 – 10 

սg/L (supplemental 

iodide should not exceed 2 

սg/L).

Ammonia Not determined 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 

Fluoride Not determined Perchlorate Not determined Chlorate Not determined Alkalinity (mg/L as CaCO3) 

Conductivity (սS/cm) 

80 

1700 

92 

1900 

861

18001

Measured in replicate A of the control, low, and high test concentrations on Day 0 and in alternating replicates weekly. 

Page 23 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 22 of 80

DER Template Version: 22 September 2011 

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

weekly and that pH and hardness be 

measured at least at the beginning 

and end of the test.

1 Calculated by reviewer at average of minimum and maximum. 

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

Expected toxicity The doses for preliminary acute 

testing were based on the published fish acute toxicity information of EPTC (LC50 values of 14 and 18 ppm for trout and bluegill, respectively), the solubility limit of EPTC in water (370 mg/L at 20 °C), and consultation with the Study Sponsor. 

EPA recommends that the highest 

test concentration is either the 

solubility limit of the test 

compound, 100 mg/L, or 

demonstrates adequate evidence 

of toxicity (e.g., <10% mortality), 

whichever concentration is lowest.

Page 24 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 23 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species: Xenopus laevis Life stage: NF stage 51 Test duration: 4 daysAdditional details: After 96 hours of exposure, the 

LC50 and EC50 values were 18 

(95% C.I.: 16-21) and 9.1 (95% C.I.: 7.7-11) mg a.i./L, respectively, based on nominal concentrations. There was no mortality at 

13 mg a.i./L and 

100% mortality at 25 and 50 mg a.i./L. At 13 mg a.i./L, all tadpoles exhibited adverse effects (i.e., pale color, lethargy, 

or loss of equilibrium) at 96 hours. Since the analytical recoveries 

Two initial preliminary 96-hour acute range-finding studies were conducted under static-renewal conditions (renewal at 48 hours) at 1.6, 3.1, 6.3, 13, 25, and 50 mg a.i./L and 1.6, 3.1, 6.3, 13, and 25 mg a.i./L, respectively. Due to lower than expected measured EPTC concentrations and suspected volatility and degradation of the test substance in the dilution matrix, a third range-finding exposure was performed using nominal concentrations of 1.6, 3.1, 6.3, 13, 25 and 50 mg a.i./L under flow-through conditions. The final range-finding exposure was conducted using an exposure system 

Page 25 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 24 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

during the preliminary acute exposure were slightly variable, the EC50 calculation was based on nominal dose. Water quality parameters measured during the 96-hour exposure period established a dissolved oxygen range of 7.8 to 8.6 mg/L, a pH range of 8.2 to 8.3, and a temperature range of 21 to 22 °C. 

consisting of an intermittent-flow proportional diluter, a temperature controlled water bath, and a set of eight exposure aquaria with a total solution volume of 6.5 L. Two replicates were maintained for each concentration and control. Each replicate vessel contained 20 tadpoles at exposure initiation. Test organisms were monitored daily for observations of mortality and abnormal behavior. During the exposure, dissolved oxygen, pH and temperature were monitored daily. Samples from each replicate of each concentration were sampled at 24 hours and again at the 72-hour interval from replicate A of each concentration to confirm exposure dose. 

Page 26 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 25 of 80

DER Template Version: 22 September 2011 

 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; fish short-term reproductive assay with EPTC and consultation with the Study Sponsor. Highest nominal test concentration (i.e., 3.0 

mg a.i./L) was approximately 1/3 of the nominal EC50 value (9.1 

mg a.i./L). 

EPA recommends that the 

highest test concentration is 

either the solubility limit of the test 

compound, 100 mg/L, or 

demonstrates adequate evidence 

of toxicity (e.g., <10% mortality), 

whichever concentration is 

lowest.

Reference study citation (if applicable):

NA 

Page 27 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 26 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation of test concentrations: 0.2 Typically for these exposures, a 10% dilution series is 

applied with three test substance concentrations. However, based on the results of the fish short-term reproductive assay with EPTC, the dilution series was increased to 20% and a fourth concentration was included. For the fish study, the high dose was set using 1/3 of the 96-hour LC50 for adult fish, three EPTC-dosed concentrations separated by a dilution factor of 10%. During this exposure, latent lethal and sublethal responses (approximately exposure day 11) were observed in the high and mid doses. For the amphibian study, the intent of a 20% dilution series and four EPTC-dosed treatment doses was to avoid the latent responses observed in the fish study and still maintain > 2 uncompromised EPTC exposure concentrations. 

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, 

Page 28 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 27 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

plus solvent control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit of quantification (LOQ): 0.0062 mg a.i./L EPA recommends that for 

chemical test concentrations 

below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not reported Frequency of measurement: Samples were collected 

for analysis on Days 0, 7, 16, and 21. 

At exposure initiation (day 0), samples from replicate A of each treatment and control solution were removed and analyzed and from alternating replicates weekly thereafter. On exposure day 0, an interfering peak (possibly related to the matrix in an aging test system) was present in the control vessel. On first glance, this 

It is recommended that test item 

concentration be measured in 

one tank at each treatment level 

at test initiation and every week 

thereafter.

Page 29 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 28 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

appeared to be control contamination, therefore all control vessels were sampled on day 1. Upon further diagnosis, this interfering peak was found to be present in all samples. The area of this interfering peak approximated the area of the low standard and was impacting the limit of quantitation (LOQ) for this analysis. As a result of this interference, the low concentration calibration standard was removed from the standard curve and the LOQ for this analysis was subsequently increased to offset any potential impact from this interfering peak. Since the peak observed in the control vessel at day 0 was not determined to be EPTC, the day 1 sampling was not necessary and therefore, not reported. 

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

Page 30 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 29 of 80

DER Template Version: 22 September 2011 

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 

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

Page 31 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 30 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

NA EPA recommends that the 

solvent not exceed 0.02 ml/L3. 

OECD recommends that solvent 

have no effect on survival nor 

produce any other adverse 

effects and that concentration not 

be greater than 0.1 ml/L4

.

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

NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 

12:12 (light:dark).

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

Toxicology, 76, pp.69–92. 

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

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

Page 32 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 31 of 80

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Light intensity at water’s surface: 0.750-1.2 Klux EPA recommends light intensity 

0.6 – 2 Klux (at water’s surface).

Additional details: NA 

Page 33 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 32 of 80

DER Template Version: 22 September 2011 

Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with EPTC. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.00 <0.0062 NA NA 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient of 

variation (CV) 

20%. Solvent control (if 

applicable) 

NA NA NA NA 

Treatment 1 0.024 0.019 5.1 NA Treatment 2 0.12 0.10 6.5 NA Treatment 3 0.60 0.501 3.5 NA Treatment 4 3.0 2.6 4.9 NA 

Abbreviations: CV Coefficient of variation. 1 calculated as 0.49 by study author 

Page 34 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 33 of 80

DER Template Version: 22 September 2011 

 E. Observations

 Biological Endpoints: 

Day 7: Development stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLL 

(HLL:SVL) 

Day 21: Development stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathology (tadpoles from 

the mean-measured 0.49 mg a.i./L treatment group were not included in the histopathology 

analysis) 

Daily: Mortality, 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.

Page 35 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 34 of 80

DER Template Version: 22 September 2011 

II. RESULTS AND DISCUSSION 

A. Results

By test termination, mean survival was 100% in the negative control and 98, 99, 100, and 96% in the 

mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 9). 

Table 9: Larval Mortality in Xenopus laevis.

Treatment (mg a.i./L) 

[measured]

Larval Mortality 

Day 71 Day 21 

n Mortality # Mortality % n Mortality # Mortality % 

Negative Control 80 NR NR 60 0 0 

0.019 80 NR NR 60 2 2 

0.10 80 NR NR 60 1 1 

0.50 80 NR NR 60 0 0 

2.6 80 NR NR 60 32 4 

Abbreviations: NR=Not reported 

1

 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice were not reported. Further, only 

cumulative Day 21 survival was reported. The study author did not report when mortalities occurred. 

2

 Two mortalities occurred in one of the replicates. 

Page 36 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 35 of 80

DER Template Version: 22 September 2011 

Median developmental stage on Day 7 was 54 in the negative control and 0.019-0.50 mg a.i./L treatment 

groups and 53 in the mean-measured 2.6 mg a.i./L treatment group (Table 10). By Day 21, median 

development stage was 57 in the negative control and all treatment groups except the mean-measured 

0.50 mg a.i./L treatment group where median developmental stage averaged 57.5. The 10th and 90th

percentiles in Day 21 developmental stage did not differ by more than four NF stages. No asynchronous 

development was reported. 

On day 21, there was only 1 (1.7%) tadpole greater than stage 60 (i.e., stage 61) in each of the 

mean-measured 0.019 and 0.50 mg a.i./L treatment groups. These tadpoles were excluded from 

analyses with respect to body weight, snout vent length, and hind leg length (including normalized hind 

leg length) by the study author and reviewer. 

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

Treatment (mg a.i./L) 

[measured]

Developmental Stage 

Day 7 Day 21 

n 

Median 

Stage 

# Asynchronous n 

Median 

Stage 

# Asynchronous 

Negative Control 4 54 0 4 57 0 

0.019 4 54 0 4 57 0 

0.10 4 54 0 4 57 0 

0.50 4 54 0 4 57.5 0 

2.6 4 53 0 4 57 0 

Page 37 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 36 of 80

DER Template Version: 22 September 2011 

Day 7 normalized HLL averaged 0.12 in the negative control and 0.11 in the mean-measured 0.010, 0.10, 

0.49, and 2.6 mg a.i./L treatment groups (Table 11). Day 21 normalized HLL averaged 0.44 in the 

negative control and 0.45, 0.44, 0.44, and 0.36 in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg 

a.i./L treatment groups (Table 11). 

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

Treatment (mg a.i./L) 

[measured]

Hind Limb Length (HLL) 

Day 7 Day 212

n Mean

(mm) 

±SD 

HLL: 

SVL1 n Mean(mm) 

±SD 

HLL: 

SVL1

Negative control 4 2.31 0.08 0.12 4 13.64 0.37 0.44 

0.019 4 2.07 0.21 0.11 4 13.74 0.71 0.45 

0.10 4 2.05 0.18 0.11 4 13.30 0.54 0.44 

0.50 4 1.99 0.11 0.11 4 12.90 0.97 0.44 

2.6 4 1.81 0.10 0.11 4 10.39 0.44 0.36 

Abbreviations: NA Not applicable. SD Standard deviation. 

1

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

2

 Day 21 HLL:SVL values are the median values reported by the study author. 

Page 38 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 37 of 80

DER Template Version: 22 September 2011 

Mean Day 7 SVL averaged 20.13 mm in the negative control and 18.95, 18.78, 18.55, and 17.08 mm 

in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 12). 

Mean Day 21 SVL averaged 31.15 mm in the negative control and 30.45, 30.15, 29.56, and 28.50 mm 

in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively. 

Mean Day 7 body weight averaged 0.57 g in the negative control and 0.50, 0.48, 0.45, and 0.33 g 

in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 12). 

Mean Day 21 body weight averaged 1.92 g in the negative control and 1.89, 1.80, 1.69, and 1.48 g in 

the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively. 

Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[measured] 

Snout-Vent Length (SVL)2 Body Weight1

Day 7 Day 21 Day 7 Day 21 

n Mean

(mm) 

±SD n Mean

(mm) 

±SD n Mean

(g) 

±SD n Mean

(g) 

±SD 

Negative 

Control 

4 20.13 0.19 4 31.15 0.68 4 0.57 0.07 4 1.92 0.11 

0.019 4 18.95 1.03 4 30.45 0.24 4 0.50 0.07 4 1.89 0.02 

0.10 4 18.78 0.67 4 30.15 0.52 4 0.48 0.04 4 1.80 0.07 

0.50 4 18.55 0.66 4 29.56 0.41 4 0.45 0.04 4 1.69 0.08 

2.6 4 17.08 0.78 4 28.50 0.35 4 0.33 0.06 4 1.48 0.05 

Abbreviations: SD Standard deviation. 

1

 Also referred to as “wet weight” in the test guideline. 

2

 SVL values represent the mean of the replicate medians 

Page 39 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 38 of 80

DER Template Version: 22 September 2011 

Tadpoles selected for histological analysis were stage-matched to the median of the control (stage 57), 

if possible. If there were replicates with more than five larvae at the appropriate stage, then five larvae 

were randomly selected. If there was not a sufficient number of tadpoles at the appropriate stage, the 

next stage up was selected (stage 58), until a total of five tadpoles were selected. If an insufficient number 

of tadpoles still remained, the next stage down from the median of the control was selected until a 

total of five tadpoles were selected. 

No samples from the mean-measured 0.50 mg a.i./L treatment group were analyzed for histopathological 

effects. 

The following lesions were not present in any examined sections from study tadpoles: thyroid gland 

hypertrophy, thyroid gland atrophy, decreased follicular lumen area, colloid depletion, changes in colloid 

quality, and metamorphic pharyngeal changes. Mild follicular cell hypertrophy and hyperplasia were 

observed in the control and all treatment groups. Mild follicular lumen area increase was observed in 

the negative control 0.1 and 2.6 mg a.i./L treatment groups. The similarity in incidence and mild severity 

across groups indicates that these were not treatment-related findings, but a reflection of normal 

developmental variation (Tables 13 and 14). 

Page 40 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 39 of 80

DER Template Version: 22 September 2011 

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

Treatment 

(mg a.i./L) 

[measured] 

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 17 20 18 

1 20 0 20 0 20 3 20 2 

2 20 0 20 0 20 0 20 0 

3 20 0 20 0 20 0 20 0 

0.0192 0 19 19 19 19 19 17 19 15 

1 19 0 19 0 19 2 19 4 

2 19 0 19 0 19 0 19 0 

3 19 0 19 0 19 0 19 0 

0.10 0 20 20 20 20 20 19 20 18 

1 20 0 20 0 20 1 20 2 

2 20 0 20 0 20 0 20 0 

3 20 0 20 0 20 0 20 0 

0.503 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

2.6 0 20 20 20 20 20 18 20 17 

1 20 0 20 0 20 2 20 3 

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. 

2

 One sample was missing from this group (n = 19). 

3

 Samples were not analyzed from this group.

Page 41 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 40 of 80

DER Template Version: 22 September 2011 

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

Treatment 

(mg a.i./L) 

[measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 19 20 20 NA NA NA NA NA NA

1 20 1 20 0 NA NA NA NA NA NA

2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA

0.019 0 19 19 19 19 NA NA NA NA NA NA

1 19 0 19 0 NA NA NA NA NA NA

2 19 0 19 0 NA NA NA NA NA NA3 19 0 19 0 NA NA NA NA NA NA

0.10 0 20 18 20 20 NA NA NA NA NA NA

1 20 2 20 0 NA NA NA NA NA NA

2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA

0.50 0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

Page 42 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 41 of 80

DER Template Version: 22 September 2011 

Treatment 

(mg a.i./L) 

[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 

2 NA NA NA NA NA NA NA NA NA NA3 NA NA NA NA NA NA NA NA NA NA

2.6 0 20 19 20 20 NA NA NA NA NA NA

1 20 1 20 0 NA NA NA NA NA NA

2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA

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. 2 One sample was missing from this group (n = 19). 3 Samples were not analyzed from this group. 

Page 43 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 42 of 80

DER Template Version: 22 September 2011 

Beginning on Day 13, several tadpoles in the negative control and most treatment groups were observed 

with spinal curvature. By test termination, spinal deformities were observed for 33% of negative control 

tadpoles and 41, 44, 29, and 0% of tadpoles in the mean-measured 0.019, 0.10, 0.49, and 2.6 mg a.i./L 

treatment groups, respectively (Table 15). These observations were not considered to be 

treatment-related. For studies conducted at Smithers Viscient, there is no correlation between the 

presence of spinal curvature and the increase in % CV for potentially affected endpoints (e.g., SVL, HLL, 

wet weight). 

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[measured] 

Clinical Signs1

Type n 

Incidence at Test 

Termination (%) 

Negative Control Spinal curvature 60 33 

0.019 Spinal curvature 58 41 

0.10 Spinal curvature 59 44 

0.50 Spinal curvature 60 28 

2.6 Spinal curvature 57 0 

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

Page 44 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 43 of 80

DER Template Version: 22 September 2011 

B. Study Author’s Analysis and Conclusions

Developmental stage, SVL, HLL, and wet weight were statistically analyzed to identify significant 

reductions in the treatment organisms compared to control organisms. All statistical conclusions were 

made at the 95% level of certainty except in the case of the basic assumption tests (e.g., Shapiro-Wilks’ 

Test and Bartlett’s Test in which the 99% level of certainty was applied). When necessary, the OECD 

Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application was 

consulted (OECD, 2006). CETIS-Comprehensive Environmental Toxicity Information SystemTM (Ives, 

2011) Version 1.8.4.18 software was used to perform all statistical computations. 

Mortality was analyzed using step-down Cochran-Armitage test (step down) for monotonic trend or 

Fisher's Exact test with Bonferroni-Holm adjustment for non-monotonic trend. 

Developmental stage was analyzed using the Jonckheere-Terpstra test (step-down). When monotonic 

trends were present, HLL, SVL, and wet weight were analyzed using the Jonckheere-Terpstra test 

(step-down). When trends were non-monotonic, data were tested for normality and homogeneity of 

variance. If the data met the assumptions of parametric statistics, Dunnett’s Multiple Comparison Test 

or the Tamhane-Dunnett tests were used. If the data did not meet these assumptions, the Mann-Whitney 

test with Bonferonni-Holm adjustment was employed. 

For larval survival, Fisher's Exact Test with Bonferroni-Holm Adjustment determined no significant 

difference in any treatment level tested compared to the control. 

Day 7 data 

The day 7 developmental stage was analyzed by applying the multi-quantal Jonckheere Test to the 20th 

through the 80th percentiles for all treatment levels compared to the control. The multi-quantal procedure 

determined a significant reduction in the day 7 developmental stage among tadpoles exposed to the 2.6 

mg a.i./L treatment level compared to the control. 

Jonckheere-Terpstra Step-Down Test determined a significant difference in day 7 whole body wet weight 

and snout-vent length among tadpoles exposed to all treatment levels tested compared to the control. 

Page 45 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 44 of 80

DER Template Version: 22 September 2011 

Jonckheere-Terpstra Step-Down Test determined a significant reduction in day 7 hind limb length among 

tadpoles exposed to the 0.49 and 2.6 mg a.i./L treatment levels compared to the control. 

Dunnett's Multiple Comparison Test determined a significant reduction in day 7 hind limb length 

normalized by SVL among tadpoles exposed to the 2.6 mg a.i./L treatment level compared to the control. 

Day 21 data 

The day 21 developmental stage was analyzed by applying the multi-quantal Jonckheere Test to the 20th 

through the 80th percentiles for all treatment levels compared to the control. The multi-quantal procedure 

determined no significant reductions in the day 21 developmental stage among tadpoles exposed to any 

of the treatment levels tested compared to the control. 

Jonckheere-Terpstra Step-Down Test determined a significant difference in day 21 whole body wet weight 

and hind limb length among tadpoles exposed to treatment levels > 0.10 mg a.i./L compared to the 

control. 

Jonckheere-Terpstra Step-Down Test determined a significant difference in day 21 snout-vent length 

among tadpoles exposed to all treatment levels tested compared to the control. 

Jonckheere-Terpstra Step-Down Test determined a significant reduction in day 21 hind limb length 

normalized by SVL among tadpoles exposed to the 2.6 mg a.i./L treatment level compared to the control. 

In summary, on Day 7, both developmental stage and normalized HLL were significantly reduced at the 

highest concentration relative to the negative control. Further, median wet weight and median SVL were 

significantly inhibited on Day 7 at all treatment levels relative to the negative control, while median HLL 

was significantly reduced at the two highest treatment groups. 

Similar to Day 7, median normalized HLL at Day 21 was significantly reduced at the highest treatment 

level relative to the negative control. However, developmental stage at Day 21 was comparable between 

the negative control and all treatment groups. Day 21 wet weight and HLL were significantly reduced at 

the mean-measured 0.10 mg a.i./L treatment groups, relative to the negative control, whereas SVL was 

reduced at all treatment levels. 

Page 46 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 45 of 80

DER Template Version: 22 September 2011 

There were no treatment-related histopathological effects, and no asynchronous development was 

observed. Further, the test material did not appear to have any effect on sub-lethal effects. 

 C. Reviewer’s Analysis and Conclusions 

Statistical Methods: Day 7 wet weight, SVL, and HLL had monotonic trends and were analyzed using 

the Jonckheere-Terpstra test. Day 7 developmental endpoints (i.e., NF stage and normalized HLL) were 

not monotonic and did not meet the assumptions of normality and homogeneity of variance (i.e., 

Shapiro-Wilks’ and Bartlett’s Tests, respectively) and were analyzed using the Mann-Whitney Test. Day 

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

was analyzed with the Mann-Whitney test. All Day 21 growth parameters had a monotonic response and 

were analyzed using the Jonckheere-Terpstra test. Day 21 normalized HLL did not have a monotonic 

response but met the assumptions of parametric statistics and was therefore analyzed with Dunnett’s test. 

All analyses were conducted using SAS version 8.1. There were only two individuals from this study 

(0.024 mg a.i./L, Tank C and 0.60 mg a.i./L, Tank A) exhibiting developmental stages >60; and both 

were at the 61 stage by day 21. Replicate means and medians for body weight, snout vent length, and 

hind leg length (including normalized hind leg length) were calculated excluding all tadpoles at NF stage 

61. 

 Conclusions: 

On Day 7, the reviewer did not find significant inhibitions in developmental stage or normalized HLL (p

< 0.05). However, all growth parameters (i.e., SVL, body weight, and HLL) were significantly reduced 

in the mean-measured 0.10, 0.50, and 2.6 mg a.i./L treatment groups relative to the negative control 

(p <0.05). On Day 21, there were no significant effects on developmental stage. However, normalized 

HLL was significantly reduced at the highest treatment level (18%; Dunnett’s, p = 0.002). Day 21 weight 

was significantly reduced at the three highest treatment groups relative to the negative control (6-23% 

reductions; Jonckheere-Terpstra, p  0.020). Day 21 SVL was significantly reduced at all treatment levels 

(2-9% reductions; Jonckheere-Terpstra, p  0.04). Day 21 HLL, however, was only significantly reduced 

at the highest treatment level (24% reduction; Jonckheere-Terpstra, p < 0.001). 

Page 47 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 46 of 80

DER Template Version: 22 September 2011 

Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with EPTC. 

Treatment 

(mg a.i./L) 

[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 57 NA 0 NA 0 NA NA NA NA NA No 

0.019 54 0.48 57 0.48 -5.9 0.13 2.5 0.92 NA NA NA NA No 

0.10 54 1.0 57 1.0 -5.2 0.68 0.23 1.0 NA NA NA NA No 

0.50 54 1.0 57.5 0.22 -7.4 0.066 -0.23 1.0 NA NA NA NA No 

2.6 53 0.10 57 1.0 -8.5 0.065 -17.5 0.002 NA NA NA NA No 

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

Page 48 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 47 of 80

DER Template Version: 22 September 2011 

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). 

Page 49 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 48 of 80

DER Template Version: 22 September 2011 

Table 17: Growth Endpoints1,2 in the AMA with EPTC. 

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 

There were no major deficiencies noted in this study. Although there were minor deviations from 

guidelines, as noted in the Materials and Methods section of the DER, the study met all validity and 

performance criteria.

F. Reviewer’s Comments 

The reviewer’s results were based on replicate means and medians calculated by excluding tadpoles at 

NF stage 61. Therefore, the reviewer’s results are reported in the Executive Summary and Conclusions 

sections of this DER. 

The in-life portion of the definitive toxicity test was conducted from February 14 to March 6, 2012. 

Treatment 

(mg a.i./L) 

[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 NA 0 NA 0 NA 0 NA 

0.019 -5.8 0.07 -2.2 0.04 -12.2 0.12 -1.5 0.19 

0.10 -6.7 0.02 -3.2 0.01 -16.1 0.03 -5.9 0.02 

0.50 -7.8 0.006 -5.1 <0.001 -22.2 0.006 -12.0 <0.001 

2.6 -15.2 <0.001 -8.5 <0.001 -41.9 <0.001 -22.9 <0.001 

Statistical Test Jonckheere Jonckheere Jonckheere Jonchkeere 

Page 50 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 49 of 80

DER Template Version: 22 September 2011 

III. REFERENCES 

APHA, AWWA, WPCF, 2005. Standard Methods for the Examination of Water and Wastewater. 21st Edition, 

Washington, DC. 

ASTM, 2004. Standard Guide for Conducting the Frog Embryo Teratogenesis Assay – Xenopus (FETAX). 

American Society for Testing and Materials, ASTM E 1439-98(2004). Philadelphia, PA. 

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for Conducting 

Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society for Testing 

and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania. 

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User’s Guide. 

Tidepool Scientific Software, McKinleyville, California. 

Mount, D.I. and W.A. Brungs, 1967. A simplified dosing apparatus for fish toxicological studies. Water 

Research 1L 21-29. 

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

OECD, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. Number 

1. OECD Principles on Good Laboratory Practice (as revised in 1997). Environment Directorate Chemicals 

Group and Management Committee. ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp. 

OECD, 2006. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. 

Environmental Health and Safety Publications. Series on Testing and Assessment. No. 54. Paris, France. 

OECD, 2009. Amphibian Metamorphosis Assay. OECD Guideline for the Testing of Chemicals. No. 231. 

Paris, France. 

Page 51 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 50 of 80

DER Template Version: 22 September 2011 

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. Water 

Research 3: 793-821. 

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory 

Practices Standards; Final Rule. Office of the Federal Register, National Archives and Records 

Administration. U.S. Government Printing Office, Washington, D.C. 

U.S. EPA, 1989. Toxic Substances Control Act; Good Laboratory Practice Standards; Final Rule. (40 CFR, 

Part 792) Federal Register, Part III, 48(230): 53922-53944, August 17, 1989. 

U.S. EPA, 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline, OPPTS 

850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. "Public Draft". EPA 

712-C-96-113. April 1996. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA, 2007. Office of Waste. Test Methods for Evaluating Solid Waste, Physical/Chemical Methods 

(SW-846). February 2007. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA, 2009. Endocrine Disrupter Screening Program Test Guidelines OPPTS 890.1100: Amphibian 

Metamorphosis (Frog). EPA 740-C-09-002. October 2009. 

York, Duncan, 2012. EPTC - Short-Term Reproduction Assay with Fathead Minnow (Pimephales promelas) 

Following OPPTS 890.1350 and OECD 229 Guidelines. Smithers Viscient, Wareham,MA. Study No. 

12791.6156.

Page 52 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 51 of 80

DER Template Version: 22 September 2011 

APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

test for amphib metamorph screen study - EPTC 

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 

 0.958 0.504 0.999 0.439 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.57 0.07 0.04 12.33 0.46, 0.69 

 Dose1 4 0.50 0.07 0.04 14.76 0.39, 0.62 

 Dose2 4 0.48 0.04 0.02 7.87 0.42, 0.54 

 Dose3 4 0.45 0.04 0.02 9.56 0.38, 0.51 

 Dose4 4 0.33 0.06 0.03 16.77 0.24, 0.42 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.57 0.50 0.67 . .

 Dose1 0.50 0.42 0.59 87.80 12.20 

 Dose2 0.48 0.44 0.52 83.93 16.07 

 Dose3 0.46 0.39 0.49 77.79 22.21 

 Dose4 0.31 0.29 0.42 58.10 41.90 

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

***

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 9.23 <.001 

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 0.57 . 0.57 . . . . . . 

 Dose1 0.50 0.299 0.50 0.064 . . . . . 

 Dose2 0.48 0.122 0.48 0.024 0.981 . . . . 

 Dose3 0.45 0.024 0.45 0.004 0.638 0.908 . . . 

Page 53 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 52 of 80

DER Template Version: 22 September 2011 

 Dose4 0.33 <.001 0.33 <.001 0.007 0.019 0.093 . . 

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

***

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 13.03 0.011 

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 0.57 . .

 Dose1 0.50 0.346 0.124 

 Dose2 0.48 0.103 0.029 

 Dose3 0.46 0.067 0.006 

 Dose4 0.31 0.067 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

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

***

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 9.23 <.001 

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 -0.57 . -0.47 . . . . . . 

 Dose1 -0.50 0.299 -0.47 0.997 . . . . . 

 Dose2 -0.48 0.122 -0.47 0.998 0.981 . . . . 

 Dose3 -0.45 0.024 -0.47 0.999 0.638 0.908 . . . 

 Dose4 -0.33 <.001 -0.47 0.999 0.007 0.019 0.093 . . 

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

***

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 13.03 0.011 

Page 54 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 53 of 80

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 -0.57 . .

 Dose1 -0.50 0.346 0.876 

 Dose2 -0.48 0.103 0.971 

 Dose3 -0.46 0.067 0.994 

 Dose4 -0.31 0.067 1.000 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 55 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 54 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 0.811 0.001 6.750 0.003 USE NON-PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 54.00 0.00 0.00 0.00 . , .

 Dose1 4 53.75 0.50 0.25 0.93 52.95, 54.55 

 Dose2 4 54.00 0.00 0.00 0.00 . , .

 Dose3 4 54.00 0.00 0.00 0.00 . , .

 Dose4 4 53.25 0.50 0.25 0.94 52.45, 54.05 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 54.00 54.00 54.00 . .

 Dose1 54.00 53.00 54.00 99.54 0.46 

 Dose2 54.00 54.00 54.00 100.00 0.00 

 Dose3 54.00 54.00 54.00 100.00 0.00 

 Dose4 53.00 53.00 54.00 98.61 1.39 

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

***

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 4.25 0.017 

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 54.00 . 54.00 . . . . . . 

 Dose1 53.75 0.638 53.92 0.424 . . . . . 

 Dose2 54.00 1.000 53.92 0.453 0.795 . . . . 

 Dose3 54.00 1.000 53.92 0.468 0.795 1.000 . . . 

 Dose4 53.25 0.015 53.25 0.003 0.219 0.030 0.030 . . 

Page 56 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 55 of 80

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 10.09 0.039 

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 54.00 . .

 Dose1 54.00 0.478 0.159 

 Dose2 54.00 1.000 0.500 

 Dose3 54.00 1.000 0.673 

 Dose4 53.00 0.102 0.027 

 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 4.25 0.017 

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 -54.00 . -53.80 . . . . . . 

 Dose1 -53.75 0.638 -53.80 0.879 . . . . . 

 Dose2 -54.00 1.000 -53.80 0.903 0.795 . . . . 

 Dose3 -54.00 1.000 -53.80 0.915 0.795 1.000 . . . 

 Dose4 -53.25 0.015 -53.80 0.922 0.219 0.030 0.030 . . 

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

***

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 10.09 0.039 

MannWhit - testing each trt median signif. different from control 

Page 57 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 56 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -54.00 . .

 Dose1 -54.00 0.478 0.841 

 Dose2 -54.00 1.000 0.500 

 Dose3 -54.00 1.000 0.327 

 Dose4 -53.00 0.102 0.973 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 58 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 57 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-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 

 0.962 0.589 0.936 0.470 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 20.13 0.19 0.09 0.94 19.82, 20.43 

 Dose1 4 18.95 1.03 0.51 5.42 17.31, 20.59 

 Dose2 4 18.78 0.67 0.33 3.54 17.72, 19.83 

 Dose3 4 18.55 0.66 0.33 3.54 17.51, 19.59 

 Dose4 4 17.08 0.78 0.39 4.57 15.83, 18.32 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 20.05 20.00 20.40 . .

 Dose1 18.95 17.70 20.20 94.16 5.84 

 Dose2 18.60 18.20 19.70 93.29 6.71 

 Dose3 18.45 17.90 19.40 92.17 7.83 

 Dose4 17.10 16.10 18.00 84.84 15.16 

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

***

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 9.25 <.001 

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 20.13 . 20.13 . . . . . . 

 Dose1 18.95 0.107 18.95 0.020 . . . . . 

 Dose2 18.78 0.057 18.78 0.010 0.997 . . . . 

 Dose3 18.55 0.024 18.55 0.004 0.930 0.991 . . . 

 Dose4 17.08 <.001 17.08 <.001 0.016 0.030 0.070 . . 

Page 59 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 58 of 80

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 12.90 0.012 

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 20.05 . .

 Dose1 18.95 0.233 0.073 

 Dose2 18.60 0.066 0.017 

 Dose3 18.45 0.066 0.006 

 Dose4 17.10 0.066 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose2 

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

***

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 9.25 <.001 

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 -20.13 . -18.70 . . . . . . 

 Dose1 -18.95 0.107 -18.70 0.998 . . . . . 

 Dose2 -18.78 0.057 -18.70 0.999 0.997 . . . . 

 Dose3 -18.55 0.024 -18.70 0.999 0.930 0.991 . . . 

 Dose4 -17.08 <.001 -18.70 0.999 0.016 0.030 0.070 . . 

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

***

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 12.90 0.012 

MannWhit - testing each trt median signif. different from control 

Page 60 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 59 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -20.05 . .

 Dose1 -18.95 0.233 0.927 

 Dose2 -18.60 0.066 0.983 

 Dose3 -18.45 0.066 0.994 

 Dose4 -17.10 0.066 1.000 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 61 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 60 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

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 

 0.951 0.379 1.332 0.304 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 2.31 0.08 0.04 3.37 2.18, 2.43 

 Dose1 4 2.07 0.21 0.11 10.29 1.73, 2.41 

 Dose2 4 2.05 0.18 0.09 8.98 1.75, 2.34 

 Dose3 4 1.99 0.11 0.05 5.33 1.82, 2.16 

 Dose4 4 1.81 0.10 0.05 5.75 1.64, 1.98 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 2.28 2.26 2.42 . .

 Dose1 2.10 1.80 2.30 89.80 10.20 

 Dose2 2.10 1.78 2.20 88.63 11.37 

 Dose3 2.00 1.88 2.08 86.20 13.80 

 Dose4 1.81 1.69 1.93 78.42 21.58 

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

***

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 5.97 0.004 

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 2.31 . 2.31 . . . . . . 

 Dose1 2.07 0.116 2.07 0.022 . . . . . 

 Dose2 2.05 0.072 2.05 0.014 0.999 . . . . 

 Dose3 1.99 0.026 1.99 0.005 0.926 0.981 . . . 

 Dose4 1.81 <.001 1.81 <.001 0.135 0.207 0.445 . . 

Page 62 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 61 of 80

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 11.23 0.024 

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 2.28 . .

 Dose1 2.10 0.235 0.074 

 Dose2 2.10 0.067 0.020 

 Dose3 2.00 0.067 0.004 

 Dose4 1.81 0.067 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose2 

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

***

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 5.97 0.004 

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 -2.31 . -2.05 . . . . . . 

 Dose1 -2.07 0.116 -2.05 0.996 . . . . . 

 Dose2 -2.05 0.072 -2.05 0.998 0.999 . . . . 

 Dose3 -1.99 0.026 -2.05 0.998 0.926 0.981 . . . 

 Dose4 -1.81 <.001 -2.05 0.999 0.135 0.207 0.445 . . 

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

***

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 11.23 0.024 

MannWhit - testing each trt median signif. different from control 

Page 63 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 62 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -2.28 . .

 Dose1 -2.10 0.235 0.926 

 Dose2 -2.10 0.067 0.980 

 Dose3 -2.00 0.067 0.996 

 Dose4 -1.81 0.067 1.000 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 64 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 63 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 Test Stat P-value Test Stat P-value 

 0.935 0.191 5.166 0.008 USE NON-PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.12 0.00 0.00 2.28 0.11, 0.12 

 Dose1 4 0.11 0.01 0.00 5.08 0.10, 0.12 

 Dose2 4 0.11 0.01 0.00 8.53 0.09, 0.12 

 Dose3 4 0.11 0.00 0.00 2.21 0.10, 0.11 

 Dose4 4 0.11 0.00 0.00 1.64 0.10, 0.11 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.11 0.11 0.12 . .

 Dose1 0.11 0.10 0.11 94.14 5.86 

 Dose2 0.11 0.10 0.12 94.79 5.21 

 Dose3 0.11 0.11 0.11 92.62 7.38 

 Dose4 0.11 0.10 0.11 91.54 8.46 

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

***

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 2.13 0.127 

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 0.12 . 0.12 . . . . . . 

 Dose1 0.11 0.236 0.11 0.059 . . . . . 

 Dose2 0.11 0.325 0.11 0.062 1.000 . . . . 

 Dose3 0.11 0.104 0.11 0.021 0.988 0.956 . . . 

 Dose4 0.11 0.055 0.11 0.010 0.919 0.838 0.997 . . 

Page 65 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 64 of 80

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 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 0.11 . .

 Dose1 0.11 0.125 0.030 

 Dose2 0.11 0.678 0.170 

 Dose3 0.11 0.066 0.038 

 Dose4 0.11 0.065 0.010 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 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 

 4 15 2.13 0.127 

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 -0.12 . -0.11 . . . . . . 

 Dose1 -0.11 0.236 -0.11 0.975 . . . . . 

 Dose2 -0.11 0.325 -0.11 0.983 1.000 . . . . 

 Dose3 -0.11 0.104 -0.11 0.987 0.988 0.956 . . . 

 Dose4 -0.11 0.055 -0.11 0.988 0.919 0.838 0.997 . . 

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

***

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 

Page 66 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 65 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -0.11 . .

 Dose1 -0.11 0.125 0.970 

 Dose2 -0.11 0.678 0.830 

 Dose3 -0.11 0.066 0.962 

 Dose4 -0.11 0.065 0.990 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 67 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 66 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 0.822 0.002 19.000 <.001 USE NON-PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 57.00 0.00 0.00 0.00 . , .

 Dose1 4 57.25 0.50 0.25 0.87 56.45, 58.05 

 Dose2 4 57.00 0.00 0.00 0.00 . , .

 Dose3 4 57.50 0.58 0.29 1.00 56.58, 58.42 

 Dose4 4 57.00 0.00 0.00 0.00 . , .

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 57.00 57.00 57.00 . .

 Dose1 57.00 57.00 58.00 100.44 -0.44 

 Dose2 57.00 57.00 57.00 100.00 0.00 

 Dose3 57.50 57.00 58.00 100.88 -0.88 

 Dose4 57.00 57.00 57.00 100.00 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 

 4 15 1.71 0.199 

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 57.00 . 57.19 . . . . . . 

 Dose1 57.25 0.693 57.19 0.852 . . . . . 

 Dose2 57.00 1.000 57.19 0.879 0.835 . . . . 

 Dose3 57.50 0.165 57.19 0.892 0.835 0.282 . . . 

 Dose4 57.00 1.000 57.00 0.648 0.835 1.000 0.282 . . 

Page 68 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 67 of 80

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 5.96 0.202 

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 57.00 . .

 Dose1 57.00 0.478 0.841 

 Dose2 57.00 1.000 0.500 

 Dose3 57.50 0.223 0.917 

 Dose4 57.00 1.000 0.667 

 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 1.71 0.199 

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 -57.00 . -57.00 . . . . . . 

 Dose1 -57.25 0.693 -57.13 0.365 . . . . . 

 Dose2 -57.00 1.000 -57.13 0.390 0.835 . . . . 

 Dose3 -57.50 0.165 -57.25 0.209 0.835 0.282 . . . 

 Dose4 -57.00 1.000 -57.25 0.213 0.835 1.000 0.282 . . 

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

***

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 5.96 0.202 

MannWhit - testing each trt median signif. different from control 

Page 69 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 68 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -57.00 . .

 Dose1 -57.00 0.478 0.159 

 Dose2 -57.00 1.000 0.500 

 Dose3 -57.50 0.223 0.083 

 Dose4 -57.00 1.000 0.333 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 70 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 69 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 0.530 1.218 0.344 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.92 0.11 0.06 5.77 1.74, 2.09 

 Dose1 4 1.89 0.02 0.01 1.22 1.85, 1.92 

 Dose2 4 1.80 0.07 0.03 3.74 1.70, 1.91 

 Dose3 4 1.69 0.08 0.04 4.99 1.55, 1.82 

 Dose4 4 1.48 0.05 0.03 3.48 1.39, 1.56 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.93 1.77 2.03 . .

 Dose1 1.89 1.86 1.91 98.51 1.49 

 Dose2 1.80 1.73 1.89 94.11 5.89 

 Dose3 1.69 1.58 1.78 88.02 11.98 

 Dose4 1.46 1.44 1.55 77.06 22.94 

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

***

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 23.69 <.001 

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 1.92 . 1.92 . . . . . . 

 Dose1 1.89 0.949 1.89 0.353 . . . . . 

 Dose2 1.80 0.139 1.80 0.028 0.507 . . . . 

 Dose3 1.69 0.002 1.69 <.001 0.011 0.216 . . . 

 Dose4 1.48 <.001 1.48 <.001 <.001 <.001 0.008 . . 

Page 71 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 70 of 80

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 15.19 0.004 

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 1.93 . .

 Dose1 1.89 0.494 0.193 

 Dose2 1.80 0.235 0.020 

 Dose3 1.69 0.103 <.001 

 Dose4 1.46 0.067 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

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

***

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 23.69 <.001 

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 -1.92 . -1.75 . . . . . . 

 Dose1 -1.89 0.949 -1.75 0.999 . . . . . 

 Dose2 -1.80 0.139 -1.75 1.000 0.507 . . . . 

 Dose3 -1.69 0.002 -1.75 1.000 0.011 0.216 . . . 

 Dose4 -1.48 <.001 -1.75 1.000 <.001 <.001 0.008 . . 

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

***

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 15.19 0.004 

MannWhit - testing each trt median signif. different from control 

Page 72 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 71 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1.93 . .

 Dose1 -1.89 0.494 0.807 

 Dose2 -1.80 0.235 0.980 

 Dose3 -1.69 0.103 1.000 

 Dose4 -1.46 0.067 1.000 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 73 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 72 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 0.943 0.277 3.367 0.037 USE NON-PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 31.15 0.68 0.34 2.17 30.07, 32.22 

 Dose1 4 30.45 0.24 0.12 0.79 30.06, 30.83 

 Dose2 4 30.15 0.52 0.26 1.71 29.33, 30.97 

 Dose3 4 29.56 0.41 0.21 1.39 28.91, 30.21 

 Dose4 4 28.50 0.35 0.17 1.22 27.95, 29.06 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 31.17 30.50 31.74 . .

 Dose1 30.37 30.26 30.80 97.76 2.24 

 Dose2 30.07 29.67 30.78 96.80 3.20 

 Dose3 29.62 29.01 30.00 94.91 5.09 

 Dose4 28.39 28.22 29.00 91.52 8.48 

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

***

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 

 4 15 18.49 <.001 

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 31.15 . 31.15 . . . . . . 

 Dose1 30.45 0.149 30.45 0.029 . . . . . 

 Dose2 30.15 0.027 30.15 0.005 0.886 . . . . 

 Dose3 29.56 <.001 29.56 <.001 0.099 0.410 . . . 

 Dose4 28.50 <.001 28.50 <.001 <.001 0.001 0.038 . . 

Page 74 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 73 of 80

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 15.70 0.003 

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 31.17 . .

 Dose1 30.37 0.156 0.042 

 Dose2 30.07 0.156 0.014 

 Dose3 29.62 0.067 <.001 

 Dose4 28.39 0.067 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

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

***

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 18.49 <.001 

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 -31.15 . -29.96 . . . . . . 

 Dose1 -30.45 0.149 -29.96 1.000 . . . . . 

 Dose2 -30.15 0.027 -29.96 1.000 0.886 . . . . 

 Dose3 -29.56 <.001 -29.96 1.000 0.099 0.410 . . . 

 Dose4 -28.50 <.001 -29.96 1.000 <.001 0.001 0.038 . . 

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

***

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 15.70 0.003 

MannWhit - testing each trt median signif. different from control 

Page 75 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 74 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -31.17 . .

 Dose1 -30.37 0.156 0.958 

 Dose2 -30.07 0.156 0.986 

 Dose3 -29.62 0.067 1.000 

 Dose4 -28.39 0.067 1.000 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 76 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 75 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 0.975 0.850 0.721 0.591 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 13.64 0.37 0.18 2.69 13.05, 14.22 

 Dose1 4 13.74 0.71 0.36 5.19 12.61, 14.87 

 Dose2 4 13.30 0.54 0.27 4.08 12.44, 14.16 

 Dose3 4 12.90 0.97 0.48 7.52 11.36, 14.45 

 Dose4 4 10.39 0.44 0.22 4.22 9.69, 11.09 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 13.77 13.10 13.91 . .

 Dose1 13.63 12.99 14.71 100.74 -0.74 

 Dose2 13.15 12.82 14.07 97.51 2.49 

 Dose3 13.05 11.61 13.91 94.61 5.39 

 Dose4 10.44 9.83 10.85 76.16 23.84 

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

***

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 18.51 <.001 

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 13.64 . 13.69 . . . . . . 

 Dose1 13.74 0.998 13.69 0.629 . . . . . 

 Dose2 13.30 0.867 13.30 0.299 0.866 . . . . 

 Dose3 12.90 0.340 12.90 0.081 0.390 0.904 . . . 

 Dose4 10.39 <.001 10.39 <.001 <.001 <.001 <.001 . . 

Page 77 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 76 of 80

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 10.41 0.034 

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 13.77 . .

 Dose1 13.63 0.678 0.282 

 Dose2 13.15 0.494 0.121 

 Dose3 13.05 0.494 0.068 

 Dose4 10.44 0.067 <.001 

 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 18.51 <.001 

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 -13.64 . -12.79 . . . . . . 

 Dose1 -13.74 0.998 -12.79 0.982 . . . . . 

 Dose2 -13.30 0.867 -12.79 0.988 0.866 . . . . 

 Dose3 -12.90 0.340 -12.79 0.991 0.390 0.904 . . . 

 Dose4 -10.39 <.001 -12.79 0.992 <.001 <.001 <.001 . . 

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

***

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 10.41 0.034 

MannWhit - testing each trt median signif. different from control 

Page 78 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 77 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -13.77 . .

 Dose1 -13.63 0.678 0.718 

 Dose2 -13.15 0.494 0.879 

 Dose3 -13.05 0.494 0.932 

 Dose4 -10.44 0.067 0.999 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 79 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 78 of 80

DER Template Version: 22 September 2011 

test for amphib metamorph screen study - EPTC 

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 

 0.964 0.626 0.632 0.647 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.44 0.02 0.01 3.42 0.42, 0.46 

 Dose1 4 0.45 0.03 0.01 5.86 0.41, 0.49 

 Dose2 4 0.44 0.03 0.01 5.85 0.40, 0.48 

 Dose3 4 0.44 0.04 0.02 8.23 0.38, 0.50 

 Dose4 4 0.36 0.02 0.01 4.40 0.34, 0.39 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.43 0.43 0.46 . .

 Dose1 0.45 0.42 0.49 102.50 -2.50 

 Dose2 0.44 0.42 0.48 100.23 -0.23 

 Dose3 0.44 0.39 0.48 99.77 0.23 

 Dose4 0.36 0.34 0.38 82.49 17.51 

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

***

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 15 8.23 0.001 

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 0.44 . 0.45 . . . . . . 

 Dose1 0.45 0.924 0.45 0.708 . . . . . 

 Dose2 0.44 1.000 0.44 0.642 0.978 . . . . 

 Dose3 0.44 1.000 0.44 0.612 0.959 1.000 . . . 

 Dose4 0.36 0.002 0.36 <.001 0.001 0.004 0.005 . . 

Page 80 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 79 of 80

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 9.59 0.048 

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 0.43 . .

 Dose1 0.45 0.678 0.718 

 Dose2 0.44 0.889 0.442 

 Dose3 0.44 0.678 0.500 

 Dose4 0.36 0.066 0.017 

 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 8.23 0.001 

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 -0.44 . -0.43 . . . . . . 

 Dose1 -0.45 0.924 -0.43 0.844 . . . . . 

 Dose2 -0.44 1.000 -0.43 0.872 0.978 . . . . 

 Dose3 -0.44 1.000 -0.43 0.886 0.959 1.000 . . . 

 Dose4 -0.36 0.002 -0.43 0.894 0.001 0.004 0.005 . . 

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

***

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 9.59 0.048 

MannWhit - testing each trt median signif. different from control 

Page 81 of 310
Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

 EPA MRID Number 48827601

Page 80 of 80

DER Template Version: 22 September 2011 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -0.43 . .

 Dose1 -0.45 0.678 0.282 

 Dose2 -0.44 0.889 0.558 

 Dose3 -0.44 0.678 0.500 

 Dose4 -0.36 0.066 0.983 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 82 of 310
DATA EVALUATION RECORD 

EPTC 

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

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616403) 

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:

David A. McEwen, B.S. Date: 1/23/2012 

Secondary Reviewer Signature:

Michelle Sharpe-Kass, M.S Date: 1/24/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 83 of 310
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 84 of 310
EPTC I 041401 

Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page I of 16 

OCSPP 890.1150/ OECD None 

Primary Reviewer: Jessica Ryman, Ph.D., D.A.B.T 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Sig"""" : --J-+--.P......,..-.;..--

Date: -~--'~~c..L..,J..-­

Signature: -~""t""'~,L-----,----­

Date: __ ___,,,6,,,_._. lt ~}J~ Template version 08/201 t 

DATA EVALUATION RECORD 

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

PC CODE: 041401 DP BARCODE: D397177 

TXR#: 0051762 CAS No.: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: S-ethyl dipropylthiocarbamate 

CITATION: Willoughby, J.A (2011). EPTC: Androgen Receptor Binding (Rat Prostate 

Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study #: 9142V100349ARB, November 10, 2011. MRID 48616403. Unpublished. 

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

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

Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. Julyl 9, 2012. 

MRID 48843501. Unpublished. 

SPONSOR: Gowan Company, 370 S. Main Street, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48616403), 

ventral prostate cytosol from Sprague Dawley rats was used as the source of the AR. 

The saturation binding experiment (data submitted separately, MRID 48843501) was conducted 

to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radio labeled reference androgen (Rl 881 ). 

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

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

following a request by the Agency. The mean dissociation constant (Kct) for [3H]-R1881 was 

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

batch of prostate cytosol that was used in the assay. Only one of the three Kct values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for 

individual runs acceptable (R2 = 0.880-0.986), confidence in these numbers is low based on the 

large variation in Kct estimates among runs. 

Page 85 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

The competitive binding assay was conducted to measure the binding of a single concentration (1 

nM) of the radio-labeled reference androgen ([3H]-R1881) in the presence of increasing 

concentrations of EPTC (logarithmic increase from 10-10 to 10-3 M). DMSO was used as a 

vehicle at a final assay concentration of approximately 3.2%. The assay included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three 

independent runs were conducted with 3 replicates per concentration per run. 

The estimated log IC50s for R1881 and dexamethasone were within the excepted ranges at -9.0 to 

-9.9 M and -4.4 to -4.6 M, respectively. The average log IC50 was -9.3 M for R1881 and -4.5 M 

for dexamethasone, and the mean RBA for the weak positive control was 0.0016%. Confidence 

in the numbers is high due to the small variation, and the solvent control responses indicated no 

drift in the study assay. The performance criteria were met, with the following exceptions: in 

the second assay, the top specific binding (%) of EPTC was 118.2%, which slightly exceeded the 

range of the Guideline performance criteria (80 to 115%); and in the first assay, the bottom 

plateau level (% binding) for dexamethasone was not able to be accurately calculated, because 

the concentration range for dexamethasone was prepared slightly different than the guideline, in 

error. As these deviations were minor and were not considered to have affected the results of the 

study, all three runs were considered valid. 

EPTC reduced the mean specific binding of the ligand in a concentration dependent manner in 

all three runs, with maximum displacement of binding occurring at 10-3 M (50-56.5% binding). 

An IC50 and relative binding affinity (RBA) could not be calculated for EPTC. Specific binding 

was also reduced at 10-4 M (87.4-91%), but not at EPTC concentrations ≤10-5 M (≥92.8% 

binding). Specific [3H]-R1881 binding to the AR was between 50 and 75% at all concentrations 

of EPTC tested in all three runs. 

Based on the results from the three runs, EPTC is classified as Equivocal in the Androgen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150). 

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

Assurance statements were provided. 

 

Page 86 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test facility: CeeTox, Inc. 

Location: 4717 Campus Drive, Kalamazoo, MI 49008 

Study Director: J.A. Willoughby, Sr. 

Other Personnel: D. Blakeman, C. Haines, and S. McColley 

Study Period: May 25, 2011 to November 10, 2011 

2. Test substance: EPTC 

 Description: Liquid 

 Source: Gowan company 

 Batch/Lot #: P1105096 

 Purity: >99.9% (expiry date February 4, 2015) 

 Solubility: Not reported 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: Ambient 

 CAS #: 759-94-4 

 Molecular weight: 189.3 g/mol 

 Structure: 

N

S

O 3CH

3CH

CH3

3. Non-labeled ligand: R1881 

 Supplier: Sigma Aldrich, St. Louis, MO 

 Catalog #: R0908 

 Batch #: 060M4638 

 Purity: 98% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: Perkin-Elmer, Boston, MA 

 Catalog and Batch #: NET590; Lot # 653698 

 Date of production: February 24, 2011 

 Date of use: September 10, 2011 to September 17, 2011 

 Radiochemical purity: Not reported 

 Specific activity: 85.1 Ci/mol 

**The SAadjusted was 82.6 Ci/mol (10-Sept-2011), 82.5 Ci/mol (13-Sept-2011), and 92.5 

Ci/mol (17-Sept-2011). 

 Concentration in stock: 1.0 nM 

5. Positive control: Dexamethasone 

 Supplier: Sigma Aldrich, St. Louis, MO 

 Catalog #: D1756 

 Lot #: 077K1050 

 Purity: 99% 

 CAS # : 50-02-2 

Page 87 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

6. Solvent/vehicle control: DMSO 

 Justification for choice of solvent: Due to issues with pipetting and volatilization when using ethanol. 

 Final concentration: 3.2% 

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was 

purchased from Charles River Laboratories (location not reported). Male Sprague Dawley 

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

following day. 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 (10 mM Tris, 1 mM sodium 

molybdate, 1.5 mM EDTA, 10% glycerol and 1 mM DTT, 0.5% protease inhibitor) at pH 

7.4 to the ventral prostate tissues at 10 mL/g of tissue. The tissues were minced, 

homogenized at 4°C, and centrifuged for 30 min at 30,000 × g at 4°C. The supernatant was 

aliquoted (2-4 mL) in tubes and stored at -80°C until used. Protein concentration of the 

cytosol was determined to be 6.2 mg/mL using a protein kit compatible with DTT in the 

TEDG buffer (BioRad Protein Assay Kit). 

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

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

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

R1881 ligand (MRID 48843501). The conditions for the saturation binding experiment are 

summarized in Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand (as serial dilutions) 0.25 to 10 nM 

Concentration of non-labeled ligand (100X [radioligand]) 2 to 1000 nM 

Optimization of receptor concentration Sufficient to bind 25 to 35% of 

radioligand at 0.25 nM

Temperature 4o C 

Incubation time 16 to 20 hours 

Composition of assay buffer 

(TEDG) 

Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride (PMSF) 1 mM 

DTT 1 mM 

Sodium Molybdate 1 mM 

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

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

H]-R1881 (originally 

85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time 

(adjusted specific activities were not reported), and serial dilutions in low-salt 

TEDG+PMSF buffer were prepared to achieve the final concentrations in cytosol of 0.25, 

0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonPage 88 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

specific binding, solutions of non-labeled R1881 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 [3H]-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 with 0.25 nM radiolabeled R1881. 

The optimal protein concentration was determined to be 1.86 mg protein/assay tube, which 

resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic 

protein used in this assay was thawed fresh for this experiment at 4°C, and maintained at 

4°C during the binding assay. Each saturation binding experiment consisted of three noncurrent runs (conducted on September 9, 11, and 16, 2011, respectively), and each run 

contained three replicates at each concentration, resulting in the 72 samples depicted in 

Table 2. 

TABLE 2. Saturation Binding Experiment Runa,b

Total Binding Non-Specific Binding Radioligand alone 

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

[

3

H]-R1881 

Final conc. (nM) 

[

3

H]-R1881 

Final conc. (nM) 

R1881 

Final conc. (nM) 

[

3

H]-R1881 

Initial conc. (nM) 

[

3

H]-R1881 

(μL) 

0.25 0.25 25 10 7.5 

0.50 0.50 50 10 15 

0.70 0.70 70 10 21 

1.0 1.0 100 10 30 

1.5 1.5 150 10 45 

2.5 2.5 250 100 7.5 

5.0 5.0 500 100 15 

10 10 1000 100 30 

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

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

c Tubes 1-24 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. Samples were dried, 

and 300 μl of prostate cytosol were added. 

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

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

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

cytosol were added. 

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

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

nM [3

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

Following addition of triamcinolone acetonide, [3H]-R1881, and/or R1881, the tubes were 

dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at 

approximately 4°C. Samples were maintained at temperatures of approximately 4°C except 

during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) 

slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. 

The samples were then centrifuged, and the supernatant was aspirated and discarded. The 

samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed by 

vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and 

decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. 

The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples were 

Page 89 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

maintained on ice at all times between vortexing. After the final centrifugation, the ethanol 

supernatants were decanted into scintillation vials that each contained 14-mL portions of 

scintillation cocktail, and the radiation was quantified by liquid scintillation counting. A 

total of three runs were performed. 

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

binding experiment is presented in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 10-15%b

Concentration of test substance (as serial dilutions) 10-10 to 10-3 M 

Incubation Temperature 4±2°C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Protease inhibitor 0.5% (v/v) 

DTT 1 mM 

Sodium molybdate 1 mM 

a Data were obtained from page 15 of the study report. 

b Stated in Study Report on page 15. 

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

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures 

the binding of a single concentration of [3H]-R1881 (adjusted specific activity of 85.1

Ci/mol) to the AR in the presence of increasing concentrations of a test substance. 

DMSO was used as the vehicle, and the solubility of the test material at the top 

concentration of 10-3 M in 100% DMSO was determined by visual observation. 

Additionally, all test material solutions were examined for precipitation following the 

overnight incubation period. As no precipitation was observed, the suitable top 

concentration of EPTC for use in all independent runs was 10-3 M. 

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

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

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

and each run contained three replicates at each concentration, resulting in a total of 81 

samples per run. 

Page 90 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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

Solvent Control Reference standard c Weak positive control d Test Chemical None 

DMSO R1881 Dexamethasone EPTC 

Tubes 7-12 Tubes 13-18 and 19-33 e Tubes 37-60 Tubes 61-84 Tubes 1-6

1×10-12.48 1×10-11 1×10-11 1×10-10 1×10-10

1×10-11.48 1×10-10 1×10-10 1×10-9 1×10-9

1×10-10.48 1×10-9 1×10-9 1×10-8 1×10-8

1×10-9.48 1×10-8 1×10-8 1×10-7 1×10-7

1×10-8.48 1×10-7 1×10-7 1×10-6 1×10-6

1×10-7.48 1×10-6 1×10-6 1×10-5 1×10-5

-- 1×10-5 1×10-4 1×10-4

-- 1×10-4 1×10-3 1×10-3

a Data were obtained from pages 34-39 of the study report. 

b Each concentration of each chemical was run in triplicate for a total of 81 tubes per run. Tubes 7-84 contained 

50 μL of triamcinolone acetonide and 30 μL [3

H]-R1881. Samples were dried, and 300 μL of prostate 

cytosol were added. Tubes 7-84 also contained 10 μL of the solvent control, reference standard (nonradiolabeled R-1881), weak positive control, or test substance, with the exception of Tubes 13-18 that 

contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Tubes 1-6 contained 

only 30 μL of [3

H]-R1881. 

c The concentrations of R1881 used in the 1st competitive binding assay ranged from 1x10-7.48 to 1x10-12.48, 

while the standard concentrations of 1x10-6 to 1x10-11 were used in the 2nd and 3rd assays. 

d The concentrations of dexamethasone used in the 1st competitive binding assay ranged from 1x10-4 to 1x10-11, 

while concentrations of 1x10-3 to 1x10-10 were used in the 2nd and 3rd assays. 

e Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881 

4. Data Analysis: The top and bottom of the curve, Hill slope, inhibition concentration (IC50), 

and standard deviations were assessed using GraphPad PRISM (GraphPad Software, Inc., 

San Diego, CA). 

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 (i.e. a Log IC50 was obtained). 

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. 

Equivocal: The average lowest portion of curves across runs is between 50% and 75% 

radioligand binding (i.e. radioligand displacement is at least 25% but less than 50%), or 

the curve falls outside the range for the weak positive control (-0.6 to -1.4). 

b. Descriptors for receptor binding 

Bmax: Maximal binding capacity

Kd: Dissociation constants 

Page 91 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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 × 100 ÷ IC50 of test substance 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The mean Kd for [3H]-R1881 was 1.080±0.690 nM, and the 

estimated Bmax was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol 

that was used in the assay. Only one of the three individual Kd values was within the range 

reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the 

recommended range. Although the goodness of fit (R2

 = 0.880-0.986) was acceptable, 

confidence in these numbers is low based on the large variation in Kd estimates among runs.

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

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.880 0.986 0.893 0.880-0.986 

Bmax (nM) 0.010 0.008 0.011 0.010±0.002 

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

Kd (nM) 0.855 0.531 1.855 1.080±0.690 

a Data were obtained from supplemental information provided by the laboratory (MRID 48843501). 

b The range of R2

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

R2

 Goodness of fit for curve calculated for specific binding.

Figure 1 illustrates the non-specific, specific, and total binding curves for [3H]-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 [3H]-R1881 to the androgen receptor. 

Page 92 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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

Binding Experiment.

Page 93 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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

Page 94 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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

binding experiments are summarized in Table 6 and shown graphically in Figures 3-5. 

EPTC reduced the mean specific binding of the ligand, [3H]-R1881, in all three runs in a 

concentration dependent manner. At 10-3 M of EPTC, specific binding was reduced to 56.5, 

50.0, and 50.6% in the first, second, and third assays, respectively. At 10-4 M, specific 

binding was reduced to 89.4, 87.4, and 91.0% in the first, second and third assays, 

respectively. Specific binding was ≥92.8% at EPTC concentrations ≤10-5 M. As specific 

binding was not ≤50% at any concentration of EPTC, an IC50 and RBA could not be 

calculated for EPTC. All three competitive binding runs were classified as equivocal since 

the percent specific [3

H]-R1881 bound to the AR was between 50 and 75% at all 

concentrations of EPTC tested.

The estimated log IC50s for R1881 and the weak positive control (dexamethasone) were in 

the ranges of -9.0 to -9.9 M and -4.4 to -4.6 M, respectively. The mean RBA for the weak 

positive control was 0.0016%. Confidence in these numbers is high due to the small 

variation. The solvent control responses indicated no drift in the study assay. 

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

Parameter Assay 1 Assay 2 Assay 3 Mean

r

2

 (unweighted) R1881 NR NR NR NR 

Dexamethasone NR NR NR NR 

EPTC NR NR NR NR 

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

Dexamethasone -4.4 -4.4 -4.6 -4.5b

EPTC NA NA NA NA 

IC50 (M) b R1881 1.3 x 10-10 10-9 10-9 5.0 x 10-10

Dexamethasone 4.0 x 10-5 4.0 x 10-5 2.5 x 10-5 3.2 x 10-5

EPTC NA NA NA NA 

RBA (as% IC50) c Dexamethasone 0.0003 0.0025 0.0040 0.0016 

 EPTC NA NA NA NA 

a Data were obtained from page 20 of the study report. 

b Calculated by reviewer 

c Calculated by reviewer: [IC50 (in M) Dexamethasone or EPTC / IC50 (in M) R1881] x 100% 

NA Not applicable 

NR Not reported 

RBA Relative binding affinity

Page 95 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

FIGURE 3. Binding of EPTC, R1881 and Dexamethasone in Assay 1. 

 

Page 96 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

FIGURE 4. Binding of EPTC, R1881 and Dexamethasone in Assay 2. 

Page 97 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

FIGURE 5. Binding of EPTC, R1881 and Dexamethasone in Assay 3. 

Page 98 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

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

functioning properly, each run was evaluated using the following criteria (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 (% binding) 

≤15 8.7 to 9.7 X 

Test chemical Top (% binding) 80 to 115 101.5 to 118.2 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 96 to 103 X 

Bottom (% binding) -2.0 to 2.0 0 X 

Hill Slope -1.2 to -0.8 -1.0 X 

Weak positive control (dexamethasone) fitted curve parameters 

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

Bottom (% binding) -12 to 12 -1 to 7 X 

Hill Slope -1.4 to -0.6 -1.0 to -0.6 X 

Saturation Binding Experiment Kd (nM) 0.685 – 1.57 1.08 ± 0.690 X 

Non-specific binding (%) ≤20.0 9.7-14% X 

a Data were obtained from pages 23-28 of the study report.

b These values represent ranges from the validation study. 

c Calculated by reviewer from page 34-38. Avg dpm (1 ml) R1881 (NSB) / Avg dpm (1ml) Total binding 

(solvent control). 

NR Not reported 

The performance criteria were met, with the following exceptions: in the second assay, the 

top specific binding (%) of EPTC was 118.2%, which slightly exceeded the range of the 

Guideline performance criteria (80 to 115%); and in the first run assay, the bottom plateau 

level (% binding) for dexamethasone was not able to be accurately calculated, because the 

concentration range for dexamethasone was prepared slightly different than the guideline in 

error. As these deviations were minor and were not considered to have affected the results 

of the study, all three runs were considered valid. 

Additionally, the curve for the reference material showed that increasing concentrations of 

unlabeled R1881 displaced [3H]-R1881 in a manner consistent with one-site binding, as 

indicated by a Hill slope of -1.0 to -0.6. Examination across the runs indicated consistency 

of the Hill slope, placement along the X-axis, and top and bottom plateaus. 

The percentage of the total specific binding in the solvent controls was approximately 10%. 

This was within the less than the ≤ 10-15% recommended in the guideline. Ligand 

depletion was also minimal. Sufficient optimization of the number of specific binding sites 

is supported curves for controls of acceptable steepness, low non-specific binding, and low 

variability. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: All three assays achieved the “equivocal” 

criterion described in the guideline (between 50 and 75% radioligand binding). There were 

Page 99 of 310
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 16 

EPTC / 041401 OCSPP 890.1150/ OECD None

no apparent alterations in radiolabeled R1881 binding at any of the lower concentrations 

tested. EPTC was classified as “equivocal”. 

B. AGENCY COMMENTS: In the saturation binding experiment, the mean Kd for [3H]-

R1881 was 1.080 nM, and the mean estimated Bmax was 1.071 fmol/100 μg protein for the 

single batch of prostate cytosol that was used in the assay. Only one of the three individual 

Kd values was within the range reported in the EPA validation program (0.685 to 1.57 nM); 

the mean value was above the recommended range. In the competitive binding experiment, 

the average log IC50s for R1881 (-9.3 M) and the weak positive control, dexamethasone 

(-4.5 M), were within the expected ranges. The mean RBA for the weak positive control 

was 0.0016%. 

The solvent control responses indicated no drift in the study assay. The performance criteria 

were met, with the following exceptions: in the second assay, the top specific binding (%) 

of EPTC was 118.2%, which slightly exceeded the range of the Guideline performance 

criteria (80 to 115%); and in the first assay, the bottom plateau level (% binding) for 

dexamethasone was not able to be accurately calculated, because the concentration range for 

dexamethasone was prepared slightly different than the guideline in error. As these 

deviations were minor and were not considered to have affected the results of the study, all 

three runs were considered valid. 

EPTC reduced the mean specific binding of the ligand in a concentration dependent manner 

in all three runs. The maximum binding displacement occurred at 10-3 M of EPTC, with 

specific binding being reduced to 50-56.5%. Specific binding was also reduced at 10-4 M 

(87.4 – 91%), but not at EPTC concentrations ≤10-5 M (≥92.8% binding). An IC50 and RBA 

could not be calculated for EPTC. EPTC is classified as equivocal in the AR assay as 

specific radiolabeled ligand bound was between 50 and 75% up to the highest EPTC 

concentration tested in all three runs. 

C. STUDY DEFICIENCIES: The following deficiencies were noted that did not have an 

adverse effect on the results, interpretation or conclusions of this study: 

 Curves were not provided showing the average binding of each test substance across all 

three runs. 

 Non-specific binding exceeded the tolerance limits for Assays 2 and 3. This was not 

considered to have a negative impact on the study because there was still enough of a 

dynamic range in which to detect changes in binding with concentration for control and 

test chemicals. 

Page 100 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1200; Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616402) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature: 

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 12/16/2011 

Secondary Reviewer Signature: 

Michelle Sharpe-Kass, M.S. Date: 1/17/2012 

Program Manager: Signature: 

Jack D. Early, M.S. Date: 1/23/2011 

Quality Assurance: Signature: 

Jack D. Early, M.S. Date: 1/23/2011 

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

signing by CSS-Dynamac Corporation personnel. 

Page 101 of 310
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 102 of 310
EPTC/041401 

Aromatase (Human Recombinant) Assay (2011) I Page 1of13 

OCSPP 890.1200 I OECD None 

Primary Reviewer: Vincent Chen M.S. Signature: ___:....____::_ _____ _ 

Health Effects Division (7509P) Date: .2-Y :rvr-.J z..o...L.S 

Secondary Reviewer: Greg Akerman, Ph.D. Signature: ~/6__,.....,,,_..,,.A'----- Risk Assessment Branch III, Health Effects Division (7509P) Date: --~<i~/.l lt ) __ 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 041401 

TXR: 0051762 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: 

IUPAC: 

CAS: 

S-ethyl dipropylthiocarbamate 

S-ethyl N, N-dipropylcarbamothioate 

DP BARCODE: 0397177 

CAS No.: 759-94-4 

CITATION: Wilga, P.C. (201 1). EPTC: Human Recombinant Aromatase Assay. CeeTox, 

Inc [4717 Campus Drive, Kalamazoo, MI 49009, US]. Study No. 9142V100349AROM. 09 November 2011. MRID 48616402. Unpublished. 

SPONSOR: Gowan Company; 370 Main St., Yuma, AZ 85364, US 

TEST ORDER: EDSP-041401-64 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48616402), 

EPTC (>99.9% w/w, Batch# Pl 105096) in dimethyl sulfoxide (DMSO) was incubated with 

human recombinant aromatase and tritiated androstenedione ([ 1 ~- H(N) ]-androst-4-ene-3, 17-

dione; [3H]-ASDN) at increasing logarithmic concentrations from 10- 10 to 10- 3 M for 15 minutes 

at 3 7°C to assess the potential of EPTC to inhibit aromatase activity. 

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

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

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity control, a background activity control, a positive control 

series (10- 10 to 10- 5 M) using a known inhibitor (4-hydroxyandrostenedione ; 4-0H ASDN), and 

the test chemical series c10- 10 to 10-3) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.161 to 0.376 nmol·mgprotein- 1·min- 1 for the three test runs, with a mean and standard deviation of 0.261±0.077 

nmol·mg-protein- 1·min- 1. Activity in the background controls ranged from 0.40% to 0.95% and 

averaged 0.57% of the full activity controls. The response of the full activity controls and 

background controls was acceptable for each run. 

Page 103 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

Results for the positive control 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 within runs. Also, the coefficients of variation (%CVs) for replicates of each 

concentration of EPTC within a run were generally within the 15% guideline. For 4-OH ASDN, 

the estimated log IC50 averaged –7.21 M, and the slope was -0.97. 

For EPTC, aromatase activity averaged 0.242±0.068 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration of 10-10 M and 0.213±0.011 nmol·mg-protein-1·min−1

 at the highest tested 

concentration of 10−3 M (when Run 1 was excluded due to precipitation). The data for EPTC 

were not suited for modeling. The average lowest portion of the response curve across runs was 

between 50 and 75% activity (69% at 10–3 M EPTC; excluding run 1 due to precipitation) 

indicating an equivocal response. 

Based on the data from the average response curve, EPTC is classified as Equivocal for 

aromatase inhibition 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, Quality Assurance, and Data 

Confidentiality statements were provided. 

 

Page 104 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Substance: EPTC 

 Description: Liquid 

 Source: Gowan 

 Batch # (Expiration Date): P1105096 (February 4, 2015) 

 Purity: >99.9% 

 Vapor Pressure: 4500 mPa at 25°C 

 Storage Conditions: Room temperature 

 Stability: Not reported 

 Solvent: DMSO 

 Solubility: Visually soluble at 10−3M in 2/3 test runs. 

 Highest Concentration Tested: 10−3M 

 Stock Solution Preparation Methodology: Not reported 

 Molecular Weight: 189.3 

 CAS #: 759-94-4 

 Structure: 

N S

O 3CH

3CH

CH3

2. Non-Labeled Substrate: Androstenedione (ASDN) 

 CAS # : 63-05-8 

 Source: Steraloids, Inc. (Cat. # L1712) 

 Batch # (Expiration Date): L1712 (April 2016) 

 Purity: 99.8% 

3. Radiolabeled Substrate: [1β3

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

 Source: Perkin Elmer (Cat. # NET-926) 

 Batch # (Expiration Date): 619344 (January 10, 2012) 

 Radiochemical Purity (Supplier): >97% 

 Specific Activity: 26.3 Ci/mmol 

Radiochemical Purity (Vendor 

Determination): >97% 

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

 CAS # 566-48-3

 Source: Sigma-Aldrich (Cat. # F2552) 

 Batch # (Expiration Date): 081K2133 (March 2015) 

 Purity: 99.6% 

5. Solvent (Vehicle Control): DMSO 

 Source: Sigma-Aldrich (Cat. # D2650) 

 Batch # (Expiration Date): RNBB7886 (February 2013) 

 Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 

Concentration (% of Total Volume in 

Assays): 1% v/v 

Page 105 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

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

Source: BD Biosciences GentestTM (Woburn, MA; Cat. # 456260) 

Lot # (Expiration Date): 03897 (February 2014) 

Protein Concentration: 7.4 mg/mL 

Cytochrome C Reductase Activity: 290 nmol/min/mg 

Aromatase Activity: 6.0 pmol/min/pmol P450 

B. METHODS

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

[

3

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

approximately 0.1 μM, and the amount of tritium added to each incubation tube was 

0.1 μCi. 

Test chemical(s) stock solutions were prepared such that the total volume of each test 

chemical formulation used per assay was no more than 1% v/v of the total assay volume to 

minimize the potential for the solvent to inhibit the enzyme. The report specified that 

DMSO was chosen over ethanol as the solvent of choice for the following reasons: 1) 

DMSO was listed as one of the vehicles acceptable for use in OCSPP 890.1200 guideline; 2) 

DMSO was not as volatile as ethanol and so evaporation was less of a concern in the assay, 

and 3) DMSO was more accurate to pipette because of density and viscosity. 

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

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 (10-10 to 10-5 M; Table 4) were achieved by the addition of 20 μL of the 

dilution for a final assay volume of 2 mL. 

Human recombinant microsomes were purchased from BD Biosciences, and stored at -80 ± 

10oC for no longer than 7 months for this study. Before use of the microsomes, the batch 

was thawed and sub-aliquoted at 0.008 mg/mL into individual vials before being refrozen to 

minimize freeze-thaw cycles to no more than one. 

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are 

reported in Table 1. 

TABLE 1. Assay Components and Conditions 

Assay Factor Values 

Sodium phosphate buffer (pH 7.4) 0.1M 

Microsomal protein 0.004 mg/mL 

NADPH 0.3 mM 

[3H]ASDN 100 nM 

Propylene glycol 5% 

Temperature 37±2°C 

Incubation time 15 min 

2. Suitability Assessments: The protein concentration of the microsomes was supplied by the 

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

vendor as 6.0 pmol/min/pmole P450. The minimum aromatase activity in the full activity 

Page 106 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

control samples was determined to be 0.161 nmol/min/mg protein, which was greater than 

the minimum recommended aromatase activity of 0.1 nmol/mg-protein/min. 

3. Aromatase Assay: Each assay run contained 4 tubes for the full enzyme activity and 4 

tubes for the background activity controls. Two tubes of each control were run at the 

beginning of the assay, and two of each control were run at the end of the assay. A full 

concentration curve in duplicate for the positive control, and a full concentration curve in 

triplicate for the test substance were established. The aromatase assay was conducted 

generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10). 

The amount of 3

H2O 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: No information was provided concerning demonstration of 

proficiency. However, the report stated that prior to conducting this assay using EPTC, a 

full-scale assay consisting of three independent runs was conducted using the positive 

control (4-OH ASDN), econazole, fenarimol, nitrofen, and atrazine. Proficiency was 

demonstrated when the positive control met the performance criteria from the guideline and 

by the correct classification of the four proficiency chemicals in Table 6 of the guideline. 

Data were generated by CeeTox and are maintained at CeeTox. 

a. Positive Control: Data were not provided. 

(1) Initial Demonstration of Laboratory Proficiency: The positive control data 

(new/historical data for laboratory) were not reported in regards to the following criteria: 

 Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mgprotein/min. 

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

 Coefficient of variation (% CV) for replicates within each sample type and 

concentration of 4-OH ASDN was <15%. 

Performance criteria were reported for the test runs only (Table 2), and served as guidance 

in identifying runs that provided parameters in the preferred ranges. 

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

applicable): Demonstration of proficiency by a new technician, if applicable, was not 

reported. The positive control data for slope, top and bottom percent, and log IC50 met the 

criteria as listed in section (i) of OCSPP 890.1200 with the following exceptions: (i) the top 

percent for one run (run 2) was slightly below (88.48%) the recommended range; and (ii) 

the log IC50 for one run (run 3) was slightly below (-7.35) the recommended range. 

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 -1.02 -0.94 

Top (%) 90 110 88.48 104.31 

Bottom (%) -5 +6 -0.28 0.66 

Log IC50 (M) -7.3 -7.0 -7.35 -7.09 

Page 107 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

a

 Data were obtained from page 27 of the study report. 

b. Proficiency Chemicals: Data not provided. 

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Inhibitor Not reported 

Fenarimol 60168-88-9 Inhibitor Not reported 

Nitrofen 1836-75-5 Inhibitor Not reported 

Atrazine 1912-24-9 Non-inhibitor Not reported 

5. Determination of Aromatase Activity with Test Chemical: The response of aromatase 

activity to the presence of eight concentrations of EPTC per run, in triplicate, was tested 

during three independent runs. Solubility was visually assessed (presence of cloudiness or a 

precipitate). The test compound was soluble at 10–3 M in two runs, but a slight amount of 

precipitation was visible in the first run. The full enzymatic activity (≥95% for the means of 

each run) was obtained at the two lowest concentrations of EPTC, defining the top of the 

concentration-response curve. 

TABLE 4. Test Chemical Study Design for Each Test Run 

Sample Type Repetitions (Tubes) Description Reference or Chemical (M) 

Full Activity Control 4 All test componentsa 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

EPTC Conc 1 3 All test components plus EPTC 1×10–3

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

EPTC Conc 3 3 All test components plus EPTC 1×10–5

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

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

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

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

EPTC Conc 8 3 All test components plus EPTC 1×10–10

a

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

C. DATA ANALYSIS 

1. Raw Data: Raw data were converted to aromatase activity (nmol/mg protein/min) and 

percent control for the positive control and test chemical. The following raw data and 

calculated endpoints for each run were included in the report (Table 5). 

Page 108 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

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/min X 

Average aromatase activity in the full activity control tubes X 

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

DPM Disintegrations per minute

2. Statistical Methods: Statistical analyses and graphical displays were conducted using 

IDBS XLfit v5.2.0.0 (Guildford, UK). Basic statistical analyses were performed on the 

data, which included means of replicates, standard deviation of the mean, standard error of 

the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed. 

The response curve was fitted by weighted least squares nonlinear regression analysis with 

weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model 

(XLfit v5.2.0.0, Fit Model 208) and Tukey’s Bi-Weight statistical analysis for outlier 

analysis. For each run, percent of control activity were plotted versus logarithm (base 10) of 

the EPTC 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% activity

b 

Equivocal 

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

Data do not fit model --- a

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

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

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

determined to be untestable in the aromatase assay. 

II. RESULTS 

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 

0.161 to 0.376 nmol·mg-protein−1·min−1 for the three test runs, with a mean and standard 

deviation of 0.261 ± 0.077 nmol·mg-protein−1

·min−1. Activity in the background controls 

ranged from 0.40% to 0.95% and averaged 0.57% of the full activity controls. The response 

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

activity. 

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase 

activity averaged 0.257 ± 0.078 nmol·mg-protein−1·min−1 at the lowest tested concentration 

Page 109 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

(10−10 M) and 0.002 ± 0.001 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 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 within runs. 

The following minor exceptions were observed: in Run 2 of the assay, the "top of curve" 

value (e.g., 88.48%) was marginally lower than the specified range compared to the 

guideline range of 90%-110%; in Run 3 of the assay, the IC50 was marginally lower than the 

specified range (log IC50 = -7.35) compared to the guideline range of -7.3 to -7.0. The 

coefficient of variations (CVs) for replicates within each concentration of 4-OH ASDN and 

EPTC were generally within the 15% guideline, with exceptions being 18.7%, 16.1%, 

23.6%, and 18.8%. 

TABLE 7. Effect of EPTC on Aromatase Activity (as Percent of Control) from Independent Runs a

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

4-OH ASDN 

(positive control) 

-5 3 0.83 0.09 0.04 11.3 

-6 3 6.53 0.89 0.36 13.6 

 -6.5 3 16.91 2.34 0.96 13.8 

 -7 3 36.66 2.76 1.13 7.5 

 -7.5 3 64.65 3.60 1.47 5.6 

 -8 3 81.24 5.06 2.07 6.2 

 -9 3 93.92 9.84 4.02 10.5 

 -10 3 98.58 8.83 3.61 9.0 

EPTC -3 3 69.32 5.54 2.26 8.0 

 -4 3 96.53 6.40 2.61 6.6 

 -5 3 100.17 7.98 3.23 7.9 

 -6 3 94.33 8.75 3.57 9.3 

 -7 3 98.90 9.51 3.88 9.6 

 -8 3 92.24 10.32 4.21 11.2 

 -9 3 95.23 8.84 3.61 9.3 

 -10 3 93.63 12.49 5.10 13.3 a

 Values were calculated by the reviewers based on data provided on pages 41-54. 

SD Standard Deviation 

SEM Standard Error of the Mean 

CV Coefficient of Variance 

Page 110 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

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

C. TEST SUBSTANCE: For EPTC, aromatase activity averaged 0.242 ± 0.068 

nmol·mg-protein−1·min−1 at the lowest tested concentration of 10-10 M and 0.213 ± 0.011 

nmol·mg-protein-1·min−1 at the highest tested concentration of 10−3 M (when Run 1 was 

Page 111 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

excluded due to precipitation). Precipitation was only observed in Run 1 at 10−3 M, and the 

report stated that the microsomal reaction “appeared to receive no NADPH.” The mean 

aromatase activity of EPTC (expressed as % full control activity) for each concentration 

tested across all three runs is provided in Table 7 (presented above), along with the overall 

standard deviation, standard error of the mean, and %CV. Inhibition response curves for 

EPTC from each run are shown in Figure 2, and the average inhibition response curve 

across all runs is shown in Figure 3. 

Page 112 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

FIGURE 2. Inhibition Response Curves for EPTC. 

Page 113 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

FIGURE 3. Mean Inhibition Response Curve for EPTC. 

The data for EPTC were not suitable for modeling. Although the mean aromatase activity 

of 10–3 M EPTC was determined to be 69.3% of control activity (excluding Run 1 due to 

precipitation), the best fit mean inhibition response curve (Figure 3) was a straight line 

(essentially no inhibition was observed at other doses). The effect of the positive control on 

inhibition of aromatase activity is presented in Table 8. For 4-OH ASDN, the estimated log 

IC50 averaged –7.21 M, and the slope was -0.97. Confidence in these values is very high 

due to the small variation (<5% CV). 

TABLE 8. Effect of EPTC and 4-OH ASDN on Aromatase Activity (as Percent of Control) from 

Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SD %CV 

 Log IC50 (M) 

EPTC NA NA NA NA NA NA 

4-OH ASDN -7.19 -7.09 -7.35 -7.21 0.13 1.82 

 Slope 

EPTC NA NA NA NA NA NA 

4-OH ASDN -0.94 -1.02 -0.95 -0.97 0.04 4.49 

a Data were provided on page 27 of the study report. Mean, SD, and %CV were calculated by the reviewers. 

SD Standard Deviation 

CV Coefficient of Variance 

NA Not applicable. Values for EPTC were not suited for modeling. 

As aromatase activity was only 69.3% of control activity at the highest dose, EPTC was 

determined to be “equivocal” for inhibition of aromatase activity in this assay. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: EPTC was determined to be “equivocal” for 

inhibition of aromatase activity in this assay. 

Page 114 of 310
Aromatase (Human Recombinant) Assay (2011) / Page 13 of 13 

EPTC / 041401 OCSPP 890.1200 / OECD None

B. AGENCY COMMENTS: For the positive control substance (4-OH ASDN), aromatase 

activity averaged 0.257 ± 0.078 nmol·mg-protein−1·min−1 at the lowest tested concentration 

(10−10 M) and 0.002 ± 0.001 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 curve, hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. The %CVs for replicates within each concentration of 4-OH 

ASDN and EPTC were generally within the 15% guideline. Minor deviations from the 

guideline ranges did not confound the results of this study. 

For EPTC, aromatase activity averaged 0.242 ± 0.068 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration of 10-10 M and 0.213 ± 0.011 nmol·mg-protein-1·min−1 at the highest 

tested concentration of 10−3 M (Run 1 was excluded due to precipitation). Precipitation was 

only observed in Run 1 at 10−3

 M. As precipitation was observed in the first run at the 

highest dose, it is unclear why further evaluations were not carried out at 10–3.5 M. It is also 

unclear why precipitation at 10−3 M was only observed in Run 1. For 4-OH ASDN, the 

estimated log IC50 averaged –7.27 M, and the slope was -1.00. Confidence in these values 

is very high due to the small variation (<5% CV). 

The data for EPTC were not suitable for modeling. Although the mean aromatase activity 

of 10–3 M EPTC was determined to be 69.3% of control activity (excluding Run 1 due to 

precipitation. As the average lowest portion of curves across runs was between 50% and 

75% activity (69.3%), EPTC was determined to be “equivocal” for inhibition of aromatase 

activity in this assay. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

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

 Proficiency chemical data were not provided. 

Page 115 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616404) 

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: 1/10/2012 

Secondary Reviewer Signature:

David A. McEwen, B.S. Date: 1/23/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/25/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/25/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 116 of 310
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 117 of 310
EPTC/ 04140 I 

Estrogen Receptor Binding (Rat Uterine Cytosol) (20 t I) I Page I of 17 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 041401 DP BARCODE: D397177 

TXR#: 0051762 CAS No.: 759-94-4 

TEST MATERIAL (PURITY): EPTC (99.9% a.i.) 

SYNONYMS: S-Ethyl dipropylthiocarbamate 

CITATION: Willoughby, J.A. , Sr. (2011). EPTC: Estrogen Receptor Binding (Rat Uterine 

Cytosol). CeeTox, Inc. , Kalamazoo, MI. Laboratory Study Number: 9142V100349ERB, November 10, 2011. MRID 48616404. Unpublished. 

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

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

Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. Julyl 9, 2012. 

MRID 48843501. Unpublished. 

SPONSOR: Gowan Company, 370 Main Street, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48616404), 

uterine cytosol from Sprague-Dawley rats was used as the source of estrogen receptors (ER) to 

conduct binding experiments. A competitive binding experiment was conducted to measure the 

binding of a single concentration of [3H]- l 7~-estradiol ( 1 nM) in the presence of increasing 

concentrations of EPTC (logarithmic increase from 10- 10 to 10- 3 M). DMSO was used as a 

vehicle at a final concentration of 4%. The assay included 19-norethindrone as a weak positive 

control, octyltriethoxysilane as a negative control, and 17-~-estradiol as the natural ligand 

reference material. 

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

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

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

(160 to 320 μg versus 50± 10 μg) . The mean dissociation constant (Kct) for [3H]-17~-estradiol 

was 0.331±0.061 nM and the estimated Bmax was 74.55±3.03 fmol/100 μg protein for the 

Page 118 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

prepared rat uterine cytosol. The Kd for each run was within the expected Guideline range of 

0.03 to 1.5 nM. 

In the competitive binding experiment, the mean IC50 was 9.3 x 10−10 M for the natural ligand, 

17β-estradiol, and 3.2 × 10−6 M for the weak positive control, which had a relative binding 

affinity (RBA) of 0.029%. Performance criteria were met with the exception of the Hill slope for 

17β-estradiol was outside the recommended range (−1.1 to −0.7) two for the runs (−1.4 and −1.7, 

respectively and the bottom plateau of the binding curve for 17β-estradiol for run 3 (5%) 

exceeded the recommended range (−4 to 1%). 

An estimated mean IC50 was not calculated for EPTC as it did not displace 50% or the 

radiolabeled ligand from the ER. In two assay runs, EPTC reduced the specific binding of the 

radiolabeled estradiol at the highest concentration tested (10−3 M) to 96-107%; in Run 3 percent 

binding was 69.6% at 10−3 M. Based on the results from the three runs, EPTC is classified as 

Not Interactive in the Estrogen Receptor Binding Assay. 

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

Assay (OCSPP 890.1250). 

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided. 

 

Page 119 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

Location: Kalamazoo, MI 

Study Director: Jamin A. Willoughby, Sr., Ph.D 

Other Personnel: Karen Rutherford, Director of Laboratory Operations 

David Blakeman, Senior Scientist/Endocrine Group Leader 

Cameron Haines, Scientist 

Steven McColley, Scientist 

Colleen Toole, Director of Project Management 

Study Period: May 25, 2011 - November 10, 2011 

2. Test substance: EPTC 

 Description: Liquid 

 Source: Gowan Company 

 Lot/Batch #: P1105096, Exp. Feb 4, 2015

 Purity: 99.9 % w/w 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 CAS #: 759-94-4 

 Molecular weight: 189.3 g/mol

 Structure: 

N

S

O 3CH

3CH

CH3

3. Non-labeled ligand: 17β-estradiol 

 Supplier: Sigma-Aldrich, St Louis, MO 

 Catalog # E8875 

 Batch #: 110M0138V 

 Purity: 100% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-estradiol 

 Supplier: Perkin-Elmer, Boston, MA 

 Catalog #: NET517 

 Batch#: 650702 

 Radiochemical purity: NR 

 Specific activity: 130.2 Ci/mol 

 Concentration of stock: 1 nM 

5. Positive control: 19-norethindrone 

 Supplier: Sigma-Aldrich, St Louis, MO 

 Catalog # N4128 

 Batch #: 030M1359V 

 Purity: 99% 

 CAS # : 68-22-4 

 

6. Negative control: Octyltriethoxysilane 

 Supplier: Sigma-Aldrich, St Louis, MO 

Page 120 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

 Catalog # 440213 

 Batch #: 24996KK 

 Purity: 99.34% 

 CAS # : 2943-75-1 

7. Solvent/vehicle control: DMSO 

 Justification for choice of solvent: Recommended solvent according to guideline, and EPTC is soluble up to 10-3M 

 Final Concentration: 4% 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): Rat uterine tissue was obtained from Harlan 

laboratories, from female Sprague-Dawley rats that were ovariectomized 7 days prior to 

being euthanized. Animals were 12-13 weeks old at the time of euthanasia. The uteri were 

frozen and shipped overnight on dry ice. Once at the lab, uteri were inspected for signs of 

residual ovarian tissue, weighed, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + 

PMSF (phenylmethylsulfonyl fluoride) buffer and used immediately. Uteri were 

homogenized in buffer, centrifuged for 10 min at 2500 × g at 4 oC. The resulting supernatant 

was transferred and centrifuged for 60 minutes at 105,000 × g, discarding the resulting 

pellets. Protein concentration of the cytosol was determined to be 3.22 mg/mL using a 

protein kit compatible with DTT in the TEDG buffer (e.g., BioRad Protein Assay Kit). 

Cytosol was divided into aliquots (1-6 mL) for immediate use or storage at -80 oC.

2. Saturation (Radioligand) Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-17β-estradiol was performed to demonstrate 

that the ER was present in reasonable concentrations and had the appropriate affinity for the 

native ligand (MRID 48843501). The conditions for the saturation binding are summarized 

in Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat uterine cytosol 

Concentration of radioligand (as serial dilutions) 0.03 to 3 nM 

Concentration of non-labeled ligand (100X [radioligand]) 3 to 300 nM 

Concentration of receptor Sufficient to bind approximately 25 

to 35% of radioligand at 0.03 nM

Temperature 4oC 

Incubation time 16 to 20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride (PMSF) 1 mM 

DTT 1 mM 

a Data were obtained from page 1 of the study report (MRID 48843501). 

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

H]-17β-estradiol 

(originally 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over 

time (adjusted specific activities were not reported), and serial dilutions in TEDG+PMSF 

buffer were prepared to achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, 

Page 121 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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, until a concentration was 

reached that bound approximately 25 to 35% of the total radioactivity added. The final 

protein concentrations were 320 μg, 192 μg and 160 μg per assay tube for the first, second 

and third saturation binding experiments, respectively (Note: typically 50 ± 10 μg protein 

per tube). Each assay consisted of three non-concurrent runs (conducted on August 5, 6, 

and 7, 2011, respectively). Each run included three replicates of each test substance at each 

concentration, resulting in the 72 samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned

 Assay Components 

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

350 μL 300 μL --- TEDG+PMSF buffer 

50 μL 50 μL 50 μL [

3

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

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

100x each respective labeled concentration)f

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

concentration) 

500 μL 500 μL 50 μL Total volume in each assay tube 

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

b Total binding = [3

H]-17β-estradiol bound to ER 

c Non-specific binding = [3

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

d Total [3

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

e Final concentrations of [3

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

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

Tubes were incubated with gentle vortexing for 17.5-19 hours at approximately 4C. To 

separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and 

vortexed (3 times with 5-minute intervals). Subsequently, the contents of each tube were 

washed three times as follows: 2-mL portions of TEDG+PMSF buffer were added, 

vortexed, centrifuged for 10 minutes at 1000 x g, and the supernatant decanted and 

discarded. After the final centrifugation, ethanol (1.5 mL) was added to the HAP pellet 

remaining in each tube to extract the [3

H]-17β-estradiol, followed by vortexing, and 

centrifugation for 10 minutes at 1000 x g. Aliquots (1 mL) of supernatant were 

radioassayed by scintillation counting. The temperature was maintained at approximately 

4oC throughout the assay prior to extraction with ethanol. 

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

competitive binding experiment is included in Table 3. 

Page 122 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a 

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 10-15% of radioligand b 

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

Temperature 4 ± 2 C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Protease Inhibitor 0.5% (v/v) 

DTT 1 mM 

a Data were obtained from page 15 of the study report. 

b Calculated by reviewer from data on pages 35, 38 and 41 

A solubility test was performed to determine the appropriate solvent (DMSO) using visual 

observation to examine for precipitation (use of magnification as not reported). On the day 

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

H]-17β-estradiol was adjusted for 

decay over time, and diluted in TEDG + PMSF buffer to achieve a final concentration of 

1 nM. For each batch of cytosol, the optimal protein concentration was determined by 

testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a 

concentration was reached that bound 10-15% of the total radioactivity added (80 μg protein 

per tube). Serial dilutions of EPTC, the weak positive control (19-norethindrone), the 

negative control (octyltriethoxysilane), and the reference material (non-labeled 17β-estradiol) 

were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three 

runs, and each run contained three replicates at each concentration, six replicates measuring 

total activity (50μL master mix) plus three measuring total binding (solvent control) resulting 

in a total of 306 samples. 

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

EPTC Weak Positive control Negative control Reference Chemical 

19-Norethindrone Octyltriethoxysilane Non-labeled 17β-estradiol 

Tubes 79-102 Tubes 31-54 Tubes 55-78 Tubes 1-30 

10−10 10−8.5 10−10 Solvent control/master mix c

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 24, 26, 28, and 35-43 of the study report. 

Page 123 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

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

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

cytosol (with ER), for a total of 500 μL. 

c Solvent is DMSO in Tubes 7-9. 

Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 C. To separate bound 

from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and the tubes were 

vortexed (3 times with 5-minute intervals). Subsequently, the contents of each tube were 

washed three times as follows: TEDG+PI buffer was added, vortexed, centrifuged for 10 

min at 1000 × g, and the supernatant decanted and discarded. Ethanol was added to the HAP 

pellet remaining in each tube to extract the [3H]-17β-estradiol, allowed to sit at room 

temperature for 15-20 min with vortexing, and centrifugation for 10 min at 1000×g. An 

aliquot of supernatant was radioassayed by scintillation counting. The temperature was 

maintained at 4±2 oC throughout the assay prior to extraction with ethanol. 

C. DATA ANALYSIS: For the saturation binding experiment, total binding and non-specific 

binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 

(GraphPad Software, Inc., La Jolla, CA), incorporating automatic outlier elimination 

according to the method of Motulsky and Brown (2006)1

 implemented by using the ROUT 

procedure in Prism v. 5 with a Q value of 1.0. Receptor binding data plots were corrected for 

ligand depletion with the method of Swillens (1995)2

. For the competitive binding assay, 

GraphPad Prism was used to generate nonlinear regression used to fit a curve (for 17βestradiol, the positive control, and the test substance) to the Hill equation formula, which 

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

competitive binding experiment (log IC50 and RBA), mean and standard deviation were 

calculated for each run and mean and standard error were calculated for the composite three 

runs using XLfit. 

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 above75% (i.e., 

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

binding curve cannot be fitted and the lowest average percent binding among 

concentration groups in the data is above 75%. 

 

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regression: a new 

method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123-142. 

2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for 

computer analysis. Molec. Pharmacol. 47(6):1197-1203. 

Page 124 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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 at which 50% of the radioligand is displaced from 

the receptor

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment 

parameters are presented in Table 5. The mean Kd for [3H]-17β-estradiol was 

0.331±0.061 nM, and the estimated Bmax was 74.55±3.03 fmol/100 μg protein for the 

prepared rat uterine cytosol. The Kd for each run was within the expected range of 0.03 

to 1.5 nM. Although the Scatchard plots fit straight lines to the data, the concavity 

observed in the data sets may indicate issues with ligand depletion. 

Page 125 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

TABLE 5. Saturation Binding Experiment of [3

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

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.976 0.982 0.980 0.976-0.982 

Bmax (nM) 0.148 0.102 0.080 0.110±0.035 

Bmax (fmol/100 μg protein) 69.25 79.76 74.65 74.55±3.03 

Kd (nM) 0.453 0.286 0.255 0.331±0.061 

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

b The range of R2

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

R2

 Goodness of fit for curve calculated for specific binding. 

Figure 1 illustrates the non-specific, specific, and total binding curves for [3H]-17β-estradiol to 

the ER 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]- 17β-estradiol to the ER. 

 

Page 126 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

Binding Experiment. 

Page 127 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-estradiol to the Estrogen Receptor. 

Page 128 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

binding experiments are summarized in Table 6 and presented graphically in Figures 3-5. 

EPTC had no effect on specific binding at concentrations of ≤10−3 M in Run 2 and at ≤10−4 in 

Runs 1 and 3. However, EPTC at 10-3 M reduced specific binding slightly in Run 1 (96.2%) 

and by 69.6% in Run 3. The estimated mean log IC50 was not calculated for EPTC as the 

percent displacement of labeled ligand binding did not reach 50% for any run. The mean 

IC50 for the positive control was 3.2 × 10−6

 M. The mean RBA was 0.029% for the weak 

positive control, and a RBA could not be calculated for EPTC. 

As EPTC displaced <25% of the radiolabeled estradiol at concentrations up to 10−3

 M in two 

runs (score of 0) and between 25 and 50% in the third run (score of 1), the overall 

classification for EPTC is not interactive (mean score of 0.3) in this assay (Table 7). 

TABLE 6. Competitive Binding Assay of EPTC with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean

r

2

 (unweighted), 17β-estradiol NR NR NR NR

Positive control NR NR NR NR

EPTC NR NR NR NR

Log IC50 (M), 17β-estradiol −9.0 −9.1 −9.0 −9.0 

19-norethindrone −5.6 −5.4 −5.5 −5.5 

EPTC NA NA NA NA 

IC50 (M), 17β-estradiol 1 x 10−9 7.9 x 10−10 1 x 10−9 9.3 x 10−10

19-norethindrone 2.5 x 10−6 4.0 x 10−6 3.2 x 10−6 3.2 x 10−6

EPTC NA NA NA NA 

Log (RBA) 19-norethindrone −1.4 −1.7 −1.5 −1.5 

 EPTC NA NA NA NA 

RBA (%), 19-norethindrone 0.040 0.020 0.032 0.029 

EPTC NA NA NA NA 

a Data were obtained from page 21 of the study report. IC50, RBA, and Log (RBA) were calculated by 

reviewer. 

r

2

 Goodness of fit 

RBA Relative binding affinity 

NR Not reported 

NA Not applicable

TABLE 7. Binding Classification of EPTC with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d

Classification category value b 0 0 1 0.3 Not Interactive 

a Data were calculated by the reviewer. 

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 

Page 129 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

EPTC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1 

Page 130 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

EPTC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 2 

Page 131 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

ETPC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 3

Page 132 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

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

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

TABLE 8. Performance Criterion a Tolerance 

Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge residual SD ≤2.35 1.20-1.95 X 

Top (% binding) 94 to 111 95-109 X 

Bottom (% binding) −4 to 1 −1 to 5 X 

Hill Slope (log10(M)-1) −1.1 to −0.7 −1.7 to −1.0 X 

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

Loge residual SD` NA 0.91-1.59 

Top (% binding) NA 49-100 

Bottom (% binding) NA −9 to 0 

Hill Slope (log10(M)-1) NA −2.5 to −0.9 

Solvent concentration

DMSO ≤10% 4% X 

Negative control (octyltriethoxysilane) does not displace more 

than 25% of [3

H]-17β-estradiol from the ER on average across all 

concentrations 

≤25% <4% X 

a Data were obtained from pages 25, 27 and 29 of the study report. 

b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were 

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

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

NA Not applicable 

The performance criteria for the competitive binding assay were generally met with the 

exception two parameters for the reference chemical 17β-estradiol: The bottom plateau (5%) 

for the third run exceeded the recommended range (−4 to 1%) and the Hill slope fell outside 

the recommended range (−1.1 to −0.7) for runs 2 and 3 (−1.4 and −1.7, respectively). 

EPTC was tested over a concentration range that fully defined the top of the curve. The 

maximum percent binding 97.3-108.2% was within 25 percentage points of the lowest 

concentration of the estradiol standard 95.1-107.4%. Examination across the runs indicated 

that the Hill slope for 17β-estradiol decreased with each run and the Hill slope for 19-

norethindrone was much higher in the second run than the first or third. Placements along 

the X-axis, and top and bottom plateaus were consistent across runs. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In the first independent run the mean specific 

binding was >96% at every soluble concentration tested to EPTC. In the second independent 

run the mean specific binding was <101% at every soluble concentration tested. In the third 

independent run the mean specific binding for EPTC at 10−3 M was 69.6% of control. 

Since EPTC was classified as "not interactive" for the first two independent runs and 

"equivocal" in the third run, EPTC has a final classification of not interactive. 

Page 133 of 310
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 17 

EPTC/ 041401 OCSPP 890.1250/ OECD None

B. AGENCY COMMENTS: In the saturation binding experiment, the protein concentrations 

used varied greatly between each run, and were approximately 3- to 6-fold greater than 

recommended. The mean Kd for [3H]-17β-estradiol was 0.331 nM and the estimated Bmax

was 74.55 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd for each run was 

within the expected Guideline range of 0.03 to 1.5 nM. 

For the competitive binding experiment, EPTC reduced the specific binding only at the 

maximum concentration tested (10−3 M) in Runs 1 (96.2%) and Run 3 (69.6%). The 

estimated mean IC50 was not calculated for EPTC as it did not achieve 50% binding. The 

mean IC50 for the positive control was 3.2 × 10−6 M. The mean RBA was 0.029% for the 

weak positive control. 

In two assay runs, EPTC reduced the specific binding of the radiolabeled estradiol at the 

highest concentration tested (10−3 M) from 95-107% in Runs 1 and 2 and in Run 3 the 

percent binding was reduced to 69.6% at 10−3 M. EPTC was classified as not interactive in 

in the first two runs and equivocal in the third run. Overall, EPTC is classified as not 

interactive in this assay. 

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

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

 The weak positive control, 19-norethindrone, showed a top plateau of 49% binding in the 

second assay. The Reviewer noted that the weak positive control data were well outside 

the guidelines for acceptance, however, in the same run the positive and negative controls 

were within the acceptable criteria. 

Page 134 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616405) 

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: 1/6/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 1/18/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/23/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/23/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 135 of 310
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 136 of 310
EPTC/ 041401 

Estrogen Receptor Transcriptional Activation (2011) I Page I of 10 

OCSPP 890.1300/ OECD 455 

Primary Reviewer: =S=h=ei=la"--=H=e=al"-"'"y_,_,-=-P=h=.D=-·=---------S ig ~ ur// : /l/1A ) :;;---

Health Effects Division Date: ~ fJJ!?];?> 

Secondary 

Health Effects 

Reviewer: 

Division 

Minerva Mercado-Feliciano, Ph.D. DABT Signature:

Date: e--

~~ 

6 -,-'t_cz=J!E- Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455. 

PC CODE: 041401 DP BARCODE: D397177 

TXR#: 0051762 CAS No.: 759-94-4 

TEST MATERIAL (PURITY): EPTC 99.9% a.i. 

SYNONYMS: S-ethyl N,N-dipropylcarbamothioate 

CIT A TI ON: Willoughby, J .A. (2011 ). EPTC: Estrogen Receptor Transcriptional Activation 

(Human Cell Line (HeLa-9903)). CeeTox, Kalamazoo, MI. Laboratory report 

number 9142V-100349ERTA, Nov 10, 2011. MRID 48616405. Unpublished. 

SPONSOR: Gowan Company, 370 S. Main Street, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation (ERTA) assay 

(MRID 48616405), hERa-HeLa-9903 cells cultured in vitro were exposed to EPTC (99.9% a.i., 

Batch # Pl 105096) at logarithmically increasing concentrations from 10- 10 M to 10- 3 Min 

DMSO (0.1 %) for 24 hours. The experiments were performed using 96-well plates and each 

EPTC concentration was tested in 6 replicates/plate. Cells were exposed to the test agents for 24 

± 2 hr to induce reporter (luciferase) gene products. Luciferase expression in response to 

activation of the estrogen receptor was measured using a proprietary method. 

EPTC was tested up to the guideline maximum concentration of 10-3 M. No cytotoxicity was 

observed at any concentration of EPTC tested. The mean RPCMax for EPTC was 4.1 % for the 

first assay and 3.6% for the second assay, and the associated PCMax was 10- 5 and 10-6 M, 

respectively. The reference chemicals 17~-estradiol and 17a-estradiol all performed within the 

acceptable range. However, cytotoxicity was observed at the highest concentrations of 

corticosterone (10-4 M) and 17-a methyl testosterone (10-5 M) in both assays. 17amethyltestosterone did not perform as expected, having RPCMax of 13.7% in the first assay and 

6.2% for the second assay; Log PCso and Log PC10 could not be calculated. 

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

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

Page 137 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

very weak activity, EPTC responses were similar to those of the negative control corticosterone 

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

maximum of 6.2-13.7% PC. EPTC was only able to reach a maximum of 3.6-4.1% PC when 

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

PC10 in both assay runs, EPTC was considered negative for estrogen receptor transcriptional 

activation in this test system. 

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor 

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided. 

 

Page 138 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS 

A. MATERIALS 

1. Test Substance: EPTC (S-ethyl N,N-dipropylcarbamothioate) 

 Description: Liquid, mw 189.3 g/mol 

 Source: Gowan Company 

 Lot/Batch #: P1105096, Exp. Feb 4 2015

 Purity: 99.9 % w/w 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 Vapor pressure: NA 

 CAS #: 759-94-4 

 Structure: 

N

S

O 3CH

3CH

CH3

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 58-18-4 

 

3. Vehicle(s)

 Solvent: DMSO, Supplier and lot not reported 

 Solvent control (final concentration): 0.1% 

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma 

infection using a DNA fluorochrome procedure. Cells were maintained in Eagles Minimum 

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

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

Page 139 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

under 5% CO2 at 37oC. Upon reaching 75-90% confluence, cells were subcultured prior to 

exposure to the test material. Cells used in the range finding study were passage 21. New 

cell vials were brought up from cryopreservation and maintained for the two valid runs 

(8/23/2011 and 8/25/2011) and were passages 19 and 20 prior to seeding into plates. 

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 

approximately 1 × 104 cells/100 μL medium/well in a 96 well plate and allowed to attach for 

at least 3 hours. The growth media was replaced with media containing serial log dilutions 

of EPTC in DMSO. Cells were incubated for 24 ± 2 hours at 37±1°C. The total final 

concentration of DMSO was 0.1%. Cytotoxicity was determined by propidium iodide (PI) 

uptake, with 125μM digitonin as a positive control for cytotoxicity. 

It was stated that the transcriptional activation of the estrogen receptor was determined as 

described in CeeTox Standard Operating Protocol (SOP) 2041. A list of reagents was 

provided, but the assay reagent was classified as proprietary information, and the SOP was 

not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10−3

 to 

10−6.5 M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. Solubility was determined by 

visual inspection of the test chemicals and controls final concentrations in culture medium. 

b. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, 

April 12, and April 28, 2011 (MRID 48843501), using cells at Passage 15, 25 and 28, 

respectively. Based on the assay dates and cell passage numbers, it is unlikely that the cells 

used for proficiency testing are from the same frozen stock as the cells used in the main 

assay. The cells were tested using the following set of proficiency chemicals, and each 

chemical was tested in duplicate on separate days: 

Compound CAS No. Concentration 

Range (M) 

Expected 

Response a Notes 

Diethylstilbestrol (DES) 56-53-1 10−14 to 10−8 Positive --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-hydroxyphenyl)benzene c 15797-52-1 10−12 to 10−5 Positive Cytotoxic at 100μM. PCmax approx. 

50% of PC. Binds to hERα and has 

ER antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that 

should be the highest tested 

concentration 

a Positive = RPCmax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCmax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

c Compound selected to challenge solubility and cytotoxicity. 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

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

therefore unacceptable. 

 

Page 140 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included on separate 

plates in the current assay: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−14 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen 

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

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive 

control (PC), the luminescence signals from the concurrent plate were analyzed by 

subtracting the mean value of the vehicle control from each well value to normalize the data; 

each normalized value was then divided by the mean value of the normalized PC. The 

resulting value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. The maximum response relative to the positive control (RPCmax) for 

EPTC was calculated, but the software used was not reported. The test material was defined 

as negative for inducing estrogen receptor transcriptional activation if the RPCMax < PC10 in 

at least 2 of 2 (or 2 of 3) runs. LogEC50 and Hill slope values are calculated only if a 

positive response is observed. Coefficients of variation (CV) were calculated for the 

luminescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of 

insolubility were excluded from analyses. 

4. Definitions 

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

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Precipitation was not observed in any of the EPTC solutions. The results of the 

cytotoxicity test are presented in Table 1. No cytotoxicity was observed for EPTA at any 

concentration tested. Based on these results, concentrations of 10 −10 M to 10−3

 M were 

selected for the assay. 

Page 141 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

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

Concentration (M) % Viability b Comments 

10−3 82 

No precipitation observed for any concentration 

10−3.5 87 

10−4 91 

10−4.5 92 

10−5 97 

10−5.5 97 

10−6 97 

10−6.5 99 

E2 1nM NR 

VC b NR 

a Data were obtained from page 18 of the study report. 

b VC = Vehicle control 

B. Positive and Negative Reference Chemicals 

1. Proficiency Chemicals: The laboratory proficiency assays using the required reference 

compounds were not included in the original study report, but were provided to the Agency 

at a later date (MRID 48843501). The responsiveness of cells to the required proficiency 

chemicals was performed in duplicate on different days for each chemical. The reported 

responses are in Table 2. 

TABLE 2. Proficiency Chemicals 

Compound Expected Response Lab Response 

Run 1 Run 2 Run 3 

Diethylstilbestrol Positive Positive Positive NA 

17α-Ethynyl estradiol Positive Positive Positive NA 

Hexestrol Positive Positive Positive NA 

Genistein Positive Positive Positive NA 

Estrone Positive Positive Positive NA 

Butyl paraben Positive Positive Positive NA 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive 

Dibutyl phthalate Negative Negative Negative NA 

Atrazine Negative Negative NA Negative 

Corticosterone Negative Negative NA Negative 

NA = not applicable. The chemical was not tested at this time. 

2. Reference Chemicals: Values derived from the concentration response curve (e.g., Log

PC50, Log PC10, Log EC50, Hill slope) for the four concurrently run reference materials are 

included in Table 3. The reference chemicals 17β-estradiol and 17α-estradiol all performed 

within the acceptable range. However, cytotoxicity was observed at the highest 

concentrations of corticosterone (10−4 M) and 17-α methyltestosterone (10−5 M) in both 

assays. The Log PC50 and Log PC10 for 17α-methyltestosterone were not calculated. Visual 

inspection of the raw data indicated that 17α-methyltestosterone had an RPCMax of 13.7% in 

the first assay and 6.2% for the second assay. The PC10 for 17α-methyltestosterone was not 

calculated, and no reason was given for not calculating it despite reaching >10% PC. The 

Reviewers assume it was because higher concentrations did not result in increased 

transcriptional activation, as would be expected. Based on this, a PC10 and PC50 could not 

be calculated for 17α-methyltestosterone. It was stated that these deviations from the ranges 

suggested in the OCSPP guideline were minor and not considered to impact the 

interpretation of results. 

Page 142 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −10.4 −10.2 X 

 Log PC10 <−11 −11.3 −11.3 X 

 Log EC50 −11.3 to −10.1 −10.3 −10.2 X 

 Hill Slope 0.7 to 1.5 1.1 1.1 X 

 Test range 10−14 to 10−8 M 10−15 to 10−8 M 10−15 to 10−8 M X 

17α-estradiol

 Log PC50 −9.6 to −8.1 −8.4 −8.6 X 

 Log PC10 −10.7 to −9.3 −9.5 −9.6 X 

 Log EC50 −9.6 to −8.4 −8.4 −8.6 X 

 Hill Slope 0.9 to 2.0 1.2 1.5 X 

 Test range 10−12 to 10−6 M 10−13 to 10−6 M 10−13 to 10−6 M X 

Corticosterone

 Test range 10−10 to 10−4 M 10−11 to 10−4 M 10−11 to 10−4 M X 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 NC NC X 

 Log PC10 −8.0 to −6.2 NC NC X 

 Test range 10−11 to 10−5 M 10−12 to 10−5 M 10−12 to 10−5 M X 

a Data were obtained from page 21 of the study report. 

NC = not calculable. 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The overall mean TA value, expressed in arbitrary light units, for the vehicle 

control was 9779 for the first assay and 11742 for the second assay. The mean value for the 

positive control was 205092 for the first assay and 187875 for the second assay. The PC50

(50% of the maximum response) for E2 in this assay is 107435 for the first assay and 99808 

for the second assay and the PC10 (10% of the maximum response) is 29310 for the first 

assay and 29355 for the second assay.

 TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a 

Sample Vehicle Control Positive Control b 

Runs Mean SD Mean SD Fold Induction c 

1 9779 1490 205092 40377 21 

2 11742 1469 187875 66332 16 

a Data were obtained from pages 29-30 of the study report. 

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation are presented in Figure 

1 below. The mean RPCMax was 4.1% at 10−5

 M for the first assay and 3.6% at 10−6 M for 

the second assay. However, the lack of responsiveness of the assay to the weak agonist 

17α-methyltestosterone precluded the detection of a weak agonist. 

Page 143 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

TABLE 5. Relative Transcriptional Activation (RTA) of EPTCa 

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 2 

Conc. (M) Mean SD Mean SD 

10−3 3.0 1.2 2.6 1.2 

10−4 3.3 1.2 3.3 1.5 

10−5 4.1 1.2 3.4 0.4 

10−6 3.8 1.3 3.6 1.5 

10−7 0.5 1.5 1.4 0.7 

10−8 1.4 1.4 1.4 1.7 

10−9 0.4 1.4 0.7 1.6 

10−10 0.4 0.9 1.0 1.3 

Log EC50b NA NA 

Hill Slopeb NA NA 

RPCMax 4.1% 3.6% 

PCMax 10−5 10−6 

PC50 NA NA 

PC10 NA NA 

a Data were obtained from pages 19 and 20 of the study report. 

NA= Not Applicable 

Page 144 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of EPTC. 

3. Performance Criteria: The laboratory proficiency assays using the required reference 

compounds were not included in the study report and were not available to the reviewer. 

17β-estradiol and 17α-estradiol all performed within the acceptable range. However, 

cytotoxicity was observed at the highest concentrations of corticosterone (10−4 M) and 

Page 145 of 310
Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10 

EPTC/ 041401 OCSPP 890.1300/ OECD 455 

17-α methyltestosterone (10−5 M) in both assays. The Log PC50 and Log PC10 for 

17α-methyltestosterone was not calculated. Visual inspection of the raw data indicated that 

17α-methyltestosterone had an RPCMax of 13.7% in the first assay and 6.2% for the second 

assay. In the first assay, 17α-methyltestosterone induced transcriptional activation greater 

than 10% PC at 10-8 M, which is within the range of acceptable PC10 values; however higher 

concentrations did not result in further increased activity. The second assay shows a dosedependent increase in luciferase activity from 17α-methyltestosterone, however while 

maximum induction does reach the acceptable range of PC10 values it does not reach the 

acceptable range of PC50 values. Because of this, proper PC10 and PC50 values could not be 

calculated for 17α-methyltestosterone. Triplicate samples of the PC and the VC were 

included in each plate, and the mean luciferase activity of the PC was greater than 4-fold 

that of the mean vehicle control on each plate. Based on calculations performed by the 

Reviewers, the fold-induction corresponding to PC10 of concurrent PC was greater than 1+2 

standard deviations of the fold-induction value of the concurrent VC, as expected. There 

was some inter- and intra-assay variability, with some instances of the CV being greater 

than 20%, indicating an unreliable calculation of the PC10. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of EPTC for use in 

the transcriptional activation assays was 10−3 M. Cytotoxicity (>20% reduction in cell 

viability) was not observed at any test concentration of EPTC. In two valid independent 

runs of the transcriptional activation assay, EPTC did not result in an increase in luciferase 

activity at any of the viable concentrations tested (RPCmax<10%). The highest mean 

induction value was 4.1% compared to vehicle control. EPTC was tested across a soluble 

concentration range (10−10 to 10−3 M) and was determined to not be an agonist of human 

estrogen receptor alpha (hERa) using the HeLa-9903 cell line. 

B. AGENCY COMMENTS: EPTC was tested up to the guideline maximum, 10−3 M. No 

cytotoxicity was observed at any concentration of EPTC tested. The mean RPCMax was 

4.1% at 10−5

 M for the first assay and 3.6% at 10−6 M for the second assay. Although the 

conditions of this assay were not optimal to detect very weak activity, EPTC responses were 

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

of 17α-methyltestosterone, which was able to reach a maximum of 6.2-13.7% PC. EPTC 

was only able to reach a maximum of 3.6-4.1% PC when tested up to the highest 

concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay 

runs, EPTC was considered negative for estrogen receptor transcriptional activation in this 

test system. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

 17α-methyltestosterone did not induce transcriptional activation equal to 50% or 10% 

of the positive control in either run; therefore a PC10 and PC 50 could not be calculated. 

This indicates weak responsiveness of the assay to the very weak agonist. 

 Cytotoxicity was observed in the reference chemicals 17α-methyltestosterone and 

corticosterone; cytotoxicity should not occur in any of the recommended reference 

chemical concentrations. 

Page 146 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 1 of 89 

Data Requirement: EPA DP Barcode 402838 

OECD Data Point 229 

EPA MRID 48827602 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: EPTC Purity: >99.9% 

Common name 

Chemical name: IUPAC: S-ethyl dipropyl(thiocarbamate) 

 CAS name: 

 CAS No.: 759-94-4 

 Synonyms: Eptam® Technical. 

 EPA PC Code: 041401 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 9/25/12 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc Date: 9/27/12 

Primary Reviewer: Frank T. Farruggia, Ph.D. Date: 3/22/13 

EPA/OPP/EFED/ERB3 

Secondary Reviewer(s): Amy Blankinship Date: 6/5/15 

EPA/OPP/EFED/ERB3 

Date Evaluation Completed: 6/5/15

CITATION: York, D.O. 2012. EPTC - Short-term Reproduction Assay with the Fathead Minnow (Pimephales promelas). 

Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 12791.6156. Submitted by Gowan 

Company, Yuma, Arizona. Completion date May 4, 2012. 

FRANK 

FARRUGGIA

Digitally signed by FRANK FARRUGGIA 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=FRANK 

FARRUGGIA, dnQualifier=0000048865 

Date: 2015.06.11 08:16:39 -04'00'

AMY 

BLANKINSHIP

Digitally signed by AMY BLANKINSHIP 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, dnQualifier=0000040917 

Date: 2015.06.15 17:34:16 -04'00'

Page 147 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 2 of 89 

Note: 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). 

Guideline recommendations are provided in italics; these recommendations should remain visible in the 

completed DER. 

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

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

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

Page 148 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 3 of 89 

EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of EPTC with fathead minnows (Pimephales promelas) was 

conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each 

group; 6 months old) were exposed to EPTC (>99.9% purity) at nominal concentrations of 0 (control), 0.08, 

0.80, and 8.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.014), 0.069, 0.69, and 6.9 mg 

a.i./L. The test system was maintained at 24 to 25o

C and a pH of 6.7 to 7.4. 

Mean survival was 100, 100, 75, and 13% for males and 94, 100, 38 and 6% for females in the control, low, 

mid, and high treatment groups, respectively; overall survival (both sexes) was 96, 100, 50, and 9% 

respectively. Due to the high mortality at the high concentration, these data were not used for statistical 

analysis. Mortality was significantly different in the mid and high treatment groups compared to the negative 

control (p<0.05; Fisher’s Exact Test). There were no notable observations with regards to behavior or changes 

in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad, in the control or 

treated groups. There were no notable observations of secondary sex characteristics or clinical signs for any 

treatment group compared to the controls. Male and female growth parameters (wet weight and total length) 

were not significantly different from the control. 

Spawning in each replicate occurred at least every 4 days and mean fecundity in controls was 22 

eggs/female/day/replicate (range 18-25 E/F/D); fertilization success in the control group averaged 96%. 

Fecundity was significantly (p<0.05; Dunnett’s) reduced by 75% at the mid treatment concentration. 

There were no significant differences between controls and the low and mid treatment groups for male or 

female gonado-somatic index (GSI). Male tubercle scores was marginally significantly (p=0.057; MannWhitney) reduced compared to the control (26 vs 41 in control) at the 0.69 mg a.i./L treatment level and 

was approaching significance (p=0.086) in the 0.069 mg ai/L treatment level (32 vs. 41); none of the 

surviving females were found to have tubercles. 

There were no treatment-related histopathologic observations in males. In female fish, there was an increase 

in incidence of minimal to moderate oocyte atresia at the mid treatment level compared to the control; however, 

the smaller sample size (n=6) prevented further interpretation of this finding. There were no treatment-related 

effects at any test level for gonadal staging. There were no other significant effects on other endpoints. For 

Page 149 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 4 of 89 

male vitellogenin (VTG), the reported control values were unusually high (comparable to control female values) 

even after re-analysis; the values in the other treatment groups were also comparable to female VTG values. 

No obvious reasons for these high values were reported and as such, these data are not considered reliable 

for interpretation. Sex steroids were not measured in this study. 

The validity criteria were met except that dissolved oxygen fell slightly below the recommended 60% of 

saturation (58%) in one test chamber; the chambers were cleaned and the levels returned to above the 

recommended value, and this deviation did not sufficiently impact the interpretation of the study. 

In regards to male VTG, the inability to evaluate this endpoint is a major limitation in this study. However, it is 

noted that there were no effects on female VTG or male GSI or histopathology. This information along with 

the information provided in the remaining Tier 1 battery were considered collectively for evaluating potential 

effects in male fish from EPTC exposure. Based on this information, additional information regarding impacts 

to male VTG is not requested. 

As such, this assay satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction 

Assay (OCSPP Guideline 890.1350). 

Results Synopsis

Test Organism age at test initiation: 6 months 

Mean body weight at test initiation (if measured): Male 2.9 g; Female 1.6 g 

Mean length at test initiation (if measured): Not reported. 

 Test Type (Flow-through, Static, Static Renewal): Flow-through

Page 150 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 5 of 89 

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

Treatment 

 (mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

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

M F M F M F M4 F M F M F 

0.069 No No Yes No No No No No NRL No NA NA NA NA 

0.69 Yes No No No No No No No NRL No NA NA NA NA 

6.93 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable, NRL Not reliable,. 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 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 4 Due to unusually high control male VTG values, these data are not considered reliable for interpretation.

Page 151 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 6 of 89 

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. The unionized ammonia and residual chlorine in the test water were not 

reported. The OCSPP 890.1350 performance criteria establish maximum levels 

for these values and it is unclear if the maximum recommendations were 

exceeded.

2. The dissolved oxygen fell to 58% of saturation in the control replicate B on Day 

10 of the exposure which is slightly below the recommended 60% of saturation. 

The test chambers were cleaned and the levels were above 60% by the next 

day. 

 

 These deviations do not have an impact on the acceptability of this study. 

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

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

pertinent U.S. EPA Good Laboratory Practice regulations with the following 

exceptions: routine dilution water and food contaminant screening analyses 

were conducted following standard validated methods, this exception has no 

impact on the study results. 

A. TEST MATERIAL: EPTC; CAS# 759-94-4.

Description: Not reported.

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. : P1105096 

Page 152 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 7 of 89 

Purity: >99.9% 

Impurities: None reported

Stability of Compound: Analytical verification of the test material from time 0, 6, 13, and 21 days 

yielded recoveries of 78.8% to 91.3% of nominal concentrations. Stock solution fortified with EPTC 

at concentrations of 0.04, 0.8, and 8.0 mg a.i./L yielded recoveries of 83.6 to 107%. There was 

no pattern of decline during the study period, indicating the test material was stable under the test 

conditions. 

Storage Conditions of Test Chemicals: Stored at room temperature in dark, ventilated cabinet. 

Page 153 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 8 of 89 

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 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: 13 days EPA recommends a minimum two-week 

acclimation period. Note that the 

acclimation period is different from the 

subsequent, in situ pre-exposure phase. 

Page 154 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 9 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: 1 mortality (<1%) Mortalities during the 7 days prior to 

pre-exposure period were <1%. Fish did not receive any treatment for disease during acclimation period. Behavioral abnormalities or clinical signs were not reported.

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be 

less than 5% of the culture population. If 

mortality during these 7 days is greater than 

10%, EPA recommends that the fish be 

rejected. If mortality is between 5-10%, 

EPA recommends that fish be held another 

7 days. If mortalities greater than 5% occur 

during this extended acclimation period, 

EPA recommends that the fish not be used. 

Type of food: Frozen brine shrimp EPA recommends that fish be fed frozen 

brine shrimp twice per day to promote 

active reproduction and maintain body 

condition. 

Source of food: Not reported Representative samples of the food source 

were periodically analyzed for pesticides, PCBs, and toxic metals by GeoLabs, Inc. and analyte levels were below LOC specified by ASTM (2005).

Frequency of feeding: 2 times/day Details on feeding: None. 

Page 155 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 10 of 89 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 6 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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

2.9 g Based on 20 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): 

2.4 to 3.4 Individual weights within ± 20% of the 

estimated mean. 

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

1.6 g Based on 20 females used to stock 

aquaria for pre-exposure phase. 

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

1.3 to 2.0 Individual weights within ± 26% of the 

estimated mean. 

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

Not reported 

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

Not reported 

Page 156 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 11 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Duration of pre-exposure phase: 13 days EPA recommends a minimum of 14 days. 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: 36 Extra replicates were established to 

account for a potential lack of spawning in some chambers and/or mortality during this phase. 

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. 

Page 157 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 12 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of spawns during preexposure: >two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the 

definitive test. 

Details on pre-exposure: None. 

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 flowthrough 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). 

Page 158 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 13 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system: 

Intermittent flow proportional diluters 

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: Ca. 45 mL/minute or 2.7 

L/hr and 6.6 volume replacements/day. 

Calculated by the reviewer based on 0.5 L/cycle, 132 cycles/24 hrs and 60 minutes/hr.

Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

Details on toxicant mixing for flow-through systems: 

Flow-splitting chambers were used between diluter cells and the 4 replicate test vessels. Treatment recoveries were not measured prior to test initiation to indicate if diluter had reached equilibrium. Flow splitting accuracy not reported. 

Recommended toxicant mixing for flowthrough 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? Not reported EPA recommends aeration if dissolved 

oxygen reaches <4.9 mg/L (<60% 

saturation).

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

Page 159 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 14 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Well water EPA recommends natural or reconstituted 

water; it is recommended that natural 

water be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants. OECD accepts 

any water in which the test species show 

control survival at least as good as 

indicated in the test guideline.

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

Yes 

Test vessel type/materials: Glass aquaria with 

silicone sealant and nylon. 

EPA and OECD recommend that watercontact 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: 20 cm wide x 39 cm 

long 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).

Page 160 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 15 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: 4-inch diameter aged 

PVC cut in half (arches) and into 7 cm sections. 

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 7 cm sections. 

Each substrate placed on screened glass collection tray (13 x 11 x 3 cm, L x W x H), covered with 0.5 cm2 stainless steel 

mesh. 

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 25 24.5 1 Continuously 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 6.7 7.4 7.1 1 Daily EPA recommends pH 6.5 to 9.0.

Page 161 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 16 of 89 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Dissolved oxygen (mg/L) 4.8 8.1 6.5 1 Daily EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 

20 26 23 1 Weekly EPA recommends total alkalinity >20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 60 72 66 1 Weekly Total organic carbon (mg/L) 0.11 0.16 0.14 Monthly 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 <10 Pg/L. 

Page 162 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 17 of 89 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Conductivity (Pmhos/cm) 330 450 390 1 Weekly 

General recommendations for frequency 

of measurements: EPA recommends that 

temperature, pH, and dissolved oxygen 

be measured in all test tanks at least 

weekly and that hardness and alkalinity 

be measured in controls and in one tank 

at the highest test concentration at least 

weekly. In addition, continuous 

temperature monitoring of at least one 

tank is encouraged. 

Abbreviations: NA Not applicable. 1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels. 

Page 163 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 18 of 89 

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? Yes 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 rangefinder? 

0 (control), 2.5, 5.0, 10, 20, and 40 mg a.i./L

EPA recommends that the highest test 

concentration be selected based on toxicity 

data for other fish studies or species, if 

available. Otherwise, either the solubility 

limit of the test compound or 100 mg/L 

(whichever is lower) is appropriate.

Species: Pimephales promelasLife stage: Not reported 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: 96 hours EPA recommends a 96-hour exposure. 

Page 164 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 19 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: At test termination, 

17% and 100% mortality was observed in the 20 and 40 mg a.i./L treatment level. 

EPA recommends conducting a range-finder 

with five test concentrations plus a control 

(six total treatment levels), with four 

females and two males per exposure tank 

(36 fish total). The number of mortalities 

that occur may be used to develop a 

concentration-response curve. 

Based upon the results, the highest 

concentration that does not result in 

increased mortality or signs of overt 

morbidity compared to controls, or 1/3 the 

derived 96-hr LC50, may be selected as the 

highest exposure concentration in the 21-

day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

Page 165 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 20 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

The acute toxicity 96-hr LC50

value with fathead minnows was reported determined to be 25 mg a.i./L (95% CI of 20 to 31 mg a.i./L). Nominal concentrations selected based on range finding study results and consultation with sponsor. 

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., 1/3 the 96-hour LC50), whichever 

concentration is lowest.

Reference study citation (if applicable):

Not reported 

Separation of test concentrations:

0 (control), 0.8, 0.80, and 80 mg a.i./L 

EPA suggests that a concentration 

separation of between 0.33 (or three-fold) 

and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

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

concentrations and a control, plus solvent 

control if appropriate.

Page 166 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 21 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean measured 

Limit of quantification (LOQ): <0.014 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, 6, 13, and 21 days It is recommended that test item 

concentration be measured prior to the 

addition of fish in all tanks and at least 

weekly thereafter in two replicates per 

treatment level.

Page 167 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 22 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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 preexposure fecundity, and the tanks with the 

highest fecundity are randomly assigned to 

a definitive test treatment and block first. 

Each block contains one replicate of each 

treatment, including controls. 

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is 

used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Page 168 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 23 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

that solvent have no effect on survival nor 

produce any other adverse effects and that 

concentration not be greater than 0.1 ml/L4.

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

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

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

Toxicology, 76, pp.69–92. 

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

Assessment. No. 23. Paris, France. 

Page 169 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 24 of 89 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Light intensity at water’s surface: 

560-980 lux Light was provided by Sylvania 

Oktron® fluorescent bulbs and 

intensity was measured with a VWR light meter.

EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: All exposure solutions were 

reported as clear and colorless with no undissolved test substance observed. 

Page 170 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 25 of 89 

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0 <LOQ NA 

EPA recommends that test item 

concentrations be maintained at a coefficient 

of variation (CV) 20%. Treatment 1 0.08 0.069 1.7 

Treatment 2 0.8 0.69 6.2 Treatment 3 8.0 6.9 2.1 Diluter Stock NA NA NA 

Abbreviations: CV Coefficient of variation. LOQ=0.014 mg a.i./L. E. Observations

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

secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology, and plasma VTG were evaluated. 

Page 171 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 26 of 89 

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 17ȕ-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad 

weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. 

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior. 

I. RESULTS AND DISCUSSION 

 For all endpoints, the high treatment level was not statistically analyzed due to high mortality. A. Results 

Mean male survival values were 100, 100, 75, and 13% and female survival values were 94, 100, 38 and 6% in the mean-measured <LOQ (control), 0.069, 0.69, and 6.9 mg a.i./L treatment levels, respectively (Table 9). Overall survival (both sexes) was 96, 100, 50, and 9% in the mean-measured <LOQ (control), 0.069, 0.69, and 6.9 mg a.i./L treatment levels, respectively. 

Page 172 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 27 of 89 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival N1 # Surviving % Survival 

Control (<LOQ) 8 8 100 16 15 94 

0.069 8 8 100 16 16 100 

0.69 8 6 75 16 6 38 

6.92 8 2 13 16 1 6 

Abbreviations: NA Not applicable. 1 Total number of fish at test initiation. 2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L. 

Page 173 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 28 of 89 

Mean male body weight values were 3.73, 3.53, and 3.52 g and mean female body weight values were 1.39, 1.43 and 1.64 g in the meanmeasured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 65.7, 64.0 

and 64.4 mm and female body length values were 50.7, 51.6 and 52.0 mm in the mean-measured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively. 

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Control (<LOQ) 4 3.73 0.18 4 1.39 0.17 4 65.7 1.4 4 50.7 1.1 

0.069 4 3.53 0.61 4 1.43 0.11 4 64.0 4.8 4 51.6 0.66 

0.69 4 3.52 0.22 4 1.64 0.11 4 64.4 2.8 4 52.0 0.51 

6.91 4 NA NA 4 NA NA 4 NA NA 4 NA NA 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 1 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L. 

Page 174 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 29 of 89 

Mean fecundity values were 22, 27 and 5.6 and fertilization success was 96, 96, and 85 in the meanmeasured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 11). 

Table 11: Fecundity and Fertilization Success in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 Fertilization Success (%)2

Mean ±SD Mean ±SD 

Control (<LOQ) 22 3.0 96 1.4 

0.069 27 5.0 96 1.8 

0.69 5.6 3.1 85 20 

6.93 NA NA 

Abbreviations: NA Not applicable. ND Not determined. 

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. 

3

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 175 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 30 of 89 

Median male tubercle scores were 41, 32 and 26 in the mean-measured <LOQ (control), 0.069, and 

0.69 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) 

[mean-measured]

Males Females 

n 

Median 

Tubercle Score 

n 

Median 

Tubercle Score 

Control (<LOQ) 4 41 4 0 

0.069 4 32 4 0 

0.69 4 26 4 0 

6.91 4 NA 4 NA 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 

1

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 176 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 31 of 89 

Mean male GSI was 1.5, 1.2 and 1.9 and mean female GSI was 13, 12 and 16 in the meanmeasured <LOQ (control), 0.069, and 0.69 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) 

[mean-measured]

Males Females 

n 

Mean GSI1

(%) 

±SD n 

Mean GSI1

(%) 

±SD 

Control (<LOQ) 4 1.5 0.32 4 13 0.80 

0.069 4 1.2 0.29 4 12 2.2 

0.69 4 1.9 0.17 4 16 1.7 

6.92 4 NA NA 4 NA NA 

Abbreviations: NA Not applicable. 

1

 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 

100. 

2

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 177 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 32 of 89 

Median male and female gonadal stages were 2, 2 and 3 in the mean-measured <LOQ (control), 0.069, 

and 0.69 mg a.i./L treatment levels, respectively (Table 14). The study authors did not indicate treatmentrelated affects at any test level for gonadal staging. 

Table 14: Gonadal Staging in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Control (<LOQ) 8 2 15 2 

0.069 8 2 16 2 

0.69 6 3 6 3 

6.93 NA NA NA NA 

Abbreviations: J Juvenile. NA Not applicable. ND Not determined. UTS Unable to stage. 

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. 

3

 The one male and one female were not examined by the pathologist. 

LOQ=0.014 mg a.i./L. 

In male fish, there was one incidence of mild testicular degeneration at the 0.069 mg a.i./L group. 

Otherwise, there were no treatment-related histological effects or differences in gonadal stages in testes in 

any of the treatment groups (Table 15). In female fish, there was an increase in incidence of minimal to 

moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control (Table 17). 

However, the smaller sample size (n=6) prevented further interpretation of this finding. 

Page 178 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 33 of 89 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Duct 

Mineralization 

Interstitial cell 

(Leydig) 

hypertrophy/hype

rplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 8 8 8 8 8 8 NA NA 8 8 

1 8 0 8 0 8 0 NA NA 8 0 

2 8 0 8 0 8 0 NA NA 8 0 

3 8 0 8 0 8 0 NA NA 8 0 

4 8 0 8 0 8 0 NA NA 8 0 

0.069 0 8 8 8 8 8 7 NA NA 8 8 

1 8 0 8 0 8 0 NA NA 8 0 

2 8 0 8 0 8 1 NA NA 8 0 

3 8 0 8 0 8 0 NA NA 8 0 

4 8 0 8 0 8 0 NA NA 8 0 

0.69 0 6 6 6 6 6 6 NA NA 6 6 

1 6 0 6 0 6 0 NA NA 6 0 

2 6 0 6 0 6 0 NA NA 6 0 

3 6 0 6 0 6 0 NA NA 6 0 

4 6 0 6 0 6 0 NA NA 6 0 

6.9 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 

4 NA NA NA NA NA NA NA NA NA NA 

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 

1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

LOQ=0.014 mg a.i./L.

Page 179 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 34 of 89 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

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

0.069 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

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

Page 180 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 35 of 89 

Treatment 

(mg a.i./L) 

[mean-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 

0.69 0 6 6 6 6 6 6 6 6 6 6 

1 6 0 6 0 6 0 6 0 6 0 

2 6 0 6 0 6 0 6 0 6 0 3 6 0 6 0 6 0 6 0 6 0 4 6 0 6 0 6 0 6 0 6 0 

6.92 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 4 NA NA NA NA NA NA NA NA NA NA 

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

the test guideline for reference. 

2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 181 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 36 of 89 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Aggregates of 

macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 14 12 14 14 14 14 NA NA 

1 14 2 14 0 14 0 NA NA 

2 14 0 14 0 14 0 NA NA 

3 14 0 14 0 14 0 NA NA 

4 14 0 14 0 14 0 NA NA 

0.069 0 16 13 16 16 16 16 NA NA 

1 16 1 16 0 16 0 NA NA 

2 16 1 16 0 16 0 NA NA 

3 16 1 16 0 16 0 NA NA 

4 16 0 16 0 16 0 NA NA 

0.69 0 6 1 6 6 6 6 NA NA 

1 6 2 6 0 6 0 NA NA 

2 6 1 6 0 6 0 NA NA 

3 6 2 6 0 6 0 NA NA 

4 6 0 6 0 6 0 NA NA 

6.92 0 NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA 

3 NA NA NA NA NA NA NA NA 

4 NA NA NA NA NA NA NA NA 

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

2

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L 

Page 182 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 37 of 89 

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

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

Severity 

Interstitium/multifo

cal inflammation 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 14 14 14 14 14 13 14 14 

1 14 0 14 0 14 1 14 0 

2 14 0 14 0 14 0 14 0 

3 14 0 14 0 14 0 14 0 

4 14 0 14 0 14 0 14 0 

0.069 0 16 16 16 16 16 15 16 16 

1 16 0 16 0 16 1 16 0 

2 16 0 16 0 16 0 16 0 

3 16 0 16 0 16 0 16 0 

4 16 0 16 0 16 0 16 0 

0.69 0 6 6 6 5 6 4 6 6 

1 6 0 6 0 6 2 6 0 

2 6 0 6 1 6 0 6 0 

3 6 0 6 0 6 0 6 0 

4 6 0 6 0 6 0 6 0 

6.92 0 NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA 

3 NA NA NA NA NA NA NA NA 

4 NA NA NA NA NA NA NA NA 

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

2

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L 

Page 183 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 38 of 89 

Median male VTG was 9.2 x 105

, 4.2 x 105 and 8.3 x 103 and mean female VTG was 8.3 x 105, 1.2 x 

106

 and 6.4 x 105 in the mean-measured <LOQ (control), 0.069 and 6.9 mg a.i./L treatment levels, 

respectively (Table 19). The male VTG values are unusually high and further discussion of this is 

presented in the Study Author’s analysis and conclusions section. 

Table 19: Plasma Vitellogenin in Fathead Minnow (Pimephales promelas).

Treatment 

 (mg a.i./L) 

[mean-measured]

Plasma Vitellogenin (VTG) 

Males Females 

n 

Median

(ng/mL plasma) 

±SD1 n 

Mean

(ng/mL plasma) 

±SD1

Control (<LOQ) 4 9.2 x 105 7.0 x 105 4 8.1 x 105 2.2 x 105

0.069 4 4.2 x 105 2.7 x 105 4 1.2 x 106 6.0 x 105

0.69 4 8.3 x 103* 9.1 x 103 4 6.4 x 105 1.0 x 106

6.92 4 NA NA 4 NA NA 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 

1

 Standard deviations calculated by the reviewer. 

2

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 184 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 39 of 89 

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

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Plasma Testosterone (T) Plasma 17ȕ-estradiol (E2) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD 

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

0.069 NA NA NA NA NA NA NA NA NA NA NA NA 

0.69 NA NA NA NA NA NA NA NA NA NA NA NA 

6.9 NA NA NA NA NA NA NA NA NA NA NA NA 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 

LOQ=0.014 mg a.i./L. 

There were no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups (Table 21). 

Page 185 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 40 of 89 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs at Test Termination 

Males Females 

Type n1 Incidence2 Type N1 Incidence2 

Control (<LOQ) Color bars, 

8 

1 

None, Papillae present 15 4 Territorial aggression 4 

Fatpad, tubercles 4 

0.069 

Color bars, 

8 

1 None, Papillae present 

16 

2 

Territorial aggression 4 Papillae present 1 

Fatpad, tubercles 4 None 1 

0.69 

Territorial aggression 

6 

4 Papillae present 

6 

2 

Fatpad, tubercles 4 None 1 

6.9 Fatpad, tubercles 1 1 lethargic 1 1 

LOQ=0.014 mg a.i./L. 1 The value reflects the total number of individual fish surviving at test termination 2 The individual number of fish displaying the reported characteristics/signs was not reported, therefore, the numerical value in the row reflects the number of replicates 

reported to be displaying the characteristic.

Page 186 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 41 of 89 

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 controls. 

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 and Levene’s Tests, which 

were based on p<0.01 (CETIS, Version 1.8.4.20). Survival data were analyzed using a CochranArmitage Trend Step-down test. Male weight, female VTG and GSI were analyzed using ANOVA 

and a Dunnett’s Multiple comparison test. Fertility and fecundity were analyzed by Dunnett’s T3 

Multiple Comparison test. Male VTG and female weight were analyzed by ANOVA and JonckheereTerpstra test. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Levene’s to test for 

normality and homogeneity of variances, respectively. If normality and homogeneity tests passed 

(p>0.01), a parametric analysis was performed using the transformed data. If non-normality or 

unequal variance were indicated (p<0.01), 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 (both sexes) was 96, 100, 50 and 9% in the <LOQ (control), 0.069, 0.69, and 6.9 

mg a.i./L. There was a statistically significant difference in the 0.69 mg a.i./L treatment group 

compared to the negative control (p<0.05; Cochran-Armitage Trend Step-Down test). Due to the 

high mortality at the 6.9 mg a.i./L treatment group, other endpoints were not statistically analyzed 

(Table 9). There was a statistically significant difference in the 0.69 mg a.i./L treatment level 

compared to the negative control for the fecundity (p<0.05; Dunnett’s Multiple Comparison test; 

Table 11). There was a statistically significant difference between the 0.69 mg a.i./L treatment level 

compared to the negative control for the median female body weight (p<0.05; Jonckheere-Terpstra 

Step-Down test; Table 10). There was a statistically significant difference in median male tubercle 

scores at 0.69 mg a.i./L compared to the control (p<0.05; Jonckheere-Terpstra Step-Down Test; 

Table 12). None of the surviving females were found to have tubercles. There was a statistically 

significant difference in the 0.69 mg a.i./L treatment level compared to the negative control for the 

median male VTG (p<0.05; Jonckheere-Terpstra; Table 19). There were no significant differences 

between treated groups and controls for any other endpoint. 

Page 187 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 42 of 89 

In male fish, there was one incidence of mild testicular degeneration. Otherwise, there were no 

treatment-related histological effects or differences in gonadal stages in testes in any of the 

treatment groups (Table 15). In female fish, there was an increase in incidence of minimal to 

moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control (Table 17). 

However, the smaller sample size (n=6) prevented further interpretation of this finding. The study 

authors did not indicate a treatment-related effect at any test level for gonadal staging. There were 

no notable observations with regards to behavior or changes in appearance, specifically 

color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups 

(Table 21). 

In regards to the male VTG, the study authors did note that the values reported for the control and 

EPTC treated fish were unusually high. As such, they re-analyzed VTG from male fish in replicate A 

for the control and low and mid treatment groups using an ELISA kit from a different lot than the 

original analysis. They reported that the results from the second analysis were generally in 

agreement with the original values and were not included in the statistical analysis. They also noted 

that in the analysis all blanks and standard curves were consistent with historical expectations. The 

report also noted that there were no obvious explanation for the male VTG levels; there were no 

changes in the delivery system or dilution water supply or culturing practices and other data from 

these fish were within historical ranges. 

C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via 

Toxstat 3.5. Given that there were only two treatment groups available at test termination, the 

reviewer concluded that inferences about data exhibiting a trend are limited. Therefore, all endpoints 

were analyzed using non-trend statistical tests. All data were tested for normality using ShapiroWilks 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 MannWhitney U test. 

Page 188 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 43 of 89 

Fertility data was not parametric and were analyzed using Mann-Whitney. All other data was 

parametric and were analyzed using Dunnett’s. Male tubercle score was also analyzed using MannWhitney as it is not a continuous endpoint. None of the surviving females were found to have 

tubercles. The unusually high VTG values reported in the control males makes interpretation of this 

endpoint difficult and not reliable. 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 author’s and reviewer relied on the mean-measured concentrations to discuss 

effects in this study. 

 

 Conclusions: 

Adult survival of combined sexes was 96, 100, 50 and 9% at <LOQ (control), 0.069, 0.69, and 

6.9 mg a.i./L. There was a statistically significant difference in the 0.69 and 6.9 mg a.i./L 

treatment group compared to the negative control (p<0.05; Fisher’s Exact Test). Due to the high 

mortality at the 6.9 mg a.i./L treatment group, data were not reported for statistically analysis (Table 

9). Male tubercle score was marginally significantly (p=057) reduced by 33% of the control at the 

0.69 mg a.i./L treatment level and was approaching significance (p=0.086) with a reduction of 21% 

at the 0.069 mg a.i./L treatment level. Fecundity (Table 23) was significantly (p<0.05) reduced 

75% in the 0.69 mg a.i./L treatment. There were no other significant effects on endpoints. Although 

not analyzed statistically, there were no treatment-related histopathologic observations in males. In 

female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the 0.69 

mg a.i./L treatment level compared to the control. However, the smaller sample size (n=6) 

prevented further interpretation of this finding. There were no treatment-related effects at any test 

level for gonadal staging. There were no notable observations with regards to behavior or changes 

in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the 

control or treated groups. There were no notable observations of secondary sex characteristics or 

clinical signs for any treatment group compared to the controls. 

Page 189 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 44 of 89 

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

Treatment 

(mg a.i./L) 

[mean-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 

Control (<LOQ) 40.5 NA 0 NA No 0 NA NA NA NA NA 

0.069 32.0 0.086 -19.67 0.245 No 

-54.35 

(NRL) 

NA NA NA NA NA 

0.69 26.0 0.057 22.95 0.165 No 

-99.10 

(NRL) 

NA NA NA NA NA 

6.93 NA NA NA NA NA NA NA NA NA NA NA 

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

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA

Not applicable. NRL Due to the unusually high male VTG values in the control, the results for this endpoint are not reliable for interpretation 

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 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 190 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 45 of 89 

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

Treatment 

(mg a.i./L) 

[meanmeasured]

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 

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

0.069 19.10 0.248 0.00 1.00 0 NA -7.69 0.625 No 47.68 0.625 NA NA NA NA 

0.69 -74.83 <0.001 -11.72 1.00 0 NA 23.08 0.080 No -21.07 0.918 NA NA NA NA 

6.93 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

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

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

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

Page 191 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 46 of 89 

Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with EPTC.

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

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

Control (<LOQ) 

0 NA 0 NA 0 NA 0 NA 

0.069 -5.56 0.685 2.70 0.896 -2.66 0.688 1.68 0.289 

0.69 -5.63 0.679 18.44 0.065 -1.98 0.808 2.45 0.136 

6.93 NA NA NA NA NA NA NA NA 

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

Abbreviations: Diff. Difference. NA Not applicable. 

1

 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) 

control. 

2

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

3

 Endpoints at the high treatment level were not statistically analyzed due to high mortality. 

LOQ=0.014 mg a.i./L. 

E. Study Deficiencies 

OCSPP 890.1350 validity and performance criteria were met for reported parameters except that 

dissolved oxygen fell slightly below the recommended 60% of saturation (58%) in one test chamber; the 

chambers were cleaned and the levels returned to above the recommended value. In addition, the 

unionized ammonia and residual chlorine in the test water were not reported. The OCSPP 890.1350 

performance criteria establish maximum levels for these values and it is unclear if the maximum 

recommendations were exceeded. 

Additionally, endpoints for fish exposed to the highest treatment level (6.9 mg a.i./L) could not be 

evaluated due to mortality; only one male and one female survived at study termination. Therefore, only 

2 test treatment levels were analyzed in this study; OCSPP Guideline 890.1350 recommends a 

Page 192 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 47 of 89 

minimum of 3 test concentration plus a negative control. There was also significant mortality (50%) at 

the 0.69 mg a.i./L treatment level thereby leaving only one test level without the presence of overt 

toxicity. This limits the ability to interpret the EPTC effects in fish. 

While these deviations to the performance criteria were observed, they did not impact the ability to 

interpret the results of the assay.

F. Reviewer’s Comments 

The reviewer’s and the study authors’ results were in agreement regarding effects on survival. Both the 

study author’s and reviewer’s analysis detected statistically significant reductions (p<0.05) in survival at 

the 0.69 and 6.9 mg a.i./L treatment level, using either the study author’s analysis method (CochranArmitage Trend test) or the reviewer’s analysis method (Fisher’s Exact test). The reviewer’s analysis 

additionally detected a marginal significant reduction in median male tubercle score at the 0.069 mg 

a.i./L treatment level (p=0.053; Dunnett’s test), which the study author did not report. The reviewer’s 

conclusions based on the OCSPP 890.1350 flowchart are presented in the Executive Summary and 

Conclusions sections of this DER. 

In regards to male VTG, given the unusually high values reported in the control, the reviewer determined 

that the results from this endpoint were not reliable for interpretation. Additionally, in the pathologist’s 

report, it was reported that after histological assessment, for one sample submitted as a testis in the 

control, it was revealed to be a normal ovary. Based on the total number of samples submitted as 

female in the control, the reviewer believes this one sample was mis-labeled, as its inclusion as a 

female would have provided the correct number of female fish gonadal samples in the control. 

The method recoveries of EPTC in stock solution fortified at 0.005, 0.500 and 25.0 mg a.i./L was 106 

± 5.7% using a Agilent Zorbax SB-C18 3.5P column. 

The study author reported dissolved oxygen (DO) levels fell to 58% of nominal in the control, therefore 

all vessels were cleaned and the DO returned to the required range. Due to the short duration, the 

deviation is not likely to have a negative impact on the results or interpretation of this study. 

Page 193 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 48 of 89 

II. REFERENCES 

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and 

evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ 

Toxicol Chem 20:1276-1290. 

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th 

Edition, Washington, DC, 2-23-25. 

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for 

Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American 

Society for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, 

Pennsylvania. 

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User's 

Guide. Tidepool Scientific Software, McKinleyville, California. 

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. 

Water Research 1: 21 -29. 

Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. 

Water Res. 3: 793-821. 

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: 

Test No. 229. Paris, France. 40 pp 

U.S. EPA, 1989. 40CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good 

Laboratory Practices Standards; Final Rule. Office of the Federal Register, National Archives and 

Records Administration. U.S. Government Printing Office, Washington, D.C. U.S. EPA, 2002. Shortterm methods for estimating the chronic toxicity of effluents and receiving waters to marine and 

Page 194 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 49 of 89 

estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency, 

Washington, DC 20460. EPA/82 l/R-02/014. 

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disrupter 

Screening Program Test Guideline, OCSPP 890.1350. Fish Short-Term Reproduction Assay. EPA 

40-C-09-007. October 2009. U.S. Environmental Protection Agency. Washington, D.C. 

Page 195 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Page 50 of 89 

APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Reviewer’s statistical verification table 

Endpoint Monotonic?1 Parametric? 890.1350 EFED CommentsFemale body weight 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight 

NA 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 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male VTG NA No NA NA Data not reliable Female GSI NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male GSI NA 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 

Male tubercle score 

NA Yes Mann-Whitney 

Dose 1 p=0.057 

dunnett’s Dose 1 p=0.057 

EDSP and EFED same conclusions, marginal significant (p=0.057) reductions at the 0.069 mg a.i./L doses. 

Fecundity NA Yes Dunnett’s: 

Dose 2 p<0.001 

Dunnett’s: Dose 2 p<0.001 

EDSP and EFED same conclusions, a significant (p<0.001) 75% reduction at the 0.69 mg a.i./L dose. 

Fertility NA No Mann Whitney-U 

n.s. p>0.05 

Mann Whitney-U 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 

Page 196 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

-

 CONTROL 8 0 8 

 0.069 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 

 0.69 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. 

Page 197 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

-

 CONTROL 8 0 8 

 6.9 2 6 8 

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

-

 TOTAL 10 6 16 

============================================================================

 Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 2. 

 Since b is less than or equal to 4.0 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 8 0 

 1 0.069 8 0 

 2 0.69 8 2 

 3 6.9 8 6 * 

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

-

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION DEAD ALIVE TOTAL ANIMALS

Page 198 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

-

 CONTROL 1 15 16 

 0.069 0 16 16 

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

-

 TOTAL 1 31 32 

============================================================================

 Critical Fisher's value (16,16,1) (alpha=0.05) is negative. b value is 0. 

 no significant difference 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

-

 CONTROL 15 1 16 

 0.69 6 10 16 

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

-

 TOTAL 21 11 32 

============================================================================

 Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 6. 

 Since b is less than or equal to 10.0 there is a significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

Page 199 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

-

 CONTROL 15 1 16 

 6.9 1 15 16 

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

-

 TOTAL 16 16 32 

============================================================================

 Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 1. 

 Since b is less than or equal to 10.0 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 1 

 1 0.069 16 0 

 2 0.69 16 10 * 

 3 6.9 16 15 * 

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

-

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION DEAD ALIVE TOTAL ANIMALS

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

-

 CONTROL 1 23 24 

 0.069 0 24 24 

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

-

 TOTAL 1 47 48 

============================================================================

Page 200 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Critical Fisher's value (24,24,1) (alpha=0.05) is negative. b value is 0. 

 no significant difference 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

-

 CONTROL 23 1 24 

 0.69 12 12 24 

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

-

 TOTAL 35 13 48 

============================================================================

 Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 12. 

 Since b is less than or equal to 18.0 there is a significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 Fisher's Exact Test 

============================================================================

 NUMBER OF 

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

-

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

-

 CONTROL 23 1 24 

 6.9 3 21 24 

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

-

 TOTAL 26 22 48 

============================================================================

Page 201 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 3. 

 Since b is less than or equal to 18.0 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 1 

 1 0.069 24 0 

 2 0.69 24 12 * 

 3 6.9 24 21 * 

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

-

Page 202 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

test for fish screen study - EPTC 

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) -- alphalevel=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 

 0.903 0.203 0.447 0.655 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.39 0.17 0.08 11.96 1.12, 1.65 

 Dose1 4 1.43 0.11 0.06 7.87 1.25, 1.60 

 Dose2 3 1.64 0.11 0.06 6.51 1.38, 1.91 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 1.32 1.28 1.63 . .

 Dose1 1.43 1.29 1.56 102.70 -2.70 

 Dose2 1.62 1.55 1.76 118.44 -18.44 

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

***

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 

 2 8 3.50 0.081 

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 1.39 . 1.47 . . . . .

.

 Dose1 1.43 0.896 1.47 0.870 . . . .

.

 Dose2 1.64 0.065 1.47 0.882 0.144 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 3.68 0.159 

Page 203 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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 1.32 . .

 Dose1 1.43 0.491 0.808 

 Dose2 1.62 0.258 0.978 

 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 

 2 8 3.50 0.081 

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 -1.39 . -1.39 . . . . .

.

 Dose1 -1.43 0.896 -1.43 0.417 . . . .

.

 Dose2 -1.64 0.065 -1.64 0.022 0.144 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 3.68 0.159 

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 -1.32 . .

 Dose1 -1.43 0.491 0.192 

 Dose2 -1.62 0.258 0.022 

Page 204 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams Dose2 

 Jonckheere Dose2 

test for fish screen study - EPTC 

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) -- alphalevel=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 

 0.887 0.108 2.910 0.106 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 3.73 0.18 0.09 4.93 3.44, 4.03 

 Dose1 4 3.53 0.61 0.31 17.44 2.55, 4.50 

 Dose2 4 3.52 0.22 0.11 6.34 3.17, 3.88 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 3.69 3.56 3.99 . .

 Dose1 3.31 3.06 4.43 94.44 5.56 

 Dose2 3.58 3.22 3.71 94.37 5.63 

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

***

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 

 2 9 0.38 0.696 

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 3.73 . 3.73 . . . . .

.

 Dose1 3.53 0.685 3.53 0.283 . . . .

.

 Dose2 3.52 0.679 3.52 0.299 1.000 . . .

.

Page 205 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 2.00 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 3.69 . .

 Dose1 3.31 0.346 0.124 

 Dose2 3.58 0.346 0.190 

 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 

 2 9 0.38 0.696 

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 -3.73 . -3.59 . . . . .

.

 Dose1 -3.53 0.685 -3.59 0.772 . . . .

.

 Dose2 -3.52 0.679 -3.59 0.804 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 

 2 2.00 0.368 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING 

trend

Page 206 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -3.69 . .

 Dose1 -3.31 0.346 0.876 

 Dose2 -3.58 0.346 0.810 

 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 - EPTC 

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) -- alphalevel=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 0.047 0.965 0.421 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 50.73 1.06 0.53 2.09 49.04, 52.41 

 Dose1 4 51.58 0.66 0.33 1.28 50.52, 52.63 

 Dose2 3 51.97 0.51 0.30 0.99 50.69, 53.24 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 50.30 50.00 52.30 . .

 Dose1 51.35 51.10 52.50 101.68 -1.68 

 Dose2 52.10 51.40 52.40 102.45 -2.45 

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

***

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 

 2 8 2.23 0.170 

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

Page 207 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Ctrl 50.73 . 51.37 . . . . .

.

 Dose1 51.58 0.289 51.37 0.915 . . . .

.

 Dose2 51.97 0.136 51.37 0.923 0.805 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 3.17 0.205 

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 50.30 . .

 Dose1 51.35 0.230 0.928 

 Dose2 52.10 0.258 0.967 

 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 

 2 8 2.23 0.170 

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 -50.73 . -50.73 . . . . .

.

 Dose1 -51.58 0.289 -51.58 0.103 . . . .

.

 Dose2 -51.97 0.136 -51.97 0.048 0.805 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

Page 208 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Degrees of Freedom TestStat P-value 

 2 3.17 0.205 

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 -50.30 . .

 Dose1 -51.35 0.230 0.072 

 Dose2 -52.10 0.258 0.033 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams Dose2 

 Jonckheere Dose2 

test for fish screen study - EPTC 

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) -- alphalevel=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 0.864 1.433 0.288 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 65.73 1.46 0.73 2.22 63.41, 68.04 

 Dose1 4 63.98 4.82 2.41 7.54 56.30, 71.65 

 Dose2 4 64.43 2.80 1.40 4.35 59.97, 68.88 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 65.60 64.10 67.60 . .

 Dose1 63.20 59.00 70.50 97.34 2.66 

 Dose2 64.25 61.50 67.70 98.02 1.98 

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

***

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 

 2 9 0.30 0.749 

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 209 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

 Ctrl 65.73 . 65.73 . . . . .

.

 Dose1 63.98 0.688 64.20 0.318 . . . .

.

 Dose2 64.43 0.808 64.20 0.340 0.980 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.19 0.551 

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 65.60 . .

 Dose1 63.20 0.346 0.124 

 Dose2 64.25 0.678 0.232 

 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 

 2 9 0.30 0.749 

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 -65.73 . -64.71 . . . . .

.

 Dose1 -63.98 0.688 -64.71 0.749 . . . .

.

 Dose2 -64.43 0.808 -64.71 0.782 0.980 . . .

.

Page 210 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.19 0.551 

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 -65.60 . .

 Dose1 -63.20 0.346 0.876 

 Dose2 -64.25 0.678 0.768 

 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 - EPTC 

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) -- alphalevel=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 

 0.905 0.212 3.622 0.076 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 807500.0 224109.3 112054.7 27.75 450892.0, 1164108 

 Dose1 4 1192500 599075.7 299537.8 50.24 239236.9, 2145763 

 Dose2 3 637333.3 1008346 582168.7 158.21 -1867537, 3142203 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 825000.0 580000.0 1000000 . .

 Dose1 1100000 570000.0 2000000 147.68 -47.68 

 Dose2 110000.0 2000.00 1800000 78.93 21.07 

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

***

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 

Page 211 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 2 8 0.72 0.518 

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 807500.0 . 1000000 . . . . .

.

 Dose1 1192500 0.625 1000000 0.746 . . . .

.

 Dose2637333.3 0.918 637333.3 0.465 0.519 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.63 0.443 

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 825000.0 . .

 Dose1 1100000 0.484 0.813 

 Dose2 110000.0 0.612 0.368 

 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 

 2 8 0.72 0.518 

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

Page 212 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Ctrl -807500 . -807500 . . . . .

.

 Dose1-1192500 0.625 -954571 0.446 . . . .

.

 Dose2 -637333 0.918 -954571 0.486 0.519 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.63 0.443 

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 -825000 . .

 Dose1 -1100000 0.484 0.187 

 Dose2 -110000 0.612 0.632 

 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 - EPTC 

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) -- alphalevel=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 0.597 17.716 <.001 USE NON-PARAMETRIC 

TESTS

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 920000.0 697471.6 348735.8 75.81 -189833, 2029833 

 Dose1 4 420000.0 274469.2 137234.6 65.35 -16741.7,856741.7 

 Dose2 4 8252.50 9099.29 4549.65 110.26 -6226.51,22731.51 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 885000.0 210000.0 1700000 . .

Page 213 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Dose1 300000.0 250000.0 830000.0 45.65 54.35 

 Dose2 7445.00 120.00 18000.00 0.90 99.10 

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

***

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 

 2 9 4.45 0.045 

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 920000.0 . 920000.0 . . . . .

.

 Dose1420000.0 0.229 420000.0 0.081 . . . .

.

 Dose2 8252.50 0.028 8252.50 0.009 0.407 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 7.73 0.021 

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 885000.0 . .

 Dose1 300000.0 0.494 0.193 

 Dose2 7445.00 0.067 0.003 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

 Williams Dose2 

 Jonckheere Dose2 

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

***

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 

 2 9 4.45 0.045 

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 

Page 214 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

 Ctrl -920000 . -449418 . . . . .

.

 Dose1 -420000 0.229 -449418 0.960 . . . .

.

 Dose2-8252.50 0.028 -449418 0.971 0.407 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 7.73 0.021 

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 -885000 . .

 Dose1 -300000 0.494 0.807 

 Dose2 -7445.00 0.067 0.997 

 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 - EPTC 

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) -- alphalevel=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 

 0.904 0.207 1.345 0.314 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 13.00 0.82 0.41 6.28 11.70, 14.30 

 Dose1 4 12.00 2.16 1.08 18.00 8.56, 15.44 

 Dose2 3 16.00 1.73 1.00 10.83 11.70, 20.30 

Page 215 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 13.00 12.00 14.00 . .

 Dose1 11.50 10.00 15.00 92.31 7.69 

 Dose2 15.00 15.00 18.00 123.08 -23.08 

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

***

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 

 2 8 5.22 0.035 

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 13.00 . 13.45 . . . . .

.

 Dose1 12.00 0.625 13.45 0.733 . . . .

.

 Dose2 16.00 0.080 13.45 0.757 0.032 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 5.60 0.061 

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 13.00 . .

 Dose1 11.50 0.410 0.153 

 Dose2 15.00 0.095 0.898 

 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 

Page 216 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Numerator df Denominator df F-stat P-value 

 2 8 5.22 0.035 

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 -13.00 . -12.50 . . . . .

.

 Dose1 -12.00 0.625 -12.50 0.746 . . . .

.

 Dose2 -16.00 0.080 -16.00 0.028 0.032 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 5.60 0.061 

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 -13.00 . .

 Dose1 -11.50 0.410 0.847 

 Dose2 -15.00 0.095 0.102 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams Dose2 

 Jonckheere >highest dose (no sign. 

differences)

test for fish screen study - EPTC 

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) -- alphalevel=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 

 0.909 0.210 2.800 0.113 USE PARAMETRIC TESTS 

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

***

Page 217 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.53 0.32 0.16 20.99 1.02, 2.03 

 Dose1 4 1.23 0.29 0.14 23.45 0.77, 1.68 

 Dose2 4 1.88 0.17 0.09 9.11 1.60, 2.15 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 1.55 1.20 1.80 . .

 Dose1 1.15 1.00 1.60 80.33 19.67 

 Dose2 1.85 1.70 2.10 122.95 -22.95 

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

***

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 

 2 9 5.93 0.023 

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 1.53 . 1.54 . . . . .

.

 Dose1 1.23 0.245 1.54 0.620 . . . .

.

 Dose2 1.88 0.165 1.54 0.655 0.018 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 6.39 0.041 

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 1.55 . .

 Dose1 1.15 0.278 0.093 

 Dose2 1.85 0.225 0.896 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

 Williams >highest dose (no sign. 

differences)

 Jonckheere >highest dose (no sign. 

differences)

Page 218 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

***

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 

 2 9 5.93 0.023 

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 -1.53 . -1.38 . . . . .

.

 Dose1 -1.23 0.245 -1.38 0.852 . . . .

.

 Dose2 -1.88 0.165 -1.88 0.060 0.018 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 6.39 0.041 

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 -1.55 . .

 Dose1 -1.15 0.278 0.907 

 Dose2 -1.85 0.225 0.104 

 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 - EPTC 

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) -- alphalevel=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric 

analyses.

Page 219 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 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 . . , .

 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 . .

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

***

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 

 . . . 9 . . . . . .

.

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 . . . . . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 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 .

Page 220 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Dose2 0.00 1.000 .

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

 Williams Dose1 

 Jonckheere Dose1 

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

***

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 

 . . . 9 . . . . . .

.

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 . . . . . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 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 .

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams Dose1 

 Jonckheere Dose1 

test for fish screen study - EPTC 

ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) ) 

Page 221 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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) -- alphalevel=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 0.850 3.440 0.078 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 39.75 5.12 2.56 12.89 31.60, 47.90 

 Dose1 4 31.50 1.91 0.96 6.08 28.45, 34.55 

 Dose2 4 26.75 5.62 2.81 21.01 17.81, 35.69 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 40.50 33.00 45.00 . .

 Dose1 32.00 29.00 33.00 79.25 20.75 

 Dose2 26.00 22.00 33.00 67.30 32.70 

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

***

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 

 2 9 8.44 0.009 

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 39.75 . 39.75 . . . . .

.

 Dose1 31.50 0.053 31.50 0.017 . . . .

.

 Dose2 26.75 0.005 26.75 0.002 0.343 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 7.03 0.030 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

Page 222 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 40.50 . .

 Dose1 32.00 0.096 0.019 

 Dose2 26.00 0.080 0.003 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

 Williams Dose1 

 Jonckheere Dose1 

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

***

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 

 2 9 8.44 0.009 

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 -39.75 . -32.67 . . . . .

.

 Dose1 -31.50 0.053 -32.67 0.989 . . . .

.

 Dose2 -26.75 0.005 -32.67 0.993 0.343 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 7.03 0.030 

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 -40.50 . .

 Dose1 -32.00 0.096 0.981 

 Dose2 -26.00 0.080 0.997 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. 

differences)

 Jonckheere >highest dose (no sign. 

differences)

Page 223 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

test for fish screen study - EPTC 

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) -- alphalevel=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 0.916 0.482 0.632 USE PARAMETRIC TESTS 

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 22.25 2.99 1.49 13.42 17.50, 27.00 

 Dose1 4 26.50 5.00 2.50 18.87 18.54, 34.46 

 Dose2 4 5.60 3.09 1.55 55.23 0.68, 10.52 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 23.00 18.00 25.00 . .

 Dose1 26.00 21.00 33.00 119.10 -19.10 

 Dose2 5.50 2.00 9.40 25.17 74.83 

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

***

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 

 2 9 33.67 <.001 

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 22.25 . 24.38 . . . . .

.

 Dose1 26.50 0.248 24.38 0.851 . . . .

.

 Dose2 5.60 <.001 5.60 <.001 <.001 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

Page 224 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 2 8.22 0.016 

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 23.00 . .

 Dose1 26.00 0.281 0.906 

 Dose2 5.50 0.066 0.046 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

 Williams Dose2 

 Jonckheere Dose2 

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

***

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 

 2 9 33.67 <.001 

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 -22.25 . -18.12 . . . . .

.

 Dose1 -26.50 0.248 -18.12 0.959 . . . .

.

 Dose2 -5.60 <.001 -18.12 0.971 <.001 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 8.22 0.016 

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 -23.00 . .

 Dose1 -26.00 0.281 0.094 

 Dose2 -5.50 0.066 0.954 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Page 225 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Williams >highest dose (no sign. 

differences)

 Jonckheere >highest dose (no sign. 

differences)

test for fish screen study - EPTC 

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) -- alphalevel=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 

 0.732 0.002 6.115 0.021 USE NON-PARAMETRIC 

TESTS

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

***

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 96.00 1.41 0.71 1.47 93.75, 98.25 

 Dose1 4 96.00 1.83 0.91 1.90 93.09, 98.91 

 Dose2 4 84.75 20.39 10.19 24.05 52.31, 117.19 

 Level Median Min Max %of Control(means)

%Reduction(means)

 Ctrl 96.50 94.00 97.00 . .

 Dose1 96.00 94.00 98.00 100.00 0.00 

 Dose2 93.00 55.00 98.00 88.28 11.72 

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

***

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 

 2 9 1.20 0.344 

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 96.00 . 96.00 . . . . .

.

 Dose1 96.00 1.000 96.00 0.583 . . . .

.

 Dose2 84.75 0.344 84.75 0.134 0.408 . . .

.

Page 226 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 0.07 0.966 

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 96.50 . .

 Dose1 96.00 1.000 0.559 

 Dose2 93.00 1.000 0.470 

 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 

 2 9 1.20 0.344 

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 -96.00 . -92.25 . . . . .

.

 Dose1 -96.00 1.000 -92.25 0.754 . . . .

.

 Dose2 -84.75 0.344 -92.25 0.787 0.408 . . .

.

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

***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 0.07 0.966 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING 

trend

Page 227 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -96.50 . .

 Dose1 -96.00 1.000 0.441 

 Dose2 -93.00 1.000 0.530 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

 Williams >highest dose (no sign. 

differences)

 Jonckheere >highest dose (no sign. 

differences)

Male Tubercle Score (Mann-Whitney) 

Obs conc endpoint

1 0 39

2 0 33

3 0 42

4 0 45

5 1 33

6 1 33

7 1 31

8 1 29

9 2 30

10 2 33

11 2 22

12 2 22

13 3 . 14 3 . 15 3 . 16 3 .

Page 228 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores

Expected

Under H0

Std Dev

Under H0

Mean 

Score 

0 4 40.50 26.0 5.773503 10.1250 

1 4 23.00 26.0 5.773503 5.7500 

2 4 14.50 26.0 5.773503 3.6250 

Average scores were used for ties. 

Kruskal-Wallis Test 

Chi-Square 7.0300

DF 2

Asymptotic Pr > ChiSquare 

0.0297

Exact Pr >= ChiSquare 

0.0152

Page 229 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 230 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores

Expected

Under H0

Std Dev

Under H0

Mean 

Score 

0 4 25.0 18.0 3.380617 6.250 

1 4 11.0 18.0 3.380617 2.750 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 25.0000

Normal Approximation

Z 1.9227

One-Sided Pr > Z 0.0273

Two-Sided Pr > |Z| 0.0545

t Approximation 

One-Sided Pr > Z 0.0480

Two-Sided Pr > |Z| 0.0959

Exact Test 

One-Sided Pr >= S 0.0429

Two-Sided Pr >= |S -

Mean| 

0.0857

Z includes a continuity 

correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 4.2875

DF 1

Pr > ChiSquare 

0.0384

Page 231 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 232 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores

Expected

Under H0

Std Dev

Under H0

Mean 

Score 

0 4 25.50 18.0 3.422614 6.3750 

2 4 10.50 18.0 3.422614 2.6250 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 25.5000

Normal Approximation

Z 2.0452

One-Sided Pr > Z 0.0204

Two-Sided Pr > |Z| 0.0408

t Approximation 

One-Sided Pr > Z 0.0400

Two-Sided Pr > |Z| 0.0801

Exact Test 

One-Sided Pr >= S 0.0286

Two-Sided Pr >= |S -

Mean| 

0.0571

Z includes a continuity 

correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 4.8018

DF 1

Pr > ChiSquare 

0.0284

Page 233 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 234 of 310
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

Page 235 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616406) 

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: 1/19/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 1/23/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

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

signing by CSS-Dynamac Corporation personnel. 

 

Page 236 of 310
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 237 of 310
In vivo Hershberger Assay (2011)/ Page 1of13 

EPTC/041401 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

DAT A EVALUATION RECORD 

OCSPP 890.1400/ OECD 441 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 041401 DP BARCODE: D397 l 77 

TXR#: 0051762 CAS#: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: S-ethyl N,N-dipropylcarbamothioate 

CITATION: Zorrilla, L. (2011). The Hershberger Assay with EPTC in Castrated 

CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. 

Laboratory Project Study ID: C201-200, November 4, 2011. MRID 48616406. 

Unpublished. 

SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In a Hershberger assay (MRID 48616406) screening for 

androgenic activity, EPTC (>99.9% a.i., lot # Pl 105096) in corn oil was administered daily via 

oral gavage to groups of 60- to 61-day old, castrated male Sprague Dawley rats (8/group) at dose 

levels of 0 (vehicle), 50, or 250 mg/kg/day. An androgenic positive control group consisting of 8 

castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) 

injection was also included as a positive control. 

To screen for potential anti-androgenic activity, EPTC (>99.9% a.i., lot # P 1105096) in corn oil 

was administered daily via oral gavage to 60- to 61-day old, castrated male Sprague Dawley rats 

(8/group) at dose levels of 50, 150, or 250 mg/kg/day in conjunction with a daily dose of 

reference androgen TP at 0.4 mg/kg/day by s.c. injection. The control group consisted of eight 

castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the anti-androgenic 

positive control group consisted of eight castrated male rats dosed orally with flutamide (FT) in 

corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for I 0 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

One positive control (TP only) rat died after the first day of dosing due to a gavage error. All 

other animals survived until scheduled termination. There were no treatment-related clinical 

signs of toxicity. 

Page 238 of 310
In vivo Hershberger Assay (2011)/ Page 2 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

In the androgen agonist assay, body weights for the 50 and 250 mg/kg/day EPTC-treatment 

groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, 

overall (Days 1-11) body weight gains were decreased (p≤0.05) by 40%. Animals dosed with 

TP had a 10% increase (p≤0.05) in body weights on Day 11 and a 69% increase in overall body 

weight gains compared to controls. Animals in the positive control (TP) group had increases 

(p≤0.05) in accessory sex organ weights as follows: 1578% in seminal vesicles; 1542% in 

ventral prostate; 259% in the levator ani-bulbocavernosus (LABC) muscle complex; 550% in 

Cowper’s gland; and 70% in glans penis. The maximum guideline-specified %CV was slightly 

exceeded for the seminal vesicle in the 250 mg/kg/day (43% vs. 40%) group, but all other %CV 

values were less than the maximum permissible values. 

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were comparable 

to controls throughout the duration of the assay. At 250 mg/kg/day, overall body weight gains 

were decreased (p≤0.05) by 34%. For the positive control group (TP + FT), overall body weight 

gains were decreased (not significant, NS) by 20%. At 50 mg/kg/day of EPTC, the weights of 

seminal vesicles were decreased by 23% (p≤0.05). The LABC weights were decreased by 10% 

(p≤0.05) at 150 mg/kg/day and by 16% (p≤0.05) at 250 mg/kg/day. Animals dosed with TP + 

FT (positive control) had decreases (p≤0.05) in accessory sex organ weights as follows: 88% in 

seminal vesicles; 93% in ventral prostate; 55% in LABC; and 65% in Cowper’s gland. The 

maximum guideline-specified %CV was slightly exceeded for the glans penis in the 250 

mg/kg/day (19% vs. 17%) group. All other %CV values were less than the maximum 

permissible values. 

Based on the decreases in overall body weight gain observed at the high-dose (250 mg/kg/day) in 

both the androgenic and anti-androgenic assays, the dosing was considered to be adequate. 

Statistically significant changes in organ weights were not seen in two or more of the five 

androgen sensitive tissues. EPTC was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

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

890.1400). 

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

provided. 

 

Page 239 of 310
In vivo Hershberger Assay (2011)/ Page 3 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Systems, Inc. 

Location: Durham, NC

Study Director: L. Zorrilla 

Other Personnel: Not provided 

Study Period: July 15-26, 2011 

2. Test Substance: EPTC 

 Description: Off white solid 

 Source: Gowan Company (commercial technical product) 

 Lot/Batch #: P1105096, expires February 2, 2015 

 Purity: >99.9 % a.i. 

 Stability: Stable in the vehicle for up to 25 days at 1-10°C (Smithers Viscient Inc., 

Study No. 12791-6162) 

 CAS #: 759-94-4 

 Structure: 

3. Reference Androgen: Testosterone propionate (TP) 

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

 Batch #: 048K1328 

 Purity: 100.2 % 

 CAS # : 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

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

 Lot #: 107K1293 

 Purity: >99 % 

 CAS # : 13311-84-7 

5. Solvent/Vehicle Control: Corn Oil 

 Supplier: MP Biomedicals, LLC (Solon, OH) 

 Lot/Batch #: 7862K 

 Rationale (if other than water) : Not provided 

 Final concentration: Not applicable 

6. Test Animals: 

 Species: Rat (castrated males) 

 Strain: Sprague Dawley (Crl:CD®[SD] IGS) 

 Age/weight at dose initiation: Post-natal day (PND) 60/61; 249.2-303.7 g 

Source: Charles River Laboratories International, Inc., Raleigh, NC 

Housing: Rats were housed up to 2/cage in polycarbonate cages with absorbent heattreated hardwood bedding (Northeastern Products Corp., Warrensburg, NY). 

 Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad 

libitum; low phytoestrogen with total genistein equivalents = 8.2 μg/g diet

 Water: Reverse osmosis tap water, ad libitum

 Environmental conditions: Temperature: 22-25oC

Humidity: 34-60% 

Air changes: Not reported

Photoperiod: 12 hrs light/12 hrs dark

Acclimation period: At least 8 days 

S N

CH3

CH3

O

3

CH

Page 240 of 310
In vivo Hershberger Assay (2011)/ Page 4 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

B. STUDY DESIGN

1. In-life dates: Start: 7/15/2011 End: 7/26/2011 

2. Study Design: The current Hershberger Assay was conducted to screen for both potential 

androgenic and anti-androgenic activity. In the test for androgenic activity, EPTC was 

administered daily via oral gavage to groups of castrated male rats. Positive androgenic 

activity is defined as a significant increase in two or more target organ weights compared to 

the vehicle control group. The positive control group for androgenic activity is a group of 

castrated male rats dosed by s.c. injection with TP at 0.4 mg/kg/day. In the test for antiandrogenic activity, EPTC was administered daily via oral gavage to groups of castrated 

male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection. Anti-androgenic 

activity is indicated by a statistically significant decrease in two or more target organ 

weights of the treated groups (test substance + TP) compared to the TP-only control group. 

The positive control group for anti-androgenic activity is a group of castrated male rats 

dosed by s.c. injection with TP (0.4 mg/kg/day) in conjunction with an oral dose of FT (3.0 

mg/kg/day). For both assays, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration for measurement of 

target organ weights. 

3. Study Schedule: Following an 8-day acclimation period at the test facility, castrated male 

rats (surgical manipulation performed by Supplier) were administered the test substances 

from PND 60/61 to PND 69/70. Rats were euthanized on PND 70/71, approximately 24 

hours after the last dose and necropsied for target organ weight measurements. 

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences in group means at study initiation. 

TABLE 1. Study design a

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (negative control) 0 8 

Low EPTC 50 8 

High EPTC 250 8 

Testosterone propionate (TP) (s.c.), positive 

control b 

0.4 8 

Anti-Androgen Assay 

Testosterone propionate (negative control, s.c.) b 0 + 0.4 8 

Low EPTC +TP 50 + 0.4 8 

Mid EPTC +TP 150 + 0.4 8 

High EPTC +TP) 250 + .04 8 

Flutamide (oral gavage) + TP (s.c.), positive 

control 

3 = 0.4 8 

a Data were obtained from page 15 of the study report. 

b Same animals dosed for both androgen agonist (served as positive control) and anti-androgen (served as 

vehicle control) assays. This did not affect the outcome or conclusions of this study. 

Page 241 of 310
In vivo Hershberger Assay (2011)/ Page 5 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

5. Dose Selection Rationale: The EPTC dose levels were selected based on the results of a 

subchronic neurotoxicity study and a developmental toxicity range-finding study (study 

report numbers not provided). In the neurotoxicity study, Alpk:APfSd rats were 

administered EPTC in the diet at dose levels of 100, 500, or 2500 ppm (equivalent to 7.9, 

34.9, and 195.1 mg/kg/day) for 13 weeks. It was stated that bodyweights for the 2500 ppm 

animals were slightly reduced (↓4% in males and ↓3.3% in females) during Week 2. In the 

developmental toxicity range-finding study, female rats were administered EPTC in corn oil 

by daily oral gavage at dose levels of 50, 90, 160, 280, or 500 mg/kg/day during gestation 

days 6-15. It was stated that all 500 mg/kg/day animals died during dosing. At 160 

mg/kg/day and above, body weight gains were decreased by 29-62%, food consumption was 

reduced, and clinical signs (yellow urogenital matting and staining and red matter around 

the nasal area) were observed. Based on these results, doses of ≤250 mg/kg/day were used 

for both the androgen agonist and anti-androgen assays. 

6. (a) Dose Preparation: Dose formulations were prepared once prior to initiation of 

treatment by suspending the appropriate amounts of test substance in corn oil. Flutamide 

and TP were prepared once prior to initiation of treatment by mixing in corn oil. 

Homogeneity and concentration analyses were performed on all dose formulations. No 

stability analyses were performed with the current study. 

In a previous storage stability study1, EPTC (lot number not reported) was determined to be 

stable in corn oil for up to 25 days at 1-10°C. 

(b) Dose Analysis

Results

Homogeneity (%CV): 1.14-5.01%

Stability (% of nominal): Data not provided 

Concentration (% of nominal): 101-104%

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 of EPTC were administered to the animals daily 

via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, 

and FT was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily 

based on the concurrent body weight measurement. 

8. Statistics: Final body weights, body weight gains and accessory sex organ weights were 

analyzed using studentized residual plots for outliers and Levene’s test for homogeneity of 

variance. Heterogeneous data (Cowper’s gland, anti-androgen assay) were transformed 

(logarithm, square root, multiplicative inverse transformations) and re-analyzed using 

 

1 Reibach, R. 2011b. Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers 

Viscient Inc., Study No. 12791-6162. 

Page 242 of 310
In vivo Hershberger Assay (2011)/ Page 6 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

Kruskal-Wallis and Dunn’s test. Final body weights, body weight gains, and accessory sex 

organ weights were analyzed using one-way ANOVA followed by Dunnett’s t-tests. The 

positive controls were compared to the vehicle controls using the t-test. Significance was 

denoted at p≤0.05. The statistical analyses were considered adequate. Data were analyzed 

using SAS version 9.2 software. 

C. METHODS

1. Clinical Examinations: Animals were checked for mortality or moribundity twice daily on 

weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity 

prior to dose administration and at termination. 

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

the dosing period, and at termination. 

3. Food Consumption (Optional): Food consumption was not determined. 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 70-71 

(approximately 24 hours after the final administration of the test substance), all rats were 

weighed, anesthetized by CO2 inhalation followed by cervical dislocation and subjected to a 

gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal 

vesicles with coagulating glands and fluid, LABC, Cowper’s gland, and glans penis) were 

excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the 

standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). 

II. RESULTS

A. OBSERVATIONS

1. Mortality: One positive control (TP only) rat died after the first day of dosing due to a 

gavage error. All other 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. 

In the androgen agonist assay, one 250 mg/kg/day rat was observed with rough coat and red 

discoloration of nose fur. In the anti-androgen assay, observations included one 50 

mg/kg/day rat with a facial scab (Days 7-8) and also red discoloration of nose fur (Day 11) 

and one 250 mg/kg/day rat with red discoloration of nose fur (Day 11). Salivating rats were 

observed after dosing on Day 3/4 for the 250 mg/kg/day group (3 rats) of the androgen 

agonist assay, and the 50 mg/kg/day (1 rat) and 150 mg/kg/day (2 rats) groups of the antiandrogen assay. These observations were not considered treatment-related. 

Page 243 of 310
In vivo Hershberger Assay (2011)/ Page 7 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gains 

for the androgen agonist assay are presented in Table 2. Body weights for the 50 and 250 

mg/kg/day treatment groups were comparable to controls throughout the duration of the 

assay. At 250 mg/kg/day, overall (Days 1-11) body weight gains were decreased (p≤0.05) 

by 40%. Animals dosed with TP had increased (p≤0.05) body weights on Day 11 (↑10%) 

and increased overall body weight gains (↑69%) compared to controls. 

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the 

Androgen Agonist Assay a

Study Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.4) b EPTC (50) EPTC (250) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 275.1 15.1 8 277.0 18.9 8 279.8 17.5 8 278.4 16.2 

4 8 282.7 20.3 8 297.2 20.2 8 291.1 23.8 8 284.2 15.7 

7 8 296.3 18.6 8 317.9 25.9 8 306.4 26.5 8 297.4 19.0 

11 8 315.7 20.6 8 345.9* 

(↑10) 

33.9 8 323.2 31.7 8 303.0 31.4 

BWG 

(Days 1-11) 

8 40.7 20.6 8 68.9* 

(↑69) 

19.5 8 43.5 15.0 8 24.6* 

(↓40) 

23.6 

a Data were obtained from Table 4 on page 20 and Appendix V on page 74 of the study report. Percent differences from 

controls (calculated by the reviewers) are included in parentheses. 

b The positive control group for the androgen agonist is same as the vehicle control for the anti-androgen. This did not affect 

the outcome or conclusions of this study. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p≤0.05. 

Body weight and body weight gain data for the anti-androgen assay are presented in Table 

3. Body weights in the 50 and 150 mg/kg/day groups were comparable to controls 

throughout the duration of the assay. At 250 mg/kg/day, overall body weight gains were 

decreased (p≤0.05) by 34%. For the positive control group (TP + FT), overall body weight 

gains were decreased (NS) by 20%. 

Page 244 of 310
In vivo Hershberger Assay (2011)/ Page 8 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day # 

Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.4) b

Positive Control

TP + FT 

(0.4 + 3.0) 

EPTC+TP 

50 + 0.4) 

EPTC+TP 

(150 + 0.4) 

EPTC+TP 

(250 + 0.4) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 277.0 18.9 8 277.6 17.7 8 278.2 16.8 8 277.7 16.2 8 278.9 12.1

4 8 297.2 20.2 8 293.2 20.8 8 296.2 18.6 8 289.5 18.7 8 291.7 13.6

7 8 317.9 25.9 8 309.0 23.1 8 318.0 22.7 8 313.5 23.4 8 311.9 16.0

11 8 345.9 33.9 8 332.8 25.7 8 345.9 28.4 8 339.5 22.5 8 324.1 16.7

BWG 

(days 1-11) 

8 68.9 19.5 8 55.2 

(↓20) 

9.3 8 67.6 15.6 8 61.8 15.5 8 45.3* 

(↓34) 

12.3

a Data were obtained from Table 5 on page 21 and Appendix V on page 74-75 of the study report. Percent differences from 

controls (calculated by the reviewers) are included in parentheses. 

b The vehicle control for the anti-androgen is same as the positive control group for the androgen agonist. This did not affect 

the outcome or conclusions of this study. 

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 determined. 

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone concentrations 

were not determined. 

E. ORGAN WEIGHTS: Accessory sex organ weights for the androgen agonist assay are 

presented in Table 4. There were also no treatment-related effects in any of the accessory 

sex organs of animals dosed with EPTC. Animals in the TP group had increases (p≤0.05) in 

accessory sex organ weights as follows: 1578% in seminal vesicles; 1542% in ventral 

prostate; 259% in LABC; 550% in Cowper’s gland; and 70% in glans penis. 

The maximum guideline-specified %CV was slightly exceeded for the seminal vesicle %CV 

in the 250 mg/kg/day EPTC group (43% vs. 40%). All other %CV values were less than the 

maximum permissible values. 

Page 245 of 310
In vivo Hershberge

EPTC/041401 O

TABLE 4. Accessory Sex Organ Weights (g) from Androgen Agonist Assay in Sprague-Dawley Rats a

Organ 

Dose (mg/kg/day) 

Vehicle control EPTC (50) EPTC (250) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N 

Seminal vesicles 

8 0.0427 0.0101 24 8 0.0357 0.0075 21 8 0.0372 0.0158 43 8 

Ventral prostate 

8 0.0115 0.0050 43 c 8 0.0089 0.0029 33 8 0.0080 0.0028 35 8 

LABC 8 0.1513 0.0242 16 8 0.1689 0.0264 16 8 0.1411 0.0265 19 8 Cowper’s glands 

8 0.0064 0.0020 31 8 0.0062 0.0012 20 8 0.0059 0.0020 34 8 

Glans penis 8 0.0503 0.0086 17 8 0.0506 0.0074 15 8 0.0507 0.0096 19 8 

a Data were obtained from Table 6 on page 23 and Appendix VI on page 77 of the study report. Percent differences from controls were calculated 

included in parentheses. 

b The positive control group for the androgen agonist is same as the vehicle control for the anti-androgen. This did not affect the outcome or conc

c The %CV exceeds the maximum acceptable %CV. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation NA Not applicable * Significantly different from controls at p≤0.05 
In vivo Hershberger Assay (2011)/ Page 10 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

Accessory sex organ weights for the anti-androgen assay are presented in Table 5. At 50 

mg/kg/day, the weights of seminal vesicles were decreased by 23% (p≤0.05). The LABC 

weights were decreased by 10% (p≤0.05) at 150 mg/kg/day and 16% (p≤0.05) at 250 

mg/kg/day. Animals dosed with TP + FT (positive control) had decreases (p≤0.05) in 

accessory sex organ weights as follows: 88% in seminal vesicles; 93% in ventral prostate; 

55% in LABC; and 65% in Cowper’s gland. 

The maximum guideline-specified CV was slightly exceeded for the glans penis in the 250 

mg/kg/day (19% vs. 17%) group. All other CV values were less than the maximum 

permissible values.

Page 247 of 310
In vivo Hershberger 

EPTC/041401 O

TABLE 5. Accessory Sex Organ Weights (g) from Anti-Androgen Agonist Assay in Sprague-Dawley Rats a

Organ

Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.4) b 

EPTC+TP 

(50+0.4) 

EPTC+TP 

(150+0.4) 

EPTC+TP 

(250+0.4) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N 

Seminal vesicles 

8 0.7163 0.1581 22 8 0.5486* 

(↓23) 

0.1383 25 8 0.5766 0.0674 12 8 0.6224 0.1470 24 8 

Ventral prostate 

8 0.1888 0.0473 25 8 0.1715 0.0319 19 8 0.1612 0.0247 15 8 0.1653 0.0260 16 8 

LABC 8 0.5431 0.0529 10 8 0.4956 0.0501 10 8 0.4912* (↓10) 0.0484 10 8 0.4537* (↓16) 0.0340 7 8 Cowper’s glands 8 0.0416 0.0098 24 8 0.0417 0.0059 14 8 0.0396 0.0039 10 8 0.0387 0.0066 17 8 Glans penis 8 0.0855 0.0075 9 8 0.0948 0.0130 14 8 0.0990 0.0096 10 8 0.0911 0.0169 19 c 8 

a Data were obtained from Table 7 on page 24 and Appendix VI on page 78 of the study report. Percent differences from controls were calculated 

included in parentheses. 

b The vehicle control for the anti-androgen is same as the positive control group for the androgen agonist. This did not affect the outcome or conclc The %CV exceeds the maximum acceptable %CV. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation NA Not applicable * Significantly different from controls at p≤0.05 
In vivo Hershberger Assay (2011)/ Page 12 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In the agonist assay, the final mean body weight 

of animals administered 250 mg/kg/day EPTC was 96% of the vehicle control as a result of 

the mean body weight gain during the dosing interval being statistically significantly lower 

than the control. EPTC administered at dose levels of 50 and 250 mg/kg/day did not 

increase androgen dependent tissue weights. 

In the androgen-antagonist assay, final mean body weights of animals administered 

250 mg/kg/day EPTC plus TP were 94% of the control (vehicle and TP) as a result of the 

mean body weight gain during the dosing interval being statistically significantly decreased 

compared to control. A significant reduction in seminal vesicle weight was observed in 

animals administered 50 mg/kg/day EPTC plus TP when compared to the TP-control group, 

however, no change in the weight of this tissue occurred in the two higher dose levels 

(150 and 250 mg/kg/day) to suggest a biological effect of the change in this tissue with 

administration of EPTC plus TP. EPTC co-administered with TP caused a statistically 

significant dose-dependent decrease in LABC weights (10% and 17% for the 150 and 

250 mg/kg/day dose groups, respectively) as compared to the control group, however no 

other androgen-dependent weights were reduced at these dose levels, therefore, the 

Hershberger Bioassay is negative for EPTC at doses up to 250 mg/kg/day. 

Oral administration of EPTC of up to 250 mg/kg/day for 10 consecutive days, to castrated 

adult rats, did not result in androgen agonist or antagonist activity and 5-α reductase 

inhibition as shown by lack of effects on multiple androgen-dependent tissue weights. 

B. AGENCY COMMENTS: One positive control (TP only) rat died after the first day of 

dosing due to a gavage error. All other animals survived until scheduled termination. There 

were no treatment-related clinical signs of toxicity. 

In the androgen agonist assay, body weights for the 50 and 250 mg/kg/day treatment groups 

were comparable to vehicle controls throughout the duration of the assay. At 250 

mg/kg/day, overall (Days 1-11) body weight gains were decreased (p≤0.05) by 40%. 

Animals dosed with TP had increased (p≤0.05) body weights on Day 11 (↑10%) and 

increased overall body weight gains (↑69%) compared to controls. Animals in the positive 

control (TP) group had increases (p≤0.05) in accessory sex organ weights as follows: 

1578% in seminal vesicles; 1542% in ventral prostate; 259% in LABC; 550% in Cowper’s 

gland; and 70% in glans penis. The maximum guideline-specified %CV was slightly 

exceeded for the seminal vesicle %CV in the 250 mg/kg/day EPTC group (43% vs. 40%). 

All other %CV values were less than the maximum permissible values. 

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were 

comparable to controls throughout the duration of the assay. At 250 mg/kg/day, overall 

body weight gains were decreased (p≤0.05) by 34%. For the positive control group (TP + 

FT), overall body weight gains were decreased (NS) by 20%. At 50 mg/kg/day, the weights 

of seminal vesicles were decreased by 23% (p≤0.05). The LABC weights were decreased 

by 10% (p≤0.05) at 150 mg/kg/day and 16% (p≤0.05) at 250 mg/kg/day. Animals dosed 

with TP + FT (positive control) had decreases (p≤0.05) in accessory sex organ weights as 

follows: 88% in seminal vesicles; 93% in ventral prostate; 55% in LABC; and 65% in 

Page 249 of 310
In vivo Hershberger Assay (2011)/ Page 13 of 13 

EPTC/041401 OCSPP 890.1400/ OECD 441

Cowper’s gland. The maximum guideline-specified %CV was slightly exceeded for the 

glans penis in the 250 mg/kg/day (19% vs. 17%) group. All other %CV values were less 

than the maximum permissible values. 

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

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

Hershberger assay. 

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

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

 In the androgen agonist assay, the maximum guideline-specified %CV was slightly 

exceeded for the seminal vesicle in the 250 mg/kg/day EPTC group (43% vs. 40%). 

All other %CV values were less than the maximum recommended values. 

 In the anti-androgen assay, the maximum recommended %CV value was slightly 

exceeded for the glans penis in the 250 mg/kg/day EPTC group (19% vs. 17%) group. 

All other %CV values were less than the maximum recommended values. 

Page 250 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-05-2012 (MRID 48827603) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/23/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 8/13/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 8/14/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 8/14/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 251 of 310
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 252 of 310
EPTC I 041401 

Primary Reviewer: Chester Rodriguez, Ph.D. __ _ 

Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Health Effects Division 

Female Pubertal Assay (2012) I Page I of 16 

OCSPP 890.1450/ OECD None 

SignaM~/~~ Date: . 

Signature: 

Date: 

C~ ~ j -

Template~~ 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 041401 DP BARCODE: D402841 

TXR #: 0051762 CAS #: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: S-ethyl N.N-dipropylcarbamothioate 

CITATION: Zorrilla, L. (2012) Pubertal Development and Thyroid Function with EPTC 

in Juvenile/Peripubertal CRL: CD (SD) IGS Male and Female Rats. 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study No.: 

C201-300, May 1, 2012. MRID 48827603. Unpublished. 

SPONSOR: Gowan Company, P.O. Box 5569, Yuma, A2 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID# 48827603), 16 female 

Sprague-Dawley (Crl:CD®(SD) IGS) rats/dose group were treated daily via oral gavage with 

EPTC (99.9% a.i., Lot #: Pl 105096) in corn oil at doses of 0, 137.5, or 275 mg/kg/day from 

post-natal day (PND) 22 to 42 or 43 . Animals were examined for vaginal opening (VO) daily 

beginning on PND 22, and age and weight at day of attainment were recorded. Following 

sacrifice on PND 42 or 43, blood was collected for clinical chemistry and hormone analyses. 

Total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using 

radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed and 

examined macroscopically. The uterus, ovary, thyroid, and kidney were examined 

microscopically. 

No adverse, treatment-related related effects were observed on survival, clinical signs, organ 

weights, clinical chemistry, gross pathology, uterine or renal histology. 

The following decreases (p:S0.05) were noted in growth between the EPTC-treated groups and 

the controls(% difference): (i) unadjusted and adjusted body weight at VO at 275 mg/kg/day 

(12%; adjusted means not reported); (ii) adjusted final body weight at 137.5 mg/kg/day (7%) and 

unadjusted and adjusted final body weight at 275 mg/kg/day (8% each); and (iii) unadjusted and 

adjusted overall bodyweight gain at 137.5 (11% each) and 275 mg/kg/day (12-13%). 

Page 253 of 310
Female Pubertal Assay (2012) / Page 2 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

The relative adrenal weight was increased (p≤0.001) by 22% at 275 mg/kg/day; however, 

absolute and adjusted adrenal weights in the treated groups were similar to controls. Therefore, 

this effect was not considered adverse. All other organ weights in the treated groups were 

similar to controls. 

Ages at initial and complete VO in the treated groups were similar to control, and VO occurred 

in all animals. No treatment-related effect was observed on the mean age of first vaginal estrus, 

mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy. The number 

of antral follicles in the ovaries was increased (p≤0.05) by 145% at 275 mg/kg/day (4.9 vs. 2 in 

controls), and a similar trend (not significant, NS) was noted in the number of antral follicles at 

137.5 mg/kg/day (3.7). 

Compared to the control, there was a significant decrease (p<0.05) in mean colloid area in the 

thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar trends 

(NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. The number 

of animals with colloid areas at Grade 4 decreased from 13/15 in the control to 10/16 and 5/16 at 

137.5 and 275 mg/kg/day, respectively. Animals with follicular cell heights at Grade 3 increased 

from 2/15 in the control, to 6/16 and 10/16 at 137.5 and 275 mg/kg/day, respectively. However, 

the biological significance of these thyroid changes is unclear as there were no associated 

changes in serum T4 and TSH levels or thyroid weight. 

The dose level tested were determined to be adequate based on the decrease in body weight and 

body weight gain at 275 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 254 of 310
Female Pubertal Assay (2012) / Page 3 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratories Systems, Inc. 

Location: Durham, NC 

Study Directors: Leah Zorilla, Ph.D. 

Other Personnel: Susan Borghoff, Ph.D., DABT; study toxicologist 

Dipak, K. Giri, DVM PhD, DACVP 

Seena Polivy; principal investigator for hormone and clinical chemistry analyses 

Study Period: July 21, 2011 through May 1, 2012 

2. Test Substance: EPTC; S-ethyl N,N-dipropylcarbamothioate 

 Description: Off white solid 

 Source: Gowan Company 

 Lot #: P1105096 (expires February 2, 2015) 

 Purity: >99.9%. 

 Stability: Report stated that the compound is stable in the vehicle at 1-10°C for up to 25 days 

 CAS #: 759-94-4 

 Structure: 

N

S

O 3CH

3CH

CH3

3. Vehicle: Corn oil 

4. Test Animals:

Species: Rat (females) 

Strain: Sprague-Dawley (Crl:CD®(SD) IGS) 

Age/ Weight at 

Study Initiation: PND 22 / 48.0-67.1 g 

Source: Charles River Laboratories International, Inc. (Raleigh, NC) 

Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and 

absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning 

on PND 21. After weaning, up to 2 females were housed per cage. 

Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was 

stated that the total genistein equivalents of genistein plus daidzein was determined to be 

8.0-8.6 μg/g of feed.

Water: Reverse osmosis-purified tap water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

17-23°C 

33-77% 

Not reported 

14 h light / 10 h dark

B. STUDY DESIGN

1. In-Life Dates: Start: August 26, 2011 End: September 17, 2011 

2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the 

supplier on gestation day 8 and were allowed to deliver naturally and rear their pups to PND 

21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were 

standardized to 8 pups with equal numbers of males and females. 

Page 255 of 310
Female Pubertal Assay (2012) / Page 4 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using 

a procedure that stratifies animals across groups by body weight such that mean body 

weight of each group was not statistically different from any other group using analysis of 

variance. Care was taken to avoid placing littermates in the same dose group. The test 

groups are provided in Table 1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 16 

Low 137.5 16 

High 275 16 

a Data were obtained from Table 1 on page 15 of the study report. 

4. Dose Selection Rationale: The doses were selected based on the results from a range 

finding study1 and other previously conducted studies in female rats with orally 

administered EPTC. In the range finding study, juvenile female rats were given EPTC at 

dose levels of 0, 200, 400, or 600 mg/kg/day for 14 days beginning on PND 22. Mortality 

occurred at 600 mg/kg/day, and the mean body weights on the last day of dosing (PND 35) 

for the 400 and 200 mg/kg/day groups were 84% and 103%, respectively, of the vehicle 

control. Maternal mortality was observed on a previous developmental toxicity study at 

doses >300 mg/kg/day EPTC, albeit using older animals. Therefore, considering the young 

animal model used in this assay, and the number of days the animals are dosed, a high dose 

of 275 mg/kg/day was chosen for the female pubertal assay. The second dose (137.5 

mg/kg/day) was one-half the high dose. 

5. Dose Preparation and Analysis: EPTC dose formulations were prepared at ILS in corn oil 

twice (August 18, 2011 and September 6, 2011) during the study at concentrations of 27.5 

and 55 mg/mL. EPTC and corn oil were adjusted based on their density, weighed, and were 

mixed on a stir plate until dissolved. All EPTC formulation concentrations were solutions. 

Formulations were dispensed into vials to be used daily during the study. 

Samples of each dose formulation were collected from the top, middle, and bottom of the 

formulations and sent to Smithers Viscient (Wareham, MA) for analysis. Smithers Viscient 

analyzed, in duplicate, samples received for concentration and homogeneity. The Report 

stated that the compound is stable in the vehicle at 1-10°C for up to 25 days on the basis of 

previous study2

; however, data were not presented. 

 

1

 Zorrilla, L. (2012). Range Finder Study for the In Vivo Pubertal Assays for EPTC. Unpublished study report 

prepared by ILS, Inc. Study No. C201-500. 

2

 Dix, R. (2011). Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers Viscient, 

LLC. Study No. 12791-6162. 

Page 256 of 310
Female Pubertal Assay (2012) / Page 5 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

Results 

Homogeneity (CV): 0.564-3.34% 

Stability (% of time zero): Not reported

Concentration (% of nominal): 97.2-103% 

The analytical data indicated that the mixing procedure was adequate and the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Dose formulations were administered once daily by oral gavage 

in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 21 

or 22 consecutive days from PND 22-42/43. Dosing occurred 24-hours (± 2 hours) from the 

previous dose. Dose volume was adjusted daily based on individual animal body weight. 

The daily dosing sequence was stratified across all dose groups; 1 animal from each group 

and then repeated until all animals were dosed. 

7. Statistics: Descriptive statistics (mean, standard deviation, coefficient of variance, and 

sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets 

listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, 

Cary, NC). Studentized residual plots were used to detect possible outliers in the data and 

Levene’s test was used to assess homogeneity of variance. Heterogeneous data were 

transformed (logarithm, square root, and multiplicative inverse) and if still heterogeneous 

analyzed using the non-parametric Kruskal-Wallis and Dunn’s test. 

 

Classification Procedure 

Initial body weights 

Final body weight (use last day all body weights collected) 

Cumulative body weight gain (use last day all body weights 

collected) 

Age and body weight at VO 

Age at first estrus 

Cycle length 

Ovarian counts (follicles, cysts, corpora lutea) 

Homogeneous data sets: 

One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed 

t-tests 

Body weight 

Organ weight 

Tests for linear trend were performed and reported when 

significant (p<0.05) for endpoints that did not show any 

significant pair-wise comparisons. 

Tissue weights 

Relative tissue weights (liver, kidneys, pituitary, and 

adrenals) 

Hormone levels 

Clinical chemistry levels 

Two-way ANOVA with treatment and necropsy day (if >1 

day) as main effects. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests. 

Final body weight (use last day all body weights collected) 

Final body weight gain (use last day all body weights 

collected) 

Age and body weight at VO 

Tissue weights 

Analysis of covariance (ANCOVA) with PND 21 body 

weight (weight on day of allocation) as the covariable. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests. 

Cycling status Chi-square analysis 

Page 257 of 310
Female Pubertal Assay (2012) / Page 6 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

Classification Procedure 

Percent of animals cycling regularly 

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant, 

followed by Kruskal-Wallis and Dunn's test. 

Significance was reported as exact p-values, except no p-value was reported for Dunn's Test 

(p<0.05). The statistical analyses were considered appropriate. 

C. METHODS

1. Mortality and Clinical Examinations: Animals were observed twice daily for mortality 

and moribundity on weekdays and once daily on weekends and holidays. Rats were 

observed prior to allocation of animals to study groups, daily prior to dose administration, 

and prior to euthanasia. 

2. Body Weight: Body weights were recorded weekly following birth until allocation of 

animals to study groups. Then body weights were recorded daily prior to dosing, and final 

body weight was recorded just prior to euthanasia. 

3. Food Consumption: Food consumption was not measured. 

4. Vaginal Opening (VO): Beginning on PND 22, females were examined daily 

(approximately the same time each day) following dosing for VO. VO was recorded as: VO 

not initiated, appearance of a pin hole, vaginal thread, or complete VO.

5. Estrous Cyclicity: Beginning on the day of complete VO through euthanasia, vaginal 

smears were prepared daily after dose administration (approximately the same time each 

day), evaluated, and classified as diestrus, proestrus, or estrus. Metestrus was classified as 

diestrus. The overall cycling pattern for each female was characterized as regular, irregular, 

or not cycling. Regular cycling was defined as having recurring 4- to 5-day cycles. 

Animals were considered not to be cycling if there was ≥3 consecutive days of estrus or ≥5 

consecutive days of diestrus. 

6. Sacrifice and Pathology: Animals were moved to the necropsy holding room at least 2 

hours before euthanasia. Animals were humanely euthanized by decapitation 2-5 hours 

post-dose, in the same order as they were dosed. After euthanasia and prior to tissue 

collection, trunk blood was collected, and serum was obtained. All serum samples were 

stored at or below −70°C. Serum samples were shipped on dry ice to AniLytics Inc. 

(Gaithersburg, MD) for analysis. 

a. Hormone Analysis: Total T4 and TSH were measured in serum using radioimmunoassays. 

Samples were assayed in duplicate and in conjunction with multiple quality control samples. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

ELECTROLYTES OTHER 

Page 258 of 310
Female Pubertal Assay (2012) / Page 7 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

X Calcium X Albumin 

X Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

X Phosphorus Total cholesterol 

X Potassium Globulins 

X Sodium Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALP) X Total protein 

 Cholinesterase (ChE) Triglycerides 

 Creatine phosphokinase Albumin/globulin ratio (calculated) 

 Lactic acid dehydrogenase (LDH) Bile acids 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

X Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed and grossly observed from all study animals. The (XX) organs, in 

addition, were subjected to histological examination. 

 UROGENITAL OTHER 

XX Ovaries (paired, without oviducts)*+ XX Thyroid a*+

XX Uterus*+ X Liver* 

XX Kidneys (paired)*+ X Adrenals (paired)* 

 X Pituitary* 

a Thyroid was weighed post-fixation. 

* Weights required based on guideline 890.1450. 

+ Histopathological examination required based on guideline 890.1450. 

Uterus (without fluid) left ovary, left kidney, and thyroid (with attached trachea) were fixed 

in 10% neutral buffered formalin for at least 24-hours. Following fixation, the thyroid was 

dissected free of the trachea and weighed. After fixation, tissues were transferred to 70% 

histology grade alcohol, routinely processed into paraffin blocks, sectioned, and stained with 

hematoxylin and eosin. Histologically-processed tissues (uterus without fluid, left ovary, 

thyroid, and left kidney) from all animals were microscopically evaluated. Evaluation of 

each ovarian section included: follicular development (including presence/absence of 

tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum 

development, and changes in number of both primary and atretic follicles) in addition to any 

abnormalities/lesions, such as ovarian atrophy. Two sections of each of the 2 lobes of the 

thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point 

grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were 

evaluated relative to photomicrographs supplied by the U.S. EPA. 

II. RESULTS 

Page 259 of 310
Female Pubertal Assay (2012) / Page 8 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

A. MORTALITY: One rat (No. 14) administered vehicle control (Group 1) was found dead 

on PND 42 after receiving its daily dose. Upon necropsy, the animal had oil droplets in the 

lung lavage, and this death was attributed to a gavage error. All remaining animals survived 

to study termination. 

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clinical signs were observed. 

Rough coat was noted on at least 1 day in 3 control animals, 6 animals at 137.5 mg/kg/day, 

and 4 animals at 275 mg/kg/day. However, this finding was considered incidental as these 

animals were normal all other days of the study. All other rats in these groups appeared 

normal throughout the study. 

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, 

age of attainment of VO, weight at VO, and proportion/incidence of unopened are presented 

in Table 2. The following decreases (p≤0.05) were noted between the EPTC-treated groups 

and the controls (% difference): (i) unadjusted and adjusted body weight at VO at 275 

mg/kg/day (12%; adjusted means not reported); (ii) adjusted final body weight at 137.5 

mg/kg/day (7%) and unadjusted and adjusted final body weight at 275 mg/kg/day (8% 

each); and (iii) unadjusted and adjusted overall bodyweight gain at 137.5 (11% each) and 

275 mg/kg/day (12-13%). Initial body weights and ages at initial and complete VO in the 

treated groups were similar to control. VO occurred in all animals. 

The Guideline performance criteria were met for age and weight at VO and final body 

weight. 

Page 260 of 310
Female Pubertal Assay (2012) / Page 9 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

TABLE 2. General Growth and Vaginal Opening (VO)a

Parameter Evaluated 

Dose levels 

Vehicle Control EPTC 

137.5 mg/kg/day 

EPTC 

275 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial body weight 

(PND 22; g) 

U 16 57.2 4.1 7.1 16 57.5 4.4 7.6 16 57.6 3.8 6.6 

A 16 57.4 --- --- 16 57.4 --- --- 16 57.5 --- --- 

Body weight at 

VO (g) 

U 16 114.9 8.7 7.6 16 111.8 13.0 11.6 16 100.6*** 

(↓12) 6.9 6.8 

A 16 NR --- --- 16 NR --- --- 16 NR*** --- --- 

Final body weight 

(g) 

U 15 169.7 14.0 8.3 16 157.8 11.7 7.4 16 155.9* 

(↓8) 18.1 11.6 

A 15 170.0 --- --- 16 157.7* 

(↓7) --- --- 16 155.8** 

(↓8) --- --- 

Final body weight 

(% of control)

U 15 --- --- --- 16 93.0 --- --- 16 91.9 --- --- 

A 15 --- --- --- 16 92.8 --- --- 16 91.6 --- --- 

Body weight gain 

(final – initial; g) 

U 15 112.3 11.9 10.6 16 100.4* 

(↓11) 9.9 9.9 16 98.4** (↓12) 15.6 15.9 

A 15 112.5 --- --- 16 100.3* 

(↓11) --- --- 16 98.3** (↓13) --- --- 

Age at initial VO 

(PND) 

U 16 32.5 1.6 4.9 16 32.6 2.1 6.4 16 31.4 1.0 3.3 

A 16 32.5 --- --- 16 32.6 --- --- 16 31.4 --- --- 

Age at complete 

VO (PND) 

U 16 32.5 1.6 4.9 16 32.6 2.1 6.4 16 31.4 1.0 3.3 

A 16 32.5 --- --- 16 32.6 --- --- 16 31.4 --- --- 

Proportion unopened 

(#/N) 0/16 0/16 0/16 

a Data were obtained from Table 5 on page 26 of the study report. Percent differences from controls (calculated by 

reviewers) are included in parentheses. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

SD Standard Deviation 

SE Standard Error; used for means adjusted for body weight on PND 21 

CV Coefficient of Variation 

NA Not applicable 

NR No value was reported 

* Significantly different from controls at p≤0.05. 

** Significantly different from controls at p≤0.01. 

*** Significantly different from controls at p≤0.001. 

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. The relative 

adrenal weight was increased (p≤0.001) by 22% at 275 mg/kg/day compared to controls. 

All other organ weights in the treated groups were similar to controls. 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1450), with the exception 

of the mean adrenal weight of 38.0 mg (acceptable range: 38.34-48.84 mg). 

Page 261 of 310
Female Pubertal Assay (2012) / Page 10 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

TABLE 3. Organ Weights at Necropsy a

Organ 

Vehicle Control 137.5 mg/kg/day EPTC 275 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Liver 

(g) 

U 15 8.05 0.95 11.8 16 7.41 0.80 10.8 16 7.72 1.04 13.5 

A 15 8.07 --- --- 16 7.41 --- --- 16 7.71 --- --- 

R 15 4.74 0.34 7.3 16 4.69 0.27 5.7 16 4.94 0.23 4.6 

Kidneys 

(g) 

U 15 1.3 0.08 6.3 16 1.26 0.14 11 16 1.26 0.14 10.8 

A 15 1.3 --- --- 16 1.26 --- --- 16 1.26 --- --- 

R 15 0.77 0.06 7.4 16 0.8 0.06 7 16 0.81 0.06 7.7 

Pituitary 

(mg) 

U 15 9.4 1.0 10.3 16 8.7 1.2 13.3 16 8.8 1.1 13.0 

A 15 9.4 --- --- 16 8.7 --- --- 16 8.8 --- --- 

R 15 5.6 0.9 15.2 16 5.5 0.7 12.3 16 5.7 0.8 13.9 

Adrenals 

(mg) 

U 15 38.0 4.9 13 16 39 5.3 13.5 16 42.6 6.7 15.7 

A 15 38.1 --- --- 16 39 --- --- 16 42.6 --- --- 

R 15 22.5 2.6 11.6 16 24.8 3.0 12.2 16 27.4*** 

(↑22) 3.5 12.8 

Ovaries 

(mg) 

U 15 74.4 10.1 13.6 16 74.5 12.5 16.8 16 80.7 16.7 20.7 

A 15 74.6 --- --- 16 74.4 --- --- 16 80.6 --- --- 

Uterus, wet 

(mg) 

U 15 342.4 127.1 37.1 16 325 137.2 42.2 15 343.8 130.4 37.9 

A 15 342.4 --- --- 16 325 --- --- 15 343.8 --- --- 

Uterus, 

blotted 

(mg) 

U 15 294.4 75.6 25.7 16 268.6 75.1 28.0 16 284.6 65.9 23.2 

A 15 294.4 --- --- 16 268.6 --- --- 16 284.6 --- --- 

Thyroid 

(mg) 

U 15 13.6 2.3 16.8 16 13.4 2.8 20.6 16 13.4 2.0 14.7 

A 15 13.6 --- --- 16 13.4 --- --- 16 13.4 --- --- 

a Data were obtained from Table 6 on page 28 of the study report. Percent difference from controls (calculated by reviewers) 

is presented parenthetically. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

R Organ-to-body weight ratio (relative to body weight) 

SD Standard Deviation 

CV Coefficient of Variation 

*** Significantly different from controls at p<0.001. 

E. ESTROUS CYCLICITY: Estrous cycle data are provided in Table 4. No treatmentrelated effect was observed on the mean age of first vaginal estrus, mean cycle length, 

% cycling, % regularly cycling, and the cycle status at necropsy. 

Page 262 of 310
Female Pubertal Assay (2012) / Page 11 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

TABLE 4. Estrous Cyclicitya

Treatment 

Groups N

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# Females) 

Diestrus Proestrus Estrus

Not 

Cycling 

Vehicle 16 32.8 4.6 100 100 9 3 3 0 

EPTC 

(137.5 mg/kg/day) 16 33.4 4.8 100 87.5 (n=14) 9 3 3 0 

EPTC 

(275 mg/kg/day) 16 31.9 4.6 100 100 9 2 4 0 

a Data were obtained from Table 6 on page 28 and Table 7 on page 29 of the study report. No differences (p≤0.05) from the 

controls were detected in the treated groups. 

F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Clinical chemistry and 

hormone levels are presented in Table 5. Normal ranges for these values were provided. 

Serum T4 and TSH levels in the treated groups were similar to the controls. The following 

clinical chemistry parameters differed (p≤0.01) in the treated groups compared to controls: 

(i) sodium increased by 2-4% at both doses; (ii) chloride increased by 3-4% at both doses; 

(iii) calcium decreased by 3% at 275 mg/kg/day; (iv) blood urea nitrogen decreased by 18% 

at 275 mg/kg/day; (v) SDH increased by 32% at 275 mg/kg/day; and (vi) albumin decreased 

by 7% at 275 mg/kg/day. These differences were minor and, with the exception of sodium 

and blood urea nitrogen, were within the reported normal range. 

Serum T4 mean and CV in the control group were within the acceptable range of the 

performance criteria provided in the Guideline (890.1450). 

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control 137.5 mg/kg/day EPTC 275 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4

(μg/dL) 15 3.41 0.96 28.2 16 2.93 0.41 14.0 16 3.46 0.98 28.2 

Serum TSH 

(ng/mL) 15 2.21 0.73 33.2 16 2.08 1.14 55.0 16 2.24 1.13 50.8 

Clinical Chemistry 

Sodium 

(mEq/L) 15 135 2 1.4 16 138*** 

(↑2) 2 1.6 16 140*** (↑4) 2 1.2 

Potassium 

(mEq/L) 15 7.9 0.6 7.3 16 8 1 6.6 16 7.9 0.5 5.9 

Chloride 

(mEq/L) 15 101 2 1.6 16 104*** 

(↑3) 1 1.4 16 105*** 

(↑4) 1 1.4 

Calcium 

(mg/dL) 15 10.2 0.3 2.7 16 10.1 0.2 2.4 16 9.9** 

(↓3) 0.4 3.7 

Phosphorus 

(mg/dL) 15 9.2 0.7 8 16 9.3 0.5 5.5 16 9.5 0.9 9.1 

AST (U/L) 15 293 74 25.2 16 282 38 13.6 16 273 45 16.3 

ALT (U/L) 15 110 21 19.1 16 104 12 11.3 16 104 17 16 

GGT (U/L) 15 1 1 186 16 1 1 174 16 0 0 215 

ALP (U/L) 15 365 92 25.2 16 331 56 16.8 16 323 84 26.1 

Page 263 of 310
Female Pubertal Assay (2012) / Page 12 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control 137.5 mg/kg/day EPTC 275 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

BUN 

(mg/dL) 15 11 2 19.1 16 10 2 16.5 16 9*** 

(↓18) 2 18.2 

Creatinine 

(mg/dL) 15 0.4 0 13.3 16 0.4 0.1 14.4 16 0.3 0.1 14.9 

Total 

Bilirubin 

(mg/dL) 

15 0.01 0.03 387 16 0 0 --- 16 0.03 0.04 179 

SDH (U/L) 15 19 5 25.9 16 23 5 20.4 16 25** 

(↑32) 5 20.6 

Total 

Protein 

(g/dL) 

15 5.7 0.2 4.2 16 5.8 0.3 5.2 16 5.6 0.2 3.5 

Albumin 

(g/dL) 15 4.3 0.2 4.8 16 4.3 0.2 4.9 16 4** 

(↓7) 0.2 5.8 

a Data were obtained from Table 8 on page 29 and Table 9 on page 31 of the study report. Percent difference from controls 

(calculated by reviewers) is presented parenthetically. 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01. 

*** Significantly different from controls at p<0.001. 

G. GROSS PATHOLOGY: There were no macroscopic findings observed in the uterus, 

ovary, thyroid gland, or kidney reported in any animal. 

H. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 6; follicular cell shape was not reported. A trend toward 

decreasing colloid area and increasing follicular cell height was noted in the thyroids of 

EPTC-treated animals. The number of animals with colloid areas at Grade 4 decreased from 

13/15 in the control to 10/16 and 5/16 at 137.5 and 275 mg/kg/day, respectively. Animals 

with follicular cell heights at Grade 3 increased from 2/15 in the control, to 6/16 and 10/16 

at 137.5 and 275 mg/kg/day, respectively. The changes in colloid area and cell height 

attained significance (p<0.05) at the 275 mg/kg/day dose. 

Page 264 of 310
Female Pubertal Assay (2012) / Page 13 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

(Increase) 

Severity b Incidence Grade b Incidence 

O E O E 

Vehicle Control 

1 0 15 1 0 15 

2 0 15 2 13 15 

3 2 15 3 2 15 

4 13 15 4 0 15 

5 0 15 5 0 15 

137.5 mg/kg/day 

EPTC 

1 0 16 1 0 16 

2 0 16 2 10 16 

3 6 16 3 6 16 

4 10 16 4 0 16 

5 0 16 5 0 16 

275 mg/kg/day 

EPTC* 

1 0 16 1 0 16 

2 1 16 2 5 16 

3 10 16 3 10 16 

4 5 16 4 1 16 

5 0 16 5 0 16 

a Data were obtained from Table 12 on page 34 of the study report. 

b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used. 

O No. Observed 

E No. Examined 

* Significantly different from controls at p<0.05. 

The incidences of histopathological findings of the ovaries are presented in Table 7. The 

number of antral follicles in the ovaries was increased (p≤0.05) by 145% at 275 mg/kg/day 

(4.9 vs. 2 in controls), and a similar trend (NS) was noted in the number of antral follicles at 

137.5 mg/kg/day (3.7). The incidences of other ovarian findings were similar to controls. 

TABLE 7. Number of Ovarian Follicles, Follicular Cysts, and Corpora Lutea a

Parameter 

Evaluated 

Vehicle Control 137.5 mg/kg/day EPTC 275 mg/kg/day EPTC 

N b Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Small follicles 14 35.1 34.4 98 16 38.7 26.3 68 16 27.3 24.3 89 

Medium follicles 14 13.5 9 67 16 18.5 8.2 44 16 13.6 9.1 67 

Antral follicles 14 2 2 100 16 3.7 1.6 43 16 4.9* 

(↑145) 3.9 80 

Atretic follicles 14 7.5 6.2 83 16 10.3 6.4 62 16 8.2 6.5 79 

Follicular cysts 14 0.07 0.27 386 16 0 0 - 16 0 0 - 

Corpora lutea 14 6.7 2.2 33 16 8.6 2.8 33 16 8.4 2.8 33 

a Data were obtained from Table 11 on page 33 of the study report. Percent difference from controls (calculated by 

reviewers) is presented parenthetically. 

b One animal died prior to necropsy, and another animal had predominantly stromal tissue and was not included. 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p≤0.05. 

Page 265 of 310
Female Pubertal Assay (2012) / Page 14 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

The incidences of histopathological findings of the uterus and kidneys are presented in Table 8, 

and no treatment-related findings were observed. 

TABLE 8. Histopathological Findings in the Uterus and Kidneys 

Parameter Vehicle Control 

EPTC 

137.5 mg/kg/day

EPTC 

275 mg/kg/day

Uterus Dilatation 

 Minimal 2 6 7 

 Mild 3 0 0 

 Moderate 0 0 1 

 Eosinophilic material, Minimal 0 1 1 

 Inflammatory cells/debris 

 Minimal 0 1 1 

 Mild 0 1 0 

 Endometrial folding 

 Minimal 8 8 8 

 Mild 0 0 1 

 Endometrium: inflammatory cells 

 Minimal 7 1 0 

 Mild 0 1 1 

Kidney 

Nephropathy, Minimal 8 5 9 

 Cysts present 2 0 2 

a Data (n=16, except n=15 for controls) were obtained from Appendix 2 on page 128 and Appendix 12 on page 138 

of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Oral administration of EPTC up to 275 

mg/kg/day (approaching the MTD) to intact juvenile/peripubertal female rats for at least 21 

days, did not appear to affect pubertal development or thyroid function. 

B. AGENCY COMMENTS: No adverse, treatment-related related effects were observed on 

survival, clinical signs, organ weights, clinical chemistry, gross pathology, or uterine or 

renal histology. One control rat was found dead on PND 42 due to gavage error. 

The following decreases were noted in growth between the treated groups and the controls: 

(i) unadjusted and adjusted body weight at VO at 275 mg/kg/day; (ii) adjusted final body 

weight at 137.5 mg/kg/day and unadjusted and adjusted final body weight at 275 mg/kg/day; 

and (iii) unadjusted and adjusted overall bodyweight gain at 137.5 and 275 mg/kg/day. 

The relative adrenal weight was increased at 275 mg/kg/day; however, the absolute and 

adjusted adrenal weights in the treated groups were similar to controls. Therefore, this 

effect was not considered adverse. All other organ weights in the treated groups were 

similar to controls. 

The following clinical chemistry parameters differed in the treated groups compared to 

controls: (i) sodium increased at both doses; (ii) chloride increased at both doses; (iii) 

calcium decreased at 275 mg/kg/day; (iv) blood urea nitrogen decreased at 275 mg/kg/day; 

Page 266 of 310
Female Pubertal Assay (2012) / Page 15 of 16 

EPTC / 041401 OCSPP 890.1450/ OECD None 

(v) SDH increased at 275 mg/kg/day; and (vi) albumin decreased at 275 mg/kg/day. These 

differences were minor and, with the exception of sodium and blood urea nitrogen, were 

within the reported normal range. 

Ages at initial and complete VO in the treated groups were similar to control, and VO 

occurred in all animals. No treatment-related effect was observed on the mean age of first 

vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at 

necropsy. Histological examination of the ovaries indicated a trend toward increases in the 

number of antral follicles of EPTC-treated rats, which attained significance (p≤0.05) at 275 

mg/kg/day. 

 Compared to the control, there was a significant decrease (p<0.05) in mean colloid area in 

the thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar 

trends (NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. 

However, the biological significance of these thyroid changes is unclear as there were no 

associated changes in serum T4 and TSH levels or thyroid weight. 

Based on the decrease in body weight and body weight gain, the high dose was considered 

adequate. 

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: 

 

 All performance criteria provided in the Guideline (890.1450) were met, with the 

exception of the mean adrenal weight of 38.0 mg (acceptable range: 38.34-48.84 mg). 

Page 267 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-05-2012 (MRID 48827603) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/23/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 8/13/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 8/14/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 8/14/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 268 of 310
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 269 of 310
EPTC I 041401 

Primary Reviewer: Chester Rodriguez, Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Male Pubertal Assay (2012) I Page I of 15 

CS P 890.1500/ OECD None 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None. 

PC CODE: 041401 DP BARCODE: D40284 l 

TXR #: 0051762 CAS #: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: S-ethyl N,N-dipropylcarbamothioate 

CITATION: Zorrilla, L. (2012) Pubertal Development and Thyroid Function with EPTC 

in Juvenile/Peripubertal CRL: CD (SD) IGS Female and Male Rats. 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study No.: 

C201-300, May I, 2012. MRID 48827603. Unpublished. 

SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48827603), 16 male SpragueDawley (Crl:CD®(SD) IGS) rats/dose group were treated daily via oral gavage with EPTC 

(99.9% a.i., Lot#: Pl 105096) in com oil at doses of 0, 62.5, or 125 mg/kg/day from post-natal 

day (PND) 23 to 53 or 54. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30, and age and body weight at day of attainment was recorded. Following 

sacrifice on PND 53 or 54, blood was collected for hormone assays and clinical chemistry 

analyses. Total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) 

levels were analyzed using radioimmunoassays. Thyroid, liver, adrenals, pituitary, and 

urogenital organs were weighed and examined macroscopically. The thyroid, the left kidney, 

epididymides, and testis were examined microscopically. 

No treatment-related related effects were observed on survival, clinical signs, body weights, final 

body weight, body weight gains, organ weights, clinical chemistry, gross pathology, or renal 

histology. 

No adverse, treatment-related related effects were observed on body weight or age at PPS, age at 

partial PPS, proportion/incidence of unseparated preputia, androgen-dependent tissue organ 

weights, or serum testosterone and thyroid hormone levels. The colloid area and follicular cell 

height of the thyroid in the treated groups were similar to the control group. The incidences of 

Page 270 of 310
Male Pubertal Assay (2012) / Page 2 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

histopathological findings of the testes and epididymides in the treated groups were similar to 

controls. 

The LABC weights showed a linear trend of decreased (p≤0.05) weight with increasing dose; 

however, no statistically significant effect was detected between the treated groups and the 

control group. No other statistically significant effect or trend was detected for the sex tissues or 

any other organ weighed. Therefore, this linear trend was considered incidental. 

Although no toxicity was noted at the high dose (125 mg/kg/day) and terminal body weight was 

only decreased by 5% (NS), relative to the control, the high dose is considered adequate for 

purposes of this assay based data from on a range-finding study using juvenile males in which 

dosing at 200 and 400 mg/kg/day resulted in body weight decreases of 14 and 15%, respectively 

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 271 of 310
Male Pubertal Assay (2012) / Page 3 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratories Systems, Inc. 

Location: Durham, NC 

Study Directors: Leah Zorilla, Ph.D. 

Other Personnel: Susan Borghoff, Ph.D., DABT; study toxicologist 

Dipak, K. Giri, DVM PhD, DACVP 

Seena Polivy; principal investigator for hormone and clinical chemistry analyses 

Study Period: July 21, 2011 through May 1, 2012 

2. Test Substance: EPTC; S-ethyl N,N-dipropylcarbamothioate 

 Description: Off white solid 

 Source: Gowan Company 

 Lot #: P1105096 (expires February 2, 2015) 

 Purity: >99.9%. 

 Stability: Report stated that the compound is stable in the vehicle at 1-10°C for up to 25 days 

 CAS #: 759-94-4 

 Structure: 

N

S

O 3CH

3CH

CH3

3. Vehicle: Corn oil 

4. Test Animals:

Species: Rat (males) 

Strain: Sprague-Dawley (Crl:CD®(SD) IGS) 

Age/ Weight at 

Study Initiation: PND 23 / 48.1-75.1 g 

Source: Charles River Laboratories International, Inc. (Raleigh, NC) 

Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and 

absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning 

on PND 21. After weaning, up to 2 males were housed per cage. 

Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was 

stated that the total genistein equivalents of genistein plus daidzein was determined to be 

8.0-8.6 μg/g of feed.

Water: Reverse osmosis-purified tap water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

17-23°C 

33-77% 

Not reported 

14 h light / 10 h dark

B. STUDY DESIGN

1. In-Life Dates: Start: August 26, 2011 End: September 27, 2011 

2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the 

supplier on gestation day 8 and were allowed to deliver naturally and rear their pups to PND 

21. Between PND 3 and 5, litters with the same date of birth were standardized to 8 pups 

with equal numbers of males and females. 

Page 272 of 310
Male Pubertal Assay (2012) / Page 4 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using 

a procedure that stratifies animals across groups by body weight such that mean body 

weight of each group was not statistically different from any other group using analysis of 

variance. Care was taken to avoid placing littermates in the same dose group. The test 

groups are provided in Table 1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males 

Control 0 16 

Low 62.5 16 

High 125 16 

a Data were obtained from Table 2 on page 15 of the study report. 

4. Dose Selection Rationale: The doses were selected based on the results from a range 

finding study1 and a Hershberger Assay2 in juvenile male rats with orally administered 

EPTC. In the range finding study, rats were given EPTC at dose levels of 0, 200, 400, or 

600 mg/kg/day for 14 days beginning on PND 22. Mortality occurred at 600 mg/kg/day, 

and the mean body weights on the last day of dosing (PND 35) for the 400 and 200 

mg/kg/day groups were 86% and 85%, respectively, of the vehicle control. In the 

Hershberger Bioassay, there was a statistically significant lower body weight gain after 10 

days of administration of 250 mg/kg/day EPTC compared to vehicle control. Therefore, 

considering the young animal model used in this assay, and the number of days the animals 

are dosed, a high dose of 125 mg/kg/day was chosen for the male pubertal assay to meet the 

criteria for targeting the maximum tolerated dose. The second dose (62.5 mg/kg/day) was 

one-half the high dose as described by the guidelines. 

5. Dose Preparation and Analysis: EPTC dose formulations were prepared at ILS in corn oil 

twice (August 18, 2011 and September 6, 2011) during the study at concentrations of 12.5 

and 25 mg/mL. EPTC and corn oil were adjusted based on their density, weighed, and were 

mixed on a stir plate until dissolved. All EPTC formulation concentrations were solutions. 

Formulations were dispensed into vials to be used daily during the study. 

Samples of each dose formulation were collected from the top, middle, and bottom of the 

formulations and sent to Smithers Viscient (Wareham, MA) for analysis. Smithers Viscient 

analyzed, in duplicate, samples received for concentration and homogeneity. The Report 

stated that the compound is stable in the vehicle at 1-10°C for up to 25 days on the basis of 

previous study3

. 

 

1

 Zorrilla, L. (2012). Range Finder Study for the In Vivo Pubertal Assays for EPTC. Unpublished study report 

prepared by ILS, Inc. Study No. C201-500. 

2

 Zorrilla, L. (2011). The Hershberger Assay with EPTC in Castrated CRL:CD (SD) IGS Rats. Unpublished study 

report prepared by ILS, Inc. Study No. C201-200. 

3

 Dix , R. (2011). Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers Viscient, 

LLC. Study No. 12791-6162. 

Page 273 of 310
Male Pubertal Assay (2012) / Page 5 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

Results 

Homogeneity (CV): 0.603-3.79% 

Stability (% of time zero): Not reported

Concentration (% of nominal): 99.3-104% 

The analytical data indicated that the mixing procedure was adequate and the variation 

between nominal and actual dosage to the animals was acceptable. Stability data should be 

submitted for independent verification. 

6. Dosage Administration: Dose formulations were administered once daily by oral gavage 

in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31 

or 32 consecutive days from PND 23-53/54. Dosing occurred 24-hours (± 2 hours) from the 

previous dose. Dose volume was adjusted daily based on individual animal body weight. 

The daily dosing sequence was stratified across all dose groups; 1 animal from each group 

and then repeated until all animals were dosed. 

7. Statistics: Descriptive statistics (mean, standard deviation, coefficient of variance, and 

sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets 

listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, 

Cary, NC). Studentized residual plots were used to detect possible outliers in the data and 

Levene’s test was used to assess homogeneity of variance. Heterogeneous data were 

transformed (logarithm, square root, and multiplicative inverse) and if still heterogeneous 

analyzed using the non-parametric Kruskal-Wallis and Dunn’s test. 

Page 274 of 310
Male Pubertal Assay (2012) / Page 6 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

Classification Procedure 

Initial body weights 

Final body weight (use last day all body weights collected) 

Cumulative body weight gain (use last day all body weights 

collected) 

Age and body weight at PPS 

Homogeneous data sets: 

One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed 

t-tests 

Body weight 

Organ weight 

Tests for linear trend were performed and reported when 

significant (p<0.05) for endpoints that did not show any 

significant pair-wise comparisons. 

Tissue weights 

Relative tissue weights (liver, kidneys, pituitary, and 

adrenals) 

Hormone levels 

Clinical chemistry levels 

Two-way ANOVA with treatment and necropsy day (if >1 

day) as main effects. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests. 

Final body weight (use last day all body weights collected) 

Final body weight gain (use last day all body weights 

collected) 

Age and body weight at PPS 

Tissue weights 

Analysis of covariance (ANCOVA) with PND 21 body 

weight (weight on day of allocation) as the covariable. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests. 

In the instances where PPS had not occurred prior to 

necropsy, the last day of examination plus 1 was used as the 

age at PPS and terminal body weight was used for body 

weight at PPS. In instances when at least 1 animal in any 

group exhibited incomplete PPS, including partial threads 

for > 3 days, the day partial separation was first recorded 

was used in the analyses. 

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant, 

followed by Kruskal-Wallis and Dunn's test. 

Significance was reported as exact p-values, except no p-value was reported for Dunn's Test 

(p<0.05). The statistical analyses were considered appropriate. 

C. METHODS

1. Mortality and Clinical Examinations: Animals were observed twice daily for mortality 

and moribundity on weekdays and once daily on weekends and holidays. Rats were 

observed prior to allocation of animals to study groups, daily prior to dose administration, 

and prior to euthanasia. 

2. Body Weight: Body weights were recorded weekly following birth until allocation of 

animals to the study groups. Then body weights were recorded daily prior to dosing, and 

final body weight was recorded just prior to euthanasia. 

3. Food Consumption: Food consumption was not measured. 

4. Preputial Separation (PPS): Beginning on PND 30, males were examined daily following 

dosing, and PPS was noted as: progression not initiated, partial separation, a persistent 

thread of tissue between the glans and prepuce, or complete PPS. 

5. Sacrifice and Pathology: Animals were moved to the necropsy holding room at least 2 

hours before euthanasia. Animals were humanely euthanized by decapitation 2-5 hours 

Page 275 of 310
Male Pubertal Assay (2012) / Page 7 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

post-dose, in the same order as they were dosed. After euthanasia and prior to tissue 

collection, trunk blood was collected, and serum was obtained. All serum samples were 

stored at or below −70°C. Serum samples were shipped on dry ice to AniLytics Inc. 

(Gaithersburg, MD) for analysis. 

a. Hormone Analysis: Testosterone, T4, and TSH were measured in serum using 

radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with 

multiple quality control samples. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

ELECTROLYTES OTHER 

X Calcium X Albumin 

X Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

X Phosphorus Total cholesterol 

X Potassium Globulins 

X Sodium Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALP) X Total protein 

 Cholinesterase (ChE) Triglycerides 

 Creatine phosphokinase Albumin/globulin ratio (calculated) 

 Lactic acid dehydrogenase (LDH) Bile acids 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

X Sorbitol dehydrogenase (SDH) 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in male rats based on guideline 890.1500. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed and grossly observed from all study animals. The (XX) organs, in 

addition, were subjected to histological examination. 

 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroid a*+

XX Epididymides (left and right separately)*+ X Liver* 

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 

X Levator ani/bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

a Thyroid was weighed post-fixation. 

* Weights required based on guideline 890.1500. 

+ Histopathological examination required based on guideline 890.1500. 

Left kidney and thyroid (with attached trachea) were fixed in 10% neutral buffered formalin 

for at least 24-hours. Following fixation, the thyroid was dissected free of the trachea and 

Page 276 of 310
Male Pubertal Assay (2012) / Page 8 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

weighed. Left testis and left epididymis were each placed in Bouin’s fixative for 18- to 24-

hours and then washed in 70% histology grade alcohol. After fixation, tissues were 

transferred to 70% histology grade alcohol, routinely processed into paraffin blocks, 

sectioned, and stained with hematoxylin and eosin. Histologically-processed tissues 

(thyroid, left kidney, left epididymis, and left testis) from all animals were microscopically 

evaluated. Two sections of each of the 2 lobes of the thyroid were examined and evaluated 

for follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = 

tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs 

supplied by the U.S. EPA. 

II. RESULTS 

A. MORTALITY: All animals survived to scheduled sacrifice. 

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clinical signs were observed. 

Rough coat was observed on at least 1 day in 3 control animals, 4 animals in the 62.5 

mg/kg/day group, and 3 animals in the 125 mg/kg/day group. However, this finding is 

considered incidental as these animals appeared normal on all other days of the study. In 

addition, 1 animal control animal exhibited scabs on the throat and near the left eye on at 

least 1 day. All other rats appeared normal throughout the study. 

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight 

gains, age of attainment of PPS, weight at PPS, and proportion/incidence of unseparated 

preputia are presented in Table 2 below. All values in the treated groups were similar to 

controls. 

The following Guideline performance criteria were not met: (i) CV=6.1% for age at PPS 

(top of acceptable range: 5.67%); (ii) CV=13.3% for weight at PPS (top of acceptable range: 

7.57%); (iii) CV=8.1% for final body weight (top of acceptable range: 7.47%); and (iv) 

mean weaning weight was 64.1 g while the acceptable range was 45.472-59.812 g. 

Page 277 of 310
Male Pubertal Assay (2012) / Page 9 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated 

Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Age at PPS (PND) 

U 15 44.9 2.7 6.1 16 44.4 1.7 3.8 15 45.1 2.1 4.7 

A 15 44.9 --- --- 16 44.4 --- --- 15 45.1 --- --- 

Body weight at 

PPS (g) 

U 15 239.6 31.9 13.3 16 226.2 25.3 11.2 15 232.9 29.4 12.6 

A 15 239.3 --- --- 16 226.3 --- --- 15 233.2 --- --- 

Age at partial PPS 

(PND) 

U 15 40.9 1.3 3.3 16 40.8 1.2 3.0 15 41.3 1.1 2.7 

A 15 40.9 --- --- 16 40.8 --- --- 15 41.3 --- --- 

Initial body weight 

(PND 23; g) 

U 16 64.1 4.3 6.6 16 62.8 7.1 11.4 16 64.7 4.8 7.5 

A 16 64.1 --- --- 16 62.8 --- --- 16 64.7 --- --- 

Final body weight 

(g) 

U 16 311.3 25.1 8.1 16 299.6 28.6 9.5 16 297.1 34.8 11.7 

A 16 311.3 --- --- 16 299.6 --- --- 16 297.1 --- --- 

Final body weight 

(% of control)

U 16 --- --- --- 16 96.2 --- --- 16 95.4 --- --- 

A 16 --- --- --- 16 96.2 --- --- 16 95.4 --- --- 

Body weight gain 

(final – initial; g) 

U 16 247.1 27.0 10.9 16 236.8 26.5 11.2 16 232.4 32.0 13.8 

A 16 247.1 --- --- 16 236.8 --- --- 16 232.4 --- --- 

Proportion 

unseparated (#/N) 1/16 0/16 1/16 

a Data were obtained from Table 16 on page 40 of the study report. No statistical differences were detected. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. The LABC 

weights showed a linear trend of decreased (p≤0.05) weight with increasing dose; however, 

no statistically significant effect was detected between the treated groups and control using 

Dunnett’s test. No other statistically significant effect or trend was detected on the weight 

of the sex tissues or any other organs examined. 

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 exception 

of: (i) mean kidney weight of 2.06 g (acceptable range: 2.242-3.050 g); (ii) mean LABC 

weight of 0.421 g (acceptable range: 0.447-0.855 g); (iii) CV=22.9% for seminal vesicles 

with coagulating gland and fluid (top acceptable range: 21.06%); and (iv) CV=22.1% for 

testis (top acceptable range: 17.62%). 

Page 278 of 310
Male Pubertal Assay (2012) / Page 10 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

TABLE 3. Organ Weights at Necropsy a

Organ 

Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Liver 

(g) 

U 16 14.45 1.21 8.4 16 14.15 2.00 14.2 16 14.01 1.86 13.3 

A 16 14.45 - - 16 14.15 - - 16 14.01 - - 

R 16 4.58 0.20 4.3 16 4.65 0.32 6.8 16 4.63 0.27 5.7 

Kidneys 

(g) 

U 16 2.06 0.19 9.2 16 1.99 0.21 10.6 16 2.03 0.21 10.4 

A 16 2.06 - - 16 1.99 - - 16 2.03 - - 

R 16 0.66 0.04 5.7 16 0.66 0.03 5.0 16 0.67 0.03 3.8 

Pituitary 

(mg) 

U 16 8.8 1.2 13.9 16 8.8 1.8 20.9 16 8.7 1.6 18.4 

A 16 8.8 - - 16 8.8 - - 16 8.7 - - 

R 16 2.8 0.4 15.4 16 2.9 0.6 19.1 16 2.9 0.3 10.1 

Adrenals 

(mg) 

U 16 43.7 4.4 10 16 45.4 6.1 13.5 16 43.3 5.6 12.9 

A 16 43.7 - - 16 45.4 - - 16 43.3 - - 

R 16 14 1.8 13.2 16 15.0 1.6 10.8 16 14.4 1.5 10.7 

Seminal vesicle + 

coagulating gland, 

with fluid (mg) 

U 16 548.3 125.5 22.9 16 557.9 93.2 16.7 16 515.7 125.6 24.4 

A 16 548.3 - - 16 557.9 - - 16 515.7 - - 

Seminal vesicle + 

coagulating gland, 

without fluid (mg) 

U 16 318.5 64.0 20.1 16 321.3 46.0 14.3 16 315.5 76.8 24.3 

A 16 318.5 - - 16 321.3 - - 16 315.5 - - 

Ventral prostate (mg) 

U 16 268.2 47.3 17.6 16 260.0 47.6 18.3 16 247.1 49.7 20.1 

A 16 268.2 - - 16 259.9 - - 16 247.1 - - 

Dorsolateral prostate 

(mg) 

U 16 147.5 29.3 19.9 16 144.5 30.8 21.3 16 137 24.8 18.1 

A 16 147.5 - - 16 144.5 - - 16 137 - - 

LABC 

(mg) b 

U 16 420.8 58.2 13.8 16 412.6 63.0 15.3 16 382.7 40.9 10.7 

A 16 420.9 - - 16 412.6 - - 16 382.6 - - 

Epididymis, left (mg) U 16 202.3 18.4 9.1 16 200.5 22.9 11.4 16 203.9 13.7 6.7 

A 16 202.3 - - 16 200.5 - - 16 203.9 - - 

Epididymis, right 

(mg) 

U 16 204.8 21.8 10.7 16 206.0 26.3 12.8 16 209.2 23.1 11.0 

A 16 204.8 - - 16 206.0 - - 16 209.2 - - 

Testis, left (mg) U 16 1530 153.7 10.0 16 1480.6 161.6 10.9 16 1495.6 132.2 8.8 

A 16 1530.1 - - 16 1480.7 - - 16 1495.4 - - 

Testis, right (mg) U 16 1726.6 584.2 33.8 16 1494.9 150.5 10.1 16 1561.1 268.4 17.2 

A 16 1726.7 - - 16 1495.1 - - 16 1560.9 - - 

Thyroid, fixed 

(mg) 

U 16 17.7 3.3 18.8 16 17.6 4.0 22.9 16 17.1 2.0 12.0 

A 16 17.7 - - 16 17.6 - - 16 17.1 - - 

a Data were obtained from Table 17 on pages 42-43 of the study report. No differences (p≤0.05) were noted between treated 

groups and controls. 

b A significant (p≤0.05) linear trend was noted. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

R Organ-to-body weight ratio (relative to body weight) 

SD Standard Deviation 

CV Coefficient of Variation 

E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Clinical chemistry and 

hormone levels are presented in Table 4. Serum T4 levels were increased (p<0.05) by 12% 

Page 279 of 310
Male Pubertal Assay (2012) / Page 11 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

in the 125 mg/kg/day group, but the concentration was within the reported normal range. 

Serum TSH and testosterone levels in the treated groups were similar to controls. 

The following clinical chemistry parameters differed (p<0.05) in the treated groups 

compared to controls: (i) chloride increased by 2% at both doses; (ii) SDH increased by 28 

and 40% at 62.5 and 125 mg/kg/day respectively; and (iii) albumin decreased by 5% at 125 

mg/kg/day. These differences were minor and were within the reported normal range. 

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), with the 

exception of: mean serum TSH level of 3.92 ng/mL (acceptable range: 4.212–24.112 

ng/mL); and CV=72.1% for serum TSH (top of acceptable range: 58.29%). 

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, 

Total 

(μg/dL) 

16 4.81 0.72 15.0 16 4.49 0.62 13.9 16 4.22* 

(↓12) 0.84 19.8 

Serum TSH 

(ng/mL) 16 3.92 2.82 72.1 16 4.18 2.65 63.4 16 2.78 1.26 45.2 

Serum 

testosterone 

(ng/mL) 

16 2.45 1.59 65.0 16 2.89 2.44 84.6 16 1.54 1.43 93.2 

Page 280 of 310
Male Pubertal Assay (2012) / Page 12 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Clinical Chemistry 

Sodium 

(mEq/L) 16 143 6 4.3 16 144 2 1.5 16 144 2 1.0 

Potassium 

(mEq/L) 16 8.1 0.5 6.4 16 8.3 0.6 7 16 8 0.6 8.1 

Chloride 

(mEq/L) 16 105 1 1.2 16 107*** 

(↑2) 2 1.5 16 107*** (↑2) 1 1.4 

Calcium 

(mg/dL) 16 10.3 0.3 3.2 16 10.3 0.5 5 16 10.3 0.2 2.4 

Phosphorus 

(mg/dL) 16 9.7 0.9 9.6 16 9.9 0.9 9 16 9.4 0.8 8.3 

AST (U/L) 16 228 40 17.4 16 263 72 27.4 16 235 39 16.6 

ALT (U/L) 16 102 12 11.8 16 108 18 16.5 16 105 10 9.3 

GGT (U/L) 16 0.1 0.3 273 16 0.4 0.8 186 16 0.3 0.9 279 

ALP (U/L) 16 523 98 18.8 16 521 95 18.2 16 475 96 20.1 

BUN 

(mg/dL) 16 11 2 16.4 16 11 2 16.8 16 9 2 21.2 

Creatinine 

(mg/dL) 16 0.3 0.1 18.3 16 0.4 0.1 19.1 16 0.4 0 13 

Total 

Bilirubin 

(mg/dL) 

16 0.02 0.04 215 16 0.04 0.06 165 16 0.04 0.06 165 

SDH (U/L) 16 25 4 14.2 16 32** 

(↑28) 9 26.2 16 35*** (↑40) 5 15.0 

Total 

Protein 

(g/dL) 

16 5.8 0.2 3.5 16 6 0.2 4.1 16 5.7 0.2 4.1 

Albumin 

(g/dL) 16 4.2 0.2 5.3 16 4.3 0.2 4.2 16 4.0* 

(↓5) 0.2 5.0 

a Data were obtained from Table 18-19 on pages 45-46 of the study report. Percent difference from controls (calculated by 

reviewers) is presented parenthetically. 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05. 

* Significantly different from controls at p<0.01. 

* Significantly different from controls at p<0.001. 

F. GROSS PATHOLOGY: There were no macroscopic findings reported in any animal. 

G. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 5; follicular cell shape was not reported. The colloid area and 

follicular cell height of the thyroid in the treated groups were similar to the control group. 

Page 281 of 310
Male Pubertal Assay (2012) / Page 13 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

TABLE 5. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

(Increase) 

Grade b Incidence Grade b Incidence 

O E O E 

Vehicle Control 

1 0 16 1 0 16 

2 2 16 2 1 16 

3 13 16 3 13 16 

4 1 16 4 2 16 

5 0 16 5 0 16 

62.5 mg/kg/day 

EPTC 

1 0 16 1 0 16 

2 0 16 2 1 16 

3 15 16 3 15 16 

4 1 16 4 0 16 

5 0 16 5 0 16 

125 mg/kg/day 

EPTC 

1 0 16 1 0 16 

2 0 16 2 3 16 

3 13 16 3 13 16 

4 3 16 4 0 16 

5 0 16 5 0 16 

a Data were obtained from Table 21 on page 48 of the study report. 

b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used. 

O No. Observed 

E No. Examined 

The incidences of histopathological findings of the testes, epididymides, and kidneys are 

presented in Table 6, and there were no treatment-related findings. 

TABLE 6. Incidence of Histopathological Lesions of the Testis, Epididymides, and Kidneya

Findings 

Dose Level 

Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC 

# 

Observed 

# 

Examined 

# 

Observed 

# 

Examined 

# 

Observed 

# 

Examined 

Testis 

Seminiferous tubules, mild dilatation 0 16 0 16 1 16 

Epididymis

Minimal aberrant cells in tubules 

lumen 0 16 8 16 3 16 

Minimal interstitium inflammation 9 16 11 16 9 16 

Kidneys

Minimal nephropathy 7 16 9 16 11 16 

Cysts present 1 16 2 16 0 16 

a Data were obtained from Appendix X on pages 141-147of the study report. 

Page 282 of 310
Male Pubertal Assay (2012) / Page 14 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Administration of EPTC to intact juvenile/ 

peripubertal male rats at a dose that met, but did not exceed the maximum tolerated dose, 

did not cause changes in any endpoints evaluated to suggest a disruption in pubertal 

development or thyroid function.

B. AGENCY COMMENTS: No treatment-related related effects were observed on survival, 

clinical signs, body weights, final body weight, body weight gains, organ weights, clinical 

chemistry, gross pathology, or renal histology. 

The following clinical chemistry parameters differed (p<0.05) in the treated groups 

compared to controls: (i) chloride increased by 2% at both doses; (ii) SDH increased by 28 

and 40% at 62.5 and 125 mg/kg/day respectively; and (iii) albumin decreased by 5% at 125 

mg/kg/day. These differences were minor, within the reported normal range, and were not 

considered adverse. 

No treatment-related related effects were observed on body weight or age at PPS, age at 

partial PPS, proportion/incidence of unseparated preputia, androgen-dependent tissue organ 

weights (except LABC), or serum testosterone and thyroid hormone levels. There was a 

small (12%) decrease in serum T4 at 125 mg/kg bw. However, the decrease was still within 

the normal range reported and there were no effects on the other thyroid hormones. The 

colloid area and follicular cell height of the thyroid in the treated groups were similar to the 

control group. The incidences of histopathological findings of the testes and epididymides 

in the treated groups were similar to controls. 

The LABC weights showed a linear trend of decreased (p≤0.05) weight with increasing 

dose; however, no statistically significant effect was detected between the treated groups 

and the control group. No other statistically significant effect or trend was detected for the 

sex tissues or any other organ weighed. Therefore, this linear trend was considered 

incidental. 

Although no toxicity was noted at the high dose (125 mg/kg/day) and terminal body weight 

was only decreased by 5% (NS), relative to the control, the high dose is considered adequate 

for purposes of this assay based on data from a range-finding study using juvenile males in 

which dosing at 200 and 400 mg/kg/day resulted in body weight decreases of 14 and 15%, 

respectively. 

In this study, there was no evidence to support an effect of EPTC on pubertal development 

and thyroid function at doses up to 125 mg/kg/day. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

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

 The stability data from Smithers Viscient, LLC. Study No. 12791-6162 were not 

submitted for independent evaluation. 

Page 283 of 310
Male Pubertal Assay (2012) / Page 15 of 15 

EPTC / 041401 OCSPP 890.1500/ OECD None 

 Additionally, a number of Guideline performance criteria were not met: 

 CV=6.1% for age at PPS (top of acceptable range: 5.67%) 

 CV=13.3% for weight at PPS (top of acceptable range: 7.57%) 

 CV=8.1% for final body weight (top of acceptable range: 7.47%) 

 Mean weaning weight was 64.1 g while the acceptable range was 45.472-59.812 g 

 Mean kidney weight of 2.06 g (acceptable range: 2.242-3.050 g) 

 Mean LABC weight of 0.421 g (acceptable range: 0.447-0.855 g) 

 CV=22.9% for seminal vesicles with coagulating gland and fluid (top acceptable range: 

21.06%) 

 CV=22.1% for testis (top acceptable range: 17.62%) 

 Mean serum TSH level of 3.92 ng/mL (acceptable range: 4.212–24.112 ng/mL) 

 CV=72.1% for serum TSH (top of acceptable range: 58.29%) 

Page 284 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616401) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 1/6/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 1/17/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/25/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/25/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 285 of 310
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 286 of 310
EPTC I 041401 

Primary Reviewer: Gregory Akerman 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

DAT A EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 041401 DP BARCODE: D397177 

TXR#: 0051762 CAS No.: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: 

CITATION: 

SPONSOR: 

S-ethyl N,N-dipropylcarbamothioate 

Wagner, H. (2011). EPTC: H295R Steroidogenesis Assay. CeeTox, 

Kalamazoo, MI. Study No. 9 l 42V-l 00349STER, November 10, 2011. 

MRID 48616401. Unpublished. 

Gowan Company, 370 S. Main Street, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48616401), H295R cells 

cultured in vitro in 24-well plates were incubated with EPTC (>99.9% purity, Batch # 

Pl 105096) at concentrations of 100, 10, 1, 0.1 , 0.01 , 0.001 , and 0.0001 μM for 48 hours in 

triplicate for four independent experiments. Run 1 was not analyzed because one of the forskolin 

concentrations on the quality control plate was incorrect (3 μM was used instead of 1 μM). 

Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC-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 inducer (forskolin) of estradiol and 

testosterone production. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

coefficients of variation, %CV), and appropriate induction and inhibition with positive controls. 

Testosterone concentration was not affected by EPTC. In three independent runs, statistically 

significant inhibition or induction of testosterone production was not observed. Decreases 

(p:S0.05) of9- 10% in estradiol were observed at EPTC concentrations of0.01 , 0.001 , and 0.0001 

μMin Run 3. When the estradiol results in Run 3 were normalized to percent viability, the 

inhibition of estradiol at 0.0001 , 0.001 , and 0.01 μM was not statistically significant. In 

Page 287 of 310
Steroidogenesis Assay (2011) / Page 2 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

addition, these decreases were minor and not reproduced in the other two runs. However, 

increases (p≤0.05) of 10–15% in estradiol concentrations were observed at the 100 μM dose of 

EPTC in Run 3 (with and without normalization to viability) and in Run 4. Although the effect 

was slight, it was statistically significant in two of three runs and was reproducible. Therefore, it 

is concluded that EPTC at 100 μM may induce of estradiol production. 

Based on the hormone responses in each of the three independent runs, EPTC treatment resulted 

in statistically significant and reproducible increases in estradiol production, but did not result in 

statistically significant and reproducible alterations in testosterone production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

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

Confidentiality statements were provided. 

 

Page 288 of 310
Steroidogenesis Assay (2011) / Page 3 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox 

 Location: Kalamazoo, MI 

 Study Director: Hilary Wagner, M.S. 

 Other Personnel: Colleen Toole, Ph.D., Director of Project Management 

Karen Rutherford, B.S., Director of Laboratory Operations 

David Blakeman, B.S., Senior Scientist/Endocrine Group Leader 

Briana Wallace, B.S., Lead Cell Culture Scientist 

Steven McColley, M.S., Scientist 

Cameron Haines, B.S., Scientist 

Francisca Wong, M.S., Scientist 

Joey Burgam, B.S., Associate Scientist 

Lisa Blakeman, B.S., Associate Scientist 

 Study Period: July 15, 2011 through November 10, 2011 

2. Test Substance: EPTC 

 Description: Liquid 

 Batch # (expiration date): P1105096 (February 4, 2015) 

 Purity: >99.9% 

 Solubility: Soluble at tested concentrations in DMSO 

 Vapor pressure: 4500 mPa at 25°C 

 Stability: Not reported 

 Storage conditions: Room temperature 

 CAS #: 759-94-4 

 Molecular weight: 189.3

 Structure: 

N

S

O 3CH

3CH

CH3

 

3. Positive Control: Forskolin 

 Description (molecular weight): White powder (410.50) 

 Source: Sigma Aldrich (St. Louis, MO) 

 Lot # (expiration date): 109K50571V (July 2016) 

 Purity: 98% 

 Solubility (in solvent): Soluble at tested concentrations in DMSO 

 Storage conditions: Room temperature 

 CAS #: 66575-29-9 

4. Negative Control: Prochloraz 

 Description (molecular weight): White powder (376.67) 

 Source: Sigma Aldrich (St. Louis, MO) 

 Lot # (expiration date): SZE6220X (August 8, 2012) 

 Purity: 99.1% 

 Solubility (in solvent): Soluble at tested concentrations in DMSO 

 Storage conditions: Room temperature 

 CAS #: 67747-09-5 

Page 289 of 310
Steroidogenesis Assay (2011) / Page 4 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) 

 Description (molecular weight): Clear liquid (78.13) 

 Source: Sigma-Aldrich (St. Louis, MO) (catalog # not provided) 

 Lot # (expiration date): RNBB7617 (Not provided) 

 Purity: Not provided 

 Storage conditions: Not provided 

 CAS #: 67-68-5 

 Justification for choice of 

solvent: 

Not provided 

 Final concentration in assay: 0.05% v/v 

6. Other Materials: 22R-Hydroxycholesterol 

 Description (molecular weight): White powder (402.65) 

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

 Lot # (expiration date): 089K4132, 060M4098 (not provided) 

 Purity: 99.0% 

 Storage conditions: Room temperature 

 Solvent used: Ethanol, final concentration in assay, 0.025% v/v 

 CAS #: 17954-98-2 

7. Stock Medium: Dulbecco’s modified Eagle’s medium/nutrient mixture F12 

Ham 

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

Lot # (expiration date): 021M8304 (not provided) 

HEPES: 15 mM; Sigma-Aldrich (St. Louis, MO); Lot # 021M8304 

Sodium bicarbonate: Sigma-Aldrich (St. Louis, MO); Lot # 068K0105; purity not reported 

Nu-Serum: 2.5%; Becton-Dickinson (Franklin Lakes, NJ); Catalog # 355500; Lot # 81515; 

tested for background hormone concentrations by performing laboratory 

ITS+premix: Becton-Dickinson (Franklin Lakes, NJ); Catalog # 354352; Lot # 05245, 09233 

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; Lot # 

7635054) at passage 7.5 – 8.5 were incubated in the stock medium, supplemented with 10 

μM 22R-hydroxycholesterol in ethanol at the time of plating, dosing, and harvest. 

Incubation conditions were at 5% CO2 and 37 ± 2°C for approximately 24 hours prior to 

exposure. 

 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: A hormone assay interference test was not performed. 

b. Hormone Extraction: See Section on “Hormone Measurement System.” 

c. Laboratory Proficiency Test: No laboratory proficiency test data were provided. 

2. Test Solutions: EPTC was dissolved in DMSO to make a 200 mM stock and then serially 

diluted in DMSO. 22R-hydroxycholesterol was dissolved in ethanol to make a 40 mM 

Page 290 of 310
Steroidogenesis Assay (2011) / Page 5 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

stock and then diluted in supplemented medium to a final concentration of 10 μM. EPTC 

was then diluted in supplemented medium containing 10 μM 22R-hydroxy-cholesterol to 

prepare mastermix solutions. Forskolin and prochloraz were dissolved in DMSO to make 

100 mM solutions and then serially diluted in DMSO. Forskolin and prochloraz were then 

diluted in supplemented medium containing 10 μM 22R-hydroxy-cholesterol. When added 

to the cell culture plates, these dilutions yielded final concentrations of 1 μM and 10 μM for 

forskolin, 0.1 μM and 1 μM for prochloraz, and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM 

for EPTC. The final concentration of DMSO in the medium was held constant at 0.05% 

(v/v). The final ethanol concentration in the supplemented medium was 0.025% (v/v). No 

precipitation was visible. 

3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described 

above. It was reported that hormone concentrations in undiluted Nu-Serum were 

3754 pg/mL testosterone and 3846 pg/mL estradiol. H295R cells were grown for five 

passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional 

passages prior to use in the assay. The culture medium was supplemented with 10 uM 

22R-hydroxycholesterol at the time of plating, dosing, and harvest. The concentration of 

22R-hydroxycholesterol was chosen based on laboratory proficiency experiments previously 

conducted at the performing laboratory. The cells were plated into wells of a 24-well cell 

culture plate at a density of approximately 300,000 cells/mL. The cells were then placed 

into a 5% CO2 incubator at 37 ± 2°C for approximately 24 hours prior to chemical exposure. 

Prior to dosing, the cells were checked microscopically for attachment and proper 

morphology. 

4. Exposure: The medium was removed from the cells and replaced with 1 mL of medium 

containing 10 μM 22R-hydroxycholesterol and concentrations of EPTC in triplicate, 

according to the schematic presented in Table 1. 

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to EPTC (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 13 of the study report. 

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 either forskolin 

or prochloraz, according to the schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Backgroundc Background Background 

B DMSO DMSO DMSO DMSO + MeOHd DMSO + MeOH DMSO + MeOH 

C Forskolin 

1 μM 

Forskolin 

1 μM 

Forskolin 

1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

Page 291 of 310
Steroidogenesis Assay (2011) / Page 6 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

D Forskolin 

10 μM 

Forskolin 

10 μM 

Forskolin 

10 μM 

Prochloraz 

1 μM 

Prochloraz 

1 μM 

Prochloraz 

1 μM 

a Data were obtained from page 13 of the study report. 

b Blank wells received medium containing 10 μM 22R-hydroxycholesterol. 

c Background wells received medium without 22R-hydroxycholesterol). 

d MeOH = methanol was added to these wells for 30 minutes at room temperature following medium removal. 

Following dosing, the plates were incubated for 48 ± 2 hours under the conditions 

previously described. After the 48 ± 2 hour exposure, each well was examined under the 

microscope. Images were taken of the vehicle control wells, as well as the two highest 

concentrations. Cytotoxicity was not observed. The media was collected from all wells in 

two equal aliquots and stored at -80 ± 10°C until shipment to the analytical laboratory for 

hormone measurement. 

5. Cell Viability/Cytotoxicity Assay: After media removal, cell viability was measured using 

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. On the QC 

plates, wells designated to receive methanol (control wells for cell death measurements) 

were rinsed twice with phosphate buffered saline (PBS; Gibco; Cat. # 10010; Lot # 979431), 

and then incubated in methanol (Sigma; Cat. # 494437; Lot # SHBB0973V) for 30 minutes 

at room temperature. After the methanol incubation, the methanol-treated wells were rinsed 

again with PBS three times. Following media removal and/or methanol treatment, 0.5 mL 

of 0.5 mg/mL MTT (Sigma; Cat. # M5655; Lot # MKBG1611V) solution in supplemented 

medium containing 10 μM 22R-hydroxycholesterol was added to each well of the test 

chemical and QC plates. The plates were incubated at 37±2°C in a 5% CO2 incubator for 

3 hours. Following the 3 hour incubation, the MTT solution was removed, and 0.5 mL of 

isopropanol was added to each well. Plates were incubated at room temperature for 

20 minutes with shaking. Following this incubation, absorbance at 570 nm and 650 nm 

were measured on a Packard Fusion plate reader. The absorbance at 650 nm was subtracted 

from the absorbance at 570 nm to calculate the MTT value for each well. The change in cell 

viability was determined by comparing treated wells to the solvent control (SC) wells. A 

greater than 20% reduction in cell viability was considered evidence of cytotoxicity. 

6. Hormone Measurement System: Testosterone and estradiol levels were measured at 

OpAns, LLC (Durham, NC). Testosterone and estradiol were extracted from H295R 

supplemented medium by liquid-liquid extraction using methyl tert-butyl ether (MTBE) 

after the addition of [2H5]-testosterone and [2H5]-estradiol as internal standards. Extracts 

were analyzed by HPLC-MS/MS using positive ion multiple reaction monitoring. This 

method was validated over the range 100 to 100,000 pg/mL for testosterone and 10 to 

10,000 pg/mL for estradiol (OpAns Document # OPR-OPP-0006). The lower limit of 

quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol using a 

300 μL aliquot of H295R supplemented medium. Freshly prepared calibration standards 

(n = 7) were prepared in supplemented medium for each run. QC samples spiked in 

supplemented medium at three concentrations (2 replicates per concentration), were 

included with each run. No study samples were re-analyzed during this study. 

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) 

Page 292 of 310
Steroidogenesis Assay (2011) / Page 7 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

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 

X Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and 

estradiol were calculated for each concentration of EPTC, reference chemicals, the solvent 

controls, blanks, and background wells. Relative changes in testosterone and estradiol 

production were calculated using the equation below: 

Relative Change = [Hormone] in each well ÷ [Hormone] of mean solvent control 

For forskolin induction of testosterone, relative change was calculated both with and without 

subtracting the background hormone production from the forskolin-treated wells, and blank 

and SC wells. Background hormone production was calculated from three wells with cells 

on the QC plate that received no 22R-hydroxycholesterol at the time of exposure. 

Normality of the data was evaluated using Shapiro-Wilk's test. Homogeneity of the 

variances between the treatment groups was evaluated using Levene's test. If the p-values 

were greater than 0.05 in both tests, statistical significance between each treatment group 

and the control group was evaluated using Dunnett's test. If the p-values were less than or 

equal to 0.05 in either the normality or the homogeneity test, a log transformation was 

performed on the data to attempt to approximate a normal distribution. If (following the 

transformation) p-values were greater than 0.05 in the normality and homogeneity tests, 

Dunnett's test was performed on the transformed data to evaluate statistical significance 

between each treatment group and the control group. If following the log transformation, pvalues were less than or equal to 0.05 in either the normality or the homogeneity test, the 

non-transformed data set was analyzed using the nonparametric Kruskal-Wallis test 

followed by Dunn's test to evaluate statistical significance between each treatment group 

and the control group. Data analysis at the analytical laboratory (OpAns, LLC) was 

conducted in Microsoft Excel. No additional software for statistical analyses or graphing 

was specified. The reviewers consider these analyses acceptable. 

II. RESULTS

A. TEST COMPOUND: Precipitation of the test compound was not observed at EPTC 

concentrations up to 100 μM. The %CVs for absolute hormone concentrations for the SC 

replicates within the test plates were 3.1–6.0% for testosterone and 0.9–3.0% for estradiol. 

The between plate CV for SC based on absolute concentrations (calculated by reviewer) was 

19.5% for testosterone and 11.3% for estradiol. These data met the recommended 

performance criteria of ≤30%. 

The Sponsor stated: “Four independent runs of the steroidogenesis assay were conducted. 

Run 1 was not analyzed because one of the forskolin concentrations on the QC plate was 

incorrect (3.33 μM was used instead of 1 μM). Data from Run 1 will not be presented in 

this report as the media was not analyzed for testosterone and estradiol.” The 

concentrations of estradiol and testosterone observed in the other three assay runs are 

summarized in Table 3 and graphically depicted in Figures 1 and 2. 

Page 293 of 310
Steroidogenesis Assay (2011) / Page 8 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

Testosterone concentration was not affected by EPTC. In three independent runs of the 

assay, statistically significant inhibition or induction of testosterone production was not 

observed. Decreases (p≤0.05) of 9–10% in estradiol were observed at the 0.01, 0.001, and 

0.0001 μM doses in Run 3. However, when the estradiol results in Run 3 were normalized 

to percent viability, the inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not 

statistically significant. Increases (p≤0.05) of 10–15% in estradiol was observed at the 

100 μM dose in Run 3 (with and without normalization to viability) and in Run 4. No effect 

was observed on testosterone or estradiol at other EPTC concentrations in Run 2. 

TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with EPTC for 48 Hours.a

Nominal 

Concentration 

(μM) 

Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Statistical 

Significance 

Testosterone (pg/mL) Fold Difference 

DMSO 2743 1845 2441 NA NA NA NA NA NA 

0.0001 3031 1810 2447 1.11 0.98 1.00 1.03 0.07 — 

0.001 2694 1787 2421 0.98 0.97 0.99 0.98 0.01 — 

0.01 2820 1772 2339 1.03 0.96 0.96 0.98 0.04 — 

0.1 2903 1846 2420 1.06 1.00 0.99 1.02 0.04 — 

1 2818 1855 2433 1.03 1.01 1.00 1.01 0.02 — 

10 2845 1805 2387 1.04 0.98 0.98 1.00 0.03 — 

100 2850 1882 2473 1.04 1.02 1.01 1.02 0.02 — 

Estradiol (pg/mL) Fold Difference 

DMSO 233 197 246 NA NA NA NA NA — 

0.0001 222 179 231 0.95 0.91* 0.94 0.93 0.02 Run 3 

0.001 213 178 231 0.91 0.90* 0.94 0.92 0.02 Run 3 

0.01 229 179 237 0.98 0.91* 0.96 0.95 0.04 Run 3 

0.1 230 187 231 0.99 0.95 0.94 0.96 0.03 — 

1 232 184 250 1.00 0.94 1.02 0.99 0.04 — 

10 238 196 251 1.02 1.00 1.02 1.01 0.01 — 

100 276 217 282 1.19 1.10* 1.15* 1.15 0.05 Runs 3 and 4 

a Data were obtained from pages 25-26 and 33-35 of the study report. The lower limit of quantification (LLQ) was 100 

pg/mL for testosterone and 10 pg/mL for estradiol. 

b Calculated by the reviewers. 

NA Not applicable

Page 294 of 310
Steroidogenesis Assay (2011) / Page 9 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

FIGURE 1. Change in Testosterone Production Relative to EPTC Concentration. 

Page 295 of 310
Steroidogenesis Assay (2011) / Page 10 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

FIGURE 2. Change in Estradiol Production Relative to EPTC Concentration. 

Page 296 of 310
Steroidogenesis Assay (2011) / Page 11 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

B. CYTOTOXICITY: Less than 5% cytotoxicity was noted in the reference and 

EPTC-treated wells (96–107% cytotoxicity, unrelated to dose), except in the methanoltreated wells. Data are summarized in Table 4 and depicted graphically in Figure 3. 

TABLE 4. Mean (±SD) MTT Cell Viability (% SC) Results after Treatment with Forskolin, Prochloraz, 

or EPTC for 48 Hours. a 

Compound Concen. 

(μM) 

Cell Viability – Run 2 Cell Viability – Run 3 Cell Viability – Run 4 

Mean SD Mean SD Mean SD 

Blank NA 99.4 10.54 100.1 4.93 103.0 1.84 

Background NA 102.5 5.95 98.7 0.69 104.0 1.76 

SC + Methanol NA 4.9 0.99 5.7 0.64 8.3 1.79 

Forskolin 1 103.9 3.88 97.2 2.20 107.3 0.91 

Forskolin 10 105.1 4.90 97.5 2.67 106.7 4.11 

Prochloraz 0.1 100.7 1.89 97.8 1.79 101.7 2.17 

Prochloraz 1 101.1 2.01 96.1 2.79 105.0 4.29 

EPTC 0.0001 99.8 1.21 97.6 2.22 100.7 1.32 

EPTC 0.001 98.6 1.07 98.7 2.04 100.1 2.12 

EPTC 0.01 96.6 0.65 96.3 0.96 102.3 2.45 

EPTC 0.1 99.6 1.85 100.8 1.81 101.2 0.66 

EPTC 1 97.6 1.00 97.5 1.66 98.5 1.53 

EPTC 10 99.4 1.07 99.1 1.36 100.2 0.38 

EPTC 100 98.2 0.61 98.4 3.45 100.2 3.25 

a Data were obtained from pages 19-20 of the study report. 

NA Not applicable 

Page 297 of 310
Steroidogenesis Assay (2011) / Page 12 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

FIGURE 3. MTT cell viability results for EPTC-treated cells. 

Page 298 of 310
Steroidogenesis Assay (2011) / Page 13 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

C. QC PLATE: The hormone concentrations after exposure to the reference chemical, SC, 

blank, and background samples, as well as the fold-difference change relative to SC 

(individual and mean ± SD) for the three suitable assay runs, are presented in Table 5. The 

minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for 

estradiol) were met in both blank and SC wells. Basal hormone production in SC wells was 

>17-fold minimum detection level (MDL) for testosterone and >17-fold MDL for estrogen. 

Compared to SC, 10 μM forskolin on average induced testosterone by 1.74-fold which is 

below the Guideline criteria of ≥2-fold; however, when the background is subtracted, 10 μM 

forskolin induced testosterone an average of 2.7-fold. Estradiol production was induced 

with 10 μM forskolin by 9.6-fold. Compared to SC, 1 μM prochloraz on average inhibited 

the synthesis of testosterone 0.48-fold and estradiol to 0.35-fold. Thus, the guideline 

requirements were met indicating that the assay was sensitive to induction and inhibition of 

testosterone and estradiol. 

The %CVs based on absolute hormone concentration in SC well replicates were 2.19-2.64% 

for testosterone and 1.89-4.01% for estradiol. The between-plate variability (calculated by 

the reviewer) for the QC plates based on the absolute hormone concentrations in the SC was 

18.8% for testosterone and 14.2% for estradiol. These data met the performance criteria 

recommended by the Guideline (≤30%). 

TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 

48 Hours.a

Concentration (μM) 

Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Meanb ± SDb

Testosterone (pg/mL) Fold Difference (Relative to DMSO) 

Background 1493 1035 1359 0.57 0.58 0.59 0.58 0.01 

Blank 2921 1799 2338 1.12 1.01 1.01 1.05 0.06 

DMSO 2612 1782 2319 1.00 1.00 1.00 1.00 -- 

1 μM Forskolin 4325 2403 3108 1.66c

 1.35 c 1.34 c 1.45 c 0.18 

10 μM Forskolin 5604 2561 3765 2.15 d

 1.44 d 1.62 d 1.74 d 0.37 

0.1 μM Prochloraz 2145 1505 1843 0.82 0.84 0.79 0.82 0.03 

1 μM Prochloraz 1351 740 1168 0.52 0.42 0.50 0.48 0.05 

Estradiol (pg/mL) Fold Difference (Relative to DMSO) 

Background 66 57 76 0.29 0.33 0.34 0.32 0.03 

Blank 244 179 242 1.07 1.02 1.08 1.06 0.03 

DMSO 229 175 223 1.00 1.00 1.00 1.00 -- 

1 μM Forskolin 1550 1014 1559 6.76 5.79 6.99 6.51 0.64 

10 μM Forskolin 2396 1363 2321 10.46 7.78 10.41 9.55 1.53 

0.1 μM Prochloraz 168 151 176 0.73 0.86 0.79 0.79 0.07 

1 μM Prochloraz 77 62 78 0.34 0.36 0.35 0.35 0.01 

a Data were obtained from pages 21–22 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for 

testosterone and 10 pg/mL for estradiol. 

b Calculated by the reviewers. 

c When the background is subtracted, the values are 2.53-, 1.83-, and 1.82-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 2.06-fold change. 

d When the background is subtracted, the values are 3.67-, 2.04-, and 2.51-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 2.74-fold change. 

Page 299 of 310
Steroidogenesis Assay (2011) / Page 14 of 14 

EPTC / 041401 OCSPP 890.1550/ OECD None 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: EPTC was tested at a soluble concentration range 

(0.0001 to 100 μM) and was determined not to affect testosterone or inhibit estradiol in this 

steroidogenesis assay in the H295R cell line. EPTC was associated with an induction of 

estradiol at 100 uM in the H295R cell line. 

B. AGENCY COMMENTS: All guideline acceptability recommendations and requirements 

were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, 

acceptable reproducibility (low %CV), and appropriate induction and inhibition with 

positive controls. 

Testosterone concentration was not affected by EPTC. In three independent runs of the 

assay, statistically significant inhibition or induction of testosterone production was not 

observed. Decreases (p≤0.05) of 9–10% in estradiol were observed at the 0.01, 0.001, and 

0.0001 μM doses in Run 3. However, when the estradiol results in Run 3 were normalized 

to percent viability, the inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not 

statistically significant. These decreases were minor and not reproduced in the other two 

runs. Increases (p≤0.05) of 10–15% in estradiol was observed at the 100 μM dose in Run 3 

(with and without normalization to viability) and in Run 4. Although the effect was slight, 

this effect was statistically significant in two of three runs and was reproducible. Therefore, 

it is concluded that EPTC may be associated with an induction of estradiol at 100 μM. 

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: 

 Stability of EPTC in the test system was not reported. 

 Total number of cell passages was 12-13, exceeding 10. 

 The laboratory proficiency test data were not provided. 

 Mean±SD and %CV for the fold-differences were not reported, but could be calculated 

from the data presented. 

Page 300 of 310
DATA EVALUATION RECORD 

EPTC 

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-13-2012 (MRID 48616407) 

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 Bryan, B.S. Date: 1/11/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 1/25/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 1/27/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 301 of 310
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 302 of 310
EPTC/ 041401 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

In vivo Uterotrophic Assay (2011) I Page I of8 

OCSPP 890.1600/ OECD 440 

Secondary Reviewer: _J_e_s_s_R_o_w_l_a_nd _________ Signature: -~~~==-::.L_,:::.__ 

Health Effects Division Date: ---~~---

Template version 09/2011 

DAT A EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 041401 DP BARCODE: D397 l 77 

TXR#: 0051762 CAS#: 759-94-4 

TEST MATERIAL (PURITY): EPTC (>99.9% a.i.) 

SYNONYMS: S-ethyl N,N-dipropylcarbamothioate 

CITATION: Zorrilla, L. (20 11) The Uterotrophic Assay with EPTC in Ovarectomized 

CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. 

Laboratory Project Study ID: C201-100, November 3, 2011. MRID 48616407. 

Unpublished. 

SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ 

TEST ORDER#: EDSP-041401-64 

EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48616407) conducted to screen 

for potential estrogenic activity, EPTC (>99.9% a.i., Lot# P 1105096) in corn oil was 

administered daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley 

rats at dose levels of 0 (vehicle), 50, or 200 mg/kg/day on post-natal days (PND) 59-61. A 

positive control group was treated with l 7a.-ethynyl estradiol (EE) by daily oral gavage at a dose 

level of 0.1 mg/kg/day. All animals were terminated and necropsied approximately 24 hours 

after the final dose (PND 62) to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

any of the EPTC treated groups. In the positive control group (EE), one animal exhibited 

alopecia on the neck on Day 3, which was considered incidental. No treatment-related effects on 

body weight or body weight gains were noted in the EPTC treated groups. In the positive control 

group (EE), an overall (Days 1-4) body weight loss of- 0.2 g (vs + 12.6 g controls) was observed. 

Uterine weights in the EPTC treated groups were comparable to the controls. Absolute wet and 

blotted uterus weights for the EE group were increased (p:S0.05) by 89% and 96%, respectively. 

No statistically significant changes were seen in uterine weight in this assay. EPTC is negative 

in the uterotrophic assay. 

Page 303 of 310
In vivo Uterotrophic Assay (2011) / Page 2 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

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

890.1600). 

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

provided. 

 

Page 304 of 310
In vivo Uterotrophic Assay (2011) / Page 3 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: Integrated Laboratory Systems, Inc. 

Location: Durham, NC

Study Director: L. Zorrilla 

Other Personnel: S. Borghoff (Study Toxicologist), 

P. Sproul (Toxicology Study Manager), 

A. Glasscock (Animal Facility Operations Manager), 

T. Hackett (Necropsy Manager), 

C. Swartz (Facility Veternarian), 

C. Cachafeiro (Health and Safety Officer), 

P. Reibach (Principal Investigator, Smithers Viscient).

Study Period: May 8-11, 2011 

2. Test Substance: EPTC 

 Description: Off white solid 

 Source: Gowan Company (commercial technical product) 

 Lot #: P1105096, expires February 2, 2015 

 Purity: >99.9 % a.i. 

 Stability: Stable in the vehicle for up to 25 days at 1-10°C (Smithers 

Viscient Inc., Study No. 12791-6162) 

 CAS #: 759-94-4 

 Structure: 

S N

CH3

CH3

O

3

CH

3. Reference Estrogen: 17α-ethynyl estradiol (EE) 

 Supplier: Sigma Aldrich, St. Louis, MO 

 Lot/Batch #: 090M1241V, expires February 2012 

 Purity: ≥98 % 

 CAS # : 57-63-6 

4. Solvent/Vehicle Control: Corn Oil 

Supplier: MP Biomedicals, LLC, Solon, OH 

Lot #: 7862K 

Rationale (if other than water): Not Provided 

Final concentration : Not Applicable 

5. Test Animals: 

 Species: Rat (ovariectomized females) 

 Strain: Sprague-Dawley (Crl:CD[SD] IGS) 

 Age/weight at dose initiation: PND 59 / 221.1-274.3 g females 

 Source: Charles River Laboratories International, Inc., Raleigh, NC

 Housing: 2/cage in polycarbonate cages with absorbent heat-treated 

hardwood bedding (Northeastern Products Corp., Warrensburg, 

NY) 

 Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, 

WI), ad libitum; low phytoestrogen with total geinstein 

equivalents = 6.2 μg/g diet.

 Water: Reverse osmosis-treated tap water, ad libitum 

Page 305 of 310
In vivo Uterotrophic Assay (2011) / Page 4 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

23-25oC 

34-50% 

Not provided 

 12 hrs light/12 hrs dark 

 Acclimation period: 10 days 

B. METHODS 

1. In Life Dates: Start: May 8, 2011 End: May 11, 2011 

2. Study Design: Following a 10-day acclimation period, ovariectomized female rats (surgery 

performed by Supplier) were administered the test substances for three consecutive days 

starting at PND 59. Rats were euthanized approximately 24 hours after the last dose and 

necropsied for uterine weight measurements. 

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences between group mean weights at study initiation. 

TABLE 1. Study Design of Estrogen Agonist Assaya

Test Group Dose (mg/kg/day) # of Females 

Vehicle Control 0 8 

Low EPTC 50 8 

High EPTC 200 8 

17α-ethynyl estradiol (EE), Reference Estrogen 0.1 8 

a Data were obtained from Table 1 on page 15 of the study report. 

4. Dose Selection Rationale: The EPTC dose levels were selected based on the results from 

two developmental toxicity studies (study numbers not provided). In a range-finding 

developmental toxicity study, EPTC in corn oil was administered by daily oral gavage to 

female COBS SD rats at dose levels of 50, 90, 160, 280, or 500 mg/kg/day from gestation 

day (GD) 6 through 15. Mortality was observed at 500 mg/kg/day. At 160 and 280 

mg/kg/day, body weight losses were noted during GD 6-9, food consumption was reduced, 

and clinical signs of toxicity (yellow urogenital matting and staining and red matter around 

the nose) were observed. In the definitive developmental study, EPTC was administered as 

before at dose levels of 30, 100, and 300 mg/kg/day. Body weight losses and decreased 

food consumption were noted at 300 mg/kg day [Stauffer Chemical Company (1983) as 

reference in study report, not a MRID study ] Based on these results, doses of 50 and 200 

mg/kg/day were selected for the current study. For the positive control, 17α-ethynyl 

estradiol (EE), a dose of 0.1 mg/kg/day was selected based on the previous experience 

within the testing laboratory as described in a baseline positive control study. 

5. Dose Preparation and Analysis: Dose formulations were prepared once prior to initiation 

of treatment by suspending the appropriate amounts of test substance in corn oil. The 

Page 306 of 310
In vivo Uterotrophic Assay (2011) / Page 5 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

reference estrogen, EE, was mixed with corn oil for dosing. Doses were adjusted daily 

based on individual body weight measurements. Homogeneity and concentration analyses 

were measured prior to administration in the 10 and 40 mg a.i./mL formulations. In a 

previous storage stability study1, EPTC (lot number not reported) was determined to be 

stable in corn oil for up to 25 days at 1-10°C. 

Results of Dose Analysis 

Homogeneity (% CV): 1.66-4.47% 

Stability (% of Day 0): Data not provided. 

Concentration (% of nominal): 93-96% (The initial low dose analysis was slightly out of 

range at 84.9% of nominal, therefore the low dose formulation used in the current study is 

the same low dose formulation as the concurrent EPTC Hershberger assay, MRID 

48616406). 

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 formulations, EE, or vehicle 

daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight. 

Dose volumes were adjusted daily based on the concurrent body weight measurement.

7. Statistics: Final body weights, body weight gains, and uterine weights were analyzed using 

studentized residual plots for outliers and Levene’s test for homogeneity of variance. Data 

were analyzed using one-way ANOVA followed by Dunnett’s one tailed t-test (uterine 

weights) and Dunnett’s two tailed t-test (final body weight and body weight gain). 

Significance was denoted at p≤0.05. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Animals were checked for mortality or moribundity twice daily on 

weekdays and once daily on weekends and holidays. Rats were also checked for clinical 

signs of toxicity prior to dose administration and three hours (±30 min) post-dosing. 

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

the dosing period, and at termination. 

3. Food Consumption (Optional): Food consumption was not measured. 

 

1 Reibach, R. (2011) Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers 

Viscient Inc., Study No. 12791-6162. 

Page 307 of 310
In vivo Uterotrophic Assay (2011) / Page 6 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

4. Necropsy and Measurement of Uterine Weight: Approximately 24 hours after final 

administration of the test substance, all surviving animals were euthanized by CO2

inhalation followed by cervical dislocation and subjected to a gross necropsy. The uterus 

and vagina were removed and excess fat and connective tissue were trimmed. The vagina 

was removed from the uterus below the cervix, and the uterus was weighed (to obtain a wet 

weight). The uterus was then pierced and blotted to remove luminal fluid, and weighed 

again (to obtain a blotted weight). 

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 clinical signs of toxicity were observed in any of the EPTC 

treated groups. In the positive control group (EE), one animal exhibited alopecia on the 

neck on Day 3, which was considered incidental. 

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are 

presented in Table 2. No treatment-related effects on body weight or body weight gains 

were noted in the EPTC treated groups. In the positive control group (EE), an overall (Days 

1-4) body weight loss (p≤0.05) of −0.2 g (vs +12.6 g controls) was observed. 

TABLE 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist 

Assay a

Study Day 

#

Dose (mg/kg/day) 

Vehicle Control EPTC (50) EPTC (200) EE (0.1) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 245.5 16.3 8 247.9 13.7 8 246.7 13.4 8 248.5 11.8 

2 8 244.8 14.7 8 247.3 14.3 8 245.7 15.0 8 246.5 13.9 

3 8 249.3 14.4 8 254.2 14.6 8 250.7 15.3 8 245.3 12.6 

4 8 258.0 15.9 8 261.0 15.5 8 257.9 16.1 8 248.3 12.7 

Body Weight 

Gain (Days 1-4) 

8 12.6 2.4 8 13.1 2.7 8 11.2 5.1 8 -0.2* 3.1 

a Data were obtained from Table 3 on page 19 and Appendix V on page 51 of the study report. Percent differences 

from controls were calculated by the reviewers and included in parentheses. 

N= No. of animals in the group 

SD = Standard Deviation 

* Significantly different from controls at p ≤0.05. 

Page 308 of 310
In vivo Uterotrophic Assay (2011) / Page 7 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

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 

EPTC treated groups were comparable to the controls. The mean blotted vehicle control 

uterine weight was 0.025% of the mean vehicle control terminal body weight, which meets 

the Guideline requirement (<0.04%).

Absolute wet and blotted uterus weights for the EE group were increased (p≤0.05) by 89% 

and 96%, respectively. These increased uterine weights were consistent with an estrogen 

receptor agonist.

TABLE 3. Uterine Weights from Estrogen Agonist Assay in SD Ratsa

Parameter 

Dose (mg/kg/day) 

Vehicle Control EPTC (50) EPTC (200) EE (0.1) 

N Mean SD N Mean SD N Mean SD N Mean SD 

Terminal BW (g) 8 258.0 15.9 8 261.0 15.5 8 257.9 16.1 8 248.3 12.7 

Wet, absolute (mg) 8 78.3 16.9 8 80.2 16.1 7 68.1 21.9 8 148.1* 

(↑89) 

24.5 

Wet, relative (%) b 8 0.030 0.007 8 0.031 0.007 8 0.026 0.008 8 0.060 0.013

Blotted, absolute (mg) 8 64.1 10.9 8 59.6 14.7 8 56.6 15.2 8 125.4* 

(↑96) 

24.6 

Blotted, relative (%) b 8 0.025 0.005 8 0.023 0.006 8 0.022 0.005 8 0.051 0.012

a Data were obtained from Table 4 on page 20 and Appendix VI on page 53 of the study report. 

b Relative wet and blotted uterine weights were calculated by the reviewer from the individual data. 

BW= body weight 

N= No. of animals in the group 

SD = Standard Deviation 

* Significantly different from vehicle control at p ≤ 0.05. 

2. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There were no clinical signs observed that were 

attributed to administration with EPTC or reference substance. EPTC administered up to 

200 mg/kg did not result in a change in body weight, body weight gain or uterine weight, 

wet or blotted, as compared to the vehicle control group. The positive control 17α-ethinyl 

estradiol, a known estrogen agonist, induced a statistically significant increase in both wet 

and blotted uterine weights compared to the vehicle control group. Oral administration of 

EPTC up to 200 mg/kg/day for three consecutive days to ovariectomized adult rats did not 

result in an estrogenic response as shown by a lack of effect on uterine weights. 

Page 309 of 310
In vivo Uterotrophic Assay (2011) / Page 8 of 8 

EPTC/ 041401 OCSPP 890.1600/ OECD 440

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical 

signs of toxicity were observed in any of the EPTC treated groups. In the positive control 

group (EE), one animal on Day 3 exhibited alopecia on the neck which was considered 

incidental. No treatment-related effects on body weight or body weight gains were noted in 

the EPTC treated groups. In the positive control group (EE), an overall body weight loss 

(p≤0.05) of −0.2 g (vs. +12.6 g controls) was observed. Uterine weights in the EPTC treated 

groups were comparable to the controls. Absolute wet and blotted uterus weights for the EE 

group were increased (p≤0.05) by 89% and 96%, respectively. No statistically significant 

changes were seen in uterine weight in this assay. EPTC is negative in the uterotrophic 

assay. 

 

C. STUDY DEFICIENCIES: None 

 

Page 310 of 310