UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

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

PC Code: 080807 

Decision No.: 460159 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0056244 

MRID No.: See Table 

DP Barcode: D398096 

Registration No.: NA 

Regulatory Action: NA 

CaseNo.: NA 

CAS No.: 122-34-9 

40CFR: NA 

FROM: Greg Akerman, Ph.D. .~ ~-----­ Immediate Office 

Health Effects Division (7509P) 

THROUGH: Jess 

Deputy 

Rowland 

DirectoiJ-

. ~ ...., 

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. 2010 

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 simazine: Test Order# CON-080807-17. 

Page 1 of2 

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Page 2 of 2 

II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for simazine.

III. MRID Table 

Chemical: Simazine PC Code: 080807 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48673601 

890.1150 Androgen Receptor Binding (Rat Prostate) 48619301 

890.1200 Aromatase Assay (Human Recombinant) NA 

890.1250 Estrogen Receptor Binding NA 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48619302 

890.1350 Fish Short-Term Reproduction 48673602 

890.1400 Hershberger (Rat) 48619303 

890.1450 Female Pubertal (Rat) NA 

890.1500 Male Pubertal (Rat) NA 

890.1550 Steroidogenesis (Human Cell Line – H295R) NA 

890.1600 Uterotrophic (Rat) NA 

Range finding study 48619304 

NA= Not Applicable (requirement satisfied by other scientifically relevant information (OSRI)) 

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Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay

EPA MRID Number 

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DER Template Version: 22 September 2011 

Data Requirement: EPA DP Barcode 408724 

 OECD Data Point 231 

 EPA MRID 48673601

 EPA Guideline 890.1100 

 Amphibian Metamorphosis Assay (Frog) 

 

Test Material: Simazine Purity (%): 97.8% 

Common Name Simazine 

Chemical Name IUPAC Not Reported 

 CAS Name Not Reported 

CAS No. 122-34-9 

Synonyms FL030055

EPA PC Code 080807

Primary Reviewer: John Marton, Ph.D. Date: 8/15/13 

Environmental Scientist, CDM Smith 

Secondary Reviewer(s): Teri S. Myers, Ph.D. Date: 8/16/13 

Environmental Scientist, CDM Smith 

Primary Reviewer: Frank T. Farruggia, Ph.D. Date: 2/14 

EPA/EFED/ERB3 

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

EPA/EFED/ERB6 

Date Evaluation Completed: 6/6/15

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 08:17:44 -04'00'

AMY 

BLANKINSHI

P

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 17:50:39 -04'00'

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Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay

EPA MRID Number 

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CITATION: Schneider, S.Z., T.Z. Kendall, H.O. Krueger. 2012. Simazine- Amphibian Metamorphosis Assay for 

the Detection of Thyroid Active Substances. Unpublished study performed by Wildlife International, Ltd., 

Easton, Maryland. Laboratory report number 528A-227. Study sponsored by Syngenta Crop Protection, 

LLC, Greensboro, North Carolina. Study completed November 8, 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). 

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Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay

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EXECUTIVE SUMMARY

The 21-day assay of simazine on amphibian metamorphosis of African clawed frog (Xenopus laevis) was 

conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control 

and treatment group) were exposed to nominal concentrations of simazine (97.8% purity) at 0 (negative 

control), 0.020, 0.20, and 2.0 mg a.i./L; mean-measured concentrations were <0.0100 (<LOQ; control), 

0.019, 0.18, and 1.9 mg a.i./L. The test system was maintained at 21.4 to 22.6°C and a pH of 8.0 to 8.2. 

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

A single mortality was observed in the low treatment group prior to Day 7; no additional non-incidental 

mortalities were observed throughout the remainder of the test. Clinical signs (spinal curvature) were observed 

in the control (8%), low (6%), and mid (9%) treatment groups and were not considered to be treatment 

related. 

Simazine significantly increased (p<0.05) normalized (for snout-vent length) Day 7 hind-limb length (HLL) by 

6% at the high treatment level and significantly decreased Day 21 normalized HLL by 14% in the same 

treatment group; the response was concentration-dependent in both cases. There was no significant acceleration 

or delay of median NF developmental stage at Day 7 or 21 in any treatment group. Further, no asynchronous 

development was observed throughout the test. A single late-stage tadpole was observed in the negative 

control (NF=61) and in the low and mid treatment groups (NF=62). These tadpoles were excluded from 

analysis of growth endpoints and normalized HLL. Concentration-dependent reductions in growth were 

observed on Day 21 for snout-vent length (SVL) and wet weight, with significant decreases of 7 and 23% at 

the high treatment level for SVL and wet weight, respectively relative to the negative control (p<0.05; 

Jonckheere-Terpstra). 

Changes in thyroid gland histopathology were observed in all treatment groups including the control; however, 

the observation of a slight increase in the incidence of animals with mild to moderate thyroid gland atrophy in 

the highest treatment (10/20) compared to the negative control (4/20) was reported to be treatment-related. 

The study authors noted that thyroid gland atrophy was generally associated with smaller sized tadpoles 

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regardless of treatment, suggesting that thyroid gland atrophy was secondary to reductions in size at the high 

concentration. 

The performance criteria were met in this study. 

This 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: 51 

Test organism total length at test initiation (optional): not measured 

Test type: flow-through 

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

Metamorphosis Assay (AMA) with Simazine. 

Treatment 

(mg a.i./L) 

[mean-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 

0.18 No No No No No No No 

1.9 No No Yes Yes No No Yes 

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 boardcertified 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 Series 890- Endocrine Disruptor Screening 

Program Test Guidelines, OCSPP Number 890.1100: Amphibian 

Metamorphosis (Frog); and the OECD Guidelines for Testing of Chemicals, 

Guideline 231: Amphibian Metamorphosis Assay. The following deviations were 

noted: 

1. Guidance recommends that at least 1,500 larvae/spawn be evaluated to determine the best 

single spawn for use in the definitive test. In the current study, only 250 eggs from each spawn 

were evaluated. 

2. Feeding rates were half of recommended rates as specified by EPA and OECD. 

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3. Ammonia, perchlorate, and chlorate concentrations in the dilution water were not reported. 

4. Hardness of the dilution water (132-152 mg/L as CaCO3) exceeded the recommended range 

(40-48 mg/L as CaCO3). 

5. The method for selecting the highest nominal concentration and the life stage of the test 

organisms in the range-finding tests were not reported. 

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 Good Laboratory 

Practice Standards as published by the U.S. Environmental Protection Agency (40 

CFR Parts 160 and 792); and OECD Principles of Good Laboratory Practice 

(ENV/MC/CHEM (98) 17), with the following exceptions: periodic analyses of 

water for potential contaminants were not performed according to GLP standards, 

but were performed using a certified laboratory and standard US EPA analytical 

methods; and, preliminary analyses of water iodide concentrations were not 

performed according to Good Laboratory Practice Standards. 

A. Test Material Simazine 

Description: White powder

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. : GBL2125GA7 

Purity: 97.8% 

Impurities: None reported 

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Stability of Compound: Certificate of analysis indicates test material is stable until end of April 

2014 if stored at <30°C.

Storage Conditions of 

Test Chemicals: Stored under ambient conditions 

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B. Test Organism

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a 

list of all of the necessary protocol 

deviations to accommodate this species is 

recommended for inclusion in the study 

report. The guideline recommends that the 

performance criteria used to support the 

reliability of the test be identified.

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? Yes. Adults were originally obtained from Xenopus I of 

Dexter, Michigan. 

Parents were maintained for at least 14 days following arrival at the laboratory. 

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): 

14 days

Details on parental feeding: Frogs were fed an adult 

Xenopus diet supplied by Zeigler Brothers, Inc. of Gardners, PA at a rate sufficient to maintain the health of the culture. 

Details on parental health: Culture frogs showed no sign of 

disease or stress prior to the test. 

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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 comixed)

Number of spawns evaluated (if applicable): 

2 

Number of eggs sampled per spawn: 

~250 Spawns were evaluated one 

day after deposition. 

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: 17 days post-fertilization (dpf) Mean total length at test initiation (if reported): 

Not measured 

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

Not measured 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the optional size selection method used? 

No 

Details on larval selection: Two male and two female adult 

frogs were injected with human chorionic gonadotropin and each pair was placed in a separate spawning tank overnight. Eggs were collected the following day and a subset of approximately 250 eggs from each spawn was evaluated for viability. 

Eggs from the best single spawn (91.2% viability) were used to provide tadpoles for the test. 

Loading rate (rearing density): 4 larvae/L EPA recommends that rearing density 

(loading rate) not exceed approximately 10 

larvae/L culturing system for flow-through 

systems or 4 tadpoles/L in static-renewal 

exposure systems.

Type of food: Sera Micron®

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of food: Sera North America, 

Montgomeryville, PA 

EPA recommends Sera Micron® throughout 

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

during the entire 21-d definitive study. If 

another diet is used, the study report should 

provide analysis of iodide content and 

potential contaminants, and the diet should 

demonstrate equal performance to Sera 

Micron® .

Iodide concentration in diet (if known): 

Not reported 

Frequency of feeding: 3 times/day EPA recommends that feeding occur at 

least twice per day.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: Feeding rates were half of 

recommended rates as specified by EPA and OECD. 

It is recommended that food rations during 

the pre-exposure period be increased along 

with larval growth to approximately 30 

mg/larva/day by test initiation. EPA and 

OECD recommend that food rations 

increase from 30 mg/larva/day at test 

initiation (Study Day 0-4) to 80 

mg/larva/day in the last week of the test 

(Study Day 15-21).

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Continuous flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable): 

~69 mL/min Flow rate was calculated based 

upon 10 volume additions per day and a 10 L fill volume. This flow rate resulted in complete volume replacement approximately every 2.4 hours. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement 

ca. every 2.7 hrs).

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Fluid metering pumps were used to deliver the single test substance stock solution to mixing chambers where it was mixed with well water. Dilution water flow was controlled using rotameters. 

All components were calibrated prior to the test. Flow rates were verified approximately weekly to ensure that rates varied by no more than ±10%. The general operation of the exposure system was checked visually at least two times per day during the test and at least once on the last day of the test. 

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%.

Renewal period for static renewal (if applicable): 

NA If static renewal is used, EPA 

recommends 24-hr renewal; renewal 

period is recommended not to exceed 72 

hours.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? No EPA recommends maintaining dissolved 

oxygen concentrations >40% air 

saturation (>3.5 mg/L). Aeration may be maintained through bubblers. It is 

recommended to set bubblers at levels 

that do not cause stress on the tadpoles.

Source of dilution water: Dilution water was freshwater 

obtained from a well approximately 40 meters deep located on WLI property. Water was passed through a sand filter to remove particles greater than ~25 սm, pumped into a tank, 

and aerated. Prior to use, water was filtered to 0.45 սm and passed through a UV sterilizer. 

EPA recommends natural or reconstituted 

water; it is recommended that natural 

water be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

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

perchlorate). OECD accepts any water in 

which the test species show control 

survival at least as good as indicated in 

the test guideline.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes. See Reviewer’s Comments, 

Iodide supplementation in water? 

No 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 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: 12 L Fill volume: 10 L 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

NA 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Hardness (mg/L as CaCO3) 

132 152 142 

Measured in one replicate of the control 

and high-concentration treatment weekly, 

with measurements alternating between 

replicates. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 8.0 8.2 8.1 

Measured weekly in each test chamber. EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials 

should not exceed 0.5.

Dissolved oxygen (mg/L) 

7.5 8.5 8.0 

Measured weekly in each test chamber. EPA recommends dissolved oxygen 

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

OECD recommends DO concentration 

>3.5 mg/L (>40% air saturation).

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 21.4 22.6 22.0 

Measured weekly in each test chamber. 

Temperature also was measured 

continuously in one replicate of the 

negative control. 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide (սg/L) 3 6 -- 

Prior to test. EPA recommends aquatic iodide range 

0.5 – 10 

սg/L (supplemental iodide 

should not exceed 2 

սg/L).

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Ammonia Not Reported General recommendations for frequency 

of measurements: EPA recommends that 

water quality parameters be measured in 

a control and at one test item 

concentration at least weekly. In static 

renewal systems, water quality 

parameters, including ammonia, should 

be measured just prior to renewal. In 

addition, EPA recommends that DO be 

measured at each concentration at least 

weekly and that temperature be 

measured continuously. OECD 

recommends that DO and temperature 

be measured at least weekly and that pH 

and hardness be measured at least at 

the beginning and end of the test.

Fluoride (mg/L) -- -- 0.85 December 28, 2011 Perchlorate Not Reported Chlorate Not Reported Alkalinity (mg/L as CaCO3) 

170 178 174 

Measured in one replicate of the control 

and high-concentration treatment weekly, 

with measurements alternating between 

replicates. 

Specific Conductance (սS/cm) 

322 387 354 

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 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. Two separate rangefinding studies were conducted. If yes, what was the method for determining the highest test concentration in the rangefinder? 

Not specified. EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., <10% mortality), whichever 

concentration is lowest.

Species: Xenopus laevis Life stage: Not reported Test duration: The first test was conducted for 

14 days and the second for 96 hours. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: First range-finding test was 

conducted for 14 days at a nominal concentration of 4.5 mg a.i./L. There were 3 mortalities by the end of Day 1 and signs of toxicity (i.e., small, pale, surfacing) observed throughout the test. A second range-finding study was conducted for 96 hours at a nominal concentration of 5.0 mg a.i./L. No mortalities occurred and only sign of toxicity was surfacing. 

The high concentration for the definitive test was selected based on the range-finding results and set at 2.0 mg a.i./L. The two lower concentrations were determined using a factor of 10. 

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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

Range-finder, solubility EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., <10% mortality), whichever 

concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0.10 EPA recommends that the maximum 

concentration separation be 0.1 and the 

minimum be 0.33. 

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

concentrations and a control, plus solvent 

control if appropriate.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit of quantification (LOQ): 0.0100 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, 14, and 21. 

It is recommended that test item 

concentration be measured in one tank at 

each treatment level at test initiation and 

every week thereafter.

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in solvent control (if applicable): 

NA EPA and OECD recommend the use of a 

concurrent solvent control when a 

solubilizing agent is used. EPA 

recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

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

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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).

Light intensity at water’s surface: 

0.934-1.572 Klux EPA recommends light intensity 0.6 – 2 

Klux (at water’s surface).

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

Aquatic Toxicology, 76, pp.69–92. 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: NA The test solutions were reported as clear 

and colorless with no evidence of precipitate 

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.0100 -- None EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 

20%.

Treatment 1 0.020 0.019 5.4 None 

Treatment 2 0.20 0.18 3.8 None Treatment 3 2.0 1.9 3.6 None 

Abbreviations: CV Coefficient of variation. 

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 E. Observations

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

Day 21- NF stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathology 

 Daily- mortality, clinical observations 

Were raw (individual) data provided? Yes

EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF 

developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (Days 7 and 

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

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

pathology observations.

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II. RESULTS AND DISCUSSION 

A. Results

A single mortality was observed in the mean-measured 0.019 mg a.i./L treatment group prior to 

Day 7 (Table 9).One tadpole in the 0.019 and 0.18 mg a.i./L treatment groups was inadvertently 

killed during siphoning of the test chambers and these mortalities were excluded from calculations of 

survival. 

Table 9: Larval Mortality in Xenopus laevis.

Treatment (mg a.i./L) [meanmeasured]

Larval Mortality 

Day 71 Day 21 

n Mortality # Mortality % n Mortality # Mortality % 

Negative Control 80 0 0 60 0 0 

0.019 80 1 1.2 582 0 0 

0.18 80 0 0 592 0 0 

1.9 80 0 0 60 0 0 

1

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

2

 One tadpole inadvertently killed during siphoning and mortality not considered in calculations of survival. 

By Day 7, median developmental stage was 54 in the control and low and mid treatment groups, 

and 53 in the high treatment group (Table 10). By test termination, median developmental stage 

was 57 in the control and all treatment groups. The 10th and 90th percentiles in the negative control 

did not differ by more than 4 NF stages and no asynchronous development was observed. A single 

late-stage tadpole was observed in the negative control (NF=61) and in the low and mid treatment 

group (NF=62). These tadpoles were excluded from analysis of growth endpoints and normalized 

hind-limb length. 

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

Treatment 

(mg a.i./L) 

[mean-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 

0.18 4 54 0 4 57 0 

1.9 4 53 0 4 57 0 

By Day 7, mean normalized HLL was 0.14 in the negative control and all treatment groups (Table 

11). At test termination, mean normalized HLL was 0.37 in the negative control and 0.36, 0.35, and 

0.32 in the mean-measured 0.019, 0.18, and 1.9 mg a.i./L treatment groups, respectively. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Hind Limb Length (HLL) 

Day 7 Day 21 

n Mean

(mm) 

±SD 

HLL: 

SVL1 n Mean(mm) 

±SD 

HLL: 

SVL1

Negative Control 4 2.08 0.17 0.14 4 7.95 0.69 0.37 

0.019 4 2.06 0.08 0.14 4 7.52 0.60 0.36 

0.18 4 2.10 0.19 0.14 4 7.29 0.60 0.35 

1.9 4 2.15 0.08 0.14 4 6.41 0.58 0.32 

Abbreviations: SD Standard deviation. 

1

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

Day 7 mean SVL ranged from 15.0 to 15.4 mm in the control and treatment groups (Table 12). By 

test termination, however, mean SVL in the highest treatment group (20.0 mm). 

Mean wet weight on Day 7 ranged from 0.211 to 0.241 g in the control and treatment groups and, 

like Day 7 SVL, (Table 12). 

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Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[meanmeasured]

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 21 Day 7 Day 21 

n Mean

(mm) 

±SD n Mean

(mm) 

±SD n Mean

(g) 

±SD n Mean

(g) 

±SD 

Negative Control 4 15.4 0.68 4 21.6 0.72 4 0.241 0.04 4 0.783 0.06 

0.019 4 15.1 0.50 4 20.9 0.56 4 0.228 0.02 4 0.724 0.05 

0.18 4 15.4 0.62 4 21.0 0.53 4 0.234 0.02 4 0.717 0.03 

1.9 4 15.0 0.39 4 20.0 0.36 4 0.211 0.01 4 0.602 0.05 

Abbreviations: SD Standard deviation. 

1

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

There appeared to be an increase in the number of tadpoles exhibiting mild to moderate thyroid 

gland atrophy in the high treatment group (Tables 13 and 14), and no tadpoles in this group 

exhibited thyroid gland hypertrophy. However, the study author reported that within the control and 

all treatment groups, thyroid gland atrophy was found predominantly in the smaller tadpoles, 

suggesting that smaller size resulted in gland atrophy. 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

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 18 20 16 20 18 -- -- 

1 20 0 20 3 20 2 -- -- 

2 20 0 20 1 20 0 -- -- 

3 20 2 20 0 20 0 -- -- 

0.019 0 20 17 20 15 20 20 -- -- 

1 20 2 20 5 20 0 -- -- 

2 20 1 20 0 20 0 -- -- 

3 20 0 20 0 20 0 -- -- 

0.18 0 20 18 20 17 20 20 -- -- 

1 20 2 20 3 20 0 -- -- 

2 20 0 20 0 20 0 -- -- 

3 20 0 20 0 20 0 -- -- 

1.9 0 20 20 20 10 20 20 -- -- 

1 20 0 20 6 20 0 -- -- 

2 20 0 20 4 20 0 -- -- 

3 20 0 20 0 20 0 -- -- 

1

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

2=Moderate, 3=Severe. See OECD No. 82 for reference.

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

Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Size 

Decrease 

Follicle Size 

Increased 

Gland Asymmetry 

Increase 

Follicular Cell 

Height (Decrease) 

Follicular 

Asymmetry 

Increased 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 14 20 16 20 15 -- -- 20 19 

1 20 3 20 0 20 5 -- -- 20 1 

2 20 3 20 3 20 0 -- -- 20 0 3 20 0 20 1 20 0 -- -- 20 0 

0.019 0 20 14 20 18 20 18 -- -- 20 16 

1 20 4 20 1 20 1 -- -- 20 2 

2 20 2 20 1 20 1 -- -- 20 2 3 20 0 20 0 20 0 -- -- 20 0 

0.18 0 20 14 20 19 20 17 -- -- 20 15 

1 20 6 20 1 20 2 -- -- 20 4 

2 20 0 20 0 20 1 -- -- 20 1 3 20 0 20 0 20 0 -- -- 20 0 

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Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Size 

Decrease 

Follicle Size 

Increased 

Gland Asymmetry 

Increase 

Follicular Cell 

Height (Decrease) 

Follicular 

Asymmetry 

Increased 

n Incidence n Incidence n Incidence n Incidence n Incidence 

1.9 0 20 10 20 20 20 18 -- -- 20 20 

1 20 5 20 0 20 2 -- -- 20 0 

2 20 5 20 0 20 0 -- -- 20 0 3 20 0 20 0 20 0 -- -- 20 0 

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

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By test termination, tail curvature was observed in the negative control (8%), the low (6%), and mid (9%) treatment groups; all surviving tadpoles in the high treatment group appeared normal and healthy (Table 15). 

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[mean-measured] 

Clinical Signs 

Type n Incidence 

Negative Control Tail Curvature 60 8 0.019 Tail Curvature 58 6 0.18 Tail Curvature 59 9 1.9 Tail Curvature 60 0 

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B. Study Author’s Analysis and Conclusions

Unless otherwise noted, the unit of statistical analysis was the replicate test chamber. Most endpoints 

were analyzed using two complementary statistical approaches. If responses appeared to be monotonic 

a step-down Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of replicate 

means to determine possible concentration responsive trends among the treatment groups. All 

endpoints except for survival and developmental stage were analyzed by performing pair-wise 

comparisons using Dunnett’s multiple comparison test to determine which treatment groups differed 

statistically from the control group. Data for endpoints analyzed by Dunnett’s test were evaluated for 

normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (Į=0.01). 

When the data did not satisfy the assumptions, a log transformation was applied to correct the data 

for non-normality or heterogeneous variances. 

Survival and developmental stage were not amenable to the statistical methods used for analysis of 

other endpoints. In particular, the most suitable unit of statistical analysis for survival was the individual 

animal. Therefore, survival was analyzed using the Fishers Exact test and Jonckheere-Terpstra trend 

test. In accordance with the recommendations of OECD TG 231 and OCSPP 890.1100, analysis of 

metamorphic stage was performed using the multi-quantile analysis developed by T. Springer and J. 

Green, and using the step-down Jonckheere-Terpstra trend test on stage quantiles in the replicate 

test chambers. Statistical tests used to evaluate treatment effects were performed at confidence level 

of Į=0.05 with SAS software. 

There were no treatment-related effects on any developmental or growth endpoints on Day 7 relative 

to the negative control. Further, there were no apparent effects on Day 21 in the low and mid treatment 

groups. There was no effect on developmental stage on Day 21, however, wet weight, SVL, and 

normalized HLL were all significantly reduced in the high treatment group on Day 21. The study author 

attributed these differences to slight variations in the distribution of stages between the groups, and 

were no longer apparent for SVL and normalized HLL, and greatly reduced for weight when frogs were 

stage matched at NF 57. The reduction in weight was still evident in the analysis on NF 57 tadpoles. 

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This was consistent with histological findings, where there was a slight increase in the number of 

tadpoles in the high treatment groups which seemed to show mild thyroid gland atrophy relative to the 

negative control group. The apparent slight increase in mild thyroid gland atrophy in the high treatment 

group appeared to be a consequence of a reduction in size of the tadpoles in that group relative to 

the control that can be attributed to other factors such as reduced feeding. It was noted that during 

the rangefinders conducted at similar concentrations, signs of overt toxicity included surfacing, 

discoloration, and small tadpoles. Other than the mild thyroid gland atrophy, there were no other 

histopathological findings that were different between the treatment groups and the negative control. 

 C. Reviewer’s Analysis and Conclusions 

 

Statistical Methods: Day 7 developmental stage did not exhibit a monotonic trend and was not 

normally distributed (Shapiro-Wilk, p=0.037) and had unequal variances (Levene’s test, p=1.0) and 

was therefore analyzed using the Mann-Whitney U Two-Sample test. Day 7 HLL and normalized 

HLL exhibited monotonic trends and were analyzed using the Jonckheere-Terpstra Step-Down test. 

Day 7 SVL and wet weight were normally distributed with equal variances and therefore analyzed 

using Dunnett’s Multiple Comparison test. Day 21 developmental stage was analyzed using the 

Mann-Whitney U Two-Sample test and all remaining Day 21 endpoints were analyzed using the 

Jonckheere-Terpstra Step-Down Test. Replicate means for growth endpoints on Day 21 were 

calculated by excluding late-stage tadpoles (NF > 60). A single late-stage tadpole was observed in 

the negative control (NF=61) and in the low and mid treatment group (NF=62). These tadpoles 

were excluded from analysis of growth endpoints and normalized hind-limb length. All analyses 

were conducted using CETIS v. 1.8.7.7, using backend settings implemented by EFED on 5/29/13. 

Given the low mortality rate in this study, statistical analysis was not conducted, rather survival was 

evaluated visually. 

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

statistically significant at p<0.05. 

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 Conclusions: 

On Day 7, normalized HLL was significantly increased in the high treatment group (6% increase, 

p=0.0334). No other endpoints on Day 7 were significantly different from the negative control. On 

Day 21, significant concentration-dependent reductions were noted for normalized HLL (14.1%, 

p=0.012) and growth parameters, including HLL (19.3%, p=0.003), wet weight (23.2%, 

p=0.0003), and SVL (7.23%, p=0.0007) in the high treatment group, relative to the negative 

control. According to the study report, there was a slight increase in the number of tadpoles in the 

high treatment groups which seemed to show mild thyroid gland atrophy relative to the negative 

control group. 

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

Treatment 

(mg a.i./L) 

[mean-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 -- 57 -- -- -- -- -- -- -- -- -- No 0.019 54 1.0 57 1.00 1.30 0.31 -3.34 0.44 -- -- -- -- No 0.18 54 1.0 57 1.00 1.49 0.31 -6.19 0.19 -- -- -- -- No 1.9 53 0.49 57 1.00 6.13 0.03 -14.1 0.01 -- -- -- -- Yes Statistical Test Mann-Whitney Mann-Whitney Jonckheere Jonckheere -- -- -- 

Abbreviations: Diff. Difference. 

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

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

2 Unless otherwise specified, effects are considered statistically significant at 

p<0.05. 

3 Hind-limb length is normalized to snout-vent length (SVL). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Snout-Vent Length Body Weight 

Day 7 Day 21 Day 7 Day 21 

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

Negative Control -- -- -- -- -- -- -- -- 

0.019 -1.95 0.78 -2.98 0.10 -4.99 0.85 -7.66 0.25 

0.18 0.00 1.00 -2.73 0.08 -2.60 0.97 -8.55 0.18 

1.9 -2.27 0.70 -7.23 0.0007 -12.2 0.30 -23.2 0.0006 

Statistical Test Dunnett Jonckheere Dunnett Jonckheere 

Abbreviations: Diff. Difference. 1 Unless otherwise indicated, effects are reported based on comparison to the 

negative (clean water) control. 

2

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

E. Study Deficiencies 

Although there were minor deviations from the guideline, as noted in the Materials and Methods section 

of the DER, the study met all validity and performance criteria. 

F. Reviewer’s Comments 

The review found a significant increase in normalized HLL on Day 7 in the high treatment group and 

considered the observed effects to be treatment-related. Therefore, the reviewer’s results are reported 

in the Executive Summary section of this DER. 

Both range-finding studies were conducted without the use of a solvent. The first trial was conducted 

using a saturator column, whereas the second was conducted by preparing a stock solution directly in 

dilution water. Analytical recoveries ranged from 73 to 88% of nominal in the first trial and 54 to 57% 

of nominal in the second. Additional trials with the saturator columns showed that columns were stable 

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for at least 11 days when prepared at a concentration of 4.0 mg a.i./L. Therefore the definitive test 

was conducted with the use of saturator columns. 

Glass saturator columns were prepared for the delivery of simazine. Each glass column (1” ID x 24” 

L) was packed with approximately 90 g of 8 micron glass wool that was pre-washed with reagent 

grade acetone and coated with a concentrated stock suspension of simazine. The stock suspension 

was prepared in methanol to achieve the nominal concentration of 62.5 mg/mL. One to two inch 

segments of pre-washed glass wool were immersed into the stock suspension and packed into the 

column. This procedure was repeated until all of the glass wool coated with the test substance 

suspension was packed evenly into the column. The column was vacuum dried to remove the methanol. 

Once the methanol was removed, the column was wrapped in aluminum foil and stored refrigerated 

until use. Columns were set up in pairs with the water exiting one column and entering the other, 

effectively extending the length of the column. Four pairs of columns were used to prepare the primary 

stock solution for the duration of the study. Since the columns were good for at least 11 days, one 

column from each series was replaced with a newly prepared column on a weekly basis. 

Results from the periodic screening analysis of the dilution water indicated the presence of the following 

analytes: calcium (34.9 mg/L), chloride (4.5 mg/L), fluoride (0.85 mg/L), magnesium (13.2 mg/L), 

potassium (7.00 mg/L), sodium (19.0 mg/L), and sulfate (5.7 mg/L). 

Tail curvature was reported in both the control and treatment groups. The temperature range and 

iodide concentrations were within guideline recommendations. The feeding rate was half of the 

recommended rate in the guideline. According to the study report, tail curvature for each animal was 

scored from 0-3 and scores that were 2 or greater were used to calculate percent tail curvature for a 

treatment group. The number of tadpoles at test termination that had a score of 1 or greater was 29, 

20, 18 and 2 in the control, low, mid and high treatment, respectively. 

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III. REFERENCES 

Nieuwkoop, P.D., Faber, J. (eds.). 1994. Normal Table of Xenopus laevis (Daudin): A Systematical and 

Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis. Garland 

Publishing Co., New York, NY USA. 

Personal Communication Dr. Timothy Springer and Dr. John Green. November 30, 2010. Dr. Timothy 

Springer, Wildlife International, Ltd., Easton MD 21601. Dr. John Green, E.I. DuPont de Nemours and 

Company, Wilmington, DE 19898, USA. 

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Report Date: 19 Jul-13 09:44 (p 1 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:30

Analysis ID: 01-4252-1629 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.019 8 NA 2 6 1.0000 Exact Non-Significant Effect

0.18 8 NA 2 6 1.0000 Exact Non-Significant Effect

1.9 12 NA 2 6 0.4857 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 1.61% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.75 0.25 3 1 0.4262 Non-Significant Effect

Error 3 0.25 12

Total 3.75 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Mod Levene Equality of Variance 0 5.95 1.0000 Equal Variances

Variances Levene Equality of Variance 0 5.95 1.0000 Equal Variances

Distribution Shapiro-Wilk W Normality 0.81 0.841 0.0037 Non-normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d Developmental Stage Summary

0 Negative Control 4 53.8 53 54.5 54 53 54 0.25 0.93% 0.0%

0.019 4 53.8 53 54.5 54 53 54 0.25 0.93% 0.0%

0.18 4 53.8 53 54.5 54 53 54 0.25 0.93% 0.0%

1.9 4 53.3 52.5 54 53 53 54 0.25 0.94% 0.93% Centered Untransformed

 Rankits

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d Developmental Stage

 C-mg ai/L

0

10

20

30

40

50

60

0 N 0.019 0.18 1.9

Graphics

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

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Report Date: 19 Jul-13 09:44 (p 2 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:30

Analysis ID: 16-6263-7199 Endpoint: 07d HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.15 2.68 0.268 6 0.9975 CDF Non-Significant Effect

0.18 0.2 2.68 0.268 6 0.9941 CDF Non-Significant Effect

1.9 0.75 2.68 0.268 6 0.7964 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 12.9% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.01859999 0.006199997 3 0.31 0.8176 Non-Significant Effect

Error 0.2398 0.01998333 12

Total 0.2584 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.51 11.3 0.3194 Equal Variances

Distribution Shapiro-Wilk W Normality 0.952 0.841 0.5234 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d HLL Summary

0 Negative Control 4 2.08 1.8 2.36 2.02 1.95 2.33 0.0865 8.32% 0.0%

0.019 4 2.06 1.94 2.19 2.05 1.98 2.17 0.0397 3.84% 0.72%

0.18 4 2.1 1.79 2.41 2.09 1.89 2.33 0.0972 9.26% -0.96%

1.9 4 2.15 2.03 2.28 2.14 2.09 2.24 0.0384 3.56% -3.61% Centered Untransformed

 Rankits

-0.05

-0.10

-0.15

-0.20

-0.25

0.00

0.05

0.10

0.15

0.20

0.25

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d HLL

 C-mg ai/L

Reject Null

Reject Null

0.0

0.5

1.0

1.5

2.0

2.5

0 N 0.019 0.18 1.9

Graphics

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

Page 47 of 196
Report Date: 19 Jul-13 09:44 (p 3 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 15:00

Analysis ID: 01-0963-3609 Endpoint: 07d HLL CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 0.438 1.64 1 -2 0.3309 Asymp Non-Significant Effect

0.18 0.44 1.64 1 -2 0.3298 Asymp Non-Significant Effect

1.9 1.4 1.64 2 -2 0.0802 Asymp Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C < T NA NA 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.01859999 0.006199997 3 0.31 0.8176 Non-Significant Effect

Error 0.2398 0.01998333 12

Total 0.2584 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.51 11.3 0.3194 Equal Variances

Distribution Shapiro-Wilk W Normality 0.952 0.841 0.5234 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d HLL Summary

0 Negative Control 4 2.08 1.8 2.36 2.02 1.95 2.33 0.0865 8.32% 0.0%

0.019 4 2.06 1.94 2.19 2.05 1.98 2.17 0.0397 3.84% 0.72%

0.18 4 2.1 1.79 2.41 2.09 1.89 2.33 0.0972 9.26% -0.96%

1.9 4 2.15 2.03 2.28 2.14 2.09 2.24 0.0384 3.56% -3.61% Centered Untransformed

 Rankits

-0.05

-0.10

-0.15

-0.20

-0.25

0.00

0.05

0.10

0.15

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0.25

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d HLL

 C-mg ai/L

0.0

0.5

1.0

1.5

2.0

2.5

0 N 0.019 0.18 1.9

Graphics

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

Page 48 of 196
Report Date: 19 Jul-13 09:44 (p 4 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:31

Analysis ID: 12-8528-5883 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Multiple Comparison Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett T3 Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.595 4.28 0.013 3 0.9044 CDF Non-Significant Effect

0.18 0.349 3.44 0.02 5 0.9775 CDF Non-Significant Effect

1.9 2.7 3.94 0.012 4 0.1414 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 8.93% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.0001565 5.216665E-05 3 1.54 0.2558 Non-Significant Effect

Error 0.0004075 3.395833E-05 12

Total 0.0005639999 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 12.7 11.3 0.0054 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.94 0.841 0.3455 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d Normalized HLL Summary

0 Negative Control 4 0.135 0.125 0.144 0.132 0.13 0.143 0.0029 4.31% 0.0%

0.019 4 0.136 0.135 0.138 0.137 0.135 0.137 0.000479 0.7% -1.3%

0.18 4 0.136 0.121 0.152 0.137 0.127 0.146 0.00494 7.24% -1.49%

1.9 4 0.143 0.14 0.146 0.144 0.14 0.144 0.000946 1.33% -6.13% Centered Untransformed

 Rankits

-0.002

-0.004

-0.006

-0.008

-0.010

0.000

0.002

0.004

0.006

0.008

0.010

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d Normalized HLL

 C-mg ai/L

Reject Null

Reject Null

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0 N 0.019 0.18 1.9

Graphics

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

Page 49 of 196
Report Date: 19 Jul-13 09:44 (p 5 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 15:00

Analysis ID: 20-1803-3840 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 12 NA -2 0.3090 Exact Non-Significant Effect

0.18 28 NA -2 0.3090 Exact Non-Significant Effect

1.9* 1.83 1.64 3 -2 0.0334 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C < T NA NA 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.0001565 5.216665E-05 3 1.54 0.2558 Non-Significant Effect

Error 0.0004075 3.395833E-05 12

Total 0.0005639999 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 12.7 11.3 0.0054 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.94 0.841 0.3455 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d Normalized HLL Summary

0 Negative Control 4 0.135 0.125 0.144 0.132 0.13 0.143 0.0029 4.31% 0.0%

0.019 4 0.136 0.135 0.138 0.137 0.135 0.137 0.000479 0.7% -1.3%

0.18 4 0.136 0.121 0.152 0.137 0.127 0.146 0.00494 7.24% -1.49%

1.9 4 0.143 0.14 0.146 0.144 0.14 0.144 0.000946 1.33% -6.13% Centered Untransformed

 Rankits

-0.002

-0.004

-0.006

-0.008

-0.010

0.000

0.002

0.004

0.006

0.008

0.010

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d Normalized HLL

 C-mg ai/L

0.00

0.02

0.04

0.06

0.08

0.10

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0.14

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0 N 0.019 0.18 1.9

Graphics

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

Page 50 of 196
Report Date: 19 Jul-13 09:44 (p 6 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:32

Analysis ID: 14-7358-1638 Endpoint: 07d SVL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.771 2.68 1.04 6 0.7837 CDF Non-Significant Effect

0.18 1.23E-06 2.68 1.04 6 1.0000 CDF Non-Significant Effect

1.9 0.9 2.68 1.04 6 0.7028 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 6.78% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.4274995 0.1424998 3 0.471 0.7080 Non-Significant Effect

Error 3.629999 0.3024999 12

Total 4.057499 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.852 11.3 0.8370 Equal Variances

Distribution Shapiro-Wilk W Normality 0.954 0.841 0.5484 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d SVL Summary

0 Negative Control 4 15.4 14.3 16.5 15.3 14.7 16.3 0.334 4.34% 0.0%

0.019 4 15.1 14.3 15.9 15 14.7 15.8 0.248 3.29% 1.95%

0.18 4 15.4 14.4 16.4 15.5 14.7 16 0.303 3.93% 0.0%

1.9 4 15 14.4 15.7 15 14.6 15.5 0.194 2.57% 2.27% Centered Untransformed

 Rankits

-0.2

-0.4

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-0.8

-1.0

0.0

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0.6

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1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d SVL

 C-mg ai/L

Reject Null

Reject Null

0

2

4

6

8

10

12

14

16

18

0 N 0.019 0.18 1.9

Graphics

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

Page 51 of 196
Report Date: 19 Jul-13 09:44 (p 7 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:33

Analysis ID: 02-4131-3903 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.654 2.68 0.049 6 0.8512 CDF Non-Significant Effect

0.18 0.341 2.68 0.049 6 0.9729 CDF Non-Significant Effect

1.9 1.59 2.68 0.049 6 0.3020 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 20.5% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.00189825 0.00063275 3 0.94 0.4519 Non-Significant Effect

Error 0.008081499 0.0006734582 12

Total 0.009979748 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 4.06 11.3 0.2546 Equal Variances

Distribution Shapiro-Wilk W Normality 0.919 0.841 0.1619 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

07d Wet Weight Summary

0 Negative Control 4 0.24 0.174 0.307 0.231 0.201 0.299 0.0208 17.3% 0.0%

0.019 4 0.229 0.196 0.261 0.22 0.215 0.259 0.0102 8.96% 4.99%

0.18 4 0.234 0.203 0.266 0.233 0.216 0.255 0.00983 8.39% 2.6%

1.9 4 0.211 0.191 0.231 0.211 0.197 0.226 0.00626 5.93% 12.2% Centered Untransformed

 Rankits

-0.01

-0.02

-0.03

-0.04

0.00

0.01

0.02

0.03

0.04

0.05

0.06

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d Wet Weight

 C-mg ai/L

Reject Null

Reject Null

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 N 0.019 0.18 1.9

Graphics

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

Page 52 of 196
Report Date: 19 Jul-13 09:44 (p 8 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:33

Analysis ID: 08-0913-9920 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.019 8 NA 1 6 1.0000 Exact Non-Significant Effect

0.18 8 NA 1 6 1.0000 Exact Non-Significant Effect

1.9 10 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 0.76% 1.9 >1.9 NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.1875 0.0625 3 1 0.4262 Non-Significant Effect

Error 0.75 0.0625 12

Total 0.9375 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Mod Levene Equality of Variance 1 5.95 0.4262 Equal Variances

Variances Levene Equality of Variance 9 5.95 0.0021 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.565 0.841 <0.0001 Non-normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d Developmental Stage Summary

0 Negative Control 4 57 57 57 57 57 57 0 0.0% 0.0%

0.019 4 57 57 57 57 57 57 0 0.0% 0.0%

0.18 4 57 57 57 57 57 57 0 0.0% 0.0%

1.9 4 56.8 56 57.5 57 56 57 0.25 0.88% 0.44% Centered Untransformed

 Rankits

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

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0.0

0.1

0.2

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21d Developmental Stage

 C-mg ai/L

0

10

20

30

40

50

60

0 N 0.019 0.18 1.9

Graphics

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

Page 53 of 196
Report Date: 19 Jul-13 09:44 (p 9 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Jul-13 9:41

Analysis ID: 19-8331-1929 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.975 2.68 1.18 6 0.6543 CDF Non-Significant Effect

0.18 1.49 2.68 1.18 6 0.3514 CDF Non-Significant Effect

1.9* 3.49 2.68 1.18 6 0.0116 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 14.8% 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 5.003119 1.667706 3 4.33 0.0275 Significant Effect

Error 4.617525 0.3847938 12

Total 9.620645 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.0891 11.3 0.9931 Equal Variances

Distribution Shapiro-Wilk W Normality 0.92 0.841 0.1702 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS HLL Summary

0 Negative Control 4 7.95 6.85 9.04 7.76 7.38 8.88 0.344 8.66% 0.0%

0.019 4 7.52 6.56 8.48 7.56 6.79 8.16 0.302 8.02% 5.38%

0.18 4 7.29 6.33 8.25 7.07 6.84 8.18 0.302 8.28% 8.21%

1.9 4 6.41 5.49 7.34 6.38 5.74 7.16 0.291 9.07% 19.3% Centered Untransformed

 Rankits

-0.2

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-0.6

-0.8

-1.0

0.0

0.2

0.4

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1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS HLL

 C-mg ai/L

Reject Null

Reject Null

0

2

4

6

8

10

0 N 0.019 0.18 1.9

Graphics

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

Page 54 of 196
Report Date: 19 Jul-13 09:44 (p 10 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Jul-13 9:42

Analysis ID: 01-0187-3339 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 11 NA -2 0.2429 Exact Non-Significant Effect

0.18 33 NA -2 0.1099 Exact Non-Significant Effect

1.9* 77 NA -2 0.0030 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 5.003119 1.667706 3 4.33 0.0275 Significant Effect

Error 4.617525 0.3847938 12

Total 9.620645 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.0891 11.3 0.9931 Equal Variances

Distribution Shapiro-Wilk W Normality 0.92 0.841 0.1702 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS HLL Summary

0 Negative Control 4 7.95 6.85 9.04 7.76 7.38 8.88 0.344 8.66% 0.0%

0.019 4 7.52 6.56 8.48 7.56 6.79 8.16 0.302 8.02% 5.38%

0.18 4 7.29 6.33 8.25 7.07 6.84 8.18 0.302 8.28% 8.21%

1.9 4 6.41 5.49 7.34 6.38 5.74 7.16 0.291 9.07% 19.3% Centered Untransformed

 Rankits

-0.2

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-0.6

-0.8

-1.0

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21d No LS HLL

 C-mg ai/L

0

1

2

3

4

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6

7

8

9

0 N 0.019 0.18 1.9

Graphics

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

Page 55 of 196
Report Date: 19 Jul-13 09:44 (p 11 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Jul-13 9:41

Analysis ID: 11-3271-7201 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 0.712 2.68 0.046 6 0.8188 CDF Non-Significant Effect

0.18 1.32 2.68 0.046 6 0.4386 CDF Non-Significant Effect

1.9* 3.01 2.68 0.046 6 0.0279 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 12.6% 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.005857191 0.001952397 3 3.3 0.0578 Non-Significant Effect

Error 0.007102752 0.000591896 12

Total 0.01295994 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.231 11.3 0.9725 Equal Variances

Distribution Shapiro-Wilk W Normality 0.942 0.841 0.3716 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS Normalized HLL Summary

0 Negative Control 4 0.367 0.322 0.413 0.368 0.332 0.402 0.0143 7.8% 0.0%

0.019 4 0.355 0.319 0.391 0.356 0.331 0.377 0.0115 6.45% 3.34%

0.18 4 0.344 0.308 0.381 0.337 0.326 0.377 0.0114 6.61% 6.19%

1.9 4 0.315 0.28 0.351 0.312 0.292 0.346 0.0112 7.11% 14.1% Centered Untransformed

 Rankits

-0.005

-0.010

-0.015

-0.020

-0.025

-0.030

-0.035

-0.040

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS Normalized HLL

 C-mg ai/L

Reject Null

Reject Null

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 N 0.019 0.18 1.9

Graphics

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

Page 56 of 196
Report Date: 19 Jul-13 09:44 (p 12 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Jul-13 9:42

Analysis ID: 16-7917-7403 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 9 NA -2 0.4429 Exact Non-Significant Effect

0.18 0.883 1.64 2 -2 0.1886 Asymp Non-Significant Effect

1.9* 2.25 1.64 2 -2 0.0123 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.005857191 0.001952397 3 3.3 0.0578 Non-Significant Effect

Error 0.007102752 0.000591896 12

Total 0.01295994 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.231 11.3 0.9725 Equal Variances

Distribution Shapiro-Wilk W Normality 0.942 0.841 0.3716 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS Normalized HLL Summary

0 Negative Control 4 0.367 0.322 0.413 0.368 0.332 0.402 0.0143 7.8% 0.0%

0.019 4 0.355 0.319 0.391 0.356 0.331 0.377 0.0115 6.45% 3.34%

0.18 4 0.344 0.308 0.381 0.337 0.326 0.377 0.0114 6.61% 6.19%

1.9 4 0.315 0.28 0.351 0.312 0.292 0.346 0.0112 7.11% 14.1% Centered Untransformed

 Rankits

-0.005

-0.010

-0.015

-0.020

-0.025

-0.030

-0.035

-0.040

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS Normalized HLL

 C-mg ai/L

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 N 0.019 0.18 1.9

Graphics

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

Page 57 of 196
Report Date: 19 Jul-13 09:44 (p 13 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:35

Analysis ID: 00-9050-1205 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 1.64 2.68 1.05 6 0.2816 CDF Non-Significant Effect

0.18 1.51 2.68 1.05 6 0.3417 CDF Non-Significant Effect

1.9* 3.99 2.68 1.05 6 0.0048 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 4.86% 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 4.979968 1.659989 3 5.42 0.0137 Significant Effect

Error 3.675923 0.3063269 12

Total 8.65589 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.13 11.3 0.7693 Equal Variances

Distribution Shapiro-Wilk W Normality 0.93 0.841 0.2412 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS SVL Summary

0 Negative Control 4 21.6 20.4 22.7 21.9 20.5 22 0.357 3.31% 0.0%

0.019 4 20.9 20.1 21.8 21 20.3 21.6 0.278 2.65% 2.98%

0.18 4 21 20.2 21.8 21 20.4 21.6 0.261 2.49% 2.73%

1.9 4 20 19.4 20.6 20.1 19.5 20.3 0.183 1.82% 7.23% Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS SVL

 C-mg ai/L

Reject Null

Reject Null

0

5

10

15

20

25

0 N 0.019 0.18 1.9

Graphics

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

Page 58 of 196
Report Date: 19 Jul-13 09:44 (p 14 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:36

Analysis ID: 04-6074-0832 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 13 NA -2 0.1000 Exact Non-Significant Effect

0.18 34 NA -2 0.0844 Exact Non-Significant Effect

1.9* 81 NA -2 0.0007 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 4.979968 1.659989 3 5.42 0.0137 Significant Effect

Error 3.675923 0.3063269 12

Total 8.65589 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.13 11.3 0.7693 Equal Variances

Distribution Shapiro-Wilk W Normality 0.93 0.841 0.2412 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS SVL Summary

0 Negative Control 4 21.6 20.4 22.7 21.9 20.5 22 0.357 3.31% 0.0%

0.019 4 20.9 20.1 21.8 21 20.3 21.6 0.278 2.65% 2.98%

0.18 4 21 20.2 21.8 21 20.4 21.6 0.261 2.49% 2.73%

1.9 4 20 19.4 20.6 20.1 19.5 20.3 0.183 1.82% 7.23% Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS SVL

 C-mg ai/L

0

5

10

15

20

25

0 N 0.019 0.18 1.9

Graphics

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

Page 59 of 196
Report Date: 19 Jul-13 09:44 (p 15 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:35

Analysis ID: 10-1450-4512 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.019 1.73 2.68 0.093 6 0.2482 CDF Non-Significant Effect

0.18 1.93 2.68 0.093 6 0.1812 CDF Non-Significant Effect

1.9* 5.24 2.68 0.093 6 0.0006 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 11.9% 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.06937101 0.02312367 3 9.57 0.0017 Significant Effect

Error 0.028999 0.002416583 12

Total 0.09837 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.69 11.3 0.6395 Equal Variances

Distribution Shapiro-Wilk W Normality 0.972 0.841 0.8667 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS Wet Weight Summary

0 Negative Control 4 0.784 0.688 0.879 0.788 0.711 0.848 0.03 7.66% 0.0%

0.019 4 0.724 0.644 0.804 0.711 0.681 0.793 0.0251 6.95% 7.66%

0.18 4 0.717 0.675 0.759 0.712 0.69 0.753 0.0132 3.68% 8.55%

1.9 4 0.602 0.517 0.687 0.62 0.524 0.644 0.0266 8.86% 23.2% Centered Untransformed

 Rankits

-0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07

-0.08

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS Wet Weight

 C-mg ai/L

Reject Null

Reject Null

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 N 0.019 0.18 1.9

Graphics

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

Page 60 of 196
Report Date: 19 Jul-13 09:44 (p 16 of 16)

Test Code: 080807 48673601 | 15-9293-2170

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 18 Jul-13 12:37

Analysis ID: 05-4485-1035 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 08-0160-9298

Start Date: 13 Jul-12

Ending Date: 03 Aug-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

Control vs C-mg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.019 13 NA -2 0.1000 Exact Non-Significant Effect

0.18 35 NA -2 0.0632 Exact Non-Significant Effect

1.9* 83 NA -2 0.0003 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.18 1.9 0.5848

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 0.06937101 0.02312367 3 9.57 0.0017 Significant Effect

Error 0.028999 0.002416583 12

Total 0.09837 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.69 11.3 0.6395 Equal Variances

Distribution Shapiro-Wilk W Normality 0.972 0.841 0.8667 Normal Distribution

C-mg ai/L Control Type Count Mean 95% LCL 95% UCL Median Min Max Std Err CV% %Effect

21d No LS Wet Weight Summary

0 Negative Control 4 0.784 0.688 0.879 0.788 0.711 0.848 0.03 7.66% 0.0%

0.019 4 0.724 0.644 0.804 0.711 0.681 0.793 0.0251 6.95% 7.66%

0.18 4 0.717 0.675 0.759 0.712 0.69 0.753 0.0132 3.68% 8.55%

1.9 4 0.602 0.517 0.687 0.62 0.524 0.644 0.0266 8.86% 23.2% Centered Untransformed

 Rankits

-0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07

-0.08

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d No LS Wet Weight

 C-mg ai/L

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 N 0.019 0.18 1.9

Graphics

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

Page 61 of 196
DATA EVALUATION RECORD 

SIMAZINE 

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

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48619301to include Saturation binding data; 

Main study was originally reviewed under TA 2-28-2012) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/19/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/19/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/26/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/26/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 62 of 196
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 63 of 196
SIMAZINE I 080807 

Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1 of 18 

OCSPP 890.1150/ OECD None 

Primary Reviewer: Vincent Chen, M.S. 

Health Effects Division, Office of Pesticide Programs 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division, Office of Pesticide Programs 

,, - Signature: ~< .-

Date: "Z.'1 "SutJ Z..OJ...5" 

Signature: @ L--

Date:,. <e ft\1k 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSP:t;> 890.1150 

PC CODE: 080807 DP BARCODE: D398096 

TXR#: 0056244 CAS No.: 122-34-9 

TEST MATERIAL <PURITY): Simazine (96.9% a.i.) 

SYNONYMS: 6-chloro-N ,N-diethyl-1,3,5-triazine-2,4-diamine 

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

Cytosol). CeeTox Inc., Kalamazoo, Ml. Laboratory Study No.: 904 7V100328ARB, December 5, 2011. MRID 48619301. 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, Ml. July 19, 2011. 

MRID 48843501. Unpublished. 

SPONSOR: Syngenta Crop Protection, LLC, 410 Swing Road, P.O. Box 18300, Greensboro, 

NC 

TEST ORDER#: CON-080807-17 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48619301), 

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

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

[3H]-R1881 (1 nM) in the presence of increasing concentrations (10-10 to 10-3 M) of simazine 

(96.9% purity, Lot# 429546). DMSO was used as the vehicle at a final assay concentration of 

~ 3 .2%. A total of three independent runs were performed, and each run included dexamethasone 

as a weak positive control, and Rl 881 as the ligand reference standard. 

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. As separate cytosolic preparations were used for each of the 

three competitive binding runs, the data from all three saturation binding experiments were 

evaluated. For the cytosolic preparation made on March 30, 2011, the mean dissociation 

constant (K-0) for [

3

H]-R1881was0.614 (±0.065) nM, and the estimated Bmax was 1.080 (±0.109) 

:frnol/100 μg protein. For the cytosolic preparation made on July 23, 2011, mean K-0 for 

Page 64 of 196
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 18 

SIMAZINE / 080807 OCSPP 890.1150/ OECD None

[

3

H]-R1881 was 1.080 nM (±0.690), and the estimated Bmax was 1.071 (±0.167) fmol/100 μg 

protein. For the cytosolic preparation made on September 24, 2011, the mean Kd for [3H]-R1881 

was 0.613 nM (±0.041), and the estimated Bmax was 0.817 fmol/100 μg protein (±0.049). The 

mean and individual Kd values were generally below the range reported in the EPA validation 

program (0.685 to 1.57 nM), although two individual values were within the range and one value 

was above the range. Confidence in these numbers is generally high based on the goodness of fit 

(R2 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs (although greater 

variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation of simazine was visually observed at the 

highest concentration tested (10−3 M) in the first and second runs. Therefore, only data from 

concentrations ≤10−4

 M were used for data assessment, and the highest concentration tested in 

the third run was 10−4

 M. Specific binding of [3H]-R1881 was 87.9-100.1% at simazine 

concentrations of 10−10 to 10−4 M in each run, with the exception of the 10−6 M concentration in 

Run 3 that had binding of 70.6%. However, this decrease in binding was not concentration 

dependent as binding was 92.1-92.7% at concentrations of 10−5

 and 10−4 M in the same run. 

Because specific binding was not reduced to ≤50% at any simazine concentration, an IC50 and 

relative binding affinity (RBA) could not be calculated for simazine. 

The estimated average log IC50s for R1881 (−9.3 M) and the weak positive control, 

dexamethasone (−4.4 M), were within the expected ranges, and the mean RBA for the weak 

positive control was 0.0024%. Confidence in these numbers was moderate due to variation in 

IC50 values for the reference standards. However, the performance criteria were generally met. 

Based on the results from the three runs, simazine is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150). 

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided. 

Page 65 of 196
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 18 

SIMAZINE / 080807 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

Location: 4717 Campus Drive, Kalamazoo, MI 49008 

Study Director: Willoughby, J.A. 

Other Personnel: Rutherford, K. (Director Laboratory Operations); Blakeman, D. (Senior Scientist); 

Haines, C. (Scientist); McColley, D. (Scientist); Toole, C. (Director of Project 

Management) 

Study Period: May 13, 2011 to December 5, 2011 

2. Test substance: Simazine 

 Description: White powder 

 Source: Syngenta Crop Protection, LLC 

 Lot/Batch #: 429546 (expiry date: April 2012)

 Purity: 96.9% w/w 

 Solubility: Not reported 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: Ambient 

 CAS #: 122-34-9 

 Molecular weight: 201.66 g/mol 

 Structure: 

N

N

N

Cl

NH NH

C2H5 C2H5

3. Non-labeled ligand: R1881 

 Supplier: Sigma Aldrich (St. Louis, MO)

 Catalog and Batch #: R0908; Lot No. 060M4638 

 Purity: 98% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: Perkin-Elmer (Boston, MA) 

 Catalog and Batch #: NET590; Lot No. 653698 

 Date of production: February 24, 2011 

 Date of use: September 20, 2011 to September 29, 2011 

 Radiochemical purity: Not reported 

 Specific activity: 85.1 Ci/mmol 

 Concentration of stock: 1 nM 

5. Positive control: Dexamethasone 

 Supplier: Sigma Aldrich (St. Louis, MO)

 Catalog and Batch #: D1756; Lot No. 077K1050 

 Purity: 99% 

 CAS # : 50-02-2 

Page 66 of 196
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 18 

SIMAZINE / 080807 OCSPP 890.1150/ OECD None

6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO) 

 Justification for choice of 

solvent: 

Simazine solutions up to 10-4 M can be prepared while limiting the final concentratio

of DMSO to ~3.2% (v/v). 

 Final Concentration: ~3.2% (v/v) 

B. METHODS

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

purchased from Charles River Laboratories (Wilmington, MA). Male Sprague Dawley rats 

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

<24 hours later and the ventral prostate tissues collected.

The ventral prostates were received at the laboratory frozen and inspected for healthy 

appearance, weighed, and placed in ice-cold low-salt TEDG pH 7.4 buffer (inclusion of 

protease inhibitor not reported), homogenized, and centrifuged for 30 min at 30,000 × g at 4 

oC. Supernatant was pooled, discarding the resulting pellets. Protein concentration of the 

cytosol was determined to be 5.7-8.8 mg/mL using a commercially available protein kit 

compatible with DTT in the TEDG buffer (Bradford Method; Bio-Rad Dye Reagent 

Concentrate). Cytosol was divided into aliquots (amounts not specified) for immediate use. 

2. Saturation Radioligand Binding Experiment: Saturation binding experiments measuring 

total and non-specific binding of [3H]-R1881 were performed for all three cytosolic 

preparations, created on July 23, March 30, and September 24, 2011, 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 reports (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 67 of 196
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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 [3

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

For each batch of cytosol, the optimal protein concentration was determined by calculating 

specific binding to differing amounts of protein per tube with 0.25 nM radiolabeled R1881. 

The optimal protein concentration was determined to be 1.86 mg protein/assay tube for all 

cytosolic preparation, 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 non-current runs (conducted on June 

13, 14, and 21, 2011; September 9, 11, and 16, 2011; or September 24, 25, and 26, 2011; 

respectively, for the batches of cytosol prepared on March 30, June 23, and September 24, 

2011). 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 1-24 c Tubes 25-48 d Tubes 49-72 e 

[

3

H]-R1881 

Final conc. (nM) 

[

3

H]-R1881 

Final conc. (nM) 

R1881 

Final conc. (nM) 

[

3

H]-R1881 

Initial conc. (nM) 

[

3

H]-R1881 

(μL) 

0.25 0.25 25 10 7.5 

0.50 0.50 50 10 15 

0.70 0.70 70 10 21 

1.0 1.0 100 10 30 

1.5 1.5 150 10 45 

2.5 2.5 250 100 7.5 

5.0 5.0 500 100 15 

10 10 1000 100 30 

a Data were obtained from page 2 or 3 of the study reports (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, [3

H]-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 

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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 

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

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

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

total of three runs were performed. 

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

binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 4.76-9.66% of 1.0 nM 

radioligandb 

Concentration of test substance (as serial dilutions) 10−11 to 10−4 Mc

Incubation Temperature 4±2 oC 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10 % (v/v) 

Protease Inhibitor (PI)d 0.5% (v/v) 

DTT 1 mM 

Sodium Molybdate 1 mM 

a Data were obtained from page 16 of the study report. 

b Values were calculated by the reviewer; data obtained from pages 34-36 of the study report. Protein 

concentrations (μg/tube) were not reported. 

c The first two runs tested the test substance at 10−10 to 10−3

 M, but the highest concentration precipitated, 

therefore the third run was conducted at 10−11 to 10−4

 M. 

d The recommended PI in the Guideline is 1 mM PMSF. The PI was identified in the study protocol as 

Calbiochem Protease Inhibitor Cocktail, Set III, EDTA free. It is a mixture of six PIs that includes 4-(2-

aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF has similar specificity to PMSF with greater 

stability at lower pH. 

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

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

the binding of a single concentration of [3

H]-R1881 (adjusted specific activity of 82.4 

Ci/mmol for Runs 1-2; 82.2 Ci/mmol for Run 3) to the AR in the presence of increasing 

concentrations of a test substance. 

DMSO was used as the vehicle at a final concentration of 3.2% v/v. Because precipitation 

was visually observed at 10−3 M simazine in the first and second run, the highest 

concentration of simazine used in the third run was 10−4 M. 

Although only limited information was provided for the saturation binding experiments, the 

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

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cytosolic protein used in the assay contains enough receptor to bind approximately 5-10% of 

the [3H]-R1881. 

Dilutions of simazine, the reference standard (R1881), a 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 plus six replicates of the [3H]-

R1881, non-specific binding (NSB), and solvent controls, resulting in a total of 84 samples 

per run. 

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

Solvent Control Reference standard Weak positive control Test Chemical None 

DMSO R1881 Dexamethasone Simazine (Hot Mix) 

Tubes 7-12 Tubes 13-33 c Tubes 37-60 Tubes 61-84 d Tubes 1-6

1×10-6 1×10-3 1×10-3 

1×10-7 1×10-4 1×10-4

1×10-8 1×10-5 1×10-5

1×10-9 1×10-6 1×10-6

1×10-10 1×10-7 1×10-7

1×10-11 1×10-8 1×10-8

-- 1×10-10 1×10-10 

 -- -- 1×10-11 

a Data were obtained from pages 34-36 of the study report. 

b The [3

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

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

30 μL [3

H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 7-84 also contained 

10 μL of the solvent control, reference standard (non-radiolabeled R-1881), weak positive control, or test 

substance, with the exception of Tubes 13-18 that contained 30 μL of non-radiolabeled R1881 (used to evaluate 

non-specific binding). Tubes 1-6 contained only 30 μL of [3

H]-R1881. 

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

d Simazine was tested at 10−10 to 10−3

 M in the first and second runs and precipitation was observed at the highest 

concentration; therefore, in Run 3 simazine was tested at 10−11 to 10−4

 M. 

Sample tubes were stored 16-20 hours at 4 °C to allow the reaction to reach equilibrium, and 

bound R1881 was separated from free R1881 by washing with HAP buffer and extraction 

with ethanol, followed by scintillation counting of bound [3

H]-R1881. 

It was noted that four independent runs of the binding assay were conducted because in the 

third run the DPM values were lower than historical values. These data were not included in 

the analysis of this report because this run was considered invalid. Only data from the three 

“valid” independent runs (September 20, 2011; September 22, 2011; and September 29, 

2011) were reported. 

4. Data Analysis: For the saturation binding experiment, total binding and non-specific 

binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 

(GraphPad Software, Inc., La Jolla, CA)], incorporating automatic outlier elimination 

according to the method of Motulsky and Brown (2006)1

 implemented by using the ROUT 

 

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. 

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procedure in Prism v. 5 with a Q value of 1.0. Receptor binding data plots were corrected for 

ligand depletion with the method of Swillens (1995)2

. For the competitive binding assay, 

similar methods of nonlinear regression were used to fit a curve (for R1881, the positive 

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

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

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

deviation were calculated for each run and mean and standard error were calculated for the 

composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, 

Redmond, WA), and mean and standard error were calculated for the composite three runs with 

Microsoft Excel 2010. 

5. Definitions 

a. Classification of test material 

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

Binder: The average curve for the test chemical across runs crosses 50% of radioligand 

bound. 

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

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

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

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

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

Untestable: If the test compound is not soluble above 1×10-6 M and the binding curve does 

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

b. Descriptors for receptor binding 

Bmax: maximal binding capacity 

Kd: dissociation constants 

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

by the competitor 

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

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. 

 

2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for 

computer analysis. Molec. Pharmacol. 47(6):1197-1203. 

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For the cytosolic preparation created on March 30, 2011, the mean Kd for [3H]-R1881 was 

0.614 (±0.065) nM, and the estimated Bmax was 1.080 (±0.109) fmol/100 μg protein. Only 

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

(0.685 to 1.57 nM); the mean value was below the recommended range. Confidence in 

these numbers is high according to the goodness of fit (R2 = 0.955-0.992) and the small 

variation in R2

 and Kd estimates among runs. 

For the cytosolic preparation created on July 23, 2011, the mean Kd for [3H]-R1881 was 

1.080 nM (±0.690), and the estimated Bmax was 1.071 (±0.167) fmol/100 μg protein. Only 

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

(0.685 to 1.57 nM); the mean value was in the recommended range. Confidence in these 

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

For the cytosolic preparation created on September 24, 2011, the mean Kd for [3

H]-R1881 

was 0.613 nM (±0.041), and the estimated Bmax was 0.817 (±0.049) fmol/100 μg protein. 

The mean and individual Kd values were below the range reported in the EPA validation 

program (0.685 to 1.57 nM). Confidence in these numbers is high according to the 

goodness of fit (R2 = 0.957-0.984) and the small variation in R2 and 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 ± SEMb

Cytosolic preparation March 30 2011 

R2 (unweighted) 0.994 0.993 0.994 0.955-0.992 

Bmax (nM) 0.010 0.009 0.009 0.009±0.001 

Bmax (fmol/100 μg protein) NR NR NR 1.080±0.109 

Kd (nM) 0.686 0.559 0.596 0.614±0.065 

Cytosolic preparation July 23 2011 

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 

Cytosolic preparation September 24 2011 

R2 (unweighted) 0.984 0.977 0.957 0.957-0.984 

Bmax (nM) 0.011 0.010 0.011 0.011±0.001 

Bmax (fmol/100 μg protein) 0.809 0.773 0.870 0.817±0.049 

Kd (nM) 0.565 0.638 0.635 0.613±0.041 

a Data were obtained from page 3 or 4 of the study reports (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 representative sets of the non-specific, specific, and total binding curves 

for [3

H]-R1881 to the androgen receptor for the three independent runs for each of the three 

saturation binding experiments. The specific binding reached a plateau in each run, and 

non-specific binding was less than 20% of total binding. Figure 2 contains representative 

Scatchard plots each of the three saturation binding experiments that illustrate the binding of 

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[

3

H]-R1881 to the androgen receptor. The representative figures from the March 30, 2011 

cytosol preparation are incorrectly labeled as 13-June-12, instead of 13-June-11. 

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

Binding Experiment. Representative runs of each set of experiments. 

 

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

Representative runs of each set of experiments. 

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

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

Precipitation of simazine was visually observed at the highest concentration (10−3 M) in the 

first and second runs. Therefore, only data from simazine concentrations ≤10−4 M were 

used to assess binding, and the highest concentration in the third run was 10−4 M simazine. 

 In Runs 1 and 2, specific binding of [3

H]-R1881 was 87.9-100.1% at simazine 

concentrations of 10−10 to 10−4 M. In the third run, binding was reduced to 70.6% at a single 

concentration of simazine (10−6). However, the effect was not concentration dependent as 

binding was 89.1-93.5% at concentrations ≤10−7 M and 92.1-92.7% at concentrations of 

10−5

 and 10−4 M. Because specific binding was not ≤50% at any simazine concentration, an 

IC50 and RBA could not be calculated for simazine. 

The estimated average log IC50 was −9.3 M for R1881 and −4.4 M for the weak positive 

control (dexamethasone). Compared to R1881, the mean RBA for dexamethasone was 

0.0024%. Confidence in these numbers is high for dexamethasone due to the small 

variation, but there was greater than a 1-log difference for the IC50 of R1881 in Run 2 

compared to Runs 1 and 3. 

Four independent runs of the binding assay were conducted because in the third run the 

DPM values were substantially different from typical data. Only data from the three “valid” 

independent runs were reported. 

TABLE 6. Competitive Binding Assay of Simazine with AR from Rat Prostate Cytosola

Parameter Run 1b Run 2b Run 3b Mean ± SEc,d

R2 (unweighted) R1881 NR NR NR NA 

Dexamethasone NR NR NR NA 

Simazine NR NR NR NA 

Log IC50 (M) R1881 −8.9 −10.1 −8.9 −9.3 ± 0.40 

Dexamethasone −4.4 −4.3 −4.5 −4.4 ± 0.06 

Simazine NA NA NA NA 

IC50 (M)d R1881 1.26 x 10−9 7.94 x 10−11 1.00 x 10−9 8.66 x 10−10 (± 3.93) 

Dexamethasone 3.98 x 10−5 5.01 x 10−5 3.16 x 10−5 4.05 x 10−5 (± 0.54) 

Simazine NA NA NA NA 

Log RBAd Dexamethasone −4.5 −5.8 −4.4 −4.9 ± 0.45 

Simazine NA NA NA NA 

RBA (%)d Dexamethasone 0.0032 0.0002 0.0040 0.0024 ± 0.0012 

Simazine NA NA NA NA 

a Data were obtained from the results discussion on page 21 of the study report.

b The mean is reported for the concurrent replicates within each run. 

c For means expressed in scientific notation, the SE values in parentheses are presented in the same order of 

magnitude as the mean value. 

d Calculated by the reviewers. 

SE Standard Error 

NA Not applicable 

NR Not reported 

R2

 Goodness of fit 

RBA Relative binding affinity

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

Radioinert R1881, Dexamethasone, or Simazine (Run 1). 

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

Radioinert R1881, Dexamethasone, or Simazine (Run 2). 

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

Radioinert R1881, Dexamethasone, or Simazine (Run 3). 

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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. 

≤15% 4.8 to 9.6% X 

Simazine Top (% binding) 80 to 115 87.9 to 100.1 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 98 to 114 X 

Bottom (% binding) −2.0 to 2.0 0 to 1 X 

Hill Slope −1.2 to −0.8 −1.0 to −0.8 X 

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 97 to 99 X 

Bottom (% binding) −12 to 12 −2 to 2 X 

Hill Slope −1.4 to −0.6 −1.0 to −0.9 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.559 to 0.686c

, 

0.531 to 1.855d

, 

0.565 to 0.638e

 X 

Non-specific binding (%) ≤10.0 0.37 to 0.48c

 X 

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

b These values represent ranges from the validation study. 

c Two values were below the limit and one value was within the tolerance limits. 

d One value was below the limit and one value was above the limit; the third value and the mean were within the 

tolerance limits. 

e All values were below the tolerance limit. 

f Calculated by the reviewers using the data from pages 34-36 of the study report. 

NR Not reported. 

The performance criteria were generally met. 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 −0.8. 

Examination across the runs indicated consistency of the Hill slope, and top and bottom 

plateaus, and the placement along the X-axis was consistent for dexamethasone. However, 

in the second run, the curve for R1881 was shifted substantially to the right, resulting in a 

log IC50 (−10.1 M) that is a full order of magnitude lower than the expected value. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Simazine was classified as a “non-binder’ in the 

first two valid independent runs and as “equivocal” in the third valid independent run. 

Therefore, simazine has a final classification of “non-binder.” 

B. AGENCY COMMENTS: For the cytosolic preparation created on March 30, 2011, the 

mean Kd for [3H]-R1881 was 0.614 (±0.065) nM, and the estimated Bmax was 1.080 (±0.109) 

fmol/100 μg protein. For the cytosolic preparation created on July 23, 2011, the mean Kd

for [3

H]-R1881 was 1.080 nM (±0.690), and the estimated Bmax was 1.071 (±0.167) 

fmol/100 μg protein. For the cytosolic preparation created on September 24, 2011, the 

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mean Kd for [3H]-R1881 was 0.613 nM (±0.041), and the estimated Bmax was 0.817 (±0.049) 

fmol/100 μg protein. The mean and individual Kd values were generally below the range 

reported in the EPA validation program (0.685 to 1.57 nM), although two individual values 

were within the range and one value was above the range. Confidence in these numbers is 

generally high according to the goodness of fit (R2

 = 0.880-0.992) and the small variation in 

R2

 and Kd estimates among runs (although greater variability was observed in the July 23, 

2011 saturation binding data). 

In the competitive binding experiment, precipitation of simazine was visually observed at 

the highest tested concentration (10−3

 M) in the first two runs. Therefore, only simazine 

concentrations ≤10−4

 M were used for data assessment, and the highest concentration tested 

in the third run was 10−4 M. Specific binding was 87.9-100.1% at simazine concentrations 

of 10−10 to 10−4

 M in all runs, with the exception of the 10−6 M concentration in Run 3 that 

had binding of 70.6%. However, this decrease in binding was not concentration dependent 

as binding was 92.1-92.7% at concentrations of 10−5

 and 10−4 M in the same run. Because 

specific binding was not reduced to ≤50% at any simazine concentration, an IC50 and RBA 

could not be calculated for simazine. As specific binding was >75% in all runs simazine is 

classified as a non-binder under the conditions of this assay. 

The estimated average log IC50s for R-1881 (−9.3 M) and the weak positive control, 

dexamethasone (−4.4 M), were within the expected ranges. The mean RBA for the weak 

positive control was 0.0024%. Confidence in these numbers was moderate due to variation 

in IC50 values observed for the reference standards. However, the performance criteria were 

generally met. 

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

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

 The protein concentration used in the assay was not reported. 

Page 81 of 196
DATA EVALUATION RECORD 

SIMAZINE 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48619302 to include laboratory proficiency data; 

Main study was originally reviewed under TA 2-28-2012) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

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

Secondary Reviewer: Signature:

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

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/25/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/25/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 82 of 196
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 83 of 196
SIMAZINE/ 080807 Estrogen Receptor Transcriptional Activation (2011) I Page 1 of 11 

OCSPP 890.1300/ OECD 455 

Primary Reviewer: Vincent Chen 

Health Effects Division 

Secondary Reviewer: Minerva Mercado-Feliciano 

Health Effects Division 

Signature:~C:::::::­ Date: 2. Y -:J"ulJ 2..0.J....5 

Signatu~ Date: 29-Jt.!fl...~ ~ /.STemplate version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455. 

PC CODE: 080807 DP BARCODE: D398096 

TXR#: 0056244 CAS No.: 122-34-9 

TEST MATERIAL (PURITY): Simazine (96.9% a.i.) 

SYNONYMS: 6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine 

CITATION: Willoughby, J.A. (2011) Simazine - estrogen receptor transcriptional activation 

(human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory 

Report No.: 9047V-100328ERTA, December 5, 2011. MRID 48619302. 

Unpublished. 

SPONSOR: 

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

Data for ERT A assays and Saturation Binding Data for AR and ER Binding 

Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, ML July 19, 2011. 

MRID 48843501. Unpublished. 

Syngenta Crop Protection, LLC, 410 Swing Road, P.O. Box 18300, Greensboro, 

NC 

TEST ORDER#: CON-080807-17 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 

48619302), hERa.-HeLa-9903 cells cultured in vitro were exposed to simazine (96.9% a.i., Lot# 

G27692U) in 0.1 % DMSO at logarithmically increasing concentrations from 10-11 to 10-4 M for 

24±2 hours in two independent runs. The experiments were performed using 96-well plates and 

each simazine concentration was tested in replicates of 6 wells/plate in each run. Cells were 

exposed to simazine to induce reporter (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor was measured upon addition of a luciferase 

substrate and detection with a luminometer with acceptable sensitivity. 

Simazine was tested up to the limit of solubility, 10-4 M. The mean RPCMax for simazine was 

3.5%, with an associated PCMax of 10-7 Min the first run; the mean RPCMax was <0% for the 

second run at concentrations up to 10-4 M. Performance criteria values were generally below the 

Page 84 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

the study. Because the RPCMax < PC10 in both runs, simazine 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 85 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: CeeTox, Inc. 

 Location: Kalamazoo, MI

 Study Director: J.A. Willoughby

 Other Personnel: K. Rutherford (Director Laboratory Operations); D. Blakeman (Senior Scientist); C. 

Haines (Scientist); S. McColley (Scientist); B. Wallace (Lead Cell Culture Scientist); 

and C. Toole (Director of Project Management)

 Study Period: May 13, 2011 to December 5, 2011 

2. Test Substance: Simazine 

 Description: White powder 

 Source (Catalog #): Syngenta Crop Protection, LLC (G27692U) 

 Lot/Batch # (Exp. Date): 429546 (April 2012) 

 Purity: 96.9% 

 Solubility: 10−3.3M in DMSO 

 Volatility/Vapor Pressure: Not reported 

 Stability: At least 7 years at room temperature 

 Storage conditions: Ambient 

 CAS #: 122-34-9 

 Structure: 

N

N

N

Cl

NH NH

C2H5 C2H5

3. Reference substances

 17β-estradiol (strong estrogen; positive control)

 Supplier: Not reported 

 Catalog and Batch #: Not reported 

 Purity: Not reported 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: Not reported 

 Catalog and Batch #: Not reported 

 Purity: Not reported 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: Not reported 

 Catalog and Batch #: Not reported 

 Purity: Not reported 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: Not reported 

 Catalog and Batch #: Not reported 

 Purity: Not reported 

 CAS # : 58-18-4 

4. Vehicle(s) 

Solvent: DMSO 

Solvent control : 0.1% v/v (final concentration) 

B. METHODS

Page 86 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

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 assay. Cells were maintained in Eagles Minimum 

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

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

incubator under 5% CO2 at 37oC. Upon reaching 75-90% confluence, cells were 

subcultured prior to exposure to the test material. The cells used in this study were passage 

32, 33 and 34. 

2. Transcriptional Activation Assays: For each test, cells were plated in 96-well plates at a 

density of 1×104 cells/100 μL and allowed to attach for at least 3 hours. Growth media was 

replaced with media containing serial log dilutions of simazine in DMSO (0.1% total final 

concentration). Cells were incubated for 24±2 hours at 37±2°C. Cytotoxicity was 

determined by a two-read propidium iodide uptake assay. Runs 1 and 2 for the main assay 

were conducted on May 26 and June 1, 2011, respectively. 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 CeeTox SOP 2041 was not provided 

a. Preliminary Test: A preliminary test evaluating simazine concentrations ranging from 

10−10.3 to 10−3.3 M was conducted to determine the appropriate concentration range and to 

determine concentrations resulting in insolubility and/or cytotoxicity. 

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. The cells used for proficiency testing and the main assay appeared to be from 

the same frozen stock, based on the dates for the assays and the passage numbers. 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 87 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

c. Reference Chemicals: To ensure the stability of the response from the cell line, seven 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−14 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 test material was defined as negative for inducing estrogen 

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

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

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

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

4. Definitions 

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

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Precipitation and/or cytotoxicity were not observed in any of the simazine 

solutions up to 10–3.3 M. The results of the preliminary tests are presented in Table 1. No 

cytotoxicity was observed at any concentration. Based on these results, logarithmically 

increasing concentrations of simazine from 10−11 to 10−4

 M were selected for the first assay. 

Page 88 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

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

Concentration (M) 

% Viability b

Commentsc

Run 1 Run 2 Run 3 

10−3.3 113 101 106 

10−4.3 96 94 94 

10−5.3 98 98 100 

10−6.3 94 95 97 

10−7.3 94 92 95 

10−8.3 98 96 94 

10−9.3 98 95 99 

10−10.3 100 100 100 

E2 1nM Not reported Not reported Not reported 

VC c Not reported Not reported Not reported 

a Data were obtained from page 21 of the study report. 

b If viability is <80%, the concentration is considered cytotoxic. 

c VC = Vehicle control 

B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS 

1. Proficiency Chemicals: The laboratory proficiency assays using the required reference 

compounds were not included in the original study report, but were provided to the Agency 

at a later date (MRID 48843501). The responsiveness of cells to the required proficiency 

chemicals was performed in duplicate on different days for each chemical. The reported 

responses are in Table 2a. In the proficiency tests, the reference chemicals 17β-estradiol, 

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

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

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

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

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

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

range, but the Hill Slope for 17α-estradiol was higher than expected. The PC-induced fold 

induction for the three reference chemicals was within the Guideline-recommended 

historical range of 4- to 30-fold in Runs 1 and 3, but fold induction was 75.1- to 84.7-fold in 

Run 2 with no explanation given for this 3- to 4-four increase. Although reportedly 

performed, the results of the cytotoxicity assay were not provided for reveiw. Raw data 

pertaining to the RTA of each chemical were not reported, but the scales of the graphs 

provided indicate Genistein and Butyl paraben had maximum RTAs well above 400%. The 

responses do not demonstrate proficiency as the assay cannot be fully evaluated by the 

reviewer.

Page 89 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

TABLE 2a. Proficiency Chemicals 

Compound Expected Response Lab Response 

Run 1 Run 2 Run 3 

Diethylstilbestrol Positive Positive Positive NA 

17α-Ethynyl estradiol Positive Positive Positive NA 

Hexestrol Positive Positive Positive NA 

Genistein Positive Positive Positive NA 

Estrone Positive Positive Positive NA 

Butyl paraben Positive Positive Positive NA 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive 

Dibutyl phthalate Negative Negative Negative NA 

Atrazine Negative Negative NA Negative 

Corticosterone Negative Negative NA Negative 

NA = not applicable. The chemical was not tested at this time. 

Table 2b. Performance Criteria for Reference Chemicals in the Proficiency test

Reference Chemical 

Parameter

Acceptable 

Range 

Values Acceptable 

Run 1 Run 2 Run 3 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −9.6 −11.3 −10.6 Run 1 

 Log PC10 <−11 −11.5 −12.5 −12.1 X 

 Log EC50 −11.3 to −10.1 −9.0 −11.3 −10.6 Run 1 

 Hill Slope 0.7 to 1.5 1.2 0.9 0.8 X 

 Test range (M) 10−14 to 10−8 10−14 to 10−8 10−14 to 10−8 10−14 to 10−8 X 

17α-estradiol

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

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

 Log EC50 −9.6 to −8.4 −8.2 −9.3 −8.9 Run 1 

 Hill Slope 0.9 to 2.0 0.9 0.9 2.9 Run 3 

 Test range (M) 10−12 to 10−6 10−12 to 10−6 10−12 to 10−6 10−12 to 10−6 X 

17α-methyltestosterone

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

 Log PC10 −8.0 to −6.2 −8.1 −6.3 −7.7 Run 1 

 Test range (M) 10−11 to 10−5 10−11 to 10−5 10−11 to 10−5 10−11 to 10−5 X 

2. Reference Chemicals: 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 

presented in Table 3. The acceptance criteria were not met for either run of the assay. In 

the first run, cytotoxicity was observed at the highest concentrations of 17α-estradiol 

(10−6

 M) and corticosterone (10−4 M), and at 10−13, 10−9, and 10−8 M for 17β-estradiol. In 

the second run, the responsiveness to 17β-estradiol at concentrations ≥10−10 M was 

117-150% of the response to the PC (10−9 M 17β-estradiol), and the response curve to 

17α-methyltestosterone mimicked the intensity of 17β-estradiol. Additionally, in both runs 

responsiveness of the cells to 17α-estradiol and 17-α methyltestosterone were lower than the 

accepted values, although the deviation from acceptable levels was minor, and indicates 

increased sensitivity to weak agonists. 

Page 90 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −11.5 b −11.7 X 

 Log PC10 <−11 −12.0 −12.6 X 

 Log EC50 −11.3 to −10.1 −11.4 b −11.7 b X 

 Hill Slope 0.7 to 1.5 1.6 1.1 Run 1 

 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 −9.9 b −9.4 b X 

 Log PC10 −10.7 to −9.3 −11.0 b −10.4 b X 

 Log EC50 −9.6 to −8.4 −10.0 b −9.3 X 

 Hill Slope 0.9 to 2.0 1.5 1.0 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 −9.6 b −8.8 b X 

 Log PC10 −8.0 to −6.2 −10.7 b −9.7 b X 

 Test range 10−11 to 10−5 M 10−12 to 10−5 M 10−12 to 10−5 M X a

 Data were obtained from pages 12, 22, 23 and 24 of the study report. 

b Parameters was out the validated range, however it indicates increased sensitivity of the assay system and is considered 

acceptable. 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The overall mean TA value for the vehicle control was 57529 for the first run and 

415829 for the second run, and the average TA value for the positive control was 1457867 

for the first run and 2660692 for the second run. The fold induction for the positive control 

was 25-fold for the first run and 6.4-fold for the second run. The mean normalized values 

for the positive control were 1400338 and 2244863 for the first and second runs, 

respectively. The PC50 (50% of the maximum response) for E2 in this assay is 700169 for 

the first run and 1122431 for the second run, and the PC10 (10% of the maximum response) 

is 140034 for the first run and 224486 for the second run.

 TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a 

Sample Vehicle Control Positive Control b Normalized Positive Control b

Runs Mean SD Mean SD Fold Induction c Mean SD 

1 57529 22775 1457867 210717 25 1400338 210717 

2 415829 65311 2660692 232915 6.4 2244863 232915 

a Data were obtained from pages 32 and 33 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 

simazine during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figure 1 

below. The report stated that three independent runs of the assay were performed because 

significant cytotoxicity issues were observed across all controls and the test compound in 

the first run; however, data were provide for only two runs in the current submission. The 

Page 91 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

mean RPCMax was 3.5%, with an associated PCMax of 10−7 M for the first run; the mean 

RPCMax was <0% for the second valid run at all concentrations. Because the RPCMax < PC10

in both runs, simazine was considered negative for estrogen receptor transcriptional 

activation in this test system. No explanation was given for the change in simazine 

concentrations used in the second run. 

TABLE 5. Relative Transcriptional Activation (RTA) of Simazinea

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 2 

Conc. (M) Mean SD Mean SD 

10−4 −0.7 2.1 −3.7 2.8 

10−4.5 -- -- −2.8 1.6 

10−5 2.9 1.6 −2.6 2.3 

10−5.5 -- -- −3.5 3.1 

10−6 2.3 2.0 −8.5 3.2 

10−6.5 -- -- −8.5 3.6 

10−7 3.5 2.1 −9.7 3.9 

10−7.5 -- -- −7.9 2.5 

10−8 −0.3 1.9 -- -- 

10−9 −1.1 1.6 -- -- 

10−10 −1.6 1.3 -- -- 

10−11 0.3 2.0 -- -- 

Log EC50b NA NA 

Hill Slopeb NA NA 

RPCMax 3.5 <0% 

PCMax 10−7 NA 

PC50 NA NA 

PC10 NA NA 

a Data were obtained from pages 22 and 23 of the study report. 

b Report if appropriate 

NA = Not Applicable

Page 92 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Simazine 

Compared to the Positive Control. 

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

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

full response of the cells to the reference chemicals was not satisfactory. The cytotoxicity of 

17β-estradiol, 17α-estradiol, and corticosterone to the cells in the first assay, and the 

responsiveness of 17β-estradiol, 17α-estradiol, and 17α-methyltestosterone greater than that 

of the positive control (117-150% PC) indicate a failure of the test system to respond 

Page 93 of 196
Estrogen Receptor Transcriptional Activation (2011) / Page 11 of 11 

SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

appropriately to its natural ligand, and two weak agonists. Cytotoxicity should not be 

observed at any level for the reference chemicals, and the maximum responsiveness should 

be observed in the positive control, 1 nM 17-estradiol. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The highest soluble concentration of simazine for 

use in the transcriptional activation assays was 10−4 M, identified in the range finder and 

both independent runs. There was no cytotoxicity observed at any of the exposure 

concentrations tested. In two independent runs of the transcriptional activation assay, 

simazine did not result in an increase in luciferase activity any of the concentrations 

assessed. Simazine is not an agonist of the human estrogen receptor alpha (hERα) in the He 

La-9903 model system. 

B. AGENCY COMMENTS: Two independent runs of the assay were performed. Simazine 

was tested up to the limit of solubility, 10−4 M. The mean RPCMax for simazine was 3.5%, 

with an associated PCMax of 10−7 M for the first run; the mean RPCMax was <0% for the 

second run at concentrations up to 10−4 M. Simazine was tested up to the limit of solubility, 

10−4 M. The mean RPCMax for simazine was 3.5%, with an associated PCMax of 10−7 M 

in the first run; the mean RPCMax was <0% for the second run at concentrations up to 10−4 

M. Performance criteria values were generally below the validated ranges, indicating that 

the current assay was more sensitive than during the validation experiments. Therefore the 

performance criteria deviations do not affect then interpretation of the study. Because the 

RPCMax < PC10 in both runs, simazine was considered negative for estrogen receptor 

transcriptional activation in this test system. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

 In the first assay, cytotoxicity was observed in cells treated with 17β-estradiol, 

17α-estradiol, and corticosterone. 

 Certificates of Analyses were not provided for the Reference Chemicals. 

 The source of the fetal bovine serum was not provided. 

Page 94 of 196
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Simazine 

EPA MRID Number 48673602 

Page 1 of 47 

Data Requirement: EPA DP Barcode 408724 

OECD Data Point 

EPA MRID 48673602 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Simazine Purity: 97.8% 

Common name 

Chemical name: IUPAC: 

 CAS name: 

 CAS No.: 122-34-9 

 Synonyms: 

 EPA PC Code: 080807 

Primary Reviewer: Joan Gaidos Signature: 

Environmental Scientist, CDM Smith Date: 5/14/13 

Secondary Reviewer: Teri S. Myers Signature: 

Environmental Scientist, CDM Smith Date: 8/15/13 

Primary Reviewer: Frank T. Farruggia, Ph.D. Date: 2/14 

EPA/OCSPP/OPP/EFED/ERB3

Additional Reviewer: Robin Sternberg Date: 2/21/14 

EPA/OCSPP/OPP/EFED/ERB1

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

EPA/OCSPP/OPP/EFED/ERB6

Date Evaluation Completed: 6/6/15 

CITATION: Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. 2012. Simazine: Fish Short-term 

Reproduction Assay with the Fathead Minnow (Pimephales promelas). Performed by Wildlife 

International, Ltd., Easton, Maryland, Report No.: 528A-226. Submitted by Syngenta, Greensboro, 

North Carolina. Completion date November 8, 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 08:19:44 -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 11:12:57 -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 17:50:17 -04'00'

Page 95 of 196
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 96 of 196
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 

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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

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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.

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I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 

890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 

(2009). The following deviations were noted: 

 

1. 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 

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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. 

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B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

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

Yes 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. 

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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. 

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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 

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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. 

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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). 

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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. 

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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).

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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.

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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. 

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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. 

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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. 

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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. 

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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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

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.

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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. 

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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.

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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 

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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. 

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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 

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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 

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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. 

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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). 

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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.

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility, fecundity, and VTG. 

Data were gender specific and analyzed in comparison to the 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. 

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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. 

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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. 

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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. 

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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. 

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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 

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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 141 of 196
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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 142 of 196
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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. 

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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 144 of 196
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 145 of 196
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

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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 147 of 196
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 

============================================================================

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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 

============================================================================

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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 * 

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

-

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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 

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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 

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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 . . .

.

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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

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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

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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 

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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 157 of 196
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 158 of 196
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 159 of 196
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 160 of 196
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 161 of 196
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 162 of 196
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 163 of 196
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 164 of 196
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 165 of 196
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 166 of 196
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 167 of 196
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 168 of 196
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 169 of 196
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 170 of 196
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 171 of 196
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 172 of 196
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 173 of 196
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 174 of 196
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 175 of 196
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 176 of 196
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 177 of 196
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 178 of 196
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 179 of 196
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 180 of 196
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 181 of 196
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

 

Page 182 of 196
Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC 

EPA MRID Number 48827602 

The NPAR1WAY 

Procedure 

Page 183 of 196
DATA EVALUATION RECORD 

SIMAZINE 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-28-2012 (MRID 48619303) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Kelly Luck, M.S. Date: 03/19/2012 

Secondary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 3/28/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/03/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/03/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 184 of 196
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 185 of 196
SIMAZINE/ 080807 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

In vivo Hershberger Assay (2011) I Page 1 of 11 

OCSPP 890.1400/ OECD 441 

Signature: -4~~,4:~=;...._-

Date: ___ co-"-'} 2-!="~J ..... 1..._r_ 

Template version 10/2011 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 080807 DP BARCODE: D398096 

TXR#: 0056244 CAS#: 122-34-9 

TEST MATERIAL (PURITY): Simazine (96.9% a.i.) 

SYNONYMS: 6-chloro-N,N' -diethyl-1,3,5-triazine-2,4-diamine 

CITATION: Sawhney Coder, P. (2011). Simazine - Hershberger Bioassay in 

Orchidoepididymectomized Rats. WIL Research Laboratories, LLC, Ashland, 

OH. Laboratory Report No.: WIL-639137, December 2, 2011. MRID 

48619303. Unpublished. 

SPONSOR: Syngenta Crop Protection, LLC, 410 Swing Road, P.O. Box 18300, Greensboro 

NC 

TEST ORDER#: CON-080807-17 

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48619303) screening for 

androgenic activity, simaiine (96.9% purity, Batch# 429546) in 0.5% (w/v) 

carboxymethylcellulose (CMC) was administered daily via oral gavage (5 mL/kg) to groups of 

six 56- to 57-day old (approximate), castrated male Sprague Dawley rats at dose levels of 0 

(vehicle), 65, or 200 mg/kg/day. An androgenic positive control group consisted of six castrated 

rats exposed to 0.2 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection 

plus vehicle (0.5% CMC) by oral gavage (5 mL/kg). 

To screen for potential anti-androgenic activity, simazine in 0.5% CMC was administered daily 

for ten days via oral gavage ( 5 mL/kg) to groups of six 56- to 57-day old (approximate), 

castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 20, 65, or 200 mg/kg/day in 

conjunction with a daily dose ofreference androgen TP at 0.2 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of six castrated rats exposed to 0.2 mg/kg/day 

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

All animals survived until scheduled termination. No animals exhibited any dose-related clinical 

signs of toxicology and there were no treatment-related gross pathological findings. Animals 

were sacrificed approximately 24 hours after the final dose administration. At necropsy, the five 

androgen-dependent tissues and liver were collected and weighed. 

In the androgen agonist assay, body weights for the 200 mg/kg/day group were decreased 

(p<0.05) by 8-10% on Days 7, 8 and 11 compared to controls, resulting in a 50% decrease 

Page 186 of 196
In vivo Hershberger Assay (2011) / Page 2 of 11 

SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441

(p<0.05) in overall (Days 1-11) body weight gain. Overall body weight gain was also decreased 

(p<0.05) by 25% in the 65 mg/kg/day group. Body weights in the positive control group were 

comparable to controls throughout the duration of the study. Overall food consumption was 

similar across all groups. There were no treatment-related effects on the weights of accessory 

sex organs or liver for any dose group. Animals in the positive TP control group had increased 

(p<0.01) accessory sex organ weights as follows: 347% in seminal vesicles; 484% in ventral 

prostate; 174% in levator ani-bulbocavernosus (LABC); 308% in Cowper’s gland; and 58% in 

glans penis. Liver weights in the positive control group were comparable to the vehicle control. 

There were no significant weight increases in androgen-dependent tissues under the conditions of 

this assay. 

Percent CVs for the vehicle control and the simazine treatment groups were compared to the 

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

recommended values, with the exception of Cowper’s glands in the control group (CV = 68.0%; 

maximum recommended CV = 55%). 

In the anti-androgen assay, overall body weight gain was decreased (p<0.05) by 36% in the 200 

mg/kg/day group. Body weights in the 20 and 65 mg/kg/day dose group and positive control 

group were comparable to controls throughout the duration of the study. Overall food 

consumption was similar across all groups. There were no treatment-related effects on the 

weights of accessory sex organs or liver for any dose group. Animals dosed with TP + FT 

(positive control) had decreased (p<0.01) accessory sex organ weights as follows: 69% in 

seminal vesicles; 75% in ventral prostate; 47% in LABC; 74% in Cowper’s gland; and 20% in 

glans penis. Liver weights in the positive control group were comparable to the vehicle control. 

The performance criteria indicated that this assay was performing as expected. There were no 

significant weight decreases in androgen-dependent tissues under the conditions of this assay. 

Based on the observed decreases in body weight gain of 36-50% at 200 mg/kg/day, the rats used 

in this assay were dosed adequately. 

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

tissues. Simazine 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 187 of 196
In vivo Hershberger Assay (2011) / Page 3 of 11 

SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Director: P. Sawhney Coder 

Other Personnel: E. S. Bodle (Assistant Director, Analytical Chemistry); G. M. Maginnis (Clinical 

Veterinarian); C. S. Wally (Group Supervisor, Sample Processing Laboratory); R. A. 

Wally (Operations Manager, Reporting & Technical Support Services); R. Green 

(Syngenta Study Monitor); L. Freshwater (Contributing Scientist, Statistical Analysis) 

Study Period: May 31 - December 2, 2011 

2. Test Substance: Simazine 

 Description: White powder 

 Source: Syngenta Crop Protection, LLC 

 Batch #: 429546 (expiration date 4/30/2012) 

 Purity: 96.9% 

 Stability: Stable in vehicle for up to 17 days at room temperature or under refrigerated storage 

 CAS #: 122-34-9 

 Structure: 

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: AK Scientific, Inc. (Mountain View, CA) 

 Batch #: 70321J (expiration date 8/9/2012) 

 Purity: 98.3% 

 CAS #: 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Batch #: 2AC0144 (expiration date 3/1/2013) 

 Purity: 100% 

 CAS #: 13311-84-7 

5. Solvent/Vehicle Control 

(test substance): 

Carboxymethylcellulose (CMC) 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot #: 1AA0199 (expiration date 1/31/2012) 

 Rationale (if other than water): Not provided 

 Final concentration: 0.5% (w/v) 

 Solvent/Vehicle Control 

(TP and FT): 

Ethanol/Corn oil 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot #: Ethanol: ZT0426 (expiration date 8/2/2013) 

Corn oil: ZT1301 (expiration date 7/1/2012) 

 Rationale (if other than water): Not applicable 

 Final concentration: TP and FT were dissolved in minimal amounts of 95% ethanol and then diluted 

with corn oil (ratio of ethanol to corn oil was not reported) 

N

N

N

Cl

NH NH

CH3 3CH

Page 188 of 196
In vivo Hershberger Assay (2011) / Page 4 of 11 

SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441

6. Test Animals: 

 Species: Rat (castrated males only) 

 Strain: Sprague Dawley [Crl:CD(SD)] 

 Age/weight at dose initiation: Post-natal day (PND) 56-57 (approximate)/ 215.3-275.6 g 

 Source: Charles River Laboratories (Raleigh, NC) 

 Housing: Rats were individually housed in stainless steel wire-mesh cages suspended 

above cage board. 

 Diet: Certified Rodent LabDiet® 5002, PMI International., ad libitum 

Phytoestrogen content was not reported 

 Water: Reverse-osmosis purified drinking water, ad libitum

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21.3-22.1 oC (mean daily temperature) 

44.9-55.0% (mean daily humidity) 

10/hr 

12 hrs light/12 hrs dark 

 Acclimation period: Rats were castrated at the supplier and received at the facility 4 days postcastration; rats were then allowed a 10-day acclimation period at the facility 

B. STUDY DESIGN

1. In life dates: Start: June 10, 2011 End: June 21, 2011 

2. Study Design: In a Hershberger Assay conducted to screen for potential androgenic 

activity, the test substance was administered daily via oral gavage to castrated male rats. 

Positive androgenic activity is defined as a significant increase in two or more target organ 

weights compared to the vehicle control. To screen for the potential anti-androgenic 

activity, the test substance was also administered daily via oral gavage to castrated male rats 

in conjunction with a daily dose of TP (0.2 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. 

For both assays, the animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration for organ weight measurements. 

3. Study Schedule: Male rats were castrated on PND 42 according to standard procedures and 

allowed >7 days for recovery and regression of accessory sex organ weights prior to 

initiation of dosing. The dose administration period was from PND 56 or 57 through PND 

66 or 67. Rats were euthanized approximately 24 hours after the last dose and necropsied 

for organ weight measurements. 

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences in group body weight means at study initiation. Furthermore, the body weight of 

each animal was within 20% of the overall mean. 

Page 189 of 196
In vivo Hershberger Assay (2011) / Page 5 of 11 

SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (negative control) 0 6 

Low (Simazine) 65 6 

High (Simazine) 200 6 

Testosterone propionate (TP), positive controlb 0.2 6 

Anti-Androgen Assay 

Low (Simazine +TP) 20 + 0.2 6 

Mid (Simazine +TP) 65 + 0.2 6 

High (Simazine +TP) 200 + 0.2 6 

Flutamide + TP, positive control 3 + 0.2 6 

a Data were obtained from page 21 of the study report. 

b This dose group served as the positive control for the androgen agonist assay and the negative control for the 

anti-androgen assay. The injected dose of TP was administered concurrently with an oral dose of 0.5% CMC 

(vehicle). 

5. Dose Selection Rationale: The dose levels were selected based on the results from a dose 

range-finding study1 in which four adult male rats/dose group were administered simazine at 

doses of 0, 125, 250, 500, or 750 mg/kg/day for 10 days. Dose-related reductions in mean 

bodyweight gain and/or mean body weight losses with corresponding reductions in mean 

food consumption were noted at all dose levels, resulting in dose-related reductions in mean 

body weight; no quantitative data were provided. Based on these findings, 200 mg/kg/day 

was selected as the high dose level. 

6. (a) Dose Preparation: Dose formulations were prepared approximately weekly by mixing 

appropriate amounts of test substance with 0.5% CMC. Dosing preparations of TP and FT 

were prepared by dissolving the compounds in minimal amounts of 95% ethanol and then 

diluting to volume with corn oil (ratio of ethanol to corn oil was not reported). Analyses to 

demonstrate homogeneity, stability, and resuspension homogeneity were conducted 

previously for dose formulations at 1 and 200 mg/mL following up to 17 days of room 

temperature and refrigerated storage.2 During the study, samples of each test substance 

dosing formulation (middle stratum of each) prepared during the in-life phase were analyzed 

for achieved concentration. 

(b) Dose Analysis: 

Results 

Homogeneity: Not provided 

Stability: It was stated that simazine was stable in aqueous 0.5% CMC for up to 17 days at 

room temperature at concentrations of 1 and 200 mg/mL. 

 

1

 Sawhney Coder, P. Simazine - A Dose Range-Finding Oral (Gavage) Toxicity Study in Young Adult Rats (Study 

No. WIL-639136). WIL Research Laboratories, LLC, Ashland, OH, 2011. 

2

 Gardner, K.L. Analytical Validation and Stability Study of Simazine in Aqueous Formulations (Study No. WIL639131). WIL Research Laboratories, LLC, Ashland, OH, Draft. 

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Concentration (% of nominal): 102-106%

The analytical data indicated that the variation between nominal and actual dosage to the 

animals was acceptable. The study referenced above should be submitted for verification of 

the homogeneity and stability findings. 

7. Dosage administration: Test formulations 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. For the control group dosed with TP 

alone, the vehicle (0.5% CMC) was administered concurrently via oral gavage at 5 ml/kg 

bw. Dose volumes were adjusted daily based on the concurrent body weight measurement. 

8. Statistics: Statistical analyses were conducted for organ weights, daily body weights, 

cumulative body weight gains, and food consumption. Each endpoint was tested for 

homogeneity of variance using Levene's test. If that test was significant at p=0.01, then a 

log transformation was applied and Levene's test conducted on the transformed data. If that 

test was still significant, then the square root transformation was applied to the raw data 

(except cumulative body weight gain) and Levene's test conducted again. If the test was still 

significant, then a nonparametric test, as described below, was used to analyze the data. 

One-sided tests were conducted for in-life data and two-sided tests were conducted for 

organ weight data. 

 If variances were homogeneous, the one- or two-sided t-test (for comparing positive control 

data to data for negative control) or an ANOVA (for comparing dose groups to the negative 

control) was performed on data; the ANOVA test was followed by a one- or two-sided 

Dunnett’s test. If the transformations were unsuccessful in making the variances 

homogeneous, the nonparametric one- or two-sided Wilcoxon rank sum test was used to 

compare data for the positive control to the negative control. For comparison of dose 

groups to the negative control, if the transformations were unsuccessful in making the 

variances homogeneous, the nonparametric Kruskal-Wallis test was used, followed by a 

one- or two-sided Dunn's test. Significance was denoted at p<0.05. Statistical analyses 

were performed using SAS software (version 9.2 or higher). The statistical analyses were 

considered adequate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily. Individual clinical observations were recorded daily (prior to dose 

administration) and each rat was also observed for signs of toxicity at the time of dose 

administration and approximately one hour following dosing. 

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

period, and on the day of termination. 

3. Food Consumption (Optional): Food consumption was measured for each animal daily. 

Values were reported in terms of g/animal/day. 

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4. Serum Hormone Measurements (Optional): At study termination, each animal was 

anesthetized with isoflurane, and blood was collected from the vena cava for potential future 

serum hormone analyses; however, serum hormone analyses were not conducted with this 

study. 

5. Necropsy and Organ Weights: On PND 66 or 67 (approximately 24 hours after the final 

administration of the test substance), all animals were anesthetized with isoflurane, 

exsanguinated, and subjected to a gross necropsy. The five mandatory androgen-dependent 

organs (ventral prostate, seminal vesicles, 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). 

Additionally, the liver was collected and weighed. The accessory sex organs and a portion 

of the liver were placed in 10% neutral buffered formalin; the right portion of the medial 

lobe of the liver was rinsed, flash frozen in liquid nitrogen, and stored at −70 °C for possible 

future biochemical analysis. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical signs of toxicity: No remarkable clinical signs of toxicity were observed in 

animals for any dose groups. Any clinical findings noted in the control and dose groups 

occurred infrequently, in a manner that was not dose related, and/or at similar frequency as 

the vehicle control group. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Body weights were decreased 

(p<0.05) compared to controls by 8% on Day 7, 9% on Day 8, and 10% on Day 11 in the 

200 mg/kg/day group, resulting in a 50% decrease (p<0.05) in overall (Days 1-11) body 

weight gain. Overall body weight gain was decreased (p<0.05) by 25% in the 65 mg/kg/day 

group. Body weights in the positive control group were comparable to controls throughout 

the duration of the study. 

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TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.2) Simazine (65) Simazine (200) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 239.0 9.33 6 244.3 19.39 6 242.7 14.92 6 241.4 13.72

7 6 280.3 14.45 6 286.4 21.48 6 267.8 19.07 6 257.1* 

(↓8) 

15.19

8 6 286.6 13.43 6 297.4 23.35 6 274.7 19.09 6 260.8* 

(↓9) 

15.95

11 6 301.5 17.68 6 316.2 24.47 6 289.3 22.65 6 272.6* 

(↓10) 

19.13

Body Weight Gain 

(Days 1-11) 

6 62.5 10.65 6 71.9 7.67 6 46.6* 

(↓25b

) 

9.94 6 31.3** 

(↓50b) 

7.37 

a Data were obtained from Tables S5, S6, S9, and S10 on pages 42, 44-46, 52, and 54-56 of the study report. 

Percent differences from controls are included in parentheses. 

b Calculated by the reviewers. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p<0.05 

** Significantly different from controls at p<0.01

Selected body weight and body weight gain data for the anti-androgen assay are presented in 

Table 3. Overall body weight gain was decreased (p<0.05) by 36% in the 200 mg/kg/day 

group. Body weights in the 20 and 65 mg/kg/day dose group and positive control group 

were comparable to controls throughout the duration of the study. 

TABLE 3. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen 

Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control + 

TP (0.2) 

Positive Control TP 

+ FT (0.2 + 3) 

Simazine + TP 

(20 + 0.2) 

Simazine + TP 

(65 + 0.2) 

Simazine + TP 

(200 + 0.2) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 244.3 19.39 6 241.0 14.68 6 241.9 17.83 6 243.1 14.11 6 236.6 15.63

4 6 263.2 19.26 6 261.8 18.67 6 260.1 22.33 6 258.3 14.73 6 246.6 14.54

7 6 286.4 21.48 6 283.3 20.37 6 282.6 24.94 6 282.1 17.25 6 264.3 15.68

11 6 316.2 24.47 6 308.5 27.15 6 307.0 26.57 6 306.9 20.06 6 281.6 21.33

Body Weight 

Gain 

(Days 1-11) 

6 71.9 7.67 6 67.5 16.07 6 65.1 12.20 6 63.8 9.85 6 45.3** 

(↓36) 

11.28

a Data were obtained from Tables S7, S8, S11, and S12 on pages 47-51 and 57- 61 of the study report. Percent differences from 

controls, calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

** Significantly different from controls at p<0.01 

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C. FOOD CONSUMPTION (Optional): There were no significant dose-related effects on 

overall food consumption in the androgen agonist assay or the anti-androgen assay. In the 

androgen agonist assay, mean food consumption in the 200 mg/kg/day group was decreased 

27% during Days 1-2 compared to the vehicle control; however, mean food consumption 

was comparable to the control for the remainder of the study. 

There were no significant differences in mean food consumption between the TP group and 

the vehicle control group, or between the TP/FT group and the TP group. 

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone 

measurements were not conducted. 

E. ORGAN WEIGHTS: Accessory sex organ and liver weights for the androgen agonist 

assay are presented in Table 4. There were no treatment related effects in accessory sex 

organs or liver for any simazine dose group. Animals in the TP group had increased 

(p<0.01) accessory sex organ weights as follows: 347% in seminal vesicles; 484% in 

ventral prostate; 174% in LABC; 308% in Cowper’s gland; and 58% in glans penis. Liver 

weights in the positive control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the simazine treatment groups were compared to the 

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

recommended values, with the exception of Cowper’s glands in the control group (CV = 

68.0%; maximum recommended CV = 55%). 

There were no treatment-related gross pathological findings.

TABLE 4. Accessory Sex Organ Weights from Androgen Agonist Assay in Sprague Dawley Ratsa

Organ 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.2) Simazine (65) Simazine (200) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles (mg) 

6 72.2 5.16 7.2 6 322.6** 

(↑347) 

85.63 26.5 6 70.6 6.93 9.8 6 77.7 12.46 16.0

Ventral 

prostate (mg) 

6 17.8 3.92 22.0 6 103.9** 

(↑484) 

28.59 27.5 6 17.2 4.62 26.8 6 18.0 2.91 16.2

LABC (mg) 6 141.0 21.84 15.5 6 386.2** 

(↑174) 

30.77 8.0 6 137.9 19.64 14.2 6 129.5 34.13 26.4

Cowper’s 

gland (mg) 

6 6.5 4.39 68.0 6 26.5** 

(↑308) 

7.75 29.2 6 4.6 1.4 30.3 6 5.7 1.4 24.6

Glans penis 

(mg) 

6 83.7 9.90 11.8 6 132.0** 

(↑58) 

6.77 5.1 6 80.5 10.97 13.6 6 80.6 7.63 9.5 

Liver (g) 6 12.3 1.12 9.1 6 12.9 1.29 10.0 6 12.3 2.17 17.7 6 11.5 1.44 12.6

a Data were obtained from Tables S19, S20, and S22 on pages 70-73, 76, and 77 of the study report. Percent differences from 

controls, calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01 

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Accessory sex organ and liver weights for the anti-androgen assay are presented in Table 5. 

There were no treatment related effects in accessory sex organs or liver for any dose group. 

Animals dosed with TP + FT (positive control) had decreased (p<0.01) accessory sex organ 

weights as follows: 69% in seminal vesicles; 75% in ventral prostate; 47% in LABC; 74% 

in Cowper’s gland; and 20% in glans penis. Liver weights in the positive control group 

were comparable to the vehicle control. 

Percent CVs for the vehicle control and the simazine treatment groups were compared to the 

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

recommended values. 

There were no treatment-related gross pathological findings.

TABLE 5. Accessory Sex Organ Weights from Anti-Androgen Agonist Assay in Sprague Dawley Ratsa

Organ

Dose (mg/kg/day) 

Vehicle Control + TP 

(0.2) 

Positive Control 

TP + FT (0.2 + 3) 

Simazine + TP 

(20 + 0.2) 

Simazine + TP 

(65 + 0.2) 

Simazine + TP 

(200 + 0.2) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

(mg) 

6 322.6 85.63 26.5 6 100.3** 

(↓69) 

16.23 16.2 6 372.7 90.42 24.3 6 351.2 49.00 14.0 6 335.8 27.28 8.1 

Ventral 

prostate 

(mg) 

6 103.9 28.59 27.5 6 25.8** 

(↓75) 

5.46 21.2 6 120.7 12.85 10.7 6 106.8 16.47 15.4 6 107.8 22.62 21.0

LABC 

(mg) 

6 386.2 30.77 8.0 6 203.9** 

(↓47) 

24.59 12.1 6 403.3 41.47 10.3 6 399.9 80.31 20.1 6 356.6 38.42 10.8

Cowper’s 

gland (mg) 

6 26.5 7.75 29.2 6 6.9** 

(↓74) 

1.70 24.9 6 28.3 4.86 17.2 6 24.4 5.49 22.5 6 24.9 2.54 10.2

Glans 

penis (mg) 

6 132.0 6.77 5.1 6 105.5** 

(↓20) 

14.5 13.8 6 135.0 14.57 10.8 6 138.4 14.20 10.3 6 138.2 22.79 16.5

Liver (g) 6 12.9 1.29 10.0 6 13.7 1.92 14.1 6 13.2 1.92 14.6 6 13.2 1.16 8.8 6 12.2 1.20 9.9 

a Data were obtained from Tables S19, S21, and S23 on pages 70, 71,74, 75, 78, and 79 of the study report. Percent differences from 

controls were calculated by the reviewers and included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the lack of effects on androgendependent organ weights, no evidence of androgen agonism or antagonism was noted at any 

dose level in this study when simazine was administered orally by gavage to male 

Crl:CD(SD) rats. The androgenic positive control substance, TP, elicited the expected 

increases in weights of androgen-dependent organs. The anti-androgenic positive control 

substance, FT, elicited the expected suppression of the androgenic effects of TP.

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B. AGENCY COMMENTS: All animals survived until scheduled termination. No animals 

exhibited any dose-related clinical signs of toxicology and there were no treatment-related 

gross pathological findings. 

In the androgen agonist assay, body weights were decreased (p<0.05) compared to controls 

by 8% on Day 7, 9% on Day 8, and 10% on Day 11 in the 200 mg/kg/day group, resulting in 

a 50% decrease (p<0.05) in overall (Days 1-11) body weight gain. Overall body weight 

gain was decreased (p<0.05) by 25% in the 65 mg/kg/day group. Body weights in the 

positive control group were comparable to controls throughout the duration of the study. 

Overall food consumption was similar across all groups. There were no treatment-related 

effects on the weights of accessory sex organs or liver for any dose group. Animals in the 

positive control group had the expected increases accessory sex organ weights. Liver 

weights in the positive control group were comparable to the vehicle control. As there were 

no significant weight increases in androgen-dependent tissues, simazine is not considered to 

be an androgen agonist at doses of ≤200 mg/kg/day under the conditions of this assay. 

Percent CVs for the vehicle control and the simazine treatment groups were compared to the 

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

recommended values, with the exception of Cowper’s glands in the control group (CV = 

68.0%; maximum recommended CV = 55%). 

In the anti-androgen assay, overall body weight gain was decreased (p<0.05) by 36% in the 

200 mg/kg/day group. Body weights in the 20 and 65 mg/kg/day dose group and positive 

control group were comparable to controls throughout the duration of the study. Overall 

food consumption was similar across all groups. There were no treatment-related effects on 

the weights of accessory sex organs or liver for any dose group. Animals dosed with TP + 

FT (positive control) had the expected decreases in accessory sex organ weights. Liver 

weights in the positive control group were comparable to the vehicle control. The 

performance criteria indicated that this assay was performing as expected. As there were no 

significant weight increases in androgen-dependent tissues, simazine is not considered to be 

an anti-androgen at doses of ≤200 mg/kg/day under the conditions of this assay. 

The rats were adequately dosed for this assay based on the observed decreases in body 

weight gain of 36-50% at 200 mg/kg/day. 

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 study author did not provide the rationale for the vehicle used for simazine dosing. 

 The exact ages of the rats used in the study were not reported; all ages were reported to 

be approximate. 

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