## 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 Rowland

.

# ~ Deputy 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

### 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 ot her scientifically relevant information (OSRI))

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 1 of 43 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'

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 2 of 43 DER Template Version: 22 September 2011 

### 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).

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 3 of 43 DER Template Version: 22 September 2011 

### 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-measu red 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. C oncentration-dependent reduc tions 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, respec tively 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

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 4 of 43 DER Template Version: 22 September 2011 

### 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 fo r 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

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 5 of 43 DER Template Version: 22 September 2011 

### Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 

### in the Amphibian Metamorphosis Assay (AMA) with Simazine.

Treatment (mg a.i./L) [mean-measured] NF Developmental Stage Hind Limb Length 3 

### Asynchronous Development Thyroid Gross
and Histopathology Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21
0.019 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 Re viewer’s Analysis and Statistical Verification sections ofthe 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.

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 6 of 43 DER Template Version: 22 September 2011 

### 3. Ammonia, perchlorate, and chlorate concentrations in the dilution water were not reported.
4. Hardness of the dilution water (132-152 mg/L as CaCO 3 

### ) exceeded the recommended range (40-48 mg/L as CaCO 3 

### ).
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

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
48673601

Page 7 of 43 DER Template Version: 22 September 2011 

### 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 conditionsData Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page8of43 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be identified.Species scientific name: Xenopus laevisSpecies strain (if stated): Not reported1U.S. Environmental Protection Agency (EPA).(2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor 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).Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page9of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?Yes. Adults were originallyobtained from Xenopus I ofDexter, Michigan.Parents were maintained for atleast 14 days following arrivalat the laboratory. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):14 daysDetails on parental feeding: Frogs were fed an adultXenopus diet supplied byZeigler Brothers, Inc. ofGardners, PA at a ratesufficient to maintain the healthof the culture.Details on parental health: Culture frogs showed no sign ofdisease or stress prior to thetest.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page10of43 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest 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 (ifapplicable):2Number of eggs sampled perspawn:~250 Spawns were evaluated oneday 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 testinitiation (if reported):Not measuredRange of total length at testinitiation (if reported):Not measuredData Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page11of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?NoDetails on larval selection: Two male and two female adultfrogs were injected with humanchorionic gonadotropin andeach pair was placed in aseparate spawning tankovernight. Eggs were collectedthe following day and a subsetof approximately 250 eggs fromeach spawn was evaluated forviability.Eggs from the best singlespawn (91.2% viability) wereused to provide tadpoles for thetest.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®Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page12of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource 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 (ifknown):Not reportedFrequency of feeding:3 times/day EPA recommends that feeding occur at least twice per day.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page13of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime: Feeding rates were half ofrecommended rates asspecified 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.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page14of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem (if applicable):Continuous flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):~69 mL/min Flow rate was calculated basedupon 10 volume additions per dayand a 10 L fill volume. This flowrate resulted in complete volumereplacement approximately every2.4 hours. Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page15of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Fluid metering pumps were usedto deliver the single testsubstance stock solution to mixingchambers where it was mixedwith well water. Dilution waterflow was controlled usingrotameters.All components were calibratedprior to the test. Flow rates wereverified approximately weekly toensure that rates varied by nomore than ±10%. The generaloperation of the exposure systemwas checked visually at least twotimes per day during the test andat least once on the last day ofthe 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 staticrenewal (if applicable):NA If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72

hours.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page16of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration? 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 freshwaterobtained from a wellapproximately 40 meters deeplocated on WLI property. Waterwas passed through a sand filterto remove particles greater than~25սm, pumped into a tank,and aerated. Prior to use, waterwas filtered to 0.45սm andpassed 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.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page17of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes. See Reviewer’s Comments,Iodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/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: GlassEPA 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 LFill volume:10 LData Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page18of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:NATable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsHardness (mg/Las CaCO3)132 152 142Measured in one replicate of the controland high-concentration treatment weekly,with measurements alternating betweenreplicates. EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH 8.08.2 8.1Measured 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 8.0Measured 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).Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page19of43 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C) 21.4 22.6 22.0Measured weekly in each test chamber.Temperature also was measuredcontinuously in one replicate of thenegative control. EPA recommends temperature 22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide (սg/L) 3 6 --Prior to test. EPA recommends aquatic iodide range

0.5 – 10 ս g/L (supplemental iodide should not exceed 2 ս g/L).Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page20of43 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsAmmonia NotReported 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) -- -- 0.85December 28, 2011Perchlorate NotReportedChlorate NotReportedAlkalinity (mg/Las CaCO3)170 178 174Measured in one replicate of the controland high-concentration treatment weekly,with measurements alternating betweenreplicates.SpecificConductance(սS/cm)322 387 354Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page21of43 DER Template Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes. Two separate rangefinding studies were conducted.If yes, what was the method fordetermining the highest testconcentration 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 laevisLife stage:Not reportedTest duration:The first test was conducted for14 days and the second for 96hours.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page22of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:First range-finding test wasconducted for 14 days at anominal concentration of 4.5mg a.i./L. There were 3mortalities by the end of Day 1and signs of toxicity (i.e., small,pale, surfacing) observedthroughout the test. A secondrange-finding study wasconducted for 96 hours at anominal concentration of 5.0mg a.i./L. No mortalitiesoccurred and only sign oftoxicity was surfacing.The high concentration for thedefinitive test was selectedbased on the range-findingresults and set at 2.0 mga.i./L. The two lowerconcentrations were determinedusing a factor of 10.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page23of43 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive 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 (ifapplicable):NASeparation of testconcentrations: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.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page24of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAre nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit 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 reportedFrequency of measurement: Samples were collectedfor 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.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page25of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):NA EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of larvae per treatmentat test initiation:80Was a solvent used?NoSolvent type (if applicable): NAData Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page26of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum 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? NoPositive control (if applicable): NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12 (light:dark).Light intensity at water’ssurface:0.934-1.572 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page27of43 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:NAThe test solutions were reported as clearand colorless with no evidence ofprecipitateTable 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Simazine.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative 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 0.0200.0195.4NoneTreatment 2 0.200.183.8NoneTreatment 3 2.01.93.6NoneAbbreviations:CVCoefficient of variation.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page28of43 DER Template Version: 22 September 2011E. ObservationsBiological Endpoints:Day 7- NF stage, wet weight, snout-vent length(SVL), hind-limb length (HLL), normalized HLLDay 21- NF stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathologyDaily- mortality, clinical observationsWere raw (individual) data provided?Yes EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-base d larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also incl udes thyroid gross

pathology observations.

### Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis Assay
EPA MRID Number
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Page 29 of 43 DER Template Version: 22 September 2011 

### 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 7 1 

### Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 0 0 60 0 0

0.019 80 1 1.2 58 2 

### 0 0

0.18 80 0 0 59 2 

### 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 10 th 

### and 90 th 

### 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:
SVL 1 n Mean (mm) ±SD HLL:
SVL 1


### 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 Weight 1



### 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 Observations 1

Severity 

### Thyroid Gland Hypertrophy Thyroid Gland Atrophy Follicular Cell Hypertrophy Follicular Cell Hyperplasia

n Incidence n Incidence n Incidence n Incidence 

### Negative
Control 0 20 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.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page34of43 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular SizeDecreaseFollicle SizeIncreasedGland AsymmetryIncreaseFollicular CellHeight (Decrease)FollicularAsymmetryIncreasednIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 14 20 16 20 15 -- -- 20 191 20 3 20 0 20 5 -- -- 20 12 20 3 20 3 20 0 -- -- 20 03 20 0 20 1 20 0 -- -- 20 00.019 0 20 14 20 18 20 18 -- -- 20 161 20 4 20 1 20 1 -- -- 20 22 20 2 20 1 20 1 -- -- 20 23 20 0 20 0 20 0 -- -- 20 00.18 0 20 14 20 19 20 17 -- -- 20 151 20 6 20 1 20 2 -- -- 20 42 20 0 20 0 20 1 -- -- 20 13 20 0 20 0 20 0 -- -- 20 0Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page35of43 DER Template Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular SizeDecreaseFollicle SizeIncreasedGland AsymmetryIncreaseFollicular CellHeight (Decrease)FollicularAsymmetryIncreasednIncidencenIncidencenIncidencenIncidencenIncidence1.9 0 20 10 20 20 20 18 -- -- 20 201 20 5 20 0 20 2 -- -- 20 02 20 5 20 0 20 0 -- -- 20 03 20 0 20 0 20 0 -- -- 20 01Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page36of43 DER Template Version: 22 September 2011By test termination, tail curvature was observed in the negative control (8%), the low (6%), and mid (9%) treatment groups; allsurviving tadpolesin 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 SignsTypenIncidenceNegative ControlTail Curvature6080.019 TailCurvature5860.18 TailCurvature5991.9 TailCurvature600

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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 hete rogeneous 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 significantl y 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, wher e 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 endp oints 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, statisti cal 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 signif icantly different from the negative control. On Day 21, significant concentration-dependent redu ctions 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.Data Evaluation Record on the Toxicity of Simazine to Amphibians, Metamorphosis AssayEPA MRID Number 48673601Page40of43 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Simazine.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control 54 -- 57 -- -- -- -- -- -- -- -- -- No0.01954 1.0 57 1.00 1.30 0.31 -3.34 0.44 -- -- -- -- No0.1854 1.0 57 1.00 1.49 0.31 -6.19 0.19 -- -- -- -- No1.953 0.49 57 1.006.13 0.03 -14.1 0.01-- -- -- -- YesStatistical TestMann-Whitney Mann-Whitney Jonckheere Jonckheere ------Abbreviations:Diff.Difference.1Unless otherwise indicated, effects arereported based on comparison tothe clean water control.Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Hind-limb length is normalized to snout-vent length (SVL).

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### Table 17: Growth Endpoints 1,2 

### 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 immers ed 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 diluti on 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.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 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 01-4252-1629 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test
DF P-Type 8 NA 2 1.0000 Non-Significant Effect Negative Control 0.019 6 Exact 8 NA 2 1.0000 Non-Significant Effect 0.18 6 Exact 12 NA 2 0.4857 Non-Significant Effect 1.9 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 1.61% NA C <> T NA Untransformed NA 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 3.75 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0 5.95 1.0000 Equal Variances Mod Levene Equality of Variance Variances 0 5.95 1.0000 Equal Variances Levene Equality of Variance Variances 0.81 0.841 0.0037 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Developmental Stage Summary

53.8 53 54 54 0 4 Negative Control 0.25 0.93% 0.0% 53 54.5 53.8 53 54 54 0.019 4 0.25 0.93% 0.0% 53 54.5 53.8 53 54 54 0.18 4 0.25 0.93% 0.0% 53 54.5 53.3 53 54 53 1.9 4 0.25 0.94% 0.93% 52.5 54CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Developmental Stage

C-mg ai/L 0 10 20 30 40 50 60

0 N 0.019 0.18 1.9 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d HLL CETIS Version: CETISv1.8.7 Analysis ID: 16-6263-7199 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 0.15 2.68 0.268 0.9975 Non-Significant Effect Negative Control 0.019 6 CDF 0.2 2.68 0.268 0.9941 Non-Significant Effect 0.18 6 CDF 0.75 2.68 0.268 0.7964 Non-Significant Effect 1.9 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 12.9% NA C <> T NA Untransformed NA 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 0.2584 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 3.51 11.3 0.3194 Equal Variances Bartlett Equality of Variance Variances 0.952 0.841 0.5234 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d HLL Summary

2.08 1.95 2.33 2.02 0 4 Negative Control 0.0865 8.32% 0.0% 1.8 2.36 2.06 1.98 2.17 2.05 0.019 4 0.0397 3.84% 0.72% 1.94 2.19 2.1 1.89 2.33 2.09 0.18 4 0.0972 9.26% -0.96% 1.79 2.41 2.15 2.09 2.24 2.14 1.9 4 0.0384 3.56% -3.61% 2.03 2.28CenteredUntransformed

Rankits -0.05

-0.10

-0.15

-0.20

-0.25 0.00 0.05 0.10 0.15 0.20 0.25

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d HLL CETIS Version: CETISv1.8.7 Analysis ID: 01-0963-3609 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 0.438 1.64 1 0.3309 Non-Significant Effect Negative Control 0.019 -2 Asymp 0.44 1.64 1 0.3298 Non-Significant Effect 0.18 -2 Asymp 1.4 1.64 2 0.0802 Non-Significant Effect 1.9 -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 NA C < T NA Untransformed NA 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 0.2584 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 3.51 11.3 0.3194 Equal Variances Bartlett Equality of Variance Variances 0.952 0.841 0.5234 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d HLL Summary

2.08 1.95 2.33 2.02 0 4 Negative Control 0.0865 8.32% 0.0% 1.8 2.36 2.06 1.98 2.17 2.05 0.019 4 0.0397 3.84% 0.72% 1.94 2.19 2.1 1.89 2.33 2.09 0.18 4 0.0972 9.26% -0.96% 1.79 2.41 2.15 2.09 2.24 2.14 1.9 4 0.0384 3.56% -3.61% 2.03 2.28CenteredUntransformed

Rankits -0.05

-0.10

-0.15

-0.20

-0.25 0.00 0.05 0.10 0.15 0.20 0.25

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 12-8528-5883 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett T3 Multiple Comparison Test
DF P-Type 0.595 4.28 0.013 0.9044 Non-Significant Effect Negative Control 0.019 3 CDF 0.349 3.44 0.02 0.9775 Non-Significant Effect 0.18 5 CDF 2.7 3.94 0.012 0.1414 Non-Significant Effect 1.9 4 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 8.93% NA C <> T NA Untransformed NA 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 0.0005639999 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 12.7 11.3 0.0054 Unequal Variances Bartlett Equality of Variance Variances 0.94 0.841 0.3455 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Normalized HLL Summary

0.135 0.13 0.143 0.132 0 4 Negative Control 0.0029 4.31% 0.0% 0.125 0.144 0.136 0.135 0.137 0.137 0.019 4 0.000479 0.7% -1.3% 0.135 0.138 0.136 0.127 0.146 0.137 0.18 4 0.00494 7.24% -1.49% 0.121 0.152 0.143 0.14 0.144 0.144 1.9 4 0.000946 1.33% -6.13% 0.14 0.146CenteredUntransformed

Rankits -0.002

-0.004

-0.006

-0.008

-0.010 0.000 0.002 0.004 0.006 0.008 0.010

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 20-1803-3840 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 12 NA 0.3090 Non-Significant Effect Negative Control 0.019 -2 Exact 28 NA 0.3090 Non-Significant Effect 0.18 -2 Exact 1.83 1.64 3 0.0334 Significant Effect 1.9* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 0.5848 C < T NA Untransformed NA 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 0.0005639999 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 12.7 11.3 0.0054 Unequal Variances Bartlett Equality of Variance Variances 0.94 0.841 0.3455 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Normalized HLL Summary

0.135 0.13 0.143 0.132 0 4 Negative Control 0.0029 4.31% 0.0% 0.125 0.144 0.136 0.135 0.137 0.137 0.019 4 0.000479 0.7% -1.3% 0.135 0.138 0.136 0.127 0.146 0.137 0.18 4 0.00494 7.24% -1.49% 0.121 0.152 0.143 0.14 0.144 0.144 1.9 4 0.000946 1.33% -6.13% 0.14 0.146CenteredUntransformed

Rankits -0.002

-0.004

-0.006

-0.008

-0.010 0.000 0.002 0.004 0.006 0.008 0.010

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Normalized HLL

C-mg ai/L 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d SVL CETIS Version: CETISv1.8.7 Analysis ID: 14-7358-1638 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 0.771 2.68 1.04 0.7837 Non-Significant Effect Negative Control 0.019 6 CDF 1.23E-06 2.68 1.04 1.0000 Non-Significant Effect 0.18 6 CDF 0.9 2.68 1.04 0.7028 Non-Significant Effect 1.9 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 6.78% NA C <> T NA Untransformed NA 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 4.057499 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0.852 11.3 0.8370 Equal Variances Bartlett Equality of Variance Variances 0.954 0.841 0.5484 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d SVL Summary

15.4 14.7 16.3 15.3 0 4 Negative Control 0.334 4.34% 0.0% 14.3 16.5 15.1 14.7 15.8 15 0.019 4 0.248 3.29% 1.95% 14.3 15.9 15.4 14.7 16 15.5 0.18 4 0.303 3.93% 0.0% 14.4 16.4 15 14.6 15.5 15 1.9 4 0.194 2.57% 2.27% 14.4 15.7CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0 0.0 0.2 0.4 0.6 0.8 1.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 02-4131-3903 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 0.654 2.68 0.049 0.8512 Non-Significant Effect Negative Control 0.019 6 CDF 0.341 2.68 0.049 0.9729 Non-Significant Effect 0.18 6 CDF 1.59 2.68 0.049 0.3020 Non-Significant Effect 1.9 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 20.5% NA C <> T NA Untransformed NA 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 0.009979748 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 4.06 11.3 0.2546 Equal Variances Bartlett Equality of Variance Variances 0.919 0.841 0.1619 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Wet Weight Summary

0.24 0.201 0.299 0.231 0 4 Negative Control 0.0208 17.3% 0.0% 0.174 0.307 0.229 0.215 0.259 0.22 0.019 4 0.0102 8.96% 4.99% 0.196 0.261 0.234 0.216 0.255 0.233 0.18 4 0.00983 8.39% 2.6% 0.203 0.266 0.211 0.197 0.226 0.211 1.9 4 0.00626 5.93% 12.2% 0.191 0.231CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04 0.00 0.01 0.02 0.03 0.04 0.05 0.06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 08-0913-9920 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test
DF P-Type 8 NA 1 1.0000 Non-Significant Effect Negative Control 0.019 6 Exact 8 NA 1 1.0000 Non-Significant Effect 0.18 6 Exact 10 NA 1 1.0000 Non-Significant Effect 1.9 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 1.9 >1.9 0.76% NA C <> T NA Untransformed NA 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 0.9375 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 1 5.95 0.4262 Equal Variances Mod Levene Equality of Variance Variances 9 5.95 0.0021 Unequal Variances Levene Equality of Variance Variances 0.565 0.841 <0.0001 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d Developmental Stage Summary

57 57 57 57 0 4 Negative Control 0 0.0% 0.0% 57 57 57 57 57 57 0.019 4 0 0.0% 0.0% 57 57 57 57 57 57 0.18 4 0 0.0% 0.0% 57 57 56.8 56 57 57 1.9 4 0.25 0.88% 0.44% 56 57.5CenteredUntransformed

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 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d Developmental Stage

C-mg ai/L 0 10 20 30 40 50 60

0 N 0.019 0.18 1.9 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7 Analysis ID: 19-8331-1929 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 0.975 2.68 1.18 0.6543 Non-Significant Effect Negative Control 0.019 6 CDF 1.49 2.68 1.18 0.3514 Non-Significant Effect 0.18 6 CDF 3.49 2.68 1.18 0.0116 Significant Effect 1.9* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 14.8% 0.5848 C <> T NA Untransformed NA NA

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 9.620645 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0.0891 11.3 0.9931 Equal Variances Bartlett Equality of Variance Variances 0.92 0.841 0.1702 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS HLL Summary

7.95 7.38 8.88 7.76 0 4 Negative Control 0.344 8.66% 0.0% 6.85 9.04 7.52 6.79 8.16 7.56 0.019 4 0.302 8.02% 5.38% 6.56 8.48 7.29 6.84 8.18 7.07 0.18 4 0.302 8.28% 8.21% 6.33 8.25 6.41 5.74 7.16 6.38 1.9 4 0.291 9.07% 19.3% 5.49 7.34CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0 0.0 0.2 0.4 0.6 0.8 1.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7 Analysis ID: 01-0187-3339 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 11 NA 0.2429 Non-Significant Effect Negative Control 0.019 -2 Exact 33 NA 0.1099 Non-Significant Effect 0.18 -2 Exact 77 NA 0.0030 Significant Effect 1.9* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 0.5848 C > T NA Untransformed NA NA

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 9.620645 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0.0891 11.3 0.9931 Equal Variances Bartlett Equality of Variance Variances 0.92 0.841 0.1702 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS HLL Summary

7.95 7.38 8.88 7.76 0 4 Negative Control 0.344 8.66% 0.0% 6.85 9.04 7.52 6.79 8.16 7.56 0.019 4 0.302 8.02% 5.38% 6.56 8.48 7.29 6.84 8.18 7.07 0.18 4 0.302 8.28% 8.21% 6.33 8.25 6.41 5.74 7.16 6.38 1.9 4 0.291 9.07% 19.3% 5.49 7.34CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0 0.0 0.2 0.4 0.6 0.8 1.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS HLL

C-mg ai/L 0 1 2 3 4 5 6 7 8 9

0 N 0.019 0.18 1.9 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 11-3271-7201 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 0.712 2.68 0.046 0.8188 Non-Significant Effect Negative Control 0.019 6 CDF 1.32 2.68 0.046 0.4386 Non-Significant Effect 0.18 6 CDF 3.01 2.68 0.046 0.0279 Significant Effect 1.9* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 12.6% 0.5848 C <> T NA Untransformed NA NA

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 0.01295994 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0.231 11.3 0.9725 Equal Variances Bartlett Equality of Variance Variances 0.942 0.841 0.3716 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Normalized HLL Summary

0.367 0.332 0.402 0.368 0 4 Negative Control 0.0143 7.8% 0.0% 0.322 0.413 0.355 0.331 0.377 0.356 0.019 4 0.0115 6.45% 3.34% 0.319 0.391 0.344 0.326 0.377 0.337 0.18 4 0.0114 6.61% 6.19% 0.308 0.381 0.315 0.292 0.346 0.312 1.9 4 0.0112 7.11% 14.1% 0.28 0.351CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 16-7917-7403 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 9 NA 0.4429 Non-Significant Effect Negative Control 0.019 -2 Exact 0.883 1.64 2 0.1886 Non-Significant Effect 0.18 -2 Asymp 2.25 1.64 2 0.0123 Significant Effect 1.9* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 0.5848 C > T NA Untransformed NA NA

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 0.01295994 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 0.231 11.3 0.9725 Equal Variances Bartlett Equality of Variance Variances 0.942 0.841 0.3716 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Normalized HLL Summary

0.367 0.332 0.402 0.368 0 4 Negative Control 0.0143 7.8% 0.0% 0.322 0.413 0.355 0.331 0.377 0.356 0.019 4 0.0115 6.45% 3.34% 0.319 0.391 0.344 0.326 0.377 0.337 0.18 4 0.0114 6.61% 6.19% 0.308 0.381 0.315 0.292 0.346 0.312 1.9 4 0.0112 7.11% 14.1% 0.28 0.351CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7 Analysis ID: 00-9050-1205 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 1.64 2.68 1.05 0.2816 Non-Significant Effect Negative Control 0.019 6 CDF 1.51 2.68 1.05 0.3417 Non-Significant Effect 0.18 6 CDF 3.99 2.68 1.05 0.0048 Significant Effect 1.9* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 4.86% 0.5848 C <> T NA Untransformed NA NA

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 8.65589 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 1.13 11.3 0.7693 Equal Variances Bartlett Equality of Variance Variances 0.93 0.841 0.2412 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS SVL Summary

21.6 20.5 22 21.9 0 4 Negative Control 0.357 3.31% 0.0% 20.4 22.7 20.9 20.3 21.6 21 0.019 4 0.278 2.65% 2.98% 20.1 21.8 21 20.4 21.6 21 0.18 4 0.261 2.49% 2.73% 20.2 21.8 20 19.5 20.3 20.1 1.9 4 0.183 1.82% 7.23% 19.4 20.6CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7 Analysis ID: 04-6074-0832 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 13 NA 0.1000 Non-Significant Effect Negative Control 0.019 -2 Exact 34 NA 0.0844 Non-Significant Effect 0.18 -2 Exact 81 NA 0.0007 Significant Effect 1.9* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 0.5848 C > T NA Untransformed NA NA

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 8.65589 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 1.13 11.3 0.7693 Equal Variances Bartlett Equality of Variance Variances 0.93 0.841 0.2412 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS SVL Summary

21.6 20.5 22 21.9 0 4 Negative Control 0.357 3.31% 0.0% 20.4 22.7 20.9 20.3 21.6 21 0.019 4 0.278 2.65% 2.98% 20.1 21.8 21 20.4 21.6 21 0.18 4 0.261 2.49% 2.73% 20.2 21.8 20 19.5 20.3 20.1 1.9 4 0.183 1.82% 7.23% 19.4 20.6CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS SVL

C-mg ai/L 0 5 10 15 20 25

0 N 0.019 0.18 1.9 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 10-1450-4512 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test
DF P-Type 1.73 2.68 0.093 0.2482 Non-Significant Effect Negative Control 0.019 6 CDF 1.93 2.68 0.093 0.1812 Non-Significant Effect 0.18 6 CDF 5.24 2.68 0.093 0.0006 Significant Effect 1.9* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 11.9% 0.5848 C <> T NA Untransformed NA NA

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 0.09837 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 1.69 11.3 0.6395 Equal Variances Bartlett Equality of Variance Variances 0.972 0.841 0.8667 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Wet Weight Summary

0.784 0.711 0.848 0.788 0 4 Negative Control 0.03 7.66% 0.0% 0.688 0.879 0.724 0.681 0.793 0.711 0.019 4 0.0251 6.95% 7.66% 0.644 0.804 0.717 0.69 0.753 0.712 0.18 4 0.0132 3.68% 8.55% 0.675 0.759 0.602 0.524 0.644 0.62 1.9 4 0.0266 8.86% 23.2% 0.517 0.687CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________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 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 05-4485-1035 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 Brine: Not Applicable Species: Xenopus laevis Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test
DF P-Type 13 NA 0.1000 Non-Significant Effect Negative Control 0.019 -2 Exact 35 NA 0.0632 Non-Significant Effect 0.18 -2 Exact 83 NA 0.0003 Significant Effect 1.9* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed 0.18 1.9 0.5848 C > T NA Untransformed NA NA

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 0.09837 15 Total

Distributional Tests
Attribute Test Test Stat Critical P-Value Decision( α :1%) 1.69 11.3 0.6395 Equal Variances Bartlett Equality of Variance Variances 0.972 0.841 0.8667 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Wet Weight Summary

0.784 0.711 0.848 0.788 0 4 Negative Control 0.03 7.66% 0.0% 0.688 0.879 0.724 0.681 0.793 0.711 0.019 4 0.0251 6.95% 7.66% 0.644 0.804 0.717 0.69 0.753 0.712 0.18 4 0.0132 3.68% 8.55% 0.675 0.759 0.602 0.524 0.644 0.62 1.9 4 0.0266 8.86% 23.2% 0.517 0.687CenteredUntransformed

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

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d 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

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

# DATA EVALUATION RECORD

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

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012
(Revisions to MRID 48619301to incl ude 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### 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 forAndrogen 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 B max 

## was 1.071 (±0.167) fmol/100 μg protein. For the cytosolic preparatio n made on September 24, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 0.613 nM (±0.041), and the estimated B max 

## was 0.817 fmol/100 μg protein (±0.049). The mean and individual K d 

## values were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within th e range and one value was above the range. Confidence in these numbers is generally high based on the goodness of fit (R 2 

## = 0.880-0.992) and the small variation in R 2 

## and K d 

## estimates among runs (although greater variability was observed in the Ju ly 23, 2011 saturation binding data).
In the competitive binding experiment, precipitat ion 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, a nd the highest concentration tested in the third run was 10
− 4 

## M. Specific binding of [ 3 

## H]-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 IC 50 

## and relative binding affinity (RBA) could not be calculated for simazine.
The estimated average log IC 50 

## s for R1881 (

− 9.3 M) and the weak positive control, dexamethasone (

− 4.4 M), were within the expected range s, and the mean RBA for the weak positive control was 0.0024%. Confidence in these numbers was moderate due to variation in IC 50 

## 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 require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided.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
C 2 

### H 5 

### C 2 

### H 5 

## 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 # : 50-02-2Androgen 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 concentrati o 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 (Wilm ington, MA). Male Sprague Dawley rats (number not reported) were castrated at approx imately 90 days of age and were euthanized <24 hours later and the ventral prostate tissues collected.
The ventral prostates were re ceived 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 repor ted), homogenized, and centrifuged for 30 min at 30,000 × g at 4 oC. Supernatant was pooled, dis carding the resulting pellets. Protein concentration of the cytosol was determined to be 5.7-8.8 mg/mL us ing a commercially available protein kit compatible with DTT in the TEDG buffer (Bradford Method; Bio-Rad Dye Reagent Concentrate). Cytosol was divided into aliquot s (amounts not specified) for immediate use.
2. Saturation Radioligand Binding Experiment: Saturation binding experiments measuring total and non-specific binding of [ 3 

## H]-R1881 were performed fo r all three cytosolic preparations, created on July 23, March 30, a nd 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 specif ic 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 seri al dilutions in low-salt TEDG+PMSF buffer were prepared to achieve th e 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 nonAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## specific binding, solutions of non-labeled R1881 were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration, resulting in final concentr ations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, th e radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

## H]-R1881 was measured.
For each batch of cytosol, the optimal protei n concentration was determined by calculating specific binding to differing amounts of protei n per tube with 0.25 nM radiolabeled R1881. The optimal protein concentration was determin ed 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 r uns (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 pr epared on March 30, June 23, and September 24, 2011). Each run contained three replicates at each concentration, result ing in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a ,b 

### Total Binding Non-Specific Binding Radioligand alone Tubes 1-24 c 

### Tubes 25-48 d 

### Tubes 49-72 e 

### [ 3 

### H]-R1881 Final conc. (nM) [ 3 

### H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 

### H]-R1881 Initial conc. (nM) [ 3 

### H]-R1881 (μL) 0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.0 1.0 100 10 30 1.5 1.5 150 10 45 2.5 2.5 250 100 7.5 5.0 5.0 500 100 15 10 10 1000 100 30 a Data were obtained from page 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 tr iamcinolone acetonide and 7.5-45 μL [ 3 

### H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. d Tubes 25-48 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 

### H]-R1881. R1881 was added in a 100-fold mola r excess of [ 3 

### H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

### H]-R1881 or 7.5, 15, 21, or 30 μL of 100 nM [ 3 

### H]-R1881 without cytosol or other component s to determine the total counts added.



## Following addition of triamcinolone 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 s upernatant was aspirate d and discarded. The samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed byAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## 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 scin tillation 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 Experiment a 

### 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 radioligand b 

### Concentration of test substance (as serial dilutions) 10

− 11 

### to 10

− 4 

### M c 

### 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 th e Guideline is 1 mM PMSF. The PI was identified in the study protocol as Calbiochem Protease Inhibitor Cocktail, Set III, EDTA fre e. It is a mixture of six PIs that includes 4-(2- aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBS F 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 singl e 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 pr esence of increasing concentrations of a test substance.

DMSO was used as the vehicle at a final c oncentration 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 fo r the saturation binding experiments, the reviewer was able to calculate from the comp etitive binding experiment that the amount ofAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## cytosolic protein used in the assay contains enough receptor to bind approximately 5-10% of the [ 3 

## H]-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 th ree independent runs on three different days, and each run contained three replicates at each concentration plus si x replicates of the [ 3 

## H]- R1881, non-specific binding (NSB), and solvent c ontrols, resulting in a total of 84 samples per run.

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

### Solvent Control Reference standard We ak positive control Test Chemical 

### None (Hot Mix) DMSO R1881 Dexamethasone Simazine 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. Tu bes 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 (n on-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 wa shing 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 histor ical 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 experi ment, 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 outlie rs 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## 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 H ill equation formula which incorporated log IC 50 

## as a parameter to be estimated. For parameters reported from the saturation binding experiment ( 

## K d 

## and B max 

## ) and competitive binding experiment (log IC 50 

## and RBA), m ean 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 non linear regression model, the test chemical is classified as:

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

− 0.6 to

− 1.4).

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

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

## M and the binding curve does not cross 50%, the chemical is judged to be untestable.
b. Descriptors for receptor binding

B max 

## : maximal binding capacity K d 

## : dissociation constants IC 50 

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

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

## of R1881 ÷ IC 50 

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

## (IC 50 

## of R1881 ÷ IC 50 

## of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5.

2 Swillens, S. (1995) Interpretation of binding curves obtain ed with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 

### 47(6):1197-1203.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## For the cytosolic preparation cr eated on March 30, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 0.614 (±0.065) nM, and the estimated B max 

## was 1.080 (±0.109) fmol/100 μg protein. Only one of the three K d 

## values was within th e range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was belo w the recommended range. Confidence in these numbers is high accord ing to the goodness of fit (R 2 

## = 0.955-0.992) and the small variation in R 2 

## and K d 

## estimates among runs.
For the cytosolic preparation cr eated on July 23, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 1.080 nM (±0.690), and the estimated B max 

## was 1.071 (±0.167) fmol/100 μg protein. Only one of the three K d 

## values was within th e 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 K d 

## estimates among runs.
For the cytosolic preparation crea ted on September 24, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 0.613 nM (±0.041), and the estimated B max 

## was 0.817 (±0.049) fmol/100 μg protein. The mean and individual K d 

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

## = 0.957-0.984) and the sm all variation in R 2 

## and K d 

## estimates among
runs.

TABLE 5. Saturation Binding Experiment of [ 3 

### H]R1881 with Androgen R eceptor from Rat Prostate Cytosol a 

### Parameter Run 1 Run 2 Run 3 Mean ± SEM b 

### Cytosolic preparation March 30 2011 R 2 

### (unweighted) 0.994 0.993 0.994 0.955-0.992 B max 

### (nM) 0.010 0.009 0.009 0.009±0.001 B max 

### (fmol/100 μ g protein) NR NR NR 1.080±0.109 K d 

### (nM) 0.686 0.559 0.596 0.614±0.065 Cytosolic preparation July 23 2011 R 2 

### (unweighted) 0.880 0.986 0.893 0.880-0.986 B max 

### (nM) 0.010 0.008 0.011 0.010±0.002 B max 

### (fmol/100 μ g protein) 1.087 0.896 1.229 1.071±0.167 K d 

### (nM) 0.855 0.531 1.855 1.080±0.690 Cytosolic preparation September 24 2011 R 2 

### (unweighted) 0.984 0.977 0.957 0.957-0.984 B max 

### (nM) 0.011 0.010 0.011 0.011±0.001 B max 

### (fmol/100 μ g protein) 0.809 0.773 0.870 0.817±0.049 K d 

### (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 R 2 

### is reported and the mean ± SEM is reported for the other parameters. R 2 

### 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 th ree 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 cont ains representative Scatchard plots each of the three saturation bindi ng experiments that illustrate the binding ofAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## [ 3 

## H]-R1881 to the androgen receptor. The representative figures from the March 30, 2011 cytosol preparation are inco rrectly labeled as 13-June- 12, instead of 13-June-11.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## FIGURE 1. Binding of [ 3 

## H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment. Representative runs of each set of experiments.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None


## FIGURE 2. Scatchard Plot s of the Binding of [ 3 

## H]-R1881 to the Androgen Receptor. Representative runs of each set of experiments.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None


## B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 3-5. Precipitation of simazine was visually obs erved 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 co ncentrations of 10 − 5 

## and 10
− 4 

## M. Because specific binding was not ≤ 50% at any simazine concentration, an IC 50 

## and RBA could not be calculated for simazine.
The estimated average log IC 50 

## 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 th an a 1-log difference for the IC 50 

## 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 ty pical data. Only data from the three “valid” independent runs were reported.

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

### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c, d 

### R 2 

### (unweighted) R1881 NR NR NR NA Dexamethasone NR NR NR NA Simazine NR NR NR NA Log IC 50 

### (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 IC 50 

### (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 RBA d 

### 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 concu rrent replicates within each run. c For means expressed in scientific notation, the SE valu es 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 R 2 

### Goodness of fit RBA Relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## FIGURE 3. Percentage [ 3 

## H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Simazine (Run 1).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None 

## FIGURE 4. Percentage [ 3 

## H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Simazine (Run 2).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None



## FIGURE 5. Percentage [ 3 

## H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Simazine (Run 3).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None


## C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the following criteria (Table 7):

TABLE 7. Criterion a 

### Tolerance Limit(s) b 

### Value Yes No Ligand depletion 

### is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 

### H]-R1881 added per assay tube. ≤ 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 K d 

### (nM) 

### 0.685 to 1.57 0.559 to 0.686 c 

### , 0.531 to 1.855 d


### , 0.565 to 0.638 e 

### X

Non-specific binding (%) 

### ≤ 10.0 0.37 to 0.48 c 

### 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 th e 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 concen trations of unlabeled R1881 displaced [ 3 

## H]-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 consis tency of the Hill slope, and top and bottom plateaus, and the placement al ong the X-axis was consistent for dexamethasone. However, in the second run, the curve for R1881 was shifte d substantially to the right, resulting in a log IC 50 

## (

− 10.1 M) that is a full order of magnit ude 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 “equivo cal” in the third vali d independent run. Therefore, simazine has a final classification of “non-binder.”

B. AGENCY COMMENTS: For the cytosolic prepara tion created on March 30, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 0.614 (±0.065) nM, and the estimated B max 

## was 1.080 (±0.109) fmol/100 μg protein. For the cytosolic prep aration created on Ju ly 23, 2011, the mean K d 

## for [ 3 

## H]-R1881 was 1.080 nM (±0.690), and the estimated B max 

## was 1.071 (±0.167) fmol/100 μg protein. For the cytosolic pr eparation created on September 24, 2011, theAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 18 of 18 SIMAZINE / 080807 OCSPP 890.1150/ OECD None



## mean K d 

## for [ 3 

## H]-R1881 was 0.613 nM (±0.041), and the estimated B max 

## was 0.817 (±0.049) fmol/100 μg protein. The mean and individual K d 

## 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 (R 2 

## = 0.880-0.992) and the small variation in R 2 

## and K d 

## estimates among runs (a lthough greater variab ility was observed in the July 23, 2011 saturation binding data).
In the competitive binding experiment, precipit ation 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 IC 50 

## and RBA could not be calculated for simazine. As speci fic binding was >75% in all runs simazine is classified as a non-binder under th e conditions of this assay.
The estimated average log IC 50 

## s 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 IC 50 

## 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.

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### 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 theEstrogen Receptor Transc riptional Activation (2011) / Page 2 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

## the study. Because the RPC Max 

## < PC 10 

## 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.Estrogen Receptor Transc riptional 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. McColl ey (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

− 3.3 M 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
C 2 H 5 C 2 H 5 

## 3. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Not reported Catalog and Batch #: Not reported Purity: Not reported CAS # : 50-28-2


## 17 α -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


## 17 α -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. METHODSEstrogen Receptor Transc riptional Activation (2011) / Page 4 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455



## 1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank and were verified to be free of mycoplasma infection using a DNA Fluorochrome assay. Ce lls were maintained in Eagles Minimum Essential Medium without phenol red, supplem ented with 60 mg/L kanamycin and 10% dextran-coated charcoal-treat ed fetal bovine serum (DCC-FBS; source not reported), in an incubator under 5% CO 2 

## 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 pl ated in 96-well plates at a density of 1×10 4 

## cells/100 μL and allowed to attach fo r at least 3 hours. Growth media was replaced with media containing serial log dilutions of simazi ne in DMSO (0.1% total final concentration). Cells were incubated fo r 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, respec tively. Transcriptional activation of the estrogen receptor was determined as descri bed 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 si mazine concentrations ranging from 10 − 10.3 

## to 10 − 3.3 

## M was conducted to determine the appr opriate 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 th e assays and the passage numbers. The cells were tested using the following set of profic iency 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 --- 17 α -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 0.01 b , 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 0.1 b and 1 mM 1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC. Binds to hER α 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 = RPC max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC max 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 ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.Estrogen Receptor Transc riptional 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 w ith the test chemical:

Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10

− 14 to 10

− 8 Strong estrogen 17 α -estradiol 57-91-0 10

− 12 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative compound 17 α -methyltestosterone 58-18-4 10

− 11 to 10

− 5 Very weak agonist


## 3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed 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 expres s relative transcriptional activity as a percentage of the PC. The test material was defined as negative for inducing estrogen receptor transcriptional activation if the RPC Max 

## <PC 10 

## in at least 2 of 2 runs. Log EC 50 

## and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the lumi nescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of insolubility were excluded from analyses.

4. Definitions

EC 50 

## = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response

PC 10 

## = 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

PC 50 

## = 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

RPC Max 

## = 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

PC Max 

## = concentration of a test chemical inducing the RPC Max



## 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 obser ved 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 concentra tion. Based on these re sults, logarithmically increasing concentrations of simazine from 10
− 11 

## to 10 − 4 

## M were selected for the first assay.Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455


### TABLE 1. Preliminary Test for Solubility, Cytoto xicity, and Concentration-Selection for Simazine a

Concentration (M) % Viability b Comments c 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 assa ys using the required reference compounds were not included in th e original study report, but were provided to the Agency at a later date (MRID 48843501). The responsiven ess 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 prof iciency tests, the reference chemicals 17 β -estradiol, 17 α -estradiol and 17 α -methyltestosterone were tested c oncurrently with each run of the assay (Table 2b). In the firs t run, the responsiveness of 17 β -estradiol indicated decreased sensitivity to strong agonists, and the response to 17 α -methyltestosterone showed an increased responsiveness to very weak agonists; despite the minor deviations this run is considered acceptable. Run 2 was inadequate as the PC 50 

## could not be calculated for 17 α
methyltestosterone indicating a decreased sensitivity to very weak agonists. Run 3 was acceptable as 17 β -estradiol and 17 α -methyltestosterone performed within the expected range, but the Hill Slope for 17 α -estradiol was higher than ex pected. The PC-induced fold induction for the three reference chemical s was within the Guideline-recommended historical range of 4- to 30-fold in Runs 1 a nd 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 a ssay 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 parabe n had maximum RTAs well above 400%. The responses do not demonstrate proficiency as the assay cannot be fully evaluated by the reviewer.Estrogen Receptor Transc riptional 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 17 α -Ethynyl estradiol Pos itive 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 Negativ e Negative Negative NA Atrazine Negative Ne gative NA Negative Corticosterone Negative Negative NA Negative NA = not applicable. The chemical was not tested at this time.



### Table 2b. Performance Criteria for Ref erence Chemicals in the Proficiency test Reference Chemical Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Yes No 17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 9.6

− 11.3

− 10.6 Run 1 Log PC 10 <

− 11

− 11.5

− 12.5

− 12.1 X Log EC 50 − 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 17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 8.3

− 9.4

− 8.7 X Log PC 10 − 10.7 to

− 9.3

− 9.3

− 10.5

− 9.9 X Log EC 50 − 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 17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1

− 6.2 NC

− 5.2 Run 1, 2 Log PC 10 − 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 PC 50 

## , Log PC 10 

## , Log EC 50 

## , 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 observe d at the highest concentrations of 17 α -estradiol (10
− 6 

## M) and corticosterone (10
− 4 

## M), and at 10
− 13 

## , 10 − 9 

## , and 10
− 8 

## M for 17 β -estradiol. In the second run, the responsiveness to 17 β -estradiol at concentrations ≥ 10 − 10 

## M was 117-150% of the response to the PC (10
− 9 

## M 17 β -estradiol), and the response curve to 17 α -methyltestosterone mimicked the intensity of 17 β -estradiol. Additionally, in both runs responsiveness of the cells to 17 α -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.Estrogen Receptor Transc riptional 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 17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 11.5 b

− 11.7 X Log PC 10 <

− 11

− 12.0

− 12.6 X Log EC 50 − 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 17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 9.9 b

− 9.4 b X Log PC 10 − 10.7 to

− 9.3

− 11.0 b

− 10.4 b X Log EC 50 − 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 17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1

− 9.6 b

− 8.8 b X Log PC 10 − 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 in creased sensitivity of the assa y system and is considered acceptable.



## C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive 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 PC 50 

## (50% of the maximum response) fo r E2 in this assay is 700169 for the first run and 1122431 for the second run, and the PC 10 

## (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 17 β -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 tran scriptional activation is presented in Figure 1 below. The report stated that three independe nt 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. TheEstrogen Receptor Transc riptional Activation (2011) / Page 9 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455


## mean RPC Max 

## was 3.5%, with an associated PC Max 

## of 10
− 7 

## M for the first run; the mean RPC Max 

## was <0% for the second valid run at all concentrations. Because the RPC Max 

## < PC 10 

## in both runs, simazine was considered negative for estrogen receptor transcriptional activation in this test system. No explan ation was given for the change in simazine concentrations used in the second run.

TABLE 5. Relative Transcriptional Activation (RTA) of Simazine a 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 EC 50 b NA NA Hill Slope b NA NA RPC Max 3.5 <0% PC Max 10

− 7 NA PC 50 NA NA PC 10 NA NA a Data were obtained from pages 22 and 23 of the study report. b Report if appropriate NA = Not ApplicableEstrogen Receptor Transcriptional Activation (2011) / Page 10 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455


## FIGURE 1. Fold Induction of Relative Tr anscription Activation (RTA) of Simazine Compared to the Positive Control.

3. Performance Criteria: While the Log PC 50 

## , Log PC 10 

## , Log EC 50, 

## 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 satisf actory. The cytotoxicity of 17 β -estradiol, 17 α -estradiol, and corticosterone to the cells in the first assay, and the responsiveness of 17 β -estradiol, 17 α -estradiol, and 17 α -methyltestosterone greater than that of the positive control (117-150% PC) indicate a failure of the test system to respondEstrogen Receptor Transc riptional Activation (2011) / Page 11 of 11 SIMAZINE/ 080807 OCSPP 890.1300/ OECD 455 

## appropriately to its natural ligand, and two w eak agonists. Cytotoxicity should not be observed at any level for the reference chem icals, and the maximum responsiveness should be observed in the positive cont rol, 1 nM 17-estradiol.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The highest soluble concen tration 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 cyto toxicity observed at any of the exposure concentrations tested. In tw o independent runs of the tr anscriptional activation assay, simazine did not result in an increase in lu ciferase activity any of the concentrations assessed. Simazine is not an agonist of the human estrogen receptor alpha (hER α ) 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 RPC Max 

## for simazine was 3.5%, with an associated PC Max 

## of 10
− 7 

## M for the first run; the mean RPC Max 

## 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 wa s 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 vali dated ranges, indicating that the current assay was more sensitive than duri ng the validation experime nts. Therefore the performance criteria deviations do not affect th en 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 17 β -estradiol, 17 α -estradiol, and corticosterone.

 Certificates of Analyses were not pr ovided for the Reference Chemicals.

 The source of the fetal bovine serum was not provided.

## 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, Mary land, Report No.: 528A-226. Subm itted 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'

## 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.

## 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 as say 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 25 o 

### 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 co ncentration, 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 no table observations with rega rds 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); fertiliz ation 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 orfemale 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

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 4 of 89 male vitellogenin (VTG), the reported control values were unusually high (comparable to control female values) even after re-analysis; the values in the other treatment groups were also comparable to female VTG values. No obvious reasons for these high values were reported and as such, these data are not considered reliable for interpretation. Sex steroids were not measured in this study.

The validity criteria were met except that dissolved oxygen fell slightly below the recommended 60% of saturation (58%) in one test chamber; the chambers were cleaned and the levels returned to above the recommended value, and this deviation did not sufficiently impact the interpretation of the study.

In regards to male VTG, the inability to evaluate this endpoint is a major limitation in this study. However, it is noted that there were no effects on female VTG or male GSI or histopathology. This information along with the information provided in the remaining Tier 1 battery were considered collectively for evaluating potential effects in male fish from EPTC exposure. Based on this information, additional information regarding impacts to male VTG is not requested.

As such, this assay satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350).

Results Synopsis

Test Organism age at test initiation: 6 months Mean body weight at test initiation (if measured): Male 2.9 g; Female 1.6 g Mean length at test initiation (if measured): Not reported. Test Type (Flow-through, Static, Static Renewal): Flow-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 5 of 89Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with EPTC.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFM4FMFMF0.069 No No YesNo No No No No NRLNo NA NA NA NA0.69YesNo NoNo No No No No NRLNo NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NA NA NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable,NRLNot reliable,.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.4Due to unusually high control male VTG values, these data are not considered reliable for interpretation.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 6 of 89 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 (2009). The following deviations were noted:

1. 

### The unionized ammonia and residual chlorine in the test water were not reported. The OCSPP 890.1350 performance criteria establish maximum levels
for these values and it is unclear if the maximum recommendations were
exceeded.

2. 

### The dissolved oxygen fell to 58% of saturation in the control replicate B on Day 10 of the exposure which is slightly below the recommended 60% of saturation. The test chambers were cleaned and the levels were above 60% by the next day.

These deviations do not have an impact on the acceptability of this study.

COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were provided. This study was conducted in compliance with all pertinent U.S. EPA Good Laboratory Practice regulations with the following exceptions: routine dilution water and food contaminant screening analyses were conducted following standard validated methods, this exception has no impact on the study results.

A. TEST MATERIAL: EPTC; CAS# 759-94-4.

Description: Not reported.

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow, vapor pressure of test compound, expiration date.



### Lot No./Batch No. : P1105096

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 7 of 89 Purity: >99.9%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from time 0, 6, 13, and 21 days yielded recoveries of 78.8% to 91.3% of nominal concentrations. Stock solution fortified with EPTC at concentrations of 0.04, 0.8, and 8.0 mg a.i./L yielded recoveries of 83.6 to 107%. There was no pattern of decline during the study period, indicating the test material was stable under the test
conditions.

Storage Conditions of Test Chemicals: Stored at room temperature in dark, ventilated cabinet.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 8 of 89B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated): Not reportedWere fish obtained from asingle laboratory stock?Yes EPA recommends that fish be from a single laboratory stock.Were acclimation conditionssame 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 9 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:1 mortality (<1%)Mortalities during the 7 days prior topre-exposure period were <1%.Fish did not receive any treatment fordisease during acclimation period.Behavioral abnormalities or clinical signswere 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 reportedRepresentative samples of the food sourcewere periodically analyzed for pesticides,PCBs, and toxic metals by GeoLabs, Inc.and analyte levels were below LOC specifiedby ASTM (2005).Frequency of feeding:2 times/dayDetails on feeding:None.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 10 of 89Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Mean weight of males at testinitiation (if determined):2.9 g Based on 20 males used to stockaquaria for pre-exposure phase. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):2.4 to 3.4 Individual weights within ± 20% of theestimated mean.Mean weight of females at testinitiation (if determined):1.6 g Based on 20 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):1.3 to 2.0 Individual weights within ± 26% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 11 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsDuration of pre-exposure phase: 13 days EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:36Extra replicates were established toaccount for a potential lack of spawningin some chambers and/or mortalityduring 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:2Number of females per tank:4Pre-exposure fecundity:>15 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 12 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsNumber 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.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 13 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Intermittent flow proportionaldiluters Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate: Ca. 45 mL/minute or 2.7L/hr and 6.6 volumereplacements/day.Calculated by the reviewer based on 0.5L/cycle, 132 cycles/24 hrs and 60minutes/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 forflow-through systems:Flow-splitting chambers were usedbetween diluter cells and the 4 replicatetest vessels.Treatment recoveries were not measuredprior to test initiation to indicate if diluterhad 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).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 14 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsSource 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 analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials: Glass aquaria withsilicone sealant andnylon.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 cmlong 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).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 15 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material: 4-inch diameter agedPVC 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 glasscollection tray (13 x 11 x 3 cm, L x W xH), covered with 0.5 cm2stainless steelmesh.Additional details on exposuresystem:None.Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)242524.51Continuously EPA recommends temperature 25±1 o C; inter-replicate and inter-treatment differentials should not exceed 1 o C.pH 6.77.47.11Daily EPA recommends pH 6.5 to 9.0.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 16 of 89ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsDissolved oxygen (mg/L)4.88.16.51Daily EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)20 26 231Weekly EPA recommends total alkalinity >20 mg/L as CaCO 3.Hardness (mg/L as CaCO3) 60 72 661WeeklyTotal organic carbon (mg/L) 0.11 0.160.14Monthly EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L) Not reported Not reported Not reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1

P g/L.Residual chlorine (Pg/L) Not reported Not reported Not reportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 17 of 89ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsConductivity (Pmhos/cm) 330 450 3901Weekly 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:NANot applicable.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 18 of 89D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducti ng a range-finder if 96-hour LC 50 data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 19 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:At test termination,17% and 100%mortality was observedin the 20 and 40 mga.i./L treatment level. EPA recommends conducti ng a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21-

day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 20 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:The acute toxicity 96-hr LC50value with fathead minnowswas reported determined to be25 mg a.i./L (95% CI of 20 to31 mg a.i./L).Nominal concentrations selectedbased on range finding studyresults and consultation withsponsor. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reportedSeparation of testconcentrations:0 (control), 0.8, 0.80, and 80mg a.i./L EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or ten-fold) is scientifically acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 21 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsAre nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 22 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock 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 solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 23 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 24 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:560-980 luxLight was provided by SylvaniaOktron®fluorescent bulbs andintensity was measured with aVWR light meter. EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:All exposure solutions werereported as clear and colorlesswith no undissolved testsubstance observed.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 25 of 89Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with EPTC.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0 <LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.080.0691.7Treatment 20.80.696.2Treatment 38.06.92.1Diluter StockNANANAAbbreviations:CVCoefficient of variation.LOQ=0.014 mg a.i./L.E. ObservationsBiological 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 stagingand histopathology, and plasma VTG were evaluated.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 26 of 89Were raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertiliz ation success, secondary sex characteristics, and other clinic al 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 beha vior.I.RESULTS AND DISCUSSIONFor all endpoints, the high treatment level was not statistically analyzed due to high mortality.A.ResultsMean 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 themean-measured <LOQ (control), 0.069, 0.69, and 6.9 mg a.i./L treatment levels, respectively.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 27 of 89Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesN1# Surviving% SurvivalN1# Surviving% SurvivalControl (<LOQ)881001615940.069 8 8 100 16 16 1000.69 8 6 75 16 6 386.928 2 13 16 1 6Abbreviations:NANot applicable.1Total number of fish at test initiation.2Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 28 of 89Mean 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, 64.0and 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./Ltreatment levels, respectively.Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ) 4 3.73 0.18 4 1.39 0.17 4 65.7 1.4 4 50.7 1.10.069 4 3.53 0.61 4 1.43 0.11 4 64.0 4.8 4 51.6 0.660.69 4 3.52 0.22 4 1.64 0.11 4 64.4 2.8 4 52.0 0.516.914 NA NA 4 NA NA 4 NA NA 4 NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.1Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 29 of 89Mean 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] Fecundity 1 

### 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

6.9 3 

### 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.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 30 of 89 Median male tubercle scores were 41, 32 and 26 in the mean-measured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to
have tubercles.

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L) [mean-measured] Males Females

n Median
Tubercle Score n Median
Tubercle Score

Control (<LOQ) 4 41 4 0

0.069 4 32 4 0

0.69 4 26 4 0

6.9 1 

### 4 NA 4 NA

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

1 

### Endpoints at the high treatme nt level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 31 of 89Mean 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 GSI 1 

### (%) ±SD n Mean GSI 1 

### (%) ±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

6.9 2 

### 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.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 32 of 89 Median male and female gonadal stages were 2, 2 and 3 in the mean-measured <LOQ (control), 0.069,and 0.69 mg a.i./L treatment levels, respectively (Table 14). The study authors did not indicate treatmentrelated affects at any test level for gonadal staging.

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.
Treatment (mg a.i./L) [mean-measured] Males Females

n Median Stage 1 n Median Stage 2



### Control (<LOQ) 8 2 

### 15 2



### 0.069 8 2 

### 16 2



### 0.69 6 3 

### 6 3



### 6.9 3 

### 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 effe cts 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.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 33 of 89 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1

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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 34 of 89Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ) 0 8 8 8 8 8 8 8 8 8 81 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.0690 8 8 8 8 8 8 8 8 8 81 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 35 of 89Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.690 6 6 6 6 6 6 6 6 6 61 6 0 6 0 6 0 6 0 6 02 6 0 6 0 6 0 6 0 6 03 6 0 6 0 6 0 6 0 6 04 6 0 6 0 6 0 6 0 6 06.920 NA NA NA NA NA NA NA NA NA NA1 NA NA NA NA NA NA NA NA NA NA2 NA NA NA NA NA NA NA NA NA NA3 NA NA NA NA NA NA NA NA NA NA4 NA NA NA NA NA NA NA NA NA NA1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.2Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 36 of 89 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1



### Severity Increased Oocyte
Atresia Perifollicular Cell Hyperplasia/ Hypertrophy Decreased Yolk
Formation Aggregates of macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

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
6.9 2 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

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 37 of 89 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

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
6.9 2 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

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 38 of 89 Median male VTG was 9.2 x 10 5 

### , 4.2 x 10 5 

### and 8.3 x 10 3 

### and mean female VTG was 8.3 x 10 5 

### , 1.2 x
10 6 

### and 6.4 x 10 5 

### 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) ±SD 1 

### n Mean (ng/mL plasma) ±SD 1



### Control (<LOQ) 4 9.2 x 10 5 

### 7.0 x 10 5 

### 4 8.1 x 10 5 2.2 

### x 10 5



### 0.069 4 4.2 x 10 5 

### 2.7 x 10 5 

### 4 1.2 x 10 6 6.0 

### x 10 5



### 0.69 4 8.3 x 10 3 

### * 9.1 x 10 3 

### 4 6.4 x 10 5 1.0 

### x 10 6



### 6.9 2 

### 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 39 of 89Mean 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)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)NA NA NA NA NA NA NA NA NA NA NA NA0.069NA NA NA NA NA NA NA NA NA NA NA NA0.69NA NA NA NA NA NA NA NA NA NA NA NA6.9NA NA NA NA NA NA NA NA NA NA NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.014 mg a.i./L.There were no notable observations with regards to behavior orchanges in appearance, specifically color/banding, ovipositor appearance, or size ofdorsal nape pad in the control or treated groups (Table 21).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 40 of 89Table 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 TerminationMalesFemalesTypen1Incidence2TypeN1Incidence2Control (<LOQ)Color bars,81None, Papillae present154Territorial aggression4Fatpad, tubercles40.069Color bars,81 None, Papillae present162Territorial aggression4Papillae present1Fatpad, tubercles4None10.69Territorial aggression64 Papillae present62Fatpad, tubercles4None16.9 Fatpad,tubercles11 lethargic 1 1LOQ=0.014 mg a.i./L.1The value reflects the total number of individual fish surviving at test termination2The individual number of fish displaying the reported characteristics/signs was not reported, therefore, the numerical value in the row reflects the number of replicatesreported to be displaying the characteristic.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 41 of 89 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility, fecundity, and VTG. Data were gender specific and analyzed in comparison to the controls.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant effects were detected for p<0.05 with the exception of Shapiro-Wilk’s and Levene’s Tests, whichwere 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 T3Multiple 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 fecundit y (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 cont rol (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.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 42 of 89 In male fish, there was one incidence of mild testicular degeneration. Otherwise, there were no treatment-related histological effects or differences in gonadal stages in testes in any of the treatment groups (Table 15). In female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control (Table 17). However, the smaller sample size (n=6) prevented further interpretation of this finding. The study authors did not indicate a treatment-related effect at any test level for gonadal staging. There were no notable observations with regards to behav ior 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, th ey 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 ho mogeneity of variance usin g Levene’s test. Data which met the assumptions of normality and homogeneity of variance were then analyzed using the parametric Dunnett’s test. Datawhich did not meet the parametric assumptions were analyzed using the non-parametric MannWhitney U test.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 43 of 89 Fertility data was not parametric and were analyzed using Mann-Whitney. All other data wasparametric 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 44 of 89Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with EPTC.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pControl (<LOQ) 40.5 NA 0 NA No0 NA NA NA NA NA0.069 32.0 0.086 -19.67 0.245 No-54.35(NRL)NA NA NA NA NA0.69 26.0 0.057 22.95 0.165 No-99.10(NRL)NA NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NAStatistical TestMann-WhitneyDunnett’sNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.NRLDue to the unusually high male VTG values in the control, the results for this endpoint are not reliable for interpretation1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 45 of 89Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with EPTC.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p%Diff.pControl (<LOQ)0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA0.06919.10 0.248 0.00 1.00 0 NA -7.69 0.625 No 47.68 0.625 NA NA NA NA0.69-74.83 <0.001-11.72 1.00 0 NA 23.08 0.080 No -21.07 0.918 NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NA NA NA NA NAStatistical TestDunnett’s Mann-Whitney NA Dunnett’s NA Dunnett’s NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 46 of 89 Table 24: Growth Endpoints 1,2 

### 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



### 6.9 3 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 te st 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

## 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 statisti cally significant reductions (p<0.05) in survival atthe 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 me thod (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 aut hor 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 3.5 P 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.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 48 of 89 II. REFERENCES

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ
Toxicol Chem 20:1276-1290.

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th Edition, Washington, DC, 2-23-25.

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania.

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User's Guide. Tidepool Scientific Software, McKinleyville, California.

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies.
Water Research 1: 21 -29.

Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity.
Water Res. 3: 793-821.

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: Test No. 229. Paris, France. 40 pp

U.S. EPA, 1989. 40CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory Practices Standards; Final Rule. Office of the Federal Register, National Archives andRecords 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

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 49 of 89 estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency, Washington, DC 20460. EPA/82 l/R-02/014.

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disrupter Screening Program Test Guideline, OCSPP 890.1350 . Fish Short-Term Reproduction Assay. EPA
40-C-09-007. October 2009. U.S. Environment al Protection Agency. Washington, D.C.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 50 of 89APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpoint Monotonic?1Parametric? 890.1350EFEDCommentsFemale bodyweightNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale bodyweightNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemale bodylengthNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale bodylengthNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemale VTG NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale VTG NANoNANAData not reliableFemale GSI NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale GSI NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemaletubercle scoreNA NA NA NA No scoreMale tuberclescoreNA Yes Mann-WhitneyDose 1 p=0.057dunnett’sDose 1 p=0.057EDSP and EFED same conclusions,marginal significant (p=0.057)reductions at the 0.069 mg a.i./Ldoses.Fecundity NA Yes Dunnett’s:Dose 2 p<0.001Dunnett’s:Dose 2 p<0.001EDSP and EFED same conclusions, asignificant (p<0.001) 75% reductionat the 0.69 mg a.i./L dose.Fertility NA No Mann Whitney-Un.s. p>0.05Mann Whitney-Un.s. p>0.05EDSP and EFED same conclusions, noeffectF testosterone NANANANANAM testosterone NANANANANAF estradiol NANANANANAM estradiol NANANANANA

## 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.

## 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

## 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

## 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

============================================================================

## 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

============================================================================

## 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 *

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

-

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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

## 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

## 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=0.01Levenes 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

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*** 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

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

.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

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*** 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)

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

.

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*** 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

## 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=0.01Levenes 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

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*** 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

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

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Ctrl 50.73 . 51.37 . . . . .

. Dose1 51.58 0.289 51.37 0.915 . . . .

. Dose2 51.97 0.136 51.37 0.923 0.805 . . .

.

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*** 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)

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

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

## 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=0.01Levenes 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

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*** 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

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

## 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 . . .

.

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*** 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 . . .

.

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

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*** 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=0.01Levenes 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

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*** 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

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

## 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 . . .

.

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*** 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)

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

## 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 . . .

.

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*** 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=0.01Levenes 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

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*** 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 . .

## 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

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

.

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*** 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

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

## 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 . . .

.

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*** 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=0.01Levenes 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

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*** 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

## 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

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

.

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*** 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)

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

## 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 . . .

.

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*** 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=0.01Levenes 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

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***

## 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

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

.

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*** 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)

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 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=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.

## 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 .

## 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) )

## 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=0.01Levenes 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

## 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)

## 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=0.01Levenes 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

## 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

## 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=0.01Levenes 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 . . .

.

## 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

## 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

.

## 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 

## 2Asymptotic Pr > ChiSquare 

## 0.0297

Exact Pr >= ChiSquare 

## 0.0152

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



## The NPAR1WAY Procedure

## 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 

## 1Pr > ChiSquare 

## 0.0384

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



## The NPAR1WAY Procedure

## 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 

## 1Pr > ChiSquare 

## 0.0284

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



## The NPAR1WAY Procedure

## Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



## The NPAR1WAY Procedure

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### 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% decreaseIn vivo Hershberger Assay (201 1) / Page 2 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441 

## (p<0.05) in overall (Days 1-11) body weight gain. Overall body we ight 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 treatmen t-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 contro l 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 sim azine treatment groups were compared to the performance criteria in the Guidelines. A ll %CV values were less than the maximum recommended values, with the exception of Cowp er’s glands in the control group (CV = 68.0%; maximum recommended CV = 55%).
In the anti-androgen assay, overa ll body weight gain was decreas ed (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. Th ere 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 decreas ed (p<0.01) accessory sex organ weights as follows: 69% in seminal vesicles; 75% in ventra l prostate; 47% in LABC; 74% in Cowper’s gland; and 20% in glans penis. Liver weights in the positive contro l group were comparable to the vehicle control. The performance criteria indicated that this as say was performing as expected. There were no significant weight decreases in androgen-dependen t 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 requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Hershberger Assay (201 1) / 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 Supervis or, Sample Processing Laboratory); R. A. Wally (Operations Manager, Reporting & Technical Support Se rvices); R. Green (Syngenta Study Monitor); L. Freshwater (Contributing Scie ntist, 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 Co rporation (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 Co rporation (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 Co rporation (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

CH 3 CH

3In vivo Hershberger Assay (201 1) / 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 hous ed in stainless steel wi re-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 pot ential androgenic activity, the test substance was administered daily via oral gavage to castrated male rats. Positive androgenic activity is defined as a signif icant increase in two or more target organ weights compared to the vehicle control. To screen for the po tential 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 signifi cant 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 admi nistration for organ weight measurements.
3. Study Schedule: Male rats were castrated on PND 42 acco rding to standard procedures and allowed >7 days for recovery and regressi on of accessory sex or gan weights prior to initiation of dosing. The dose administrati on 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 anal ysis indicated that th ere 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.In vivo Hershberger Assay (201 1) / Page 5 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441



### TABLE 1. Study Design a 

### Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (negative control) 0 6 Low (Simazine) 65 6 High (Simazine) 200 6 Testosterone propionate (TP), positive control b 

### 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 do se of TP was administered concurren tly 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 study 1 

## 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 da ys. Dose-related reductions in mean bodyweight gain and/or mean body weight losses with corr esponding reductions in mean food consumption were noted at all dose levels, resulting in dos e-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 prepar ed 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 re ported). Analyses to demonstrate homogeneity, st ability, and resuspension homogeneity were conducted previously for dose formulations at 1 a nd 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) prep ared 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 concentr ations 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 Validati on and Stability Study of Simazine in Aqueous Formulations (Study No. WIL639131). WIL Research Laboratories, LLC, Ashland, OH, Draft.In vivo Hershberger Assay (201 1) / Page 6 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441 

## Concentration (% of nominal): 102-106%
The analytical data indicated that the varia tion 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 administ ered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was give n via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral ga vage at 5 mL/kg bw. For th e control group dosed with TP alone, the vehicle (0.5% CMC) wa s administered concurrently via oral gavage at 5 ml/kg bw. Dose volumes were adjusted daily base d on the concurrent body weight measurement.

8. Statistics: Statistical analyses were conducted for organ weights, daily body weights, cumulative body weight gains, and food cons umption. 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 ro ot 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 descri bed below, was used to analyze the data. One-sided tests were conducted for in-life data and two-sided te sts 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 cont rol) or an ANOVA (for comparing dose groups to the negative control) was performed on da ta; the ANOVA test was follo wed by a one- or two-sided Dunnett’s test. If the tran sformations 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 tran sformations 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 wa s denoted at p<0.05. Statistical analyses were performed using SAS software (version 9.2 or higher). The statis tical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and moribundity were conducted twice daily. Individu al 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 rando mization, daily thr oughout 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.In vivo Hershberger Assay (201 1) / Page 7 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441 

## 4. Serum Hormone Measurements (Optional): At study termination, each animal was anesthetized with isoflurane, a nd 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 necrops y. 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 wei ghed. The accessory sex organs and a portion of the liver were placed in 10% neutral buffe red 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 unt il scheduled termination.
2. Clinical signs of toxicity: No remarkable clinical signs of toxicity were observed in animals for any dose groups. Any clinical fi ndings noted in the c ontrol 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 presen ted in Table 2. Body we ights were decreased (p<0.05) compared to controls by 8% on Da y 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 gr oup were comparable to controls throughout the duration of the study.In vivo Hershberger Assay (201 1) / Page 8 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441



### TABLE 2. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Androgen Agonist Assay a



### 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

B ody Weight Gain (Days 1-11) 6 62.5 10.65 6 71.9 7.67 6 46.6* ( ↓ 25 b 

### ) 9.94 6 31.3** ( ↓ 50 b 

### ) 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 decr eased (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 th roughout the duration of the study.

TABLE 3. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Anti-Androgen Assay a

Study Day # Dose (mg/kg/day) Vehicle Control + TP (0.2) 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<0.01In vivo Hershberger Assay (201 1) / Page 9 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441 

## C. FOOD CONSUMPTION (Optional): There were no signifi cant dose-related effects on overall food consumption in the androgen agonist assay or the anti-androgen assay. In the androgen agonist assay, mean food consumpti on in the 200 mg/kg/day group was decreased 27% during Days 1-2 compared to the vehicl e control; however, mean food consumption was comparable to the control fo r the remainder of the study.
There were no significant diffe rences 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 CONCE NTRATIONS (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 gr oup. Animals in the TP group had increased (p<0.01) accessory sex organ weights as fo llows: 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 sim azine treatment groups were compared to the performance criteria in the Guidelines. A ll %CV values were less than the maximum recommended values, with the exception of Co wper’s glands in the control group (CV = 68.0%; maximum recommended CV = 55%).

There were no treatment-relate d gross pathological findings.

TABLE 4. Accessory Sex Organ We ights from Androgen Agonist Assay in Sprague Dawley Rats a



### Organ Dose (mg/kg/day)
Vehicle Control Positive Control Vehicle + TP (0.2) 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, S 20, 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<0.01In vivo Hershberger Assay (201 1) / Page 10 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441 

## 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 contro l) 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 sim azine treatment groups were compared to the performance criteria in the Guidelines. A ll %CV values were less than the maximum recommended values.

There were no treatment-relate d gross pathological findings.

TABLE 5. Accessory Sex Organ Weights from Anti -Androgen Agonist Assay in Sprague Dawley Rats a



### 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, S 21, and S23 on pages 70, 71,74, 75, 78, and 79 of the study report. Percent differences from controls were calculated by the re viewers 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 androg en 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.In vivo Hershberger Assay (201 1) / Page 11 of 11 SIMAZINE/ 080807 OCSPP 890.1400/ OECD 441


## B. AGENCY COMMENTS: All animals survived until scheduled termination. No animals exhibited any dose-relate d clinical signs of toxicology a nd there were no treatment-related gross pathological findings.
In the androgen agonist assa y, 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 th e 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 compar able to the vehicle control. As there were no significant weight increases in androgen-dependent tissues, si mazine is not considered to be an androgen agonist at doses of ≤ 200 mg/kg/day under the conditi ons of this assay.

Percent CVs for the vehicle control and the sim azine treatment groups were compared to the performance criteria in the Guidelines. A ll %CV values were less than the maximum recommended values, with the exception of Co wper’s glands in the control group (CV = 68.0%; maximum recommended CV = 55%).
In the anti-androgen assay, overa ll body weight gain was decr eased (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 decr eases 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, simazi ne 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 us ed in the study were not reporte d; all ages were reported to be approximate.