### UNITED ST ATES ENVIRONMENT AL PROTECTION AGENCY WASHINGTON, 

### D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

Date: 6/30/2015

SUBJECT: Dichlobenil: Data Evaluation Records (DERs) for EDSP Tier 

### 1 Assays

PC Code: 027401 

### Decision No.: 459923 

### Petition No.: NA 

### Risk Assessmen t Type: NA 

### TXR No.: 0051691 

### MRID No.: See Table 

### DP Barcode: D397912 

### Registration No.: NA 

### Regulatory Action: NA 

### Case No.: NA 

### CAS No.: 1194-65-6 

### 40CFR: NA



### FROM: Greg Akerman, Ph

. D.

# ~**~~---** Immediate Office Health Effects Division (7509P)



### THROUGH: Jess Rowland

## d=-~**C2-=~** Deputy Director Health Effects Division



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

I. 

### ACTION REQUESTED Ver.Apr

. 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 dichlobenil: Test Order# EDSP-027401- 120.

Page I of2Page 2 of 2 

### II. RESPONSE
Attached are the EDSP Tier 1 assay DERs for dichlobenil.

III. MRID Table

Chemical: Dichlobenil PC Code: 027401 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48616101 890.1150 Androgen Receptor Binding (Rat Prostate) 48616102 890.1200 Aromatase Assay (Human Recombinant) 48616103 890.1250 Estrogen Receptor Binding 48616104 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48616105
890.1350 Fish Short-Term Reproduction 48616106 890.1400 Hershberger (Rat) 48616107 890.1450 Female Pubertal (Rat) 48616108 890.1500 Male Pubertal (Rat) 48616109 890.1550 Steroidogenesis (Human Cell Line – H295R) 48616110 890.1600 Uterotrophic (Rat) 48616111 Range finding study 48616112 Range finding study 48616113Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Data Requirement: EPA DP Barcode 397913

OECD Data Point 231

EPA MRID 48616101

EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

Test Material: Dichlobenil Purity (%): 99.3%

Common Name: Dichlobenil
Chemical Name: IUPAC: 2,6-Dichlorobenzonitrile
CAS Name: 2,6-Dichlorobenzonitrile

CAS No. 1194-65-6 Synonyms 027401

Primary Reviewer: Kindra Bozicevich

Staff Scientist, Cambridge Environmental, Inc . Date: 6/15/2012

Secondary Reviewer(s): John Marton

Staff Scientist, Cambridge Environmental, Inc. Date: 7/9/2012

Primary Reviewer (and final additional reviewer): Elyssa Arnold Biologist, EPA/OPP/EFED/ERB2 Date: 4/28/2015

Additional Reviewer(s): Catherine Aubee Biologist, EPA/OPP/EFED/ERB4 Date: 4/28/2015

Date Evaluation Completed: 28-04-2015

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DER Template Version: 22 September 2011 2015.05.29 07:57:53 -04'00'Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

CITATION: Tobor-Kaplon, M.A. 2011. Amphibian Metamorphosis (Frog) Test With Dichlobenil Technical. Unpublished study performed by Notox B.V., Hambakenwetering 7, 5231 DD’s-Hertogenbosch, The
Netherlands. Notox Project 495785. Notox Substance 202841/A. Study sponsored by Chemtura Corporation, 199 Benson Road, Middlebury, CT 06749. Study completed October 20, 2011.

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

D

isclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted.

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EPA MRID Number 48616101

EXECUTIVE SUMMARY

The 21-day assay of Dichlobenil (99.3% purity) 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 a negative control and a solvent control (0.1 mL/L; acetone) and nominal dichlobenil concentrations of 0.02, 0.2, and 2.0 mg a.i./L. Mean-measured concentrations were <0.001 (<LOQ; negative and solvent), 0.019, 0.19, and 1.9 mg a.i./L. The test system was maintained at 21.0 to 22.5°C and a pH of 7.3 to 8.2.

No mortalities were observed in the solvent control, or low or mid treatment groups. One mortality each was recorded in the negative control and high treatment group. Clinical signs ( i.e. , behavioral and other sublethal effects) including curved spine were observed in tadpoles in the solvent control and 0.019 and 0.19 mg a.i./L
treatment groups. This effect did not appear to be related to the test substance.

No significant acceleration or delay of body weight , median NF developmental stage, snout-to-vent length (SVL), hind-limb length (HLL), or normalized (for SVL) HLL was observed at Day 7 compared to the negative
control. Further, no asynchronous development was observed.

At Day 21, >20% of amphibians were at NF stage >60. When excluding organisms with NF stages greater than 60, dichlobenil significantly increased Day 21 normalized HLL at all test concentrations (7-10% greater than negative control for non-late stage amphibians, p<0.05). No significant treatment effects were detected
for Day 21 median developmental stage, body weight, or SVL for non-late stage amphibians compared to the negative control. Additionally, no significant effects on development or growth were observed when comparing treatment and negative control late-stage amphibians (NF>60). Normalized HLL at Day 21 was also significantly greater (10%, p<0.05) in the solvent control than in the negative control, as was NF development
stage.

There was a lower incidence and severity of follicular cell hypertrophy in the high treatment group compared to the negative or solvent control. No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, or follicular cell hyperplasia, and there were no differences in the amount or quality of follicular colloids.

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EPA MRID Number 48616101

All test validity and performance criteria were met in this study.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).

Results Synopsis:

Test organism NF stage at test initiation: NF 51 Test organism total length at test initiation (optional): Not Reported
Test type: flow-through

Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 in the Amphibian Metamorphosis Assay (AMA) with Dichlobenil.

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 (0.020) No No No Yes NA NA No 0.19 (0.20) No No No Yes NA NA No 1.9 (2.0) No No No Yes NA NA Yes

Abbreviations: Diff. Difference. NA Not applicable.

1 A “yes” indicates a significant difference based on comparison to the negative control.

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

the DER.

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

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I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program Test Guidelines 890.1100, Amphibian Metamorphosis (Frog), and OECD
Guideline for Testing of Chemicals No. 231, Amphibian Metamorphosis Assay. The following deviations from U.S. EPA, EDSP 890.1100 and

OECD 231 were noted:

1. Parental frogs were not maintained as in-house stock. 2. The flow-through rate was ca . 33 mL/min. EPA recommends a flow-through rate of 25

mL/min. 3. The hardness of the water used in the study ranged from 161-179 mg CaCO 3 /L. EPA recommends hardness ranging from 40 to 48 mg/L as CaCO 3 . 4. Solvent concentration (0.1 mL/L) exceeded the EPA recommended limit (0.02 mL/L), though it was within acceptable limits specified in OECD guidance (0.1 mL/L).
5. Development stage and normalized HLL were significantly increased in the solvent control compared to the negative controls at Day 21.

These deviations do not impact the results or interpretation of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance
statements were provided. This study was conducted in compliance with all pertinent OECD (OECD, 1998) Good Laboratory Practice regulations with
the following exceptions: analyses of the concentration of iodide under test conditions was performed by an outside laboratory.

A. Test Material Dichlobenil Technical

Description: White powder with lumps

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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. : AM9K12A222

Purity: 99.3% a.i.

Impurities: None reported

Stability of Compound: Mean measured concentrations yielded overall recoveries of 84-107% of

nominal with a coefficient of variation of d 10%.

Storage Conditions of

Test Chemicals: At room temperature in the dark.

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101B. 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): Daudin1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the TestGuidelines for the Endocrine Disruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3,2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Page7of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?No EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?Not applicableAcclimation period for parentalfrogs (if applicable):Days 1 to 13 postfertilizationDetails on parental feeding: During acclimation,juveniles were fed SeraMicron® twice dailyDetails on parental health: Not reportedPage8of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 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):3Number of eggs sampled perspawn:Minimum 1500NF 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:13 days postfertilization (dpf)Mean total length at testinitiation (if reported):Not measuredRange of total length at testinitiation (if reported):Not measuredPage9of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?No. Selection wasbased on viability andhatching success.Details on larval selection: Selection was basedon viability andhatching success.Loading rate (rearing density): 2 tadpoles/LTwenty tadpoles per test vessel (10 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.Page10of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsType of food:Sera Micron® EPA recommends Sera Micron ® throughout pre-exposure (after NF stage 45/46) and during the entire 21-d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food:Sera GmbH,Heinsburg, GermanyIodide concentration in diet (ifknown):Not reported 40-50 mg/kg, based on personalcommunication between the lab andDow ChemicalsFrequency of feeding:Twice per day EPA recommends that feeding occur at least twice per day.Page11of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime: During the exposureperiod, tadpolesreceived between 30and 80mg/animal/day. 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.Page12of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem (if applicable):Continuous-flow Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable): ca. 33 mL/min 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 forConducting Aquatic Laboratory Studies.Page13of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Stock solutions weredosed via a computercontrolled system intomixing chambers thenpumped into the testchambers usingperistaltic pumps.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.Page14of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(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: Adjusted ISO medium,formulated using ROwater (tap-water purifiedby reverse osmosis). 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.Page15of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesIodide supplementation inwater?Yes. KI added duringformulation for aresulting concentration of1.96Pg KI/L (1.5 mgI-/L). If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 μ g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Test vessel type/materials: Tanks consisting of glassplates sealed with a thinfilm of silicone andcovered by a removablePerspex plateEPA 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:42x13.5x19.5 cmFill volume: ca. 10 LPage16of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3) 161 179 166 Weekly EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH 7.38.27.7WeeklyEPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)5.98.88.3Weekly EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration
>3.5 mg/L (>40% air saturation).Page17of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)21.5 22.522.1Weekly EPA recommends temperature 22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide (μg/L) 3.6 3.6 NA Once, Preliminary EPA recommends aquatic iodide range

0.5 – 10 μ g/L (supplemental iodide should not exceed 2 μ g/L).Page18of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAmmonia NANANANA 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 NANANANAPerchlorate NANANA NAChlorate NANANANATotal Alkalinity (mmolHCO3-/L)0.8 1.2 1.0 WeeklySpecific Conductance(μmhos/cm)Not reported Not reported Not reported Not reportedPage19of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?NoIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?NA 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:NALife stage:NATest duration:NAAdditional details:NAPage20of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 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:Not reported 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:Dilution factor of 10 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Page21of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of test concentrations:3 plus negative andsolvent controls EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?NoIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ): 0.001 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: Measurements weremade on samplescollected on Days -2,0, 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.Page22of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):4 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:80 (20/rep)Was a solvent used?YesSolvent type (if applicable): AcetonePage23of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration(if applicable):0.1 ml/L targetconcentration 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).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquaticorganisms: A critical review. Review. Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. EnvironmentalHealth and Safety Publications. Series on Testing and Assessment. No. 23. Paris, France.Page24of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:600-1200 lux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:No anesthesia wasused during the stagingof tadpoles at testinitiation.Report indicated that no undissolvedmaterial was present in mixingchambers.Page25of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Dichlobenil.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0.0 <0.001 NA EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control (ifapplicable)0.0 <0.001 NATreatment 10.020.01910.0Treatment 20.20.195.2Treatment 32.01.94.2Abbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:snout-vent length (SVL), hind limb length (HLL), normalized HLL, body weight, anddevelopmental stage (Days 7 and 21), mortality and sub-lethal effects (daily), thyroidhistology (Days 7 and 21)Page26of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Were raw (individual) data provided?Yes EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (Days 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-based larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.Page27of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

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II. RESULTS AND DISCUSSION

A. Results

Throughout the 21-day exposure period, no mortalities were observed in the solvent control, 0.019 or 0.19 mg a.i./L treatment groups, and 1 mortality each was recorded in the negative control and 1.9 mg a.i./L
treatment group.

Table 9: Larval Mortality in X enopus laevis

.

Treatment (mg a.i./L)

[mean-measured] Larval Mortality

Day 7 1 Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 0 0 60 1 1.7

Solvent Control 80 0 0 60 0 0

0.019 80 0 0 60 0 0

0.19 80 0 0 60 0 0

1.9 80 0 0 60 1 1.7

Abbreviations: NA Not applicable.

1 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice (total across all four replicates).

At Day 7, median NF development stage was 55 in the negative and solvent controls and for all treatment groups and no tadpoles exhibited asynchronous development. By Day 21, median NF development stage ranged from 60-61 in the negative and solvent controls and all treatment groups and again no tadpoles exhibited asynchronous development. Late stage (NF>60) tadpoles were
present in all replicates of all treatment groups at Day 21, totaling 36, 58, 47, 45, and 42% of all

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surviving tadpoles in the negative control, solvent control, and 0.019, 0.19, and 1.9 mg a.i./L test groups, respectively.

Table 10: Larval Development in X enopus 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 55 0 4 60 0

Solvent Control 4 55 0 4 61 0

0.019 4 55 0 4 60.5 0

0.19 4 55 0 4 60 0

1.9 4 55 0 4 60 0

Abbreviations: NA Not applicable.

Normalized HLL on Day 7 was comparable among groups and averaged 0.19 mm in the negative and solvent controls and ranged from 0.19-0.20 mm in all treatment groups. On Day 21, normalized HLL averaged 1.15 and 1.25 mm in the negative and solvent controls, respectively, and ranged from 1.24-1.26 mm in all treatment groups.

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

Treatment (mg a.i./L)
[mean-measured] Hind Limb Length (HLL)

Day 7 Day 21 1

n Mean (mm) ±SD HLL:

SVL 2 n Mean (mm) ±SD HLL:

SVL 2

Negative Control 4 3.05 0.04 0.19 4 23.1 0.81 1.00

Solvent Control 4 2.92 0.14 0.19 4 25.2 0.96 1.10

0.019 4 3.05 0.23 0.19 4 24.7 0.96 1.07

0.19 4 3.13 0.06 0.20 4 25.3 0.74 1.09

1.9 4 3.14 0.09 0.20 4 25.1 0.67 1.11

Abbreviations: NA Not applicable. SD Standard deviation.

1 The Day 21 results include only non-late stage organisms (NF stage  60). Summary tables for late
stage amphibians are provided in Reviewer Analysis section.

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

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 12: Larval Growth in X enopus laevis.Treatment(mg a.i./L)[meanmeasured]Snout-Vent Length (SVL)Body Weight1Day 7Day 212Day 7Day 212nMean(mm)±SDnMean(mm)±SDnMean(g)±SDnMean(g)±SDNegativeControl4 15.76 0.82 4 23.1 0.41 4 0.447 0.05 4 1.71 0.05SolventControl4 15.82 0.91 4 23.0 0.92 4 0.445 0.05 4 1.76 0.210.019 4 15.92 0.95 4 23.2 0.73 4 0.452 0.06 4 1.74 0.140.19 4 16.00 0.46 4 23.3 0.43 4 0.478 0.05 4 1.79 0.121.9 4 16.07 0.22 4 22.8 0.39 4 0.491 0.02 4 1.67 0.07Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.1Also referred to as “wet weight” in the test guideline.2The Day 21 results include only non-late stage organisms (NF stage60). Summary tables for late stage amphibians are provided in ReviewerAnalysis section.Page31of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

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The only histopathology finding was a decrease in the incidence of follicular cell hypertrophy characterized and graded by the subjective % of tall columnar epithelium relative to cuboidal epithelium. There was a lower incidence and severity of follicular cell hypertrophy in the 2.0 mg a.i./L treatment group. As thyroid follicular cells normally increase in height during tadpole development, this finding suggests some retardation of metamorphosis at 2.0 mg a.i./L.

No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, follicular cell hyperplasia and there were no differences in the amount or quality of follicular colloids.

Table 13: Gross Pathology and Histopathology of the Thyroid Gland in X enopus 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 20 20 20 20 3 20 20

1 20 0 20 0 20 10 20 0

2 20 0 20 0 20 6 20 0

3 20 0 20 0 20 1 20 0

Solvent Control 0 20 20 20 20 20 1 20 20

1 20 0 20 0 20 12 20 0

2 20 0 20 0 20 7 20 0

3 20 0 20 0 20 0 20 0

0.019 0 20 20 20 20 20 5 20 20

1 20 0 20 0 20 9 20 0

2 20 0 20 0 20 6 20 0

3 20 0 20 0 20 0 20 0

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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

0.19 0 20 20 20 20 20 2 20 20

1 20 0 20 0 20 10 20 0

2 20 0 20 0 20 7 20 0

3 20 0 20 0 20 1 20 0

1.9 0 20 20 20 20 20 13 20 20

1 20 0 20 0 20 6 20 0

2 20 0 20 0 20 1 20 0

3 20 0 20 0 20 0 20 0

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

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 14: Additional Thyroid Gland Histopathology Observations in X enopus laevis.Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 20 20 20 NA NA NA NA NA NA1 20 0 20 0 NA NA NA NA NA NA2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NASolvent Control 0 20 20 20 20 NA NA NA NA NA NA1 20 0 20 0 NA NA NA NA NA NA2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA0.019 0 20 20 20 20 NA NA NA NA NA NA1 20 0 20 0 NA NA NA NA NA NA2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NAPage34of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.19 0 20 20 20 20 NA NA NA NA NA NA1 20 0 20 0 NA NA NA NA NA NA2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA1.9 0 20 20 20 20 NA NA NA NA NA NA1 20 0 20 0 NA NA NA NA NA NA2 20 0 20 0 NA NA NA NA NA NA3 20 0 20 0 NA NA NA NA NA NA1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECDNo. 82 for reference.A single tadpole in the negative control showed loss of equilibrium and was swimming at the surface from Day 16until the end of exposure. No signs of severe distress were observed. On Day 7 of exposure, tadpoles exposed todichlobenil were easier to catch than control tadpoles. This difference was not strong enough to consider it an effectand was not observed at the end of the test.Page35of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Eight percent of the tadpoles sacrificed at Day 7 of exposure had a bent tail. At Day 21, a total of 22% oforganisms had a bent tail, with 22, 28, 23, 23, and 13% exhibiting bent tail in the negative control, solvent control,0.019, 0.19 and 1.9 mg a.i./L, respectively. There was no relation between the number of incidences of tadpoleswith bent tail and the treatment.Table 15: Clinical Signs in X enopus laevis.Treatment(mg a.i./L)[mean-measured]Clinical SignsTypenIncidenceNegative ControlBent tail5913Loss of equilibrium and surfacing591Solvent ControlBent tail60170.019 Benttail 60140.19 Benttail 60141.9 Benttail 608Page36of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

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B. Study Author’s Analysis and Conclusions

Day 7 body weight and SVL and Day 7 and 21 normalized HLL were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test. One-way ANOVA with Dunnett’s test was used to analyze differences. Day 7 and Day 21 NF developmental stages were
analyzed using Jonckheere-Terpstra test. Day 21 snout-to-vent length and body weight were evaluated for normality using Shapiro-Wilk’s test per LateStage group. Nested ANOVA with Treatment and LateStage and interaction Treatment (LateStage) was used to analyze differences. Subsequently the
multiple Tukey-Kramer comparison was performed. All treatment data was compared to solvent control

data.

There was no significant treatment-related mortality. Statistically significant treatment-related effects were observed on Day 21 in the mean-measured 0.19 and 1.9 mg a.i./L treatment groups. These effects were significant decreases in development stage of Xenopus laevis tadpoles compared to the

solvent control.

A significant difference between blank and solvent control, in respect to development stage and normalized HLL, was found at Day 21. In both cases, results suggested acceleration of development in the acetone control. This was not confirmed by histopathology where no significant differences were found between controls. The study author attributes the observed difference to the intensive growth of microbial biomass in the solvent control compared to the blank control rather than to the presence of the solvent. This biomass is a source of additional food that could cause faster development.

C. Reviewer’s Analysis and Conclusions

Statistical Methods:

Data for Day 7 SVL, wet body weight, and normalized HLL exhibited monotonic responses and were analyzed using the Jonckheere-Terpstra test. Data for Day 7 normalized HLL exhibited non-monotonic responses and met assumptions of normality and variance so were analyzed using Dunnett’s test.
Data for Day 7 median values for developmental stage exhibited non-monotonic responses and were analyzed using the Mann-Whitney test. The reviewer compared the negative and solvent control for all endpoints at Day 7 using two-sided Student’s t-tests.

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At Day 21, >20% of the frogs across all treatment and control groups were at NF stage >60. After stage 60, tadpoles show a reduction in size and weight due to tissue resorption and reduction of
absolute water content. To account for this difference, a two-factor nested ANOVA with concentration, late stage (yes or no), and their interaction was used to analyze Day 21 data, with Rep(Conc) and tadpole ID(rep) as random factors, as recommended by the test guideline. All data met the normality
and variance homogeneity requirements of ANOVA. Additionally, the interaction F-test was “sliced” into two additional ANOVA F-tests, one on the mean responses across concentrations for LateStage=’No’ and another on the mean responses across concentrations for LateStage=’Yes.’ However, this statistical approach was found to be insufficient. The variance component “replicate (treatment)” was
often inestimable due to a greater between-individual variance than between-replicate variance.
Therefore, further comparisons of treatment means against negative controls were done within each level of LateStage using identical methods to the Day 7 analysis. Normalized HLL for non-late stage tadpoles exhibited a monotonic trend and was analyzed using the Jonckheere-Terpstra test. Developmental stage was analyzed using the Mann-Whitney test. The remaining endpoints were analyzed using Dunnett’s test.

Negative and solvent control groups at 21 Days were compared using a nested two-factor ANOVA. Normalized HLL was significantly greater in the solvent control than in the negative control (10%, p<0.05). Using a Student’s t-test on the median NF development stage, the reviewer detected a significant difference in the solvent control (median 61) relative to the negative control (median 60) (p<0.05). No other variables differed between the two control groups.

Due to the differences detected between the solvent and negative control groups for normalized HLL and NF stage at Day 21, statistical comparisons of the treatment groups to the solvent control were conducted for these endpoints.

No asynchronous development was observed.

Results for late stage tadpoles are presented in Table 16. Tables in the Results section of this DER for SVL, body weight, HLL, and normalized HLL present results for non-late stage tadpoles only.

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Table 16: Day 21 Larval Growth in Late-Stage X enopus laevis

.

Treatment (mg a.i./L)

[ meanmeasured] Day 21 Larval Growth for Late-stage Amphibians

Hind Limb Length (mm) Snout-Vent Length (SVL) Body Weight 1 Normalized HLL

n Mean (mm) ±SD n Mean (mm) ±SD n Mean (g) ±SD n Mean (mm) ±SD

Negative

Control 4 26.8 0.52 4 19.1 0.50 4 1.21 0.10 4 1.41 0.05

Solvent

Control 4 27.8 0.22 4 19.7 0.33 4 1.29 0.06 4 1.42 0.01

0.019 4 27.9 0.43 4 19.3 0.45 4 1.21 0.07 4 1.46 0.03

0.19 4 28.3 0.74 4 19.5 0.47 4 1.26 0.06 4 1.45 0.05

1.9 4 28.2 0.96 4 19.5 0.54 4 1.23 0.07 4 1.45 0.04

Conclusions:

There was no significant effect on mortality in the study.

At Day 7, there were no statistically significant effects for any parameter tested, compared to the negative control data. There were also no statistically significant differences between the negative and solvent control groups for any endpoint.

At Day 21, significant increases in both HLL and normalized HLL from the negative control were detected across all treatment groups using the Jonckheere-Terpstra test (p<0.05) for non-late stage
tadpoles. For late stage tadpoles, significant increases were also seen at all treatment groups for HLL (Jonckheere-Terpstra, p<0.05), but not for normalized HLL. No significant treatment effects were
detected for Day 21 median developmental stage, wet body weight, or SVL for either late stage or non-late stage tadpoles when compared to the negative control.

There was a significant increase in normalized HLL in the solvent control compared to the negative control at Day 21. When compared to the solvent control, no difference was seen in normalized HLL

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for non-late stage tadpoles in any treatment group. For late stage tadpoles, a significant increase was seen in the lowest treatment group compared to the solvent control (Jonckheere-Terpstra, p<0.05).
For the mid and high treatment groups, notable but non-significant increases were also observed (Jonckheere-Terpstra, p=0.06).

At Day 21, NF stage was greater in the solvent control (median 61) than negative control (median 60). NF stage was significantly decreased at Day 21 in all treatment groups compared to the solvent control (Jonckheere-Terpstra, p<0.05).

There was a lower incidence and severity of follicular cell hypertrophy in the 1.9 mg a.i./L treatment group. No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, follicular cell hyperplasia and there were no differences in the amount or quality of follicular colloids.

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 17: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Dichlobenil.Treatment(mg a.i./L)[meanmeasured]NF Developmental StageHind Limb Length3Asynchronous DevelopmentThyroid Gross andHistopathologyDay 7Day 214Day 7Day 21Day 7Day 21Day 21Non-Late Stage5Late-Stage6Median pMedian p% Diff. p% Diff. p% Diff. p% Diff. p%Diff. pTreatmentRelated Effects?(Yes/No)Negative Control 55 NA 60 NA 0 NA 0 NA 0 NA NA NA NA NA NoSolvent Control 55 NA61 0.0020 NA 9.62 0.030 0.46 0.826 NA NA NA NA No0.019 55 0.478 60.5 0.223 -2.56 0.8376.98 0.0293.01 0.441 NA NA NA NA No0.19 55 1.0 60 0.478 0 1.08.98 0.0052.83 0.486 NA NA NA NA No1.9 55 1.0 60 0.478 0 1.010.22 0.0022.83 0.486 NA NA NA NA YesStatistical Test Mann-Whitney Mann-Whitney DunnettJonckheereTerpstraDunnett NA NA NAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the solvent control. Conclusions regarding histopathology may be heavily weightedby the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.Page41of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 486161013Hind-limb length is normalized to snout-vent length (SVL).4Compared to the solvent control, using Jonckheere-Terpstra test for a decreasing trend: Percent differences: -1.22, -1.63, and -1.63; P-values: 0.048,0.014, 0.010; for the low, medium, and high treatment groups, respectively.5Compared to the solvent control, using Dunnett’s test: Percent differences -2.72, -0.91, and 0.23; P-values: 0.660, 0.977, 1.00; for the low, medium,and high treatment groups, respectively.6Compared to the solvent control, using Jonckheere-Terpstra test for an increasing trend: Percent differences: 2.65, 2.47, 2.47; P-values: 0.029, 0.061,0.060; for the low, medium, and high treatment groups, respectively.Page42of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101Table 18: Growth Endpoints1,2in the AMA with Dichlobenil.Treatment(mg a.i./L)[mean-measured]Snout-Vent LengthBody WeightDay 7Day 21Day 7Day 21Non-Late StageLate-StageNon-Late StageLate-Stage% Diff. p% Diff. p% Diff. p% Diff. p% Diff. p% Diff. pNegative Control 0 NA 0 NA 0 NA 0 NA 0 NA 0 NASolvent Control 0.38 0.463 -0.50 0.826 3.35 0.075 0.45 0.468 2.95 0.653 6.67 0.2130.019 1.05 0.386 0.28 0.996 1.13 0.869 1.03 0.500 1.90 0.941 0.21 1.000.19 1.52 0.232 1.12 0.818 2.43 0.432 6.85 0.232 4.68 0.562 4.15 0.6841.9 1.98 0.152 -1.27 0.759 2.15 0.522 9.71 0.056 -2.63 0.864 2.07 0.937Statistical TestJonckheereTerpstraDunnett DunnettJonckheere-TerpstraDunnett DunnettAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.2Unless otherwise specified, effects are considered statistically significant at p<0.05.Page43of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

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E. Study Deficiencies

The main study deficiencies were statistically significant differences in normalized HLL and NF development stage at Day 21 between the solvent and negative controls. Other deviations are noted in Section I. Materials and Methods were considered to be minor. Validity and performance criteria were met, including the following:

x Mortality did not exceed 10% for any given treatment;

x Three treatment levels, with all four uncompromised replicates, were available for analysis;

and

x Three treatment levels without overt toxicity were available for analysis.

F. Reviewer’s Comments

The reviewer’s conclusions differed from those reported by the study author. The reviewer’s analysis detected significant (p<0.05) increases in Day 21 normalized HLL at all treatment levels for non-late
stage tadpoles compared to the negative control. The study author did not detect these significant differences. The study author reported a statistically significant treatment-related delay (p<0.05) in
median developmental stage on Day 21 in the mean-measured 0.19 and 1.9 mg a.i./L treatment groups, relative to the solvent control. The reviewer detected this trend in the 0.019 mg a.i./L treatment group in addition to the 0.19 and 1.9 mg a.i./L treatment groups.. The study author and reviewer both detected a difference in normalized HLL between the negative and solvent controls (p<0.05). Thus,
the reviewer believes that the increase in normalized HLL seen in the treatment groups compared to the negative control may be the result of a solvent effect.

The study author attributed increased stage and normalized HLL at Day 21 in the solvent control compared to the negative control to the growth of microbial biomass in the solvent control tanks. The growth of microbial biomass is considered an effect of the solvent in the study that could impact the study endpoints and therefore needs to be evaluated across all treatment groups. Without this additional information, it is difficult to know whether effects measured in the study groups exposed to the test substance were either promoted or masked by the presence of the solvent.

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For all endpoints exhibiting monotonic trends and satisfying parametric assumptions, the OCSPP 890.1100 flowchart recommends interpretation of results using the Jonckheere-Terpstra test. However, according to historical EFED statistical guidance, the parametric William’s test should be
used to interpret statistical findings under these circumstances. For endpoints satisfying these conditions in this study, there was no difference in conclusions between the OCSPP 890.1100 flowchart and historical EFED methodologies.

III. REFERENCES

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

York.

OECD. 2007. Final report of the Validation of the Amphibian Metamorphosis Assay: Phase 2- Multichemical Interlaboratory Study. Environmental Health and Safety Publications. Series on testing and
Assessment. No. 76. Paris, France.

Cooke, A.S. 1977.. Effects of field application of the herbicides Diquat and Dichlobenil on amphibians. Environmental Pollution (12).

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity ofDichlobenil to Amphibians, Metamorphosis AssayEPA MRID Number 48616101APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:EndpointMonotonic? Parametric? 890.1100 EFEDComments7-day wet weight YesYesJonckheere:n.s. p>0.05William’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-stage median NoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-day SN-vent length YesYesJonckheere:n.s. p>0.05William’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-day hind-limblengthYes No Jonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-day normalizedHLLNo Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day stage median NoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day wet weight NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day SN-vent length NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day HLLNoYesDunnett’s:Dose 1 p=0.005Dose 2 p<0.001Dose 3 p=0.003Dunnett’s:Dose 1 p=0.005Dose 2 p<0.001Dose 3 p=0.003890.1100 and EFED same conclusions, a significant (p<0.05)promotion from control. Response was not monotonicallyincreasing, but there was an increasing trend.21-day normalizedHLLNo Yes Dunnett’s:Dose 1 p=0.020Dose 2 p=0.017Dose 3 p=0.039Dunnett’s:Dose 1 p=0.020Dose 2 p=0.017Dose 3 p=0.039890.1100 and EFED same conclusions, a significant (p<0.05)promotion from control. Response was not monotonicallyincreasing, but there was an increasing trend.Page46of107 DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

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**ANALYSIS OF 7-DAY VARIABLES**

027401 Dichlobenil 48616101 890.1100 **ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet weight (g) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.967 0.780 1.582 0.245 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

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

Ctrl 4 0.45 0.05 0.03 12.13 0.36, 0.53 Dose1 4 0.45 0.06 0.03 13.11 0.36, 0.55 Dose2 4 0.48 0.05 0.02 9.69 0.40, 0.55 Dose3 4 0.49 0.02 0.01 3.08 0.47, 0.51

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.44 0.39 0.51 . . Dose1 0.45 0.38 0.52 101.03 -1.03 Dose2 0.47 0.43 0.54 106.85 -6.85 Dose3 0.49 0.48 0.51 109.71 -9.71

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

Numerator df Denominator df F-stat P-value 3 12 0.79 0.525

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 0.45 . 0.47 . . . . .

.

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Dose1 0.45 0.998 0.47 0.801 . . . .

. Dose2 0.48 0.688 0.47 0.832 0.860 . . .

. Dose3 0.49 0.447 0.47 0.848 0.656 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 3 2.74 0.434

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 0.44 . . Dose1 0.45 1.000 0.500 Dose2 0.47 0.494 0.768 Dose3 0.49 0.235 0.944

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

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

Numerator df Denominator df F-stat P-value 3 12 0.79 0.525

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 -0.45 . -0.45 . . . . .

.

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Dose1 -0.45 0.998 -0.45 0.524 . . . .

. Dose2 -0.48 0.688 -0.48 0.239 0.860 . . .

. Dose3 -0.49 0.447 -0.49 0.141 0.656 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 3 2.74 0.434

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 -0.44 . . Dose1 -0.45 1.000 0.500 Dose2 -0.47 0.494 0.232 Dose3 -0.49 0.235 0.056

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

027401 Dichlobenil 48616101 890.1100 **ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (median) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.565 <.001 9.000 0.002 USE NON-PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

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EPA MRID Number 48616101

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

Ctrl 4 55.00 0.00 0.00 0.00 . , . Dose1 4 55.25 0.50 0.25 0.90 54.45, 56.05 Dose2 4 55.00 0.00 0.00 0.00 . , . Dose3 4 55.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 55.00 55.00 55.00 . . Dose1 55.00 55.00 56.00 100.45 -0.45 Dose2 55.00 55.00 55.00 100.00 0.00 Dose3 55.00 55.00 55.00 100.00 0.00

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

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

Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

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 55.00 . 55.13 . . . . .

. Dose1 55.25 0.389 55.13 0.833 . . . .

. Dose2 55.00 1.000 55.00 0.618 0.515 . . .

. Dose3 55.00 1.000 55.00 0.636 0.515 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.00 0.392

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

Page 50 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 55.00 . . Dose1 55.00 0.478 0.841 Dose2 55.00 1.000 0.500 Dose3 55.00 1.000 0.327

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

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

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

Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

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 -55.00 . -55.00 . . . . .

. Dose1 -55.25 0.389 -55.08 0.384 . . . .

. Dose2 -55.00 1.000 -55.08 0.410 0.515 . . .

. Dose3 -55.00 1.000 -55.08 0.425 0.515 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.00 0.392

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

Page 51 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl -55.00 . . Dose1 -55.00 0.478 0.159 Dose2 -55.00 1.000 0.500 Dose3 -55.00 1.000 0.673

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

027401 Dichlobenil 48616101 890.1100 **ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.956 0.584 2.765 0.088 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 15.76 0.82 0.41 5.21 14.45, 17.06 Dose1 4 15.92 0.95 0.48 5.99 14.41, 17.44 Dose2 4 16.00 0.46 0.23 2.85 15.27, 16.72 Dose3 4 16.07 0.22 0.11 1.35 15.73, 16.41

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 15.78 14.96 16.52 . . Dose1 15.72 15.00 17.26 101.05 -1.05 Dose2 15.89 15.59 16.63 101.52 -1.52 Dose3 16.05 15.85 16.33 101.98 -1.98

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

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

Numerator df Denominator df F-stat P-value 3 12 0.16 0.924

Page 52 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

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

Ctrl 15.76 . 15.94 . . . . .

. Dose1 15.92 0.972 15.94 0.730 . . . .

. Dose2 16.00 0.923 15.94 0.764 0.999 . . .

. Dose3 16.07 0.852 15.94 0.782 0.989 0.999 . .

.

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

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

Degrees of Freedom TestStat P-value 3 0.82 0.846

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 15.78 . . Dose1 15.72 0.889 0.614 Dose2 15.89 0.678 0.768 Dose3 16.05 1.000 0.848

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.16 0.924

Dunnett - testing each trt mean signif. different than control

Page 53 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

Ctrl -15.76 . -15.76 . . . . .

. Dose1 -15.92 0.972 -15.92 0.437 . . . .

. Dose2 -16.00 0.923 -16.00 0.398 0.999 . . .

. Dose3 -16.07 0.852 -16.07 0.348 0.989 0.999 . .

.

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

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

Degrees of Freedom TestStat P-value 3 0.82 0.846

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 -15.78 . . Dose1 -15.72 0.889 0.386 Dose2 -15.89 0.678 0.232 Dose3 -16.05 1.000 0.152

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

027401 Dichlobenil 48616101 890.1100 **ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05

Page 54 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 0.938 0.322 5.241 0.015 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 3.05 0.04 0.02 1.35 2.99, 3.12 Dose1 4 3.05 0.23 0.12 7.66 2.68, 3.42 Dose2 4 3.13 0.06 0.03 1.99 3.03, 3.23 Dose3 4 3.14 0.09 0.05 3.01 2.99, 3.29

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.06 3.00 3.09 . . Dose1 3.07 2.76 3.30 99.92 0.08 Dose2 3.15 3.04 3.18 102.54 -2.54 Dose3 3.13 3.05 3.25 102.78 -2.78

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

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

Numerator df Denominator df F-stat P-value 3 12 0.53 0.671

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 3.05 . 3.09 . . . . .

. Dose1 3.05 1.000 3.09 0.750 . . . .

. Dose2 3.13 0.745 3.09 0.783 0.824 . . .

. Dose3 3.14 0.693 3.09 0.801 0.783 1.000 . .

.

Page 55 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

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

Degrees of Freedom TestStat P-value 3 2.09 0.553

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 3.06 . . Dose1 3.07 1.000 0.500 Dose2 3.15 0.190 0.848 Dose3 3.13 0.346 0.913

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.53 0.671

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 -3.05 . -3.05 . . . . .

. Dose1 -3.05 1.000 -3.05 0.589 . . . .

. Dose2 -3.13 0.745 -3.13 0.268 0.824 . . .

. Dose3 -3.14 0.693 -3.14 0.250 0.783 1.000 . .

.

Page 56 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

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

Degrees of Freedom TestStat P-value 3 2.09 0.553

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 -3.06 . . Dose1 -3.07 1.000 0.500 Dose2 -3.15 0.190 0.152 Dose3 -3.13 0.346 0.087

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

027401 Dichlobenil 48616101 890.1100 **ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.931 0.253 1.333 0.310 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 0.20 0.01 0.01 6.62 0.17, 0.22 Dose1 4 0.19 0.01 0.01 7.44 0.17, 0.21 Dose2 4 0.20 0.01 0.00 2.96 0.19, 0.20 Dose3 4 0.20 0.01 0.00 2.96 0.19, 0.20

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

Page 57 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 0.20 0.18 0.21 . . Dose1 0.19 0.18 0.21 97.44 2.56 Dose2 0.20 0.19 0.20 100.00 0.00 Dose3 0.20 0.19 0.20 100.00 0.00

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

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

Numerator df Denominator df F-stat P-value 3 12 0.23 0.873

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 0.20 . 0.20 . . . . .

. Dose1 0.19 0.837 0.19 0.487 . . . .

. Dose2 0.20 1.000 0.19 0.518 0.903 . . .

. Dose3 0.20 1.000 0.19 0.535 0.903 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.04 0.791

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 0.20 . . Dose1 0.19 0.667 0.275 Dose2 0.20 1.000 0.560 Dose3 0.20 1.000 0.653

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL

Page 58 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

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

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

Numerator df Denominator df F-stat P-value 3 12 0.23 0.873

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 -0.20 . -0.19 . . . . .

. Dose1 -0.19 0.837 -0.19 0.718 . . . .

. Dose2 -0.20 1.000 -0.20 0.618 0.903 . . .

. Dose3 -0.20 1.000 -0.20 0.636 0.903 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.04 0.791

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 -0.20 . . Dose1 -0.19 0.667 0.725 Dose2 -0.20 1.000 0.440 Dose3 -0.20 1.000 0.347

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL

Page 59 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

Page 60 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA, NEGATIVE VS. SOLVENT CONTROLS** 

**Model Information**
**Data Set** WORK.CONTROLS **Dependent Variable BW**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 2 1 2 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 9 **Columns in Z** 12 **Subjects** 1 **Max Obs Per Subject** 119

**Number of Observations Number of Observations Read**  119 **Number of Observations Used** 119 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 13.85369605 **1** 3 11.54687535

. **2** 1 11.50307337 0.00002787 **3** 1 11.50014549 0.00000019 **4** 1 11.50012647 0.00000000

Convergence criteria met.

Page 61 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 6.051E-7 **Residual** 0.05619

**Fit Statistics -2 Res Log Likelihood**  11.5 **AIC (smaller is better)** 15.5 **AICC (smaller is better)** 15.6 **BIC (smaller is better)** 15.7

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 1 28.6 1.73 0.1988 **LateStage** 1 113 114.58 <.0001 **LateStage*Conc** 1 113 0.07 0.7939

**Model Information**
**Data Set** WORK.CONTROLS Dependent Variable **SVL Covariance Structure**  Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 2 1 2 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 9 **Columns in Z** 12 **Subjects** 1 **Max Obs Per Subject** 119

Page 62 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Number of Observations Number of Observations Read**  119 **Number of Observations Used** 119 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 416.81217773 **1** 3 416.46431653 0.00001392 **2** 1 416.46284202 0.00000004 **3** 1 416.46283803 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0.02902 **ID(Rep)** 3.316E-6 **Residual** 1.9178

**Fit Statistics -2 Res Log Likelihood**  416.5 **AIC (smaller is better)** 422.5 **AICC (smaller is better)** 422.7 **BIC (smaller is better)** 422.7

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 1 5.56 0.83 0.4009 **LateStage** 1 111 190.07 <.0001 **LateStage*Conc** 1 112 2.02 0.1575

**Model Information**
**Data Set** WORK.CONTROLS **Dependent Variable normHLL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

Page 63 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 2 1 2 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 9 **Columns in Z** 12 **Subjects** 1 **Max Obs Per Subject** 119

**Number of Observations Number of Observations Read**  119 **Number of Observations Used** 119 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 -143.34563423 **1** 4 -143.34563423 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 8.08E-21 **ID(Rep)** 0 **Residual** 0.01498

**Fit Statistics -2 Res Log Likelihood**  -143.3 **AIC (smaller is better)** -141.3 **AICC (smaller is better)** -141.3 **BIC (smaller is better)** -141.3

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 1 115 5.06 0.0263 **LateStage** 1 115 250.13 <.0001 **LateStage*Conc** 1 115 4.04 0.0467

Page 64 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Model Information**
**Data Set** WORK.CONTROLS **Dependent Variable NFStage**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 2 1 2 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 9 **Columns in Z** 12 **Subjects** 1 **Max Obs Per Subject** 119

**Number of Observations Number of Observations Read**  119 **Number of Observations Used** 119 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 148.80185351 **1** 3 147.22032577 0.00002081 **2** 1 147.21963892 0.00000004 **3** 1 147.21963753 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0.003482 **ID(Rep)** 1.396E-6 **Residual** 0.1820

Page 65 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Fit Statistics -2 Res Log Likelihood**  147.2 **AIC (smaller is better)** 153.2 **AICC (smaller is better)** 153.4 **BIC (smaller is better)** 153.5

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 1 4.86 0.36 0.5733 **LateStage** 1 111 564.00 <.0001 **LateStage*Conc** 1 111 2.40 0.1239

Page 66 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable BW**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 15 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 238

**Number of Observations Number of Observations Read**  238 **Number of Observations Used** 238 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 18.95324583 **1** 3 18.87287201 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0.000422 **ID(Rep)** 1.092E-9 **Residual** 0.05618

Page 67 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Fit Statistics -2 Res Log Likelihood**  18.9 **AIC (smaller is better)** 24.9 **AICC (smaller is better)** 25.0 **BIC (smaller is better)** 27.2

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 10.3 0.97 0.4418 **LateStage** 1 228 246.84 <.0001 **LateStage*Conc** 3 226 0.64 0.5894

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable SVL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 15 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 238

**Number of Observations Number of Observations Read**  238 **Number of Observations Used** 235 **Number of Observations Not Used** 3

Page 68 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 818.40606947 **1** 1 818.40606947 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 0 **Residual** 1.9135

**Fit Statistics -2 Res Log Likelihood**  818.4 **AIC (smaller is better)** 820.4 **AICC (smaller is better)** 820.4 **BIC (smaller is better)** 821.2

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 227 0.88 0.4507 **LateStage** 1 227 415.19 <.0001 **LateStage*Conc** 3 227 0.59 0.6192

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable normHLL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

Page 69 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Dimensions Covariance Parameters**  3 **Columns in X** 15 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 238

**Number of Observations Number of Observations Read**  238 **Number of Observations Used** 235 **Number of Observations Not Used** 3

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 -298.28177002 **1** 1 -298.28177002 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 0 **Residual** 0.01398

**Fit Statistics -2 Res Log Likelihood**  -298.3 **AIC (smaller is better)** -296.3 **AICC (smaller is better)** -296.3 **BIC (smaller is better)** -295.5

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 227 4.15 0.0069 **LateStage** 1 227 564.48 <.0001 **LateStage*Conc** 3 227 0.97 0.4055

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable NFStage**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile

Page 70 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Model Information Fixed Effects SE Method**  Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 2 No Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 15 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 238

**Number of Observations Number of Observations Read**  238 **Number of Observations Used** 238 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 316.76878266 **1** 4 316.76878266

. **2** 1 316.76447065 0.00000076 **3** 1 316.76443009 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 4.909E-9 **Residual** 0.2062

**Fit Statistics -2 Res Log Likelihood**  316.8 **AIC (smaller is better)** 320.8 **AICC (smaller is better)** 320.8 **BIC (smaller is better)** 322.3

Page 71 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 154 0.25 0.8617 **LateStage** 1 229 989.47 <.0001 **LateStage*Conc** 3 229 1.23 0.2978

Page 72 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA, SLICED BY LATESTAGE**

**LateStage=No**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable BW**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 No **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 137

**Number of Observations Number of Observations Read**  137 **Number of Observations Used** 137 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 32.75356998 **1** 3 31.68188505 0.00000124 **2** 1 31.68175253 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0.002448 **ID(Rep)** 3.019E-8 **Residual** 0.06464

Page 73 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Fit Statistics -2 Res Log Likelihood**  31.7 **AIC (smaller is better)** 37.7 **AICC (smaller is better)** 37.9 **BIC (smaller is better)** 40.0

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 11.4 1.00 0.4258 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=Yes**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable** BW **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 101

**Number of Observations Number of Observations Read**  101 **Number of Observations Used** 101 **Number of Observations Not Used** 0

Page 74 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 -20.03416014 **1** 4 -20.03416014 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 6.19E-25 **Residual** 0.04170

**Fit Statistics -2 Res Log Likelihood**  -20.0 **AIC (smaller is better)** -18.0 **AICC (smaller is better)** -18.0 **BIC (smaller is better)** -17.3

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 97 0.58 0.6307 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=No**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable SVL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 No **Conc** 4 1 3 4 5 **Rep** 4 A B C D

Page 75 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 137

**Number of Observations Number of Observations Read**  137 **Number of Observations Used** 136 **Number of Observations Not Used** 1

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 503.00923116 **1** 4 503.00923116 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 1.78E-19 **ID(Rep)** 0 **Residual** 2.3774

**Fit Statistics -2 Res Log Likelihood**  503.0 **AIC (smaller is better)** 505.0 **AICC (smaller is better)** 505.0 **BIC (smaller is better)** 505.8

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 132 0.64 0.5913 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=Yes**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable SVL**  **Covariance Structure** Variance Components **Estimation Method** REML

Page 76 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Model Information Residual Variance Method**  Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 101

**Number of Observations Number of Observations Read**  101 **Number of Observations Used** 99 **Number of Observations Not Used** 2

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 305.02510515 **1** 3 304.99527820 0.00000130 **2** 1 304.99519249 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 2.247E-7 **Residual** 1.2627

**Fit Statistics -2 Res Log Likelihood**  305.0 **AIC (smaller is better)** 309.0 **AICC (smaller is better)** 309.1 **BIC (smaller is better)** 310.5

Page 77 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 76 1.00 0.3959 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=No**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable normHLL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 No **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 137

**Number of Observations Number of Observations Read**  137 **Number of Observations Used** 136 **Number of Observations Not Used** 1

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 -165.02357623 **1** 4 -165.02357623

. **2** 1 -165.05525143 0.00000803 **3** 1 -165.05695576 0.00000003 **4** 1 -165.05696279 0.00000000

Page 78 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 1.959E-9 **Residual** 0.01502

**Fit Statistics -2 Res Log Likelihood**  -165.1 **AIC (smaller is better)** -161.1 **AICC (smaller is better)** -161.0 **BIC (smaller is better)** -159.5

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 86 5.05 0.0029 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=Yes**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable normHLL**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 101

Page 79 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Number of Observations Number of Observations Read**  101 **Number of Observations Used** 99 **Number of Observations Not Used** 2

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 -134.25470852 **1** 4 -134.25470852 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 0 **Residual** 0.01245

**Fit Statistics -2 Res Log Likelihood**  -134.3 **AIC (smaller is better)** -132.3 **AICC (smaller is better)** -132.2 **BIC (smaller is better)** -131.5

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 95 0.74 0.5296 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=No**

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable NFStage**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

Page 80 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Class Level Information Class**  **Levels Values**  **LateStage** 1 No **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 137

**Number of Observations Number of Observations Read**  137 **Number of Observations Used** 137 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 84.66233974 **1** 4 84.66233974 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 0 **Residual** 0.09951

**Fit Statistics -2 Res Log Likelihood**  84.7 **AIC (smaller is better)** 86.7 **AICC (smaller is better)** 86.7 **BIC (smaller is better)** 87.4

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 133 1.78 0.1536 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

**LateStage=Yes**

Page 81 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Model Information**
**Data Set** WORK.NEGCONT **Dependent Variable NFStage**  **Covariance Structure** Variance Components **Estimation Method** REML **Residual Variance Method** Profile **Fixed Effects SE Method** Kenward-Roger **Degrees of Freedom Method** Kenward-Roger

**Class Level Information Class**  **Levels Values**  **LateStage** 1 Yes **Conc** 4 1 3 4 5 **Rep** 4 A B C D

**Dimensions Covariance Parameters**  3 **Columns in X** 10 **Columns in Z** 20 **Subjects** 1 **Max Obs Per Subject** 101

**Number of Observations Number of Observations Read**  101 **Number of Observations Used** 101 **Number of Observations Not Used** 0

**Iteration History Iteration**  **Evaluations -2 Res Log Like**  **Criterion 0**  1 187.13083747 **1** 3 186.81514920 0.00050566 **2** 2 186.81311123 0.00000316 **3** 1 186.81309770 0.00000000

Convergence criteria met.

**Covariance Parameter Estimates**
**Cov Parm Estimate**  **Rep(Conc)** 0 **ID(Rep)** 1.963E-7 **Residual** 0.3473

Page 82 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**Fit Statistics -2 Res Log Likelihood**  186.8 **AIC (smaller is better)** 190.8 **AICC (smaller is better)** 190.9 **BIC (smaller is better)** 192.4

**Type 3 Tests of Fixed Effects Effect**  **Num DF Den DF**  **F Value Pr > F**  **Conc** 3 73.5 0.31 0.8186 **LateStage** 0

. . . **LateStage*Conc** 0

. . .

Page 83 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**ANALYSIS OF 21-DAY VARIABLES: NON-LATESTAGE ONLY**

027401 Dichlobenil 48616101 890.1100 ALL TADPOLES FOR NF STAGE ANALYSIS **ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.851 0.014 6.000 0.010 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 60.00 0.00 0.00 0.00 . , . Dose1 4 60.50 0.58 0.29 0.95 59.58, 61.42 Dose2 4 60.25 0.50 0.25 0.83 59.45, 61.05 Dose3 4 60.25 0.50 0.25 0.83 59.45, 61.05

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 60.00 60.00 60.00 . . Dose1 60.50 60.00 61.00 100.83 -0.83 Dose2 60.00 60.00 61.00 100.42 -0.42 Dose3 60.00 60.00 61.00 100.42 -0.42

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

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

Numerator df Denominator df F-stat P-value 3 12 0.80 0.517

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

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

Page 84 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 60.00 . 60.25 . . . . .

. Dose1 60.50 0.322 60.25 0.850 . . . .

. Dose2 60.25 0.782 60.25 0.877 0.864 . . .

. Dose3 60.25 0.782 60.25 0.891 0.864 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 2.50 0.475

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 60.00 . . Dose1 60.50 0.223 0.937 Dose2 60.00 0.478 0.783 Dose3 60.00 0.478 0.691

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.80 0.517

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

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

Page 85 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl -60.00 . -60.00 . . . . .

. Dose1 -60.50 0.322 -60.33 0.192 . . . .

. Dose2 -60.25 0.782 -60.33 0.205 0.864 . . .

. Dose3 -60.25 0.782 -60.33 0.212 0.864 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 2.50 0.475

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

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.968 0.813 1.347 0.305 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 1.71 0.05 0.02 2.66 1.64, 1.78 Dose1 4 1.74 0.14 0.07 7.89 1.52, 1.96 Dose2 4 1.79 0.12 0.06 6.93 1.59, 1.99 Dose3 4 1.67 0.07 0.03 4.09 1.56, 1.77

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.72 1.65 1.75 . . Dose1 1.78 1.55 1.87 101.90 -1.90 Dose2 1.79 1.65 1.94 104.68 -4.68

Page 86 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose3 1.65 1.61 1.75 97.37 2.63

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

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

Numerator df Denominator df F-stat P-value 3 12 1.09 0.392

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 1.71 . 1.75 . . . . .

. Dose1 1.74 0.941 1.75 0.780 . . . .

. Dose2 1.79 0.562 1.75 0.812 0.909 . . .

. Dose3 1.67 0.864 1.67 0.358 0.706 0.344 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.01 0.390

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 1.72 . . Dose1 1.78 0.413 0.845 Dose2 1.79 0.489 0.865 Dose3 1.65 0.410 0.336

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

Page 87 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

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

Numerator df Denominator df F-stat P-value 3 12 1.09 0.392

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 -1.71 . -1.71 . . . . .

. Dose1 -1.74 0.941 -1.73 0.449 . . . .

. Dose2 -1.79 0.562 -1.73 0.479 0.909 . . .

. Dose3 -1.67 0.864 -1.73 0.495 0.706 0.344 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.01 0.390

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 -1.72 . . Dose1 -1.78 0.413 0.155 Dose2 -1.79 0.489 0.135 Dose3 -1.65 0.410 0.664

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

Page 88 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.948 0.457 0.616 0.618 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 23.08 0.41 0.21 1.80 22.42, 23.74 Dose1 4 23.15 0.73 0.36 3.15 21.99, 24.31 Dose2 4 23.34 0.43 0.21 1.83 22.66, 24.02 Dose3 4 22.79 0.39 0.20 1.72 22.17, 23.41

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 23.11 22.56 23.56 . . Dose1 23.35 22.11 23.78 100.28 -0.28 Dose2 23.39 22.78 23.81 101.12 -1.12 Dose3 22.77 22.43 23.19 98.73 1.27

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

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

Numerator df Denominator df F-stat P-value 3 12 0.80 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 23.08 . 23.19 . . . . .

.

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DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 23.15 0.996 23.19 0.703 . . . .

. Dose2 23.34 0.818 23.19 0.737 0.949 . . .

. Dose3 22.79 0.759 22.79 0.286 0.757 0.453 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.42 0.331

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 23.11 . . Dose1 23.35 0.678 0.718 Dose2 23.39 0.494 0.810 Dose3 22.77 0.413 0.242

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.80 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

Ctrl -23.08 . -23.08 . . . . .

.

Page 90 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -23.15 0.996 -23.09 0.572 . . . .

. Dose2 -23.34 0.818 -23.09 0.606 0.949 . . .

. Dose3 -22.79 0.759 -23.09 0.624 0.757 0.453 . .

.

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

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

Degrees of Freedom TestStat P-value 3 3.42 0.331

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 -23.11 . . Dose1 -23.35 0.678 0.282 Dose2 -23.39 0.494 0.190 Dose3 -22.77 0.413 0.758

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

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.922 0.180 0.460 0.715 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Page 91 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 4 23.07 0.81 0.41 3.52 21.77, 24.36 Dose1 4 24.71 0.96 0.48 3.89 23.18, 26.24 Dose2 4 25.26 0.74 0.37 2.92 24.09, 26.44 Dose3 4 25.12 0.67 0.34 2.68 24.04, 26.19

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 23.01 22.21 24.03 . . Dose1 24.71 23.64 25.79 107.14 -7.14 Dose2 25.60 24.16 25.69 109.53 -9.53 Dose3 25.07 24.44 25.89 108.89 -8.89

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

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

Numerator df Denominator df F-stat P-value 3 12 6.33 0.008

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

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

Ctrl 23.07 . 24.54 . . . . .

. Dose1 24.71 0.034 24.54 0.996 . . . .

. Dose2 25.26 0.006 24.54 0.998 0.769 . . .

. Dose3 25.12 0.009 24.54 0.998 0.892 0.994 . .

.

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

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

Degrees of Freedom TestStat P-value 3 8.32 0.040

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 23.01 . .

Page 92 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 24.71 0.103 0.978 Dose2 25.60 0.067 0.994 Dose3 25.07 0.067 0.994

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

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

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

Numerator df Denominator df F-stat P-value 3 12 6.33 0.008

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

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

Ctrl -23.07 . -23.07 . . . . .

. Dose1 -24.71 0.034 -24.71 0.008 . . . .

. Dose2 -25.26 0.006 -25.19 0.002 0.769 . . .

. Dose3 -25.12 0.009 -25.19 0.002 0.892 0.994 . .

.

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

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

Degrees of Freedom TestStat P-value 3 8.32 0.040

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 -23.01 . .

Page 93 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -24.71 0.103 0.022 Dose2 -25.60 0.067 0.006 Dose3 -25.07 0.067 0.006

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.954 0.555 0.071 0.974 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 1.00 0.03 0.01 2.62 0.96, 1.04 Dose1 4 1.07 0.04 0.02 3.83 1.01, 1.14 Dose2 4 1.09 0.03 0.02 3.02 1.04, 1.15 Dose3 4 1.11 0.03 0.02 2.81 1.06, 1.15

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.00 0.98 1.03 . . Dose1 1.08 1.02 1.12 106.98 -6.98 Dose2 1.10 1.05 1.13 108.98 -8.98 Dose3 1.12 1.06 1.13 110.22 -10.22

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

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

Numerator df Denominator df F-stat P-value 3 12 7.54 0.004

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

Page 94 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

Ctrl 1.00 . 1.07 . . . . .

. Dose1 1.07 0.030 1.07 0.997 . . . .

. Dose2 1.09 0.006 1.07 0.998 0.830 . . .

. Dose3 1.11 0.003 1.07 0.999 0.534 0.950 . .

.

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

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

Degrees of Freedom TestStat P-value 3 8.59 0.035

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 1.00 . . Dose1 1.08 0.123 0.971 Dose2 1.10 0.066 0.995 Dose3 1.12 0.066 0.998

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

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

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

Numerator df Denominator df F-stat P-value 3 12 7.54 0.004

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

Page 95 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

Ctrl -1.00 . -1.00 . . . . .

. Dose1 -1.07 0.030 -1.07 0.007 . . . .

. Dose2 -1.09 0.006 -1.09 0.001 0.830 . . .

. Dose3 -1.11 0.003 -1.11 <.001 0.534 0.950 . .

.

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

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

Degrees of Freedom TestStat P-value 3 8.59 0.035

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 -1.00 . . Dose1 -1.08 0.123 0.029 Dose2 -1.10 0.066 0.005 Dose3 -1.12 0.066 0.002

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

Page 96 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

**ANALYSIS OF 21-DAY VARIABLES: LATESTAGE ONLY**

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.968 0.801 0.173 0.913 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 1.21 0.10 0.05 8.17 1.05, 1.36 Dose1 4 1.21 0.07 0.03 5.71 1.10, 1.32 Dose2 4 1.26 0.06 0.03 4.98 1.16, 1.35 Dose3 4 1.23 0.07 0.03 5.63 1.12, 1.34

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.20 1.09 1.33 . . Dose1 1.19 1.15 1.30 100.21 -0.21 Dose2 1.27 1.17 1.32 104.15 -4.15 Dose3 1.25 1.13 1.29 102.07 -2.07

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

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

Numerator df Denominator df F-stat P-value 3 12 0.38 0.772

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 1.21 . 1.22 . . . . .

.

Page 97 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 1.21 1.000 1.22 0.725 . . . .

. Dose2 1.26 0.684 1.22 0.759 0.814 . . .

. Dose3 1.23 0.937 1.22 0.777 0.974 0.965 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.35 0.718

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 1.20 . . Dose1 1.19 1.000 0.500 Dose2 1.27 0.678 0.810 Dose3 1.25 0.676 0.713

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.38 0.772

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 -1.21 . -1.21 . . . . .

.

Page 98 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -1.21 1.000 -1.21 0.564 . . . .

. Dose2 -1.26 0.684 -1.24 0.319 0.814 . . .

. Dose3 -1.23 0.937 -1.24 0.330 0.974 0.965 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.35 0.718

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 -1.20 . . Dose1 -1.19 1.000 0.500 Dose2 -1.27 0.678 0.190 Dose3 -1.25 0.676 0.287

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

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.961 0.675 0.024 0.995 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Page 99 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 4 19.05 0.50 0.25 2.62 18.25, 19.84 Dose1 4 19.26 0.45 0.23 2.35 18.54, 19.98 Dose2 4 19.51 0.47 0.23 2.38 18.77, 20.25 Dose3 4 19.46 0.54 0.27 2.78 18.60, 20.32

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 19.16 18.35 19.53 . . Dose1 19.20 18.86 19.79 101.13 -1.13 Dose2 19.64 18.85 19.91 102.43 -2.43 Dose3 19.37 18.90 20.19 102.15 -2.15

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

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.551

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

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

Ctrl 19.05 . 19.32 . . . . .

. Dose1 19.26 0.869 19.32 0.852 . . . .

. Dose2 19.51 0.432 19.32 0.879 0.890 . . .

. Dose3 19.46 0.522 19.32 0.893 0.941 0.999 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.74 0.627

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 19.16 . .

Page 100 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 19.20 0.889 0.614 Dose2 19.64 0.235 0.879 Dose3 19.37 0.494 0.848

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.551

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

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

Ctrl -19.05 . -19.05 . . . . .

. Dose1 -19.26 0.869 -19.26 0.326 . . . .

. Dose2 -19.51 0.432 -19.48 0.147 0.890 . . .

. Dose3 -19.46 0.522 -19.48 0.152 0.941 0.999 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.74 0.627

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -19.16 . .

Page 101 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -19.20 0.889 0.386 Dose2 -19.64 0.235 0.121 Dose3 -19.37 0.494 0.152

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

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes 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 0.956 0.584 0.970 0.439 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 26.80 0.52 0.26 1.93 25.97, 27.62 Dose1 4 27.94 0.43 0.21 1.52 27.26, 28.62 Dose2 4 28.26 0.74 0.37 2.63 27.08, 29.44 Dose3 4 28.18 0.96 0.48 3.41 26.65, 29.70

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 26.87 26.13 27.32 . . Dose1 27.97 27.40 28.44 104.28 -4.28 Dose2 28.58 27.17 28.73 105.48 -5.48 Dose3 28.11 27.10 29.39 105.15 -5.15

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

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

Numerator df Denominator df F-stat P-value 3 12 3.84 0.039

Dunnett - testing each trt mean signif. different than control

Page 102 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

Ctrl 26.80 . 27.79 . . . . .

. Dose1 27.94 0.091 27.79 0.987 . . . .

. Dose2 28.26 0.029 27.79 0.991 0.913 . . .

. Dose3 28.18 0.040 27.79 0.993 0.963 0.998 . .

.

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

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

Degrees of Freedom TestStat P-value 3 7.49 0.058

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 26.87 . . Dose1 27.97 0.067 0.990 Dose2 28.58 0.101 0.996 Dose3 28.11 0.103 0.984

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

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

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

Numerator df Denominator df F-stat P-value 3 12 3.84 0.039

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend

Page 103 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

Ctrl -26.80 . -26.80 . . . . .

. Dose1 -27.94 0.091 -27.94 0.022 . . . .

. Dose2 -28.26 0.029 -28.22 0.008 0.913 . . .

. Dose3 -28.18 0.040 -28.22 0.008 0.963 0.998 . .

.

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

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

Degrees of Freedom TestStat P-value 3 7.49 0.058

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -26.87 . . Dose1 -27.97 0.067 0.010 Dose2 -28.58 0.101 0.004 Dose3 -28.11 0.103 0.016

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

027401 Dichlobenil 48616101 890.1100 NOT LS **ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )**

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

Page 104 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

0.981 0.972 0.453 0.720 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

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

Ctrl 4 1.41 0.05 0.03 3.76 1.33, 1.50 Dose1 4 1.46 0.03 0.01 1.82 1.41, 1.50 Dose2 4 1.45 0.05 0.03 3.74 1.37, 1.54 Dose3 4 1.45 0.04 0.02 2.94 1.38, 1.52

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.40 1.37 1.49 . . Dose1 1.46 1.42 1.48 103.01 -3.01 Dose2 1.46 1.38 1.51 102.83 -2.83 Dose3 1.47 1.39 1.48 102.83 -2.83

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

*** 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 3 12 0.81 0.515

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 1.41 . 1.44 . . . . .

. Dose1 1.46 0.441 1.44 0.888 . . . .

. Dose2 1.45 0.486 1.44 0.911 1.000 . . .

. Dose3 1.45 0.486 1.44 0.922 1.000 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.38 0.711

Page 105 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 1.40 . . Dose1 1.46 0.346 0.876 Dose2 1.46 0.494 0.848 Dose3 1.47 0.577 0.860

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

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

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

Numerator df Denominator df F-stat P-value 3 12 0.81 0.515

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 -1.41 . -1.41 . . . . .

. Dose1 -1.46 0.441 -1.45 0.136 . . . .

. Dose2 -1.45 0.486 -1.45 0.145 1.000 . . .

. Dose3 -1.45 0.486 -1.45 0.150 1.000 1.000 . .

.

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

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

Degrees of Freedom TestStat P-value 3 1.38 0.711

Page 106 of 107

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 -1.40 . . Dose1 -1.46 0.346 0.124 Dose2 -1.46 0.494 0.152 Dose3 -1.47 0.577 0.140

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

Page 107 of 107

DER Template Version: 22 September 2011

# DATA EVALUATION RECORD

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

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616102)

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: Sandra Hastings Date: 3/20/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/27/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3/27/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 3/27/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL/ 027401 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1 of 14 OCSPP 890.1150/ OECD None


### Primary Reviewer: Edward Scollon
, Ph.D. 

### Health Effects Division Secondary Reviewer:**_G~re""'g""""A"--=k~erm=a=n=,-=P~h= D=-o.....**  

### ___ _ 

### Health Effects Division Template version 08/2011



### DA TA EVALUATION RECORD

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



### PC CODE: 027401 

### DP BARCODE: 0397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY) : Dichlobenil (99.3 % a.i.)



### SYNONYMS: 2
, 6-dichlorobenzonitrile



### CITATION: Meerts
, I.A

. T

. (2011). Dichlobenil Technical: Androgen Receptor Binding (Rat Prostate Cytosol )

. NOTOX B.V.
, The Netherlands. Laboratory Project Identification number : 495779
, December 9
, 2011. MRID 48616102

. U npublished

.


### SPONSOR: Chemtura Corporation
, 199 Benson Road
, Middlebury
, C T



### TEST ORDER#: EDSP-027401-120



### EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48616102)
, v entral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a

s aturation binding experiment and a competitive binding experiment. The saturation binding

e xp e riment was conducted to demonstrate that the AR in the rat prostate cytosol was present in

rea s onable number s and was functioning with appropriate affinit y for the radio-labeled reference androgen (Rl 881) prior to conducting the competitive binding experiments. The competitive bindin g assay was conducted to measure the binding of a single concentration of [3H]-Rl 881 (1 

### nM ) in the presence of increa s ing concentrations (1 

### o

- t o to 10

- 3 

### M) of dichlobenil (99.3%
, Lot # A M9 / K12A222 ). Dimeth y l s ulfoxide ( DMSO) was used a s the solvent vehicle at a final assay
c o ncentration of 3

. 3 %. A total of three independent runs were performed
, and the assay included dexamethasone as a weak positive control
, and R1881 as the ligand reference standard in each

run.

Onl y one run was considered acceptable in the saturation binding experiment
, but the estimates for the dissociation constant (Kd)
, Bm ax, and specific binding generally met the acceptance criteria
, a nd the Scatchard plot was linear (r2 

## = 0

. 994)

. For the successful run , the dissociation

con s tant ( Kd) wa s 0

. 65 nM
, and the Bm ax wa s 2

. 58 fmol/l OOμg protein. The specific binding reached a plateau
, and non-specific binding was less than 20 % of total binding (4

. 2 % ).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


### In the competitive binding experiment, dichlobe nil reduced the mean specific binding of the radiolabeled ligand in a con centration dependent manner in all three runs. The maximum reduction in [ 3 

### H]-R1881 binding occurred at th e highest concentration of dichlobenil tested (10

− 3 

### M), with specific binding being reduced to 40- 46%. Specific binding wa s also reduced at 10

− 4 

### M (52-56%), but not at concentrations ≤ 10 − 5 

### M (86-103% binding). Dichlo benil is classified as a binder in the AR assay as specific [ 3 

### H]-R1881 binding to the AR was reduced to <50% at the highest concentration tested in all three runs.
The estimated mean log IC 50 

### was
− 3.85 M for dichlobenil,

− 8.73 M for R1881 and

− 4.58 M for dexamethasone (weak positive control). Compared to R1881, the mean Relative Binding Affinity (RBA) was 0.0013% for dichlobenil and 0.0071% for dexamethasone. All performance criteria were met except that the liga nd depletion was great er (~18%) than the ≤ 10-15% criteria, and the bottom percent binding for R 1881 was low in one of the runs.
Although only one acceptable run was conducted for the saturati on binding assay, and the K d 

### was low for that run, the androgen receptor was presen t in a reasonable concentration and functioning with appropriate affinity for the ligand in the competitive binding assays based on the performance criteria and estimated IC 50 

### s for R1881 and the weak positiv e control. It is unlikely that information from additional saturation binding runs would change the interpretation of this study.

Based on the results from the three runs, dichlobe nil is classified as a Binder in the Androgen Receptor Binding Assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated Data Confidentiality, GLP, and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: NOTOX B.V. Location: Hambakenwetering 7, 5231 DD’s-Hertogenbosch, The Netherlands Study Director: I.A.T. M. Meerts Other Personnel: M. Mulder (Technical Coordinator); M.A. Thomson (Study Monitor) Study Period: May 13, 2011 to December 9, 2011



### 2. Test substance: Dichlobenil
Description: Technical; white powder with lumps Source: Chemtura Canada Lot#: AM9K12A222 (expiry date: October 12, 2012) Purity: 99.3% a.i. Solubility: Soluble in DMSO at 50 mg/mL Volatility: Not reported Stability: Stable Storage conditions: Room temperature (in the dark) CAS #: 1194-65-6 Molecular weight: 172.0 Structure:



### 3. Non-labeled ligand: R1881 Supplier: YES Pharma Ltd. (Israel) Batch #: TDNF091128 Purity: 97.7% CAS #: 965-93-5


### 4. Radioactive ligand: [ 3 

### H]-R1881 Supplier: Perkin Elmer Life and Analytic al Sciences (Boston, MA) Lot#: 614814 Date of production: July 1, 2010 Date of use: May 24, 2011 to June 14, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol (3.15 TBq/mmol) Concentration of stock: 0.1 μ M



### 5. Positive control: Dexamethasone Supplier: Sigma, Zwijndrecht, The Netherlands Catalog # Batch #: Not provided Not provided Purity: Not provided CAS # : 50-02-2



### 6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO) Justification for choice of solvent: None provided

Final Concentration: 3.3% (v:v)Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None



### B. METHODS
1. Preparation of Rat Ventral Prostate Cytosol: A total of 20 male Sprague Dawley rats (obtained from Charles Ri ver, Sulzfeld, Germany) were castr ated at 80-90 days of age and were euthanized using O 2 

### /CO 2 

### approximately 24 hours later. The ventral prostates were excised immediately after termination, wei ghed, and placed in ice-cold low-salt TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer, homogenized, and centrifuge d for 30 min at 30,000 × g at 4o C. The supernatant was pooled, discarding the resulting pell ets. Protein concentration of the cytosol (Batch P-10-1) was determined to be 4.7 mg/mL. A lthough the assay method was not specified, characterization of the cytosol used wa s performed under NOTOX project 493416. Cytosol was aliquoted into ampoules for storage at ≤− 75oC until use.

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

### H]-R1881 was performed under a separate study (NOTOX Project 493416) to de monstrate that the AR was present in reasonable concentrations and had the appropriate affi nity for the R1881 ligand. Results of the characterization of the cytosol and saturation binding experiment we re presented in an Appendix to the main study. 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 (P-10-1) Concentration of radioligand (as serial dilutions) 0.25-10 nM Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM Optimization of receptor concentration 1.3 mg/assay tube; sufficient to bind 4.2% of radioligand at 0.25 nM Temperature 4±2oC Incubation time 16-20 hours Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM a Data were obtained from Appendix 2, page 38 of the study report.



### Specifics of the saturation bindi ng experiment were not provide d. Tables of information on the rat prostate, the conditions of the study, tube conten ts, results, and graphs were provided. Based on the provided data, serial dilutions of [ 3 

### H]-R1881 in TEDG buffer were prepared to achieve the final concentrations in cy tosol of 0.25, 0.5, 0.7, 1, 1.5, 2.5, 5, and 10 nM to determine total binding. To determine non-spec ific binding, solutions of non-labeled R1881 were prepared in a similar manner to achieve co ncentrations that were 100-fold greater than each respective radiolabeled concentration, result ing in final concentra tions in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1,000 nM. In the absence of cytosol, the radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

### H]-R1881 was measured. The optimal protein concentr ation was determined by calculating specific binding to differing amounts of protein per tube, using 0.25 nM radiolabeled R1881. TheAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


### optimal protein concentration was determined to be 0.72 mg protein/as say tube. Each run contained three replicates at each concentration, re sulting 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 1.00 1.00 100 10 30 1.50 1.50 150 10 45 2.50 2.50 250 100 7.5 5.00 5.00 500 100 15 10.00 10.00 1000 100 30 a Data were obtained from Appendix 2, page 38 of the study report. 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, or 30 μL of 100 nM [ 3 H]-R1881 without cytosol or other components to determine the total counts added.


### Following addition of triamcinolone acetonide, [ 3 

### H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 16-20 hours at 4° C. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, and the supernatan t was removed using a glass pipette and discarded. The samples were washed 3 times in 50 mM TRIS bu ffer; samples were maintained on ice at all times during the washing procedure. Following the last wash and decanting of the Tris buffer, pellets were then extracted by addi tional of 2.0 mL ethanol. The samples were vortexed 3 times at 5 minute intervals, and centrifuged for 10 minutes at 600 × g . Each ethanol supernatant was then decanted into a scintillation vial, and the radiation was quantified by liquid scintillation counting. A to tal of two runs were performed; however, only one run was determined applicable as the [ 3 

### H]-R1881 batch used in the first run was too old. A third run was not conducted due to the limited am ount of cytosol.
3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is included in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat vent ral prostate cytosol (P-10-1) Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 17.6-18.7% of 1.0 nM radioligand b Concentration of test substance (as serial dilutions) 10 − 10 to 10 − 3 M Incubation Temperature 6±2oC c Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Sodium molybdate 1 μ M Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM Protease inhibitor 60 μ M a Data were obtained from pages 16 and 18 of the study report. b Data obtained from page 22 of the study repo rt; values were above the criterion of <10-15%. c Assay tubes were incubated at 6±2°C instead of 4±2°C (deviation from protocol, page 21); the slightly higher temperatures had no effect on the integrity of the study based on the results of the controls included in the assay.


### 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 (undiluted specifi c activity) to the AR in the presence of increasing concen trations of a test substance.

DMSO was used as the solvent vehicle; precipitation was not addressed in the study report. Based on the results from the saturation bindi ng experiment, the amount of cytosolic protein used for each incubation in the competi tive binding experiments was 1.3 mg/assay tube, which bound 17.6-18.7% of the radiolabeled R1881 at 1 nM (pg 22 of study report). The ligand depletion was slightly greater than the ≤ 10-15% criteria.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates for each test substance at each concentration, for a total of 75 sample tubes. In addition, six replicates of the 1 nM solution of [ 3 

### H]-R1881 in ethanol and a single ethanol bl ank were run with each assay, resulting in a total of 82 samples per run.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a b Solvent Control Reference standard We ak positive control Test Chemical None DMSO R1881 Dexamethasone Dichlobenil Tubes 1-3 and 70-72 Tubes 4-21 and 73-75 c Tubes 22-45 Tubes 46-69 Tubes unnumbered 1×10

− 7 1×10

− 3 1×10

− 3 1×10

− 8 1×10

− 4 1×10

− 4 1×10

− 9 1×10

− 5 1×10

− 5 1×10

− 10 1×10

− 6 1×10

− 6 1×10

− 11 1×10

− 7 1×10

− 7 -- 1×10

− 8 1×10

− 8 -- 1×10

− 9 1×10

− 9 -- 1×10

− 10 1×10

− 10 a Data were obtained from pages 18, 19 and 42-47 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 82 tubes per run. Tubes 1-75 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 H]-R1881. Samples were dried, and 300 μL of prostate cytosolwere added. Tubes 1-75 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tube s 4-6 and 73-75 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Six un-numbered tubes containing only 30 μL of [ 3 H]-R1881 (in ethanol) were not incubated and analyzed with an ethanol blank. c Tubes 4-6 and 73-75 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881.



### The procedures for assay incubation, separa tion of bound from free R1881 (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

### H]-R1881 were similar to those used in the saturation bi nding experiment. A total of three runs were performed as described.
4. Data Analysis: The maximal binding capacity (B max 

### ), dissociation constant (K d 

### ), and inhibition concentration (IC 50 

### ) were calculated and a Scatchard plot was also plotted for the binding data (programs not specified). The competitive binding study reported that Prism version 4.01 (GraphPad Software, San Diego, CA) was used for nonlinear regression and statistical analysis.

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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None



### 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 constant 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 × 100 ÷ IC 50 

### of test substance

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The dissociation constant (K d 

### ) for [ 3 

### H]-R1881 was 0.65 (±0.02), and the estimated B max 

### was 2.58 fmol/100 μ g protein (±0.02) for the si ngle batch of prostate cytosol that was prepared. Only two runs were conducted due to the limited amount of cytosol and only one of the runs was de termined acceptable. The goodness of fit (R 2 

### ) was 0.994 for the one acceptable run. The K d 

### was slightly lower than the range reported in the EPA validation program (0.685 to 1.57 nM), but the Scatchard plot was linear.

TABLE 5. Saturation Binding Experiment of R-1881 wi th Androgen Receptor from Rat Prostate Cytosol a Parameter Run 1 b Run 2 c Run 3 d Mean ± SE e R 2 (unweighted) NA 0.994 NA NA B max (nM) NA 0.112 ± 0.001 NA NA B max (fmol/100 μ g protein) NA 2.58 ± 0.02 NA NA K d (nM) NA 0.65 ± 0.02 NA NA a Data were obtained from Appendix 2, page 40 of the study report. b The first run was not used in evaluation of the results as the [ 3 H]-R1881 batch used for Run #1 was too old. c The mean and standard deviation are reported for the run. d A third run was not performed due to the limited amount of cytosol. e Not applicable because only on e acceptable run was evaluated. R 2 Goodness of fit for curve calculated for specific binding. NA Not applicable



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

### H]-R1881 to the androgen receptor. The specific binding reac hed a plateau, and non-specific binding was less than 20% of total binding ( 4.2%). Figure 2 is a Scatchar d plot that illustrates the binding of [ 3 

### H]-R1881 to the androgen receptor. The data fit results in a linear plot.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


### FIGURE 1. Binding of [ 3 

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

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

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

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters are presented in Table 6. Dichl obenil reduced the mean specific binding of the radiolabeled ligand in a concentration depende nt manner in all three runs. The maximum reduction in [ 3 

### H]-R1881 binding occurred at the highe st concentration tested (10

− 3 

### M), with specific binding being reduced to 40-46%. Specific binding was also reduced at 10

− 4 

### M (52-56%), but not at concentrations ≤ 10 − 5 

### M (86-103% binding). The results of each run are illustrated in Figure 3, and the average binding curve for dichlobenil across the three runs is shown in Figure 4. Dichlobenil was classifi ed as a binder in each of the three runs.
The estimated mean log IC 50 

### s were
− 3.85 M for dichlobenil,

− 8.73 M for R1881 and

− 4.58 M for the weak positive contro l (dexamethasone). Compared to R1881, the mean RBA was 0.0013% for dichlobenil and 0.0071% for the weak positive control. Confidence in these numbers is high due to the small variation. The solvent control res ponses indicated no drift in the study assay.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

TABLE 6. Competitive Binding Assay of Dichlo benil with AR from Rat Prostate Cytosol a Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c R 2 (unweighted) R1881 0.9982 0.9995 0.9986 0.9982-0.9995 Dexamethasone 0.9938 0.9964 0.9969 0.9938-0.9969 Dichlobenil 0.9639 0.9901 0.9859 0.9639-0.9901 Log IC 50 (M) R1881

− 8.72

− 8.75

− 8.73

− 8.73 ± 0.01 Dexamethasone

− 4.53

− 4.62

− 4.60

− 4.58 ± 0.03 Dichlobenil

− 3.82

− 3.82

− 3.90

− 3.85 ± 0.03 IC 50 (M) R1881 1.93 × 10

− 9 1.79 × 10

− 9 1.86 × 10

− 9 1.86 × 10

− 9 (±0.04) d Dexamethasone 2.96 × 10

− 5 2.42 × 10

− 5 2.51 × 10

− 5 2.63 × 10

− 5 (±0.17) Dichlobenil 1.51 × 10

− 4 1.52 × 10

− 4 1.26 × 10

− 4 1.43 × 10

− 4 (±0.09) Log RBA d Dexamethasone -4.19

− 4.13

− -4.13

− 4.15 ± 0.02 Dichlobenil -4.89

− 4.93

− -4.83

− 4.88 ± 0.03 RBA (%) Dexamethasone 0.00652 0.00741 0.00742 0.00711 ± 0.0003 Dichlobenil 0.00127 0.00118 0.00147 0.00131 ± 0.0001 a Data were obtained from page 24, 26 and 29 of the study report. b The means are reported for the conc urrent replicates within each run. c The range of r 2 is reported and the mean and SE are reported for the other parameters. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. d Calculated by the study reviewer. SE Standard error NA Not applicable. R 2 Goodness of fit RBA Relative Binding Affinity RBA IC 50 of R1881 ÷ IC 50 of test substanceAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


### FIGURE 3. Percentage R1881 Bound to the A ndrogen Receptor in the Presence of Test Compounds.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


### FIGURE 4. Mean Percentage R1881 Bound to th e Androgen Receptor in the Presence of Dichlobenil in Three Competitive Binding Assays.

C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluate d using the 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% 17.6 to 18.7 c X

Dichlobenil Top (% binding) 80 to 115 98 to 100 X R1881 fitted curve parameters Top (% binding) 82 to 114 100 to 104 X Bottom (% binding)

− 2.0 to 2.0

− 2.6 to

− 1.6 d X Hill Slope

− 1.2 to

− 0.8

− 1.0 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 99 to 104 X Bottom (% binding)

− 12 to 12

− 2.7 to

− 1.3 X Hill Slope

− 1.4 to

− 0.6

− 1.0 X Saturation Binding Experiment K d (nM) 0.685 to 1.57 0.65 X Non-specific binding (%) ≤ 10.0 4.35 to 4.62 X a Data were obtained from pages 22, 24, 26, 29-30 and 40 of the study report. b These values represent ranges from the validation study. c Ligand depletion was outside the limits in all three runs. d The bottom percent binding was outside the limits in one run.


### All performance criteria were met except that the ligand depletion (18-19%) was slightly greater than the ≤ 10-15% criteria, and the bottom per cent binding for R1881 was low in one of the runs. Because the other parameters were within the tole rance limits and the IC 50 

### for R1881 and dexamethasone were in accordance with values determined in the EPA validation study, these deviations are consid ered minor and likely had no effect on the results of the study.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None


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

### H]-R1881 in a manner consiste nt with one-site binding, as indicated by a Hill slope of

− 1.0. Examination across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Because the curve across runs crosses the 50% value, dichlobenil should be classified as a binder in the androge n receptor binding assay using rat prostate cytosol. The RBA of di chlobenil compared to R1881 (set at 100%) was 0.00131 ± 0.0001% (mean ± SEM). The RBA of the weak positive control dexamethasone was 0.00712 ± 0.0003%.

B. AGENCY COMMENTS: Only one run was considered acceptable in the saturation binding experiment; the [3H]-R1881 batch was too old in the 1 st 

### run and a 3 rd 

### run was not performed due to limited quantities of cy tosol. For the single acceptable run, the dissociation constant (K d 

### ) for [ 3 

### H]-R1881 was 0.65 (±0.02), and the estimated B max 

### was 2.58 fmol/100 μ g protein (±0.02) for the single batch of pr ostate cytosol that was prepared. The goodness of fit (R 2 

### ) was 0.994 for the one acceptable run. The K d 

### was slightly lower than the range reported in the EPA validation program (0.685 to 1.57 nM), but the Scatchard plot was linear. The specific binding reached a pl ateau, and non-specific binding was less than 20% of total binding (4.2%).
In the competitive binding experiment, dichlobe nil reduced the mean specific binding of the radiolabeled ligand in a con centration-dependent manner in all three runs. The maximum reduction in [ 3 

### H]-R1881 binding occurred at the highe st concentration tested (10

− 3 

### M), with specific binding being reduced to 40-46%. Specific binding was also reduced at 10

− 4 

### M (52-56%), but not at concentrations ≤ 10 − 5 

### M (86-103% binding). Di chlobenil is classified as a binder in the AR assay as specific binding was reduced to <50% at the highest concentration tested in all three runs.
The estimated mean log IC 50 

### s were
− 3.85 M for dichlobenil,

− 8.73 M for R1881, and

− 4.58 M for the weak positive contro l (dexamethasone). Compared to R1881, the mean RBA was 0.0013% for dichlobenil and 0.0071% for dexameth asone. Confidence in these numbers is high due to the small variation. The solvent c ontrol responses indicated no drift in the study assay. All performance criteria were met except that the ligand depletion was greater (~18%) than the ≤ 10-15% criteria, and the bottom per cent binding for R1881 was low in one of the runs. Because the other parameters were within the tole rance limits and the IC 50 

### for R1881 and dexamethasone were within the expe cted ranges demonstrating that the ligand was available at an appropriate level, the revi ewers consider these minor deviations and find that the performance criteria were met.
Although only one acceptable saturation binding assay was conducted and the K d 

### was low for that run, the androgen receptor was present in a reasonable concentration and functioning with appropriate affi nity for the R1881 ligand in th e competitive binding assays based on the performance criteria and estimated IC 50 

### s for R1881 and the weak positive control. It is unlikely that information from additional saturation bi nding runs would change the interpretation of this study.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 14 DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None



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

 Only one acceptable saturation bind ing assay was conducted and the K d 

### was low for that run. The ligand depletion in the competitiv e binding experiment was greater than the ≤ 10-15% criteria for all three runs.

 The bottom percent binding for R1881 was low in one of the competitive binding assays.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616103)

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: Joan Harlin, M.S. Date: 3/27/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 4/02/2012
Program Manager: Signature: Jack D. Early, M.S. Date: 4/09/2012 Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/09/2012

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).DICHLOBENIL I 027401


### Primary Reviewer: Greg Akerman, Ph.D

. 

### Health Effects Division Secondary Reviewer: John Liccione, Ph.D. 

### Health Effects Division Aromatase (Human Recombinant) Assay (20 l l) I Page l of 12 OCSPP 890.1200 I OECD None


### Signature:

..

## ~ 

# A
Date:

_ 

### {;n) JL

## Signature~ Date:
,, 

# i:m::=

DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant) ; OCSPP 890.1200



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS: 2
, 6-dichlorobenzonitrile



### CITATION: Meerts I.A.T.M. (2011). Dichlobenil technical: Aromatase assay (human recombinant). NOTOX B.V
., The Netherlands. Laboratory Report No.: 495780
, December 8
, 2011. MRID 48616103

. Unpublished.



### SPONSOR: Chemtura Corporation
, 199 Benson Road
, Middlebury
, CT



### TEST ORDER# : EDSP-027401-120



### EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assa y (MRID 48616103), dichlobenil (99.3 %
, Lot # AM9Kl2A222) was incubated with human recombinant aromatase and tritiated androstenedione**(1-~** 

### [3H(N)]-androst -4-en e-3
, 17-dione; [3H]ASDN) in ethanol at log concentrations of 10- 10 to 10

- 3 

### M for 15 minutes to assess the potential of dichlobenil to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified with liquid scintillation counting (LSC)

. Three runs were conducted
, and each run included a full activity control
, a background activity control
, a positive control series (10

- 1 0 to 10

- 5 

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

- 1 0 to 10

- 3 

### M) with three repetitions per concentration.

Aromatase activity in the full activity controls ranged from 0.4156 to 0

. 7358 nmol·mgprotein

- 1

· min

- 1 for the three test runs , with a mean and standard deviation of 0.5750 ± 

### 0.1208 nmol·mg-protein

- 1

· min

- 1

. The response of each full activity control within a run was between 90
to 110% of the mean. Activity in the background controls ranged from 0.06% to 0.39% and averaged 0.21 % of the full activity controls

. For the positive control substance ( 4-0H ASDN)
, aromatase activit y results were within the recommended ranges for the performance criteria. The estimated log IC so for 4-0H ASDN averaged

- 7

. 34 Mand the Hill slope was

- 0

. 94.Aromatase (Human Recombinant) A ssay (2011) / Page 2 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### Aromatase activity at the lowest and highest succ essfully tested concentr ations of dichlobenil (10 -10 

### and 10

− 4 

### M) were 99.6% and 87.5% of control. The highest tested concentration of dichlobenil (10

− 3 

### M) showed precipitation during the 15-mi nute incubation period; therefore, the results of the highest test concentrat ion were not used for interpretation.
Based on the data from the average response curve, dichlobenil is classified as a Non-inhibitor of aromatase activity in this assay. There is no indication in the study re port that half-log concentrations (i.e., 10 -3.5 

### M) were tested for solubility as recommended by the Test Guideline. At concentrations up to 10 -4 

### M, dichlobenil would be classified as a non-inhibitor. However, as there is no indication in the study report that 10 –3.5 

### M was evaluated for so lubility, there is less confidence in the non-inhib itor classification. It should be no ted that based on the shape of the response curve, if a soluble 10 –3.5 

### M concentration had been teste d, it is possible that the lowest portion of the curve would fall within the 50-75 % activity range resulting in an equivocal classification for this chemical.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Flagging statements were provided.Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Dichlobenil technical Description: White powder Source: Chemtura Canada Co./Cie, Ontario, Canada Lot # (expiration date): AM9K12A222 (October 12, 2012) Purity: 99.3 Wt.% Volatility: Not reported Storage conditions: At room temperature in the dark Stability: Stable Solvent: Ethanol Solubility (in test solvent): Soluble at 100 mM in ethanol Highest Concentration Tested: 10

− 3 M Stock Solution Preparation: 100 mM was freshly prepared in ethanol. Molecular weight: 172.0 CAS #: 1194-65-6 Structure:



### 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS # : 63-05-8 Source: Bio-Connect B.V., Huissen, The Netherlands (Catalog No. A637550) Batch # (expiration date): 3-FJ-51-1 (January 06, 2012) Purity: 98%



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

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

### H]ASDN) Source: Perkin Elmer, Boston, MA Batch # (expiration date): 619344 (February 14, 2012, allocated by NOTOX one year after receipt of the test substance) Radiochemical Purity (Supplier): >97% Specific activity: 0.974 TBq/mmol (26.3 Ci/mmol) Radiochemical Purity (In-lab determination): 100%



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN) CAS # 566-48-3 Source: Toronto Research Chemicals, Inc., N orth York, ON, Canada (Catalog No. F692250) Batch # (expiration date): TRC-030508 (retest date: September 14, 2012) Purity: 98%


### 5. Solvent (Vehicle Control): Ethanol Source: Batch # (expiration date): LiChrosolv, Merck, Darmstadt, Germany Not reported Justification for choice of solvent One of the solvents of choice detailed in the EPA guideline Concentration (% of total volume in assays) 1%Aromatase (Human Recombinant) A ssay (2011) / Page 4 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None



### 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes

Source: BDGentest TM , Woburn, MA (Catalog # 456260) Lot # (expiration date): 40199 (not reported) Protein concentration: 7.6 mg/mL Cytochrome C reductase activity: 290 nmol /mg protein/min Aromatase activity: 6.1 pmol/pmol P450/min



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

### H]ASDN was prepared such that the final concentration of ASDN in the assay was 0.3 mM, and the amount of tritium adde d to each incubation tube was 0.1 μ Ci.

Test chemical was formulated in ethanol such that the volume of ethanol used per assay was no more than 1% v/v of the total assay volume. A rationale for the selection of ethanol was not provided; however, dichlobenil is soluble in ethanol at the te sted concentrations and is a solvent recommended by the EPA guidelines.
A stock solution of the positive control substance, 4-OH ASDN, was formulated in ethanol. Dilutions were prepared such that the target concentrations of the pos itive control substance (10

− 10 

### to 10 − 5 

### M; Table 4) were achieved by the additi on of 5 μL of the dilution for a final assay volume of 1 mL.
Human recombinant microsomes were purchased from BD Gentest TM 

### ; microsomes were stored in aliquots at ≤− 75oC for up to 2 years. Before use, the microsomes were quickly thawed in a 37 ± 1oC water bath, then placed in an ice bath and diluted in assay buffer up to a protein concentration of 8 μg/mL. Othe r assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

TABLE 1. Assay Components and Conditions a Assay Factor Values 0.1M sodium phosphate buffer (pH 7.4) Microsomal Protein 0.004 mg/mL NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37°C Incubation Time 15 min a Data were obtained from page s 16 and 17 of the study report.


### 2. Suitability Assessments: The protein concentration of the microsomes (Lot #40199) was supplied by the vendor as 7.6 mg/mL. The micr osomal protein concentrations determined on each experimental date were 3.57, 3.64, and 3.70 mg/mL (average 3.64 ± 0.06%) with a protein assay kit (Bio-Rad Laboratories, GmbH, Munich, Germany; Lot # 110253). Because these concentrations were approximate ly 48% lower than the protein concentration provided by the supplier, the measured protein co ncentrations were used in calculations in the study. Aromatase activity of the microsom es was also provided by the vendor as 6.1 pmol/min/pmol P450.Aromatase (Human Recombinant) A ssay (2011) / Page 5 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### 3. Aromatase Assay: Each assay run contained 4 tube s for the full enzyme activity and 4 tubes for the background activity controls. Two tubes of each control were run at the beginning of the assay, and two of each control were run at the end of the assay. A full concentration curve in duplicate for the positiv e control, and a full concentration curve in triplicate for the test substa nce were established. The aromatase assay was conducted generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

### H 2 

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

− 1 

### ·min

− 1



### . 4. Demonstration of Proficiency: The demonstration of laboratory proficiency for the aromatase assay was not described or reported.

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

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

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

 The average of the background activity contro ls (n=4) within a run should fall within the range of

− 5 to 6%

 The average of the full enzyme activity cont rols (n=4) within a run should fall within the range of 100± 10%.
(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The historical positive control data fo r slope, top and bottom percent, and log IC 50 

### generally met the criteria as listed in section (i) of OCSPP 890.1200 (Table 2).

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

− 1.2

− 0.8

− 1.2

− 0.8 Top (%) 90 110 90 111 Bottom (%)

− 5 +6

− 5 6 Log IC 50 (M)

− 7.3

− 7.0

− 7.5

− 7.0 a Data were obtained from pages 18 of the study report.



### b. Proficiency Chemicals: Assays to classify proficienc y chemicals (econazole, fenarimol, nitrofen, and atrazine) were not performed.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor No Data Fenarimol 60168-88-9 Inhibitor No Data Nitrofen 1836-75-5 Inhibitor No Data Atrazine 1912-24-9 Non-inhibitor No DataAromatase (Human Recombinant) A ssay (2011) / Page 6 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None



### 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of dichl obenil per run, in triplicate, was tested during three independent runs (Table 4) . Solubility was visu ally assessed (presence of cloudiness or a precipitate). Precipitation was observed at the highest test concentration (10 -3 

### M); therefore, the results for this test c oncentration were not used. The full enzymatic activity ( ≥ 95% for the means of each run) was obtai ned in Runs 1 and 2, but not in Run 3, defining the top of the con centration-response curve.

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

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

− 6 4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10

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

− 7 4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10

− 7.5 4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10

− 8 4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10

− 9 4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10

− 10 Dichlobenil Conc 1 c 3 All test components plus dichlobenil 1×10

− 3 Dichlobenil Conc 2 c 3 All test components plus dichlobenil 1×10

− 4 Dichlobenil Conc 3 c 3 All test components plus dichlobenil 1×10

− 5 Dichlobenil Conc 4 c 3 All test components plus dichlobenil 1×10

− 6 Dichlobenil Conc 5 c 3 All test components plus dichlobenil 1×10

− 7 Dichlobenil Conc 6 c 3 All test components plus dichlobenil 1×10

− 8 Dichlobenil Conc 7 c 3 All test components plus dichlobenil 1×10

− 9 Dichlobenil Conc 8 c 3 All test components plus dichlobenil 1×10

− 10 a Data were obtained from pages 15 and 16 of the study report. b The complete assay contained buffer, pr opylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH. c Test chemical.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e activity (nmol/mg protein/min) and percent control for the positive control and test chemical. The following raw data and calculated endpoints for each run were in cluded in the report (Table 5).

TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/min X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X DPM Disintegrations per minuteAromatase (Human Recombinant) A ssay (2011) / Page 7 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None



### 2. Statistical Methods: Statistical analysis was performed on the data, which included means of replicates using the following software program: Prism version 4.0 (GraphPad Software, USA).
The response curve was fitted by weighted nonlinear regression analysis (Prism version 4.0, GraphPad Software, USA). For each run, the individual percent of control values was plotted versus the logarithm of the test substance concentratio n or positive control concentration. The fitted concentration-re sponse curve was presented in the plot. Individual plots were prepared for each test run. The average percenta ges of control values for each run were presented versus the logarithm of the test substance concentration in order to compare the percent control activity across runs.
3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

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

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

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

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

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

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically determined to be untestabl e in the aromatase assay.



### II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.4156 to 0.7358 nmol·mg-protein

− 1 

### ·min

− 1 

### for the 3 test runs, with a mean and standard deviation of 0.5750 ± 0.1208 nmol·mg-protein

− 1 

### ·min

− 1 

### . The response of each full activity control within a run was betw een 90 to 110% of the average full activity. Activity in the background controls ranged from 0.06% to 0.39 % and averaged 0.21% of the full activity controls.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.4336-0.6879 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration 10 − 10 

### M and 0.0030-0.0041 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration 10

− 5 

### M. These results were within the recomme nded ranges for the top of the curve, bottom curve, Hill slope, log IC 50 

### , and % CV for replicates of each concentration within runs. The variation in the positive control values was acceptable ( ≤ 6.6% CV). The mean aromatase activity of the positive control (expressed as % full control ac tivity) for each concentration tested across all 3 runs is presented in Tabl e 7, along with the overa ll standard deviation, SEM, and % CV. The inhibition response curv es for the positive control are shown in Figure 1.Aromatase (Human Recombinant) A ssay (2011) / Page 8 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Dichlobenil on Aromatase Activity (as percent of control) from Independent Runs a

Chemical Concen. (Log M) # Runs Overall Mean Overall SD Overall SEM Overall % CV 4-OH ASDN (positive control) − 5 3 0.65 0.05 0.03 8%

− 6 3 5.68 0.60 0.35 11%

− 6.5 3 14.6 1.23 0.71 8%

− 7 3 34.9 1.26 0.73 4%

− 7.5 3 59.0 2.10 1.21 4%

− 8 3 84.8 0.80 0.46 1%

− 9 3 97.6 5.71 3.29 6%

− 10 3 101.8 4.28 2.47 4% Dichlobenil

− 3 b 3 76.1 5.30 3.06 7%

− 4 3 87.5 10.3 5.94 12%

− 5 3 99.6 9.28 5.36 9%

− 6 3 100.8 13.44 7.76 13%

− 7 3 101.9 13.3 7.68 13%

− 8 3 100.9 10.9 6.28 11%

− 9 3 101.8 14.8 8.51 14%

− 10 3 99.6 8.25 4.76 8% a Data were obtained from pages 33- 44 of the study report. Mean, SD, SEM, and CV were calculated by the reviewers. b Precipitation occurred at the 10 -3 M concentration. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance



### C. TEST SUBSTANCE: For dichlobenil, aromatase activity averaged 0.4693-0.6728 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration 10

− 10 

### M and 0.4266-0.5839 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration 10

− 4 

### M that was used by the study author for interpretation. The highest tested concentration of dichlobenil (10

− 3 

### M; 0.3536-0.5097 nmol·mg-protein

− 1 

### ·min

− 1 

### ) was not used for interpretation as precipitation occurred during the 15-minut e incubation period. The mean aromatase activity of dichlobenil (expressed as % full control activit y) for each concentration tested across all 3 runs is presented in Table 7 (presented above), along with the overall standard deviation and % CV. The average inhibition response curv e across all runs is shown in Figure 2.Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### FIGURE 1. Inhibition Response Curves for 4-OH ASDN From Each Test Run.Aromatase (Human Recombinant) A ssay (2011) / Page 10 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### FIGURE 2. Mean Inhibition Response Curves for Dichlobenil from Each Test Run.Aromatase (Human Recombinant) A ssay (2011) / Page 11 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### The average dose-response curve indicated that the aromatase activity of dichlobenil at all concentrations tested was essent ially equivalent to the activity observed in the full activity control. These data could not be fit to the 4-parameter nonlinear regression model; therefore, log IC 50 

### and Hill slope estimates were not determined for dichlobenil. For 4-OH ASDN, the estimated log IC 50 

### averaged

− 7.34 M and the slope was

− 0.94 (Table 8). The variation in the positive control values was acceptable (<15% CV).

TABLE 8. Effect of Dichlobenil on Aromatase Activity (as Percent of Control) From Independent Runs a Chemical Run 1 Run 2 Run 3 Mean b SD b % CV b Log IC 50 (M) Dichlobenil NA NA NA NA NA NA 4-OH ASDN -7.3 -7.4 -7.3 -7.3 0.1 -1 Slope Dichlobenil NA NA NA NA NA NA 4-OH ASDN -0.96 -0.94 -0.92 -0.94 0.02 -2 a Data were obtained from Page 9 of the study report b Calculated by the reviewers from data presented in this table. NA Not applicable SD Standard Deviation CV Coefficient of Variance



### Based on the data from the average response curv e and the criteria listed above in Table 8, the results support the c onclusion that dichloben il does not inhibit catal ytic activity in the aromatase assay.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: It was not possible to calculate the mean log IC 50 

### for dichlobenil, as enzyme activity in the presence of dichloben il ranged from 71.3% to 123.6% of control in individual assay runs and bore no relationship to dichlobenil concentration. The positive control 4-OH AS DN was calculated to have a mean log IC 50 

### of

− 7.3 M. The mean slope of the 4-OH ASDN do se response curve was within the range documented in the EPA Guideline. It can be co ncluded that all criteria of this aromatase assay were met, and that Dichlobenil can be cl assified as a non-inhibitor in the aromatase assay.

B. AGENCY COMMENTS: Although data demonstrating proficiency with the positive control or recommended proficiency chemicals were not included, historical performance criteria data for the posit ive control were included.
Aromatase activity in the full activ ity controls was 0.5750 ± 0.1207 nmol·mgprotein

− 1 

### ·min

− 1 

### . The response of each full activity cont rol within a run was between 90 to 110% of the mean. Activity in the backgr ound controls ranged fr om 0.06% to 0.39% and averaged 0.21% of the full activity controls.
For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 

### , and %CV.Aromatase (Human Recombinant) A ssay (2011) / Page 12 of 12 DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None


### The estimated log IC 50 

### for 4-OH ASDN averaged

− 7.34 M and the slope was

− 0.94. The variation in the positive control values was acceptable ( ≤ 6.6% CV).
For dichlobenil, aromatase activity at th e lowest and highest successfully tested concentration, 10

− 10 

### and 10

− 4 

### M, were 99.6% and 87.5% of c ontrol. The highest test concentration of dichlobenil (10

− 3 

### M) showed precipitation in the incubation during the 15- minute incubation period; therefor e, the results of the highest test concentration were not used for interpretation. These data could not be fit to the 4-parameter nonlinear regression model.
Because the average lowest portion of th e curve at the concentrations of 10

− 4 

### M was >75% in all experiments, dichlobenil is classified as a non-inhibitor of aromatase activity in this assay. However, as there is no in dication in the study report that 10 –3.5 

### M was evaluated for solubility, there is less confidence in the non-inhib itor classification. It should be noted that based on the shape of the response curve, if a soluble 10 –3.5 

### M concentration had been tested, it is possible that the lowest porti on of the curve would fall within the 50-75% activity range resulting in an equivocal classifica tion for this chemical.

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 initial positive control proficiency test data and proficiency test chemical assay results were not provided other than historical performance criteria data for the positive control.

 The mean, standard deviation, and % coefficien ts of variation were not provided for the positive control data for the Hill Slope and log IC 50 

### . The reviewers were able to calculate these values from the individual data.

 For dichlobenil, the highes t test concentration of 10 -3 

### M was not used for interpretation in the aromatase assay results due to the presen ce of precipitation. Therefore, only seven test concentrations were used for interp retation. A mid-log concentration (i.e., 10 -4.5 

### M) should have been added in order to obtain the required eight test c oncentrations, as noted in section (j) of OCSPP 890.1200.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616104)

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: 3/21/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph. D., D.A.B.T. Date: 3/26/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/04/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/04/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL/ 027401


### Primary Reviewer: Ray Kent, Ph.D. 

### Health Effects Division Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Page I of 15 OCSPP 890.1250/ OECD None



### Secondary Reviewer: Greg Akerman, Ph.D.

# :::::~::f**~ ~~~**  Health Effects Division Date: 

### <:; / 'lC/ 

### I 1 

# rTemplate version 08/2011



### DATA EVALUATION RECORD

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



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99

. 3%)



### SYNONYMS: 2
, 6-dichlorobenzonitrile



### CITATION: Meerts
, I.A.T

. M. (2011)

. Dichlobenil Technical: Estrogen Receptor Binding Using Rat Uterine Cytosol (ER-RUC). NOTOX B.V
., Hertogenbosch
, The Netherlands

. Laboratory Report No

. : 495781
, December 9
, 2011. MRID 48616104. Unpublished



### SPONSOR: Chemtura Corporation
, 199 Benson Road, Middlebury
, CT.



### TEST ORDER#: EDSP-027401-120



### EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48616104)
, uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct saturation binding and competitive binding experiments. The saturation binding experiment was conducted
to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [3H]-17P-estradiol (1 nM) in the
presence of increasing concentrations ( 10- 1 0 

### to 10- 3 

### M) of dichlobenil (99.3% a.i., Batch # AM9K12822)

. DMSO was used as the solvent vehicle at a final concentration of 2%

. Three independent runs were performed
, and each run included 19-norethindrone as a weak positive control
, oct y ltriethoxysilane as a negative control
, and 17P-estradiol as the natural ligand

r e ference material.

F or the saturation binding experiment
, the appropriate protein concentration for the saturation binding assay was determined to be 40 μg / tube, as it bound 30

. 2% of the [3H]-17P-estradiol. The

mean K ct for [3H]-17P-estradiol was 0.12 nM and the mean Bmax was 57

. 19 fmol/100 μg protein for the prepared rat uterine cytosol. The data produced consistent and linear Scatchard plots 

### (r2

> 0

. 98 )

. In the competitive binding experiments
, the reference standard (E2)
, the weak positive control (19-nonethindrone)
, and the negative control (octyltriethoxysilane) each exhibited the expected responses. The estimated mean log IC so s were

- 9.08 and

- 5.87 M for E2 and 19-Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None 

### norethindrone, respectively, and the relative binding affinity (RBA) was 0.062% for 19- norethindrone, compared to the natural ligand.
Precipitation of dichlobenil was not observed at any concentration. In all three runs, dichlobenil showed minimal displacement (93-104% binding) of radiolabeled estradiol at dichlobenil concentrations up to 10

− 3 

### M; therefore an IC 50 

### and RBA could not be determined for dichlobenil.
Based on the results from the three competitive runs , dichlobenil is classified as Not Interactive in the Estrogen Receptor Binding Assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated Data Confidentiality, GLP, and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: NOTOX B.V.
Location: Hertogenbosch, The Netherlands Study Director: I.A.T.M Meerts Other Personnel: M.A. Thomson, Study Monitor M. Mulder, Technical Coordinator Study Period: June 14, 2011 – December 9, 2011



### 2. Test substance: Dichlobenil Description: Technical, white powder with lumps Source (cat #): NOTOX (202841/A) Lot/Batch # (Exp date): AM9K12A222 (October 12, 2012) Purity: 99.3 % Solubility: 50 mg/mL in DMSO Volatility: Not Reported (NR) Stability: Stable Storage conditions: Room temperature in the dark CAS #: 1194-65-6 Molecular weight: 172.0 Structure:



### 3. Non-labeled ligand: 17 β -estradiol Supplier: NR Catalog #: NR Batch #: NR Purity: NR CAS #: 50-28-2


### 4. Radioactive ligand: [2,4,6,7,16,17 - 3 

### H]-17 β -estradiol Supplier: NOTOX Catalog #: 202254/B Batch#: 650702 Radiochemical purity: >97% Specific activity: 4.817 TBq/mmol Concentration of stock: 37MBq/mL



### 5. Positive control: 19-norethindrone Supplier: NR Catalog #: NR Batch #: NR Purity: NR CAS #: 68-22-4Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None


### 6. Negative control: Octyltriethoxysilane Supplier: NR Catalog #: NR Batch #: NR Purity: NR CAS #: 2943-75-1



### 7. Solvent/vehicle control: DMSO
Justification for choice of solvent: Not reported

Final Concentration: 2%



### B. METHODS
1. Preparation of Rat Uterine Cytosol (RUC): A total of 20 female Sprague Dawley rats were ovariectomized, 17 of which survived th e ovariectomy. The surviving animals were euthanized 10 days later, on post-natal day 88. The uteri were weighe d, flash frozen using liquid nitrogen and stored at ≤

- 75 ° C for four months. Prior to isolation of the uterine cytosol, the uteri were thawed on ice for no more than 60 minut es. Uteri were homogenized in ice-cold TEDG (Tris, EDTA, DTT, gly cerol) + PMSF (phenylmet hylsulfonyl fluoride) buffer, and centrifuged for 10 min at 2500 × g at 4 oC. The supernat ant was transferred and centrifuged for an additional 60 minutes at 105000 × g , discarding the resulting pellet. Protein concentration of the cy tosol was determined to be 1.75 mg/mL using a protein kit compatible with DTT in the TEDG buffer. Cyto sol was divided into aliquots and stored at ≤

- 75 ° C for a maximum of 33 days (Batch U-10-1).

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

### H]-17 β -estradiol was performed to demonstrate that the ER was present in reason able concentrations and had the appropriate affinity for the native ligand. The conditions for the saturation binding are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat ut erine cytosol (Batch U-10-1) Concentration of radioligand (as serial dilutions) 0.03-3.0 nM Concentration of non-labeled ligand (100X [radioligand]) 3-300 nM Concentration of receptor 40 μg/tube. Sufficient to bind 30.2% of radioligand at 0.03 nM 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% Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM a Data were obtained from page 40 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None 

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

### H]-17 β -estradiol was adjusted for decay over time, and serial dilu tions in TEDG + PMSF buffer were prepared to achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1.0 and 3.0 nM. Solutions of non-labeled 17 β -estradiol were prepared in a simila r manner to achieve concentrations that were 100-fold greater than each respective radiolabeled concentra tion to result in final concentrations of 3, 6, 8, 10, 30, 60, 100 and 300 nM. For each batch of cytosol, the method for determining optimal protein concentra tion was not reported, but 40μg/tube bound 30.2% of radioligand at 0.03 nM. Each assay consiste d of three non-concurrent runs, and each run contained three concurrent replicates at each concentration, resu lting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a Total binding b Non-specific binding c Radioligand alone d Assay Components

Tubes 1-24 Tubes 25-48 Tubes 49-72 350 μL 300 μL --- TEDG + PMSF buffer 50 μL 50 μL 50 μL [ 3 H]-17 β -estradiol (8 se rial dilutions) e

--- 50 μL --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) f 100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from page 40 of the study report. b Total binding = [ 3 H]-17 β -estradiol bound to ER c Non-specific binding = [ 3 H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 H]-17 β -estradiol alone for dpm determination at each concentration e Final concentrations of [ 3 H]-17 β -estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. f Final concentrations of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### Tubes were incubated with gentle vortexing for 16-20 hours at 4±2  C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed (3 times with 5-minute intervals). Subsequently, the contents of each tube were washed three times as follows: TEDG +PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 x g , and the supernatant decanted and discarde d. After washing, ethanol was added to the HAP pellet remaining in each tube to extract the [ 3 

### H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 min at 1000 x g . An aliquot of supernatant was radioassayed by scintillation counting. The temp erature was maintained at 4 oC throughout the assay prior to extraction with ethanol.

3. Competitive Binding Experiment: A summary of the experiment conditions for the Competitive Binding Experiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat Ut erine Cytosol (B atch U-10-1) Concentration of radioligand 1 nM Concentration of receptor Sufficient to bind 5.90-6.54% of radioligand Concentration of test substance (as serial dilutions) 10 -10 to 10 -3 M 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% Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM a Data were obtained from pages 18-20 and 44-52 of the study report.



### A solubility test was not perfor med for dichlobenil. On the day of the assay, the specific activity of the stock solution [ 3 

### H]-17 β -estradiol was adjusted for decay over time, and diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 1.0 nM radiolabeled es tradiol, until a concentration was reached that bound 5.9-6.54% of the total radioact ivity added. Serial dilutions of the test substance, weak positive control (19-norethindrone), negative control (octyl triethoxysilane), and reference material (non-labeled 17 β -estradiol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three runs, and each run contained three replicates at each concentration, re sulting in a total of 99 samples.

TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a, b Test substance Positive control Negative control Reference Chemical 19-norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol Tubes 73-96 c Tubes 49-72 c Tubes 25-48 c Tubes 1-24, 97-99 c 10

− 10 10

− 8.5 10

− 10 Solvent control d 10

− 9 10

− 7.5 10

− 9 10

− 11 10

− 8 10

− 7 10

− 8 10

− 10 10

− 7 10

− 6.5 10

− 7 10

− 9.5 10

− 6 10

− 6 10

− 6 10

− 9 10

− 5 10

− 5.5 10

− 5 10

− 8.5 10

− 4 10

− 4.5 10

− 4 10

− 8 10

− 3 10

− 4 10

− 3 10

− 7 a Data were obtained from pages 44-52 of the study report. b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG + PMSF buffer with [ 3 H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL. c Each concentration of each chemical was run in triplicate, for a total of 99 tubes per run. d Solvent is DMSO (2%)


### The procedures for assay incubation, separati on of bound from free estradiol (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

### H]-17 β

estradiol were similar to those used in the saturation binding experiment.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None



### C. DATA ANALYSIS: For the saturation binding experi ment, total binding and non-specific binding data were modeled via non-linear regression using Graph Pad Prism v. 4.01 (GraphPad Software, Inc., San Diego, CA). For the competitive bi nding assay, similar methods of nonlinear regression were used to fit a curve (for 17 β -estradiol, the weak positive control, and the test substance) to th e Hill 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 (Microsoft Corporation, Redmond, WA).



### 1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estrad iol has been displaced from the ER).

Not interactive = there are usable data points at or above 10 -6 

### M and either the lowest point on the fitted response curve within the range of the da ta is above 75% (i.e., <25% of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Equivocal up to the limit of concentrations tested = there are no data points at or above a test chemical concentration of 10 -6 

### M and either a binding curve can be fit but ≤ 50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be f it and the lowest averag e percent binding among concentration groups in the data is >50%.

Equivocal = A run is classified as equivocal if it does not fall into any of the categories above.
The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of concentrations tested “missing”



### The values for each run were then averaged acro ss runs and the chemical classified using the following ranges:Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None

Test Material Classifi cation Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”


### b. Descriptors for receptor binding:

B max 

### : maximum specific binding number (fmol ER/1 00 μg cytosol protein) measures the concentration of active receptor sites K d 

### : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

### : concentration of the test substance at whic h 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA %): ( IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance) × 100

II. RESULTS

A. SATURATION BINDING EXPERIMENT The appropriate concentration for the saturation binding assay was determ ined to be 40 μg/tube, as it bound 30.2% of the [ 3 

### H]- 17 β -estradiol (within the Guideline range of 25- 35% binding). The results from the three saturation binding assays are summarized in Ta ble 5 and shown graphically in Figures 1 and 2. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.12 nM and the B max 

### was 57.19 fmol/100 μ g protein for the prepared rat uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. The data produced consistent and linear Scatchard plots (r 2 

### = 0.9830-0.9942).

TABLE 5. Saturation Binding Experiment of 17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol a Parameter Run 1 Run 2 Run 3 Mean ± SE

r 2 (unweighted) 0.9942 0.9830 0.9894 0.9830-0.9942 b B max (nM) 0.048 0.044 0.045 0.458 ± 0.001 B max (fmol/100 μ g protein) 60.34 55.48 55.75 57.19 ± 1.58 K d (nM) 0.144 0.092 0.125 0.120 ± 0.015 a Data were obtained from page 41 of the study report. b The r 2 is reported as a range,

r 2 Goodness of fit for curve calculated for specific bindingEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None


### FIGURE 1. Binding of [ 3 

### H]-17 β -estradiol to the Estrogen R eceptor during the Saturation Binding Experiment.

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

### H]-17 β -estradiol to the Estrogen Receptor.

B. COMPETITIVE BINDING EXPERIMENT The results from the three competitive binding runs are summarized in Table 6 and pr esented graphically in Figures 3-5. TheEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None 

### parameters determined from the competitive binding curves (r 2 

### , IC 50 

### , log IC 50 

### , and RBA) are presented in Table 7.
Precipitation of dichlobenil was not observed at any concentrati on tested. In all three runs, dichlobenil showed mini mal displacement of [ 3 

### H]-E2 from the receptor at concentrations up to 10 − 3 

### M (93-104% binding). As the percent specific binding was not decreased to ≤ 50%, an IC 50 

### and RBA were not calculated for dichloben il. The E2 reference standard and the weak positive control produced the expected binding results in all three runs, with r 2 

### values of >0.99 for each run. The estimated mean log IC 50 

### was
− 9.08 and

− 5.87 M for E2 and 19- nonethindrone, respectively, and the RBA wa s 0.062% for 19-norethindrone compared to E2. The negative control had no effect on binding.
Confidence in these numbers is high due to the small variation. As dichlobenil did not decrease specific binding for [ 3 

### H]-E2 by >25% in each run at concentrations up to 10

− 3 

### M, it is classified as not interactive in this assay (Table 8).Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None

TABLE 6. Percent Specific Binding of [ 3 H]-17 β -estradiol in Competitive Binding Assay a

Test Chemical Concentration (M) % Specific Binding Run 1 Run 2 Run 3 Mean b SE b 17 β -estradiol (E2) (reference standard) 10

− 8 7.05 8.26 7.57 7.63 0.35 10

− 8.5 19.73 18.56 22.88 20.39 1.29 10

− 9 39.63 47.33 48.86 45.27 2.86 10

− 9.5 71.18 74.31 75.64 73.71 1.32 10

− 10 85.69 92.70 89.77 89.39 2.03 10

− 11 98.32 102.40 99.22 99.98 1.24 19-nonethindrone (weak positive control) 10

− 4 0.33 -0.42 0.20 0.04 0.23 10

− 4.5 3.75 1.37 3.29 2.80 0.73 10

− 5.5 31.08 31.18 30.73 31.00 0.14 10

− 6 59.02 54.62 58.12 57.25 1.34 10

− 6.5 81.55 73.77 79.44 78.25 2.32 10

− 7 91.30 85.11 89.32 88.58 1.83 10

− 7.5 96.34 87.59 93.02 92.32 2.55 10

− 8.5 99.99 91.17 93.59 94.92 2.63 Dichlobenil (test substance) 10

− 3 93.59 98.23 94.73 95.52 1.40 10

− 4 98.47 94.22 97.76 96.82 1.31 10

− 5 97.02 92.94 99.08 96.35 1.80 10

− 6 100.56 94.40 104.00 99.65 2.81 10

− 7 102.46 96.48 102.66 100.53 2.03 10

− 8 101.77 92.24 97.61 97.21 2.76 10

− 9 102.70 91.03 102.78 98.84 3.90 10

− 10 98.42 82.66 101.13 94.07 5.76 Octyltriethoxysilane (negative control) 10

− 3 89.54 94.15 89.96 91.22 1.47 10

− 4 85.19 92.58 84.18 87.32 2.65 10

− 5 96.03 103.82 94.80 98.22 2.82 10

− 6 97.05 104.31 93.50 98.29 3.18 10

− 7 94.89 104.10 95.49 98.16 2.98 10

− 8 96.29 106.84 97.33 100.15 3.36 10

− 9 96.26 103.73 96.53 98.84 2.45 10

− 10 100.56 104.45 95.18 100.06 2.69 a Data were obtained from pages 45-52 of the study report. b Calculated by reviewerEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None


### FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Dichlobenil. Run 1

FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of Dichlobenil. Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None



### FIGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of Dichlobenil. Run 3.

TABLE 7. Competitive Binding Assa y of Dichlobenil with Estrogen Receptor from Rat Uterine Cytosol a Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

r 2 (unweighted), 17 β -estradiol 0.9966 0.9977 0.9977 0.9966-0.9977 19-norethindrone 0.9985 0.9979 0.9969 0.9969-0.9985 Dichlobenil NA NA NA NA Log IC 50 (M) 17 β -estradiol

− 9.15

− 9.07

− 9.02

− 9.08 ± 0.04 19-norethindrone

− 5.84

− 5.90

− 5.86

− 5.87 ± 0.02 Dichlobenil NA NA NA NA IC 50 (M) 17 β -estradiol 7.1 × 10 − 10 8.5 × 10 − 10 9.6 × 10 − 10 8.4 × 10 − 10 ( ± 0.72) 19-norethindrone 1.44 × 10 − 6 1.26× 10 − 6 1.38× 10 − 6 1.36× 10 − 6 ( ± 0.05) Dichlobenil NA NA NA NA Log RBA (%) c 19-norethindrone

− 1.31

− 1.17

− 1.16 -1.21 ± 0.05 Dichlobenil NA NA NA NA RBA (%) c , 19-norethindrone 0.049 0.067 0.070 0.062 ± 0.006 Dichlobenil NA NA NA NA a Data were obtained from pages 26, 30 and 33 of the study report. b The mean is reported for the concurrent replicates within each run. c The range of r 2 is reported and the mean and SE are reported for the other parameters. Calculated by reviewers. For means expressed in sc ientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. NA Not applicable.

r 2 Goodness of fit RBA (%) Relative binding affinityEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None

TABLE 8. Binding Classification of Dichlobenil with Estrogen Receptor a Run 1 2 3 Mean c Binding Classification d Classification category value b 0 0 0 0 Not interactive a Data were obtained from page 11 of the study report. b Classification category value: Interac tive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Mean of three runs expressed to the tenths place d Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5



### C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated using the following criteria in Table 9.

TABLE 9. Criterion a Tolerance Limit(s) Value Yes No

17 β -estradiol fitted curve parameters Log e residual SD ≤ 2.35 ≤ 2.22 X Top (% binding) 94 to 111 100 to 102 X Bottom (% binding) -4 to 1 -2.1 to 3.0 X (Hill) Slope (log 10 (M)

− 1 ) -1.1 to -0.7 -1 .1 to -1.0 X Weak Positive control (19-) fitted curve parameters b Log e residual SD NA ≤ 2.25 X Top (% binding) NA 91 to 99 X Bottom (% binding) NA -3.6 to -0.3 X (Hill) Slope (log 10 (M)

− 1 ) NA -1.0 to -0.9 X Solvent concentration DMSO ≤ 3% 2% X Negative control (octyltriethoxysilane) does not displace more than 25% of [ 3 H]-17 β -estradiol from the ER on average across all concentrations ≤ 25% ≤ 13% X

a Data were obtained from pages 26, 28, 30 and 33 of the study report. b The EPA Guideline does not define a set of tolerance li mits for 19-norethindrone. A cceptance criteria were only defined for norethindrone, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an acceptable weak positive control. NA Not applicable



### Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17 β -estradiol displaced [ 3 

### H]-17 β -estradiol in a manner consistent with one-site binding, as indicated by a Hill Sl ope of approximately -1.0

Dichlobenil was tested over a c oncentration range that fully de fined the top of the curve. The percent binding at this top plateau 95% to 101% was within 25 percentage points of the value for the lowest concentration of the estr adiol standard (100%). Examination of the reference standards across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 15 DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Dichlobenil technical di d not show displacement of [ 3 

### H]-E2 from the receptor at all concentrations tested. The lowest point on the fitted response curve was above 75% for all experime nts. The classification category value for dichlobenil technical is 0, indicating that dichlobenil is no t interactive with the estrogen receptor.

B. AGENCY COMMENTS: For the saturation binding experiment, the appropriate concentration for the saturation binding assay was determined to be 40 μg/tube, as it bound 30.2% of the [ 3 

### H]-17 β -estradiol. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.12 nM and the mean B max 

### was 57.19 fmol/100 μ g protein for the prepared ra t uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. The data produced consistent and linear Scatchard plots (r 2 

### = 0.9830-0.9942).
In the competitive binding experiments, the reference standard (E2), the weak positive control (19-nonethindrone), and the negative co ntrol (octyltriethoxysilane) each exhibited the expected responses. The estimated mean log IC 50 

### was
− 9.08 and

− 5.87 M for E2 and 19- nonethindrone, respectively, and the RBA was 0.062% for 19-norethindrone.
Precipitation of dichlobenil was not observed at any concentrat ion. Dichlobenil was tested over a concentration range that fully defined th e top of the curve, and the maximum percent binding with dichlobenil (95- 101%) was within 25 percenta ge points of the lowest concentration of the estradiol standard, 100%. In all three runs , dichlobenil showed minimal displacement of the radioactive ligand at concentrations up to 10

− 3 

### M (93-104% binding); therefore an IC 50 

### and RBA could not be determ ined for dichlobenil.
As dichlobenil did not decrease specific bi nding by >25% in each run at concentrations up to 10 − 3 

### M, it is classified as not interactive in this assay.

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

 One performance criterion was not met for 17 β -estradiol in the competitive binding assay. The bottom binding was 3.0%, which is highe r than the Guideline -4 to 1%. This does not negatively impact the interpretation of the study.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616105)

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: 3/20/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph. D., D.A.B.T. Date: 3/26/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/09/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/09/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL /02740 1 Estrogen Receptor Transcriptional Activation (2011) I Page I of 11 OCSPP 890.1300/ OECD 455


### Primary Reviewer: Ray Kent, Ph.D. 

### SignaDtuac;-t

- ee::

# ~~; Health Effects Division

_ _ _____L__ 

### Secondary Reviewer:

_ _cM=..c=i-=ne=r'--'-v=a-"M==er:....::c=a=do=,'--'P'"-'h=·=D...:....

____ 

### Signature: Health Effects Division Date:



## - 2q-;sTemplate version 08/2011



### DAT A EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903) ; OCSPP 890

. 1300; OECD 455

.


### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS: 2
, 6-dichlorobenzonitrile



### CITATION: Meerts
, I.A.T

. M. (2011) Dichlobenil Technical: Estrogenic Receptor Transcriptional Activation Assay (Human Cell Line (HeLa-9903)). NOTOX B

. V, Hertogenbosch, The Netherlands. Laboratory Report No.: 495782
, December 9
, 2011. MRID 48616105

. Unpublished.



### SP ONSOR: Chemtura Corporation
, 199 Benson Road
, Middlebury
, CT

.


### TEST ORDER# : EDSP-027401-120



### EXECUT IVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48616105), hERa-HeLa-9903 cells cultured 

### in vitro were exposed to dichlobenil (99.3% a.i., Batch # AM9K 128222) at logarithmically increasing concentrations from 1 x 10- 9 

### to 2.5 x 

### l 0- 4 

### Min DMSO (0

. 1 % ). The experiments were performed using 96-well plates and each dichlobenil

conc e ntration was tested in triplicate (3 wells / plate). Cells were exposed to the test agent for 20- 24 hours to determine if reporter (luciferase) gene products were induced. Luciferase expression in response to activation of the estrogen receptor was measured upon addition of a luciferase

s ubstrate and detection with a luminometer with acceptable sensitivity. A total of four independent runs of the assay were conducted. Laboratory proficiency was demonstrated by testin g the 10 chemicals listed in the test guideline.

Dichlobenil was tested up to the limit of solubility
, 2.5 x 

### 10- 4 

### M. The mean RPCMax for dichlobenil was 0.9% to 5

. 1 %
, and the associated PCM ax was 10- 8 

### or 1 

# o- 6 

### M. Performance criteria

were met for**17~-estradiol ,**  

### l 7a-estradiol and corticosterone. For l 7a-methyltestosterone
, the Log PC 

### so was not calculable for Run 1
, Run 2 and Run 4, and the Log PC 1 

### o was higher than the

e xpected range for all four Runs

. The responsiveness of the cells to the very weak positive control 17a-methyltestosterone was lower than the expected values
, indicating a decreased

s en s iti v it y of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, dichlobenil responses were similar to those of the negative control corticosterone and not comparable to the responses of l 7a-methyltestosterone
, whichEstrogen Receptor Transc riptional Activation (2011) / Page 2 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### was able to reach a maximum of 34-51% PC. Di chlobenil was only able to reach a maximum of 0.9-5.1% PC when tested up to the highest c oncentration possible ba sed on cytotoxicity. Because the RPC Max 

### < PC 10 

### in both assay runs, dichlobenil was considered negative for estrogen receptor transcriptional activa tion in this test system.
This assay satisfies the EDSP Tier 1 Test Order requi rements 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 Transcriptional Activation (2011) / Page 3 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Dichlobenil Description: Technical, white powder, MW 172.0 Source: NOTOX 202841/A Lot/Batch #: Batch # AM9K12A222 Purity: 99.3% Solubility: DMSO; 50 mg/mL Volatility: Not reported (NR) Stability: Stable (stability in DMSO not indicated) Storage conditions: Room temperature in the dark Vapor pressure: NA CAS #: 1194-65-6 Structure:



### 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Sigma, Zwijndrecht, The Netherlands Catalogue and Batch #: NR Purity: NR CAS # : 50-28-2



### 17 α -estradiol (weak estrogen) Supplier: Sigma, Zwijndrecht, The Netherlands Catalogue and Batch #: NR Purity: NR CAS # : 57-91-0 

### Corticosterone (negative compound) Supplier: Sigma, Zwijndrecht, The Netherlands Catalogue and Batch #: NR Purity: NR CAS # : 50-22-6



### 17 α -methyltestosterone (very weak agonist) Supplier: Fluka, Sigma, Zwijndrecht, The Netherlands Catalogue and Batch #: NR Purity: NR CAS # : 58-18-4


### 3. Vehicle(s)
Solvent: DMSO (SeccoSolv, Merk, Darmstadt, Germany)
Solvent control (final concentration): 0.1%Estrogen Receptor Transc riptional Activation (2011) / Page 4 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank. Cells were ma intained 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 (prepare d in-house according to NOTOX Standard Operating Procedure I VA/L/007 which is identical to the Guideline method), in an incubator under 5% CO 2 

### at 37oC. Upon reaching 75-90% confluence, cells were subcultured more than fewer than eleven tim es prior to exposure to the test material.

2. Transcriptional Activation Assays: Cells were plated at a density of 1×10 4 

### cells/100 μL medium/well in a 96-well plat e and allowed to attach for 3 hours. Growth media was replaced with media containing serial log dilutions of dichlo benil in DMSO (0.1%). Cells were incubated for 20-24 hours at 37±1°C. Cytotoxicity was determined by MTT.
Transcriptional activation of the estrogen receptor was determined using a standard luciferase assay system (Promega, Madison, W I). The plate was read with an Infinite ® 

### M200 Pro luminometer. The limit of quantitati on for this luminometer was not reported.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 2.5 ×10 -4 

### to 10 -9 

### M was conducted to determine the appropri ate concentration range and to determine concentrations resulting in insolu bility and/or cy totoxicity.

b. Proficiency Chemicals: The responsiveness of the test system was confirmed for each newly prepared batch of cell stocks taken from the frozen st ock by testing the following set of proficiency chemicals in duplicate on sepa rate days. The proficiency of the testing laboratory was demonstrated as indicated in the table below.

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 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### c. Reference Chemicals: To ensure the stability of th e response from the cell line, six 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 (ER) 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. Graph Pad Prism v. 4.01 (GraphPad Software, Inc., La Jolla, CA) was used to calculate the EC 50 

### , PC 10 

### , PC 50 

### , RPC Max 

### , and PC Max 

### for dichlobenil when applicable. 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 (o r 2 of 3) runs. Log EC 50 

### and Hill slope values are calculated only if a positive respons e is observed. Coefficien ts of variation (CV) were calculated for the luminescence data tr iplicates. Concentr ations showing >20% cytotoxicity or evidence of insolubi lity 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. The maximum stock concentration of dichlobenil in DMSO that could be prepared was a stock of 250 mM. Therefore the highest concentr ation that could be tested was 2.5 × 10 -4 

### M. The results of the cytotoxicity test are presented in Table 1. No cytotoxicity or precipitation was observed at any concentration. Based on these results,Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

### logarithmically increasing concentrations from 10 -9 

### to 2.5 ×10 -4 

### M were selected for the assay.

TABLE 1. Preliminary Test for Solubility, Cytotoxi city, and Concentration-Selection for Dichlobenil a Concentration (M) % Viability Comments 2.5 ×10

− 4 93.4 10

− 4 102.3 10

− 5 95.2 10

− 6 97.6 10

− 7 91.6 10

− 8 92.4 10

− 9 90.9 70% methanol 0 Positive control for cytotoxicity VC b 100 a Data were obtained from page 24 of the study report. b VC = Vehicle control



### B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS
1. Proficiency Chemicals: The responsiveness of cells to the required profic iency chemicals was performed in duplicate on different days. The data are reported in Table 2. The responses demonstrated the performing la boratory’s proficiency with the assay.

TABLE 2. Proficiency Chemicals a Compound Expected Response Lab Response Diethylstilbestrol Positive Positive 17 α -Ethynyl estradio l Positive Positive Hexestrol Positive Positive Genistein Positive Positive Estrone Positive Positive Butyl paraben Positive Positive 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive Dibutyl phthalate Negative Negative Atrazine Nega tive Negative Corticosterone Negative Negative a Data were obtained from page 74 of the study report.



### 2. Reference Chemicals: Values derived from the concentration response curve ( e.g ., log PC 50 

### , Log PC 10 

### , Log EC 50 

### , and Hill slope) for the four conc urrently run reference materials are included in Table 3. Perfor mance criteria were met for 17 β -estradiol, 17 α -estradiol and corticosterone. For 17 α -methyltestosterone, the Log PC 50 

### was not calculable for Runs 1, 2 and 4, and the Log PC 10 

### was higher than the acceptable ra nge in all four Runs. For 17 α

methyltestosterone, the mean RPC Max 

### range was 34-51% for all four Runs. This indicates a decreased sensitivity to very weak agonists.Estrogen Receptor Transc riptional Activation (2011) / Page 7 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455

TABLE 3. Performance Criteria for Reference Chemicals a
Reference Chemical Parameter Acceptable Range Values Acceptable Run 1 Run 2 Run 3 Run 4 Yes No 17 β -estradiol Log PC 50 − 11.4 to

− 10.1 -10.6 -10.5 -10.5 -11.2 X Log PC 10 <

− 11 -12.0 -11.5 -11.6 -12.2 X Log EC 50 − 11.3 to

− 10.1 -10.5 -10.3 -10.4 -11.3 X Hill Slope 0.7 to 1.5 1.0 0.9 0.8 1.1 X Test range 10

− 14 to 10

− 8 M 10

− 14 to 10

− 8 M X 17 α -estradiol Log PC 50 − 9.6 to

− 8.1 -8.9 -8.8 -8.9 -8.3 X Log PC 10 − 10.7 to

− 9.3 -9.7 -9.7 -9.4 -9.5 X Log EC 50 − 9.6 to

− 8.4 -8.9 -8.7 -8.9 -8.4 X Hill Slope 0.9 to 2.0 1.4 1.1 1.9 0.9 X Test range 10

− 12 to 10

− 6 M 10

− 12 to 10

− 6 M X Corticosterone Test range 10

− 10 to 10

− 4 M 10

− 10 to 10

− 4 M X 17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1 NC NC -5.0 NC X Log PC 10 − 8.0 to

− 6.2 -6.0 -5.7 -5.9 -5.9 X Test range 10

− 11 to 10

− 5 M 10

− 11 to 10

− 5 M X a Data were obtained from pages 22, 27 and 28 of the study report.



### 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 9867 – 26753, and the overall mean TA value for the positive control was 69791-221528. The induction for the positive control ranged from 7.1- to 8.7-fold. The mean normalized value for the maximum response of the positive control was 59925 - 194776. The PC 50 

### (50% of the maximum response) for E2 in this assay is 29962-97388 and the PC 10 

### (10% of the maximum response) is 5993-19478.

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 16234 1575 138332 11401 8.5 122098 11401 2 26753 NR 221528 NR 8.3 194776 NR 3 9867 NR 69791 NR 7.1 59925 NR 4 18812 NR 164310 NR 8.7 145498 NR a Data were obtained from pages 41, 42, 45, 46, 49, 50, 53 and 54 of the study report. SD calculated by reviewer. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC) NR Not reported



### 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the four a ssay runs is presented in Table 5. The concentration-response curves depicting fold induction of relative tran scriptional activation is presented in Figures 1 through 4. The mean RPC Max 

### was 0.9% to 5.1%, and the associated PC Max 

### was 10 -8 

### or 10 -6 

### M. Because the RPC Max 

### < PC 10 

### in all runs, dichlobenil was considered negative for estrogen receptor transcriptional activation in this test system.Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Tran scriptional Activation (RTA) of Dichlobenil a
Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Run 3 Run 4 Conc. (M) Mean SD Mean SD Mean SD Mean SD 2.5 ×10

− 4 0.20 1.09 -2.94 1.31 -3.11 1.23 -0.22 1.63 10

− 4 -3.41 0.32 -1.93 1.56 -2.10 0.40 1.29 0.91 10

− 5 -1.21 3.17 -2.19 0.47 -1.45 1.71 -0.49 1.15 10

− 6 2.36 0.88 1.08 1.62 0.93 1.62 2.19 1.79 10

− 7 3.21 1.24 1.67 3.50 -1.53 1.44 1.80 2.34 10

− 8 5.05 1.96 3.13 2.14 -0.59 2.70 2.49 0.73 10

− 9 4.26 3.80 1.23 1.40 -1.94 1.86 -1.03 0.71 Log EC 50 NA NA NA NA Hill Slope NA NA NA NA RPC Max 5.1 3.1 0.9 2.5 PC Max 10

− 8 10

− 8 10

− 6 10

− 8 PC 50 NA NA NA NA PC 10 NA NA NA NA a Data were obtained from pa ge 31 of the study report. NA = Not Applicable



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

VC= Vehicle Control PC= Positive Control (1 nM E2)Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### FIGURE 2. Fold Induction of Relative Tr anscription Activation (RTA) of Dichlobenil Compared to the Positive Control, Run 2.

VC= Vehicle Control PC= Positive Control (1 nM E2)



### FIGURE 3. Fold Induction of Relative Tr anscription Activation (RTA) of Dichlobenil Compared to the Positive Control, Run 3.

VC= Vehicle Control PC= Positive Control (1 nM E2)Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455



### FIGURE 4. Fold Induction of Relative Tr anscription Activation (RTA) of Dichlobenil Compared to the Positive Control, Run 4.

VC= Vehicle Control PC= Positive Control (1 nM E2)



### 3. Performance Criteria: The laboratory proficiency assays using the required reference compounds adequately demonstrated that the assay was functioning properly. Performance criteria were met for 17 β -estradiol, 17 α -estradiol and corticosterone. For 17 α methyltestosterone, the Log PC 50 

### was not calculable for Runs 1, 2 and 4, and the Log PC 10 

### was higher than the acceptable range for a ll four Runs. This indicates a decreased sensitivity to very weak agonists. However, maximum induction by 17 α -methyltestosterone was between 34-51% of PC in all runs (35, 36, 51 and 34% in the 1 st 

### , 2 nd 

### , 3 rd 

### and 4 th 

### runs, respectively), indicating that th e assay was able to detect at least some activity from 17 α

methyltestosterone. Mean luciferase activity of the PC was >4-fold that of the vehicle control for all runs, and the fo ld-induction corresponding to the PC 10 

### of the concurrent PC was greater than 1+2 standard deviations of the vehicle control on all plates.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: In this assay, four independent experiments were performed with dichlobenil at seven con centrations (from 0.001 μM to 250 μM) in triplicate. In each experiment a quality cont rol plate with the reference compounds E2, 17 α

estradiol, 17 α -methyltestosterone and corticosterone we re include. In addition on each plate concurrent positive (1nM E2) and negative (Vehicle, DMSO) controls were included in triplicate wells. The vehicle concentration was kept constant in all wells at 0.1%.
The mean luciferase activity of the positive c ontrol included in each experiment was at least 4-fold higher than the vehicl e control in all experiments. The fold induction of E2 corresponding to the PC 10 

### value was above the fold inductio n of the vehicle control +2SDEstrogen Receptor Transc riptional Activation (2011) / Page 11 of 11 DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

### (standard deviations) in all experiments. The coefficient of variation in triplicate luminescence intensity data was occasionally above the target of 20%, but this had no effects on the curve fit parameters and did not affect the integrity of the results.
The results of the reference compounds E2, 17 α -estradiol and corticoste rone were within the acceptability criteria. The curve fit values log PC 50 

### , Log PC 10 

### and Log EC 50 

### for 17 α -methyltestosterone from all four experiments were just outside the acceptability criteria mentioned in the Guideline, but were within ou r laboratory historical control data range and were thus accepted. The reference compounds were correctly classi fied as positive (E2, 17 α -estradiol and 17 α -methyltestosterone) or ne gative (corticosterone).

Dichlobenil did not show an induction of the estrogenic response at increasing concentration. This was reproducible in four independent experiments.

B. AGENCY COMMENTS: Dichlobenil was tested up to the limit of solubility, 2.5×10

− 4 

### M. The laboratory proficiency assays using th e required reference compounds adequately demonstrated that the assay was functioning pr operly. Performance criteria were met for 17 β -estradiol, 17 α -estradiol and cor ticosterone. For 17 α - methyltestosterone, the Log PC 50 

### was not calculable for Runs 1, 2 and 4, and the Log PC 10 

### was higher than the expected range for all four Runs. This indicates a decreased sensitivity to very weak agonists. The mean RPC Max 

### for dichlobenil was 0.9% to 5.1%, and the associated PC Max 

### was 10 -8 

### or 10 -6 

### M. Because the RPC Max 

### < PC 10 

### in all runs, dichlobenil was considered negative for estrogen receptor transcriptional activa tion in this test system.

C. STUDY DEFICIENCIES: The following deficiency was noted:

 The response to 17 α -methyltestosterone indicates a decreased sensitivity to weak agonists. However, the RPC Max 

### for 17 α -methyltestosterone was 30-50% compared to 0.9-5.1% for dichlobenil. It is unlikely that the decreased sensitivity changes the conclusion of these results.

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

EPA MRID Number 48616106

Data Requirement: EPA DP Barcode 397913

OECD Data Point 229

EPA MRID 48616106 EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Dichlobenil Purity: 99.3%

Common Name: Dichlobenil
Chemical Name: IUPAC: 2,6-Dichlorobenzonitrile
CAS Name: 2,6-Dichlorobenzonitrile

CAS No. 1194-65-6

EPA PC Code: 027401

Primary Reviewer: Joan Gaidos Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 6/20/12

Secondary Reviewer: Teri S. Myers Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 6/22/12

Primary Reviewer (and final additional reviewer): Elyssa Arnold Biologist, EPA/OPP/EFED/ERB2 Date: 5/29/15

Additional Reviewer: Amy Blankinship
Senior Chemist, EPA/OPP/EFED/ERB3 Date: 5/29/15

Date Evaluation Completed: 29-05-2015

Page 1 of 83 

### AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.08 10:53:51 -04'00'

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

EPA MRID Number 48616106

CITATION: Migchielsen, Ing. M.H.J. 2011. Fish Short-term Reproduction Assay with Dichlobenil Technical. Unpublished study performed by NOTOX B.V., Hambakenwetering 7, The Netherlands. Lab Study No.: 495786; Sponsor No.: EDSP-027401-120. Study sponsored by Chemtura Corporation, Middlebury, CT. Study completed September 22, 2011.

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

D

isclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted.

Page 2 of 83

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

EPA MRID Number 48616106

EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of dichlobenil with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish, 20 spawning groups (2 males and 4 females in each group; 6-7 months old) were exposed to dichlobenil (99.3% purity) at nominal concentrations of 0 (negative control), 0 (solvent control; 0.1 mL/L acetone), 0.021, 0.20, and 2.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.001; both controls), 0.021, 0.21, and 2.1 mg a.i./L. The test system was

maintained at 24.3 to 26.2 o C and a pH of 7.3 to 7.9. Unless otherwise indicated, the treatment groups were compared to the negative control.

There were no statistically significant differences (p>0.05) in mean male or female body weights or lengths
at any test level compared to the control. Overall mean survival values were 100, 96, 100, 100 and 92% in the negative control, solvent control, and low, mid, and high treatment levels, respectively. Fish exposed to the high treatment level were observed to have a lighter, more yellowish color compared to the fish in the controls and other treatment levels; however, the incidence of this effect was not reported. No other changes
in behavior or appearance were noted.

Spawning frequency in the negative and solvent control was at least every 4 days in 3 and 2 of the 4 replicates, respectively; the mean fecundity in the negative and solvent controls was 12.1 (range 6.6-15.8)and 6.5 (range 4.3-11.6) eggs/female/day/replicate. Fecundity was significantly (p<0.05; JonckheereTerpstra) reduced by 53% and 90% at the mid and high treatment levels, respectively, compared to the
negative control. The study author did not provide fertility data, but reported fertility was “more than 99.9% in all test groups” and fertility of eggs from control animals was at least 95%.

Median male tubercle score showed a statistically significant reduction from 26 in the negative control to 21, 14, and 12 in the low, mid and high treatment groups, respectively (p<0.05; Jonckheere-Terpstra). No
tubercles were observed for females, except for one female with one tubercle in the low treatment group which was reported not to be treatment related. There were no significant differences in male or female gonadal stage index (GSI) or vitellogenin (VTG).

Page 3 of 83

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

EPA MRID Number 48616106

In males, no treatment-related histopathological gonadal findings were reported. In females, there was an increased incidence and/or severity (minimal to moderate) of oocyte atresia and a shift to more mature
stages in the ovaries in the mid and high treatment groups.

Sex steroids (testosterone and 17 ȕ -estradiol) were not measured. No dorsal nape pad measurements or
other dorsal nape pad observations were reported.

While not statistically significant (p=0.078), the fecundity in the solvent control group was reduced by 46%
compared to the negative control. However, large variability was observed between the pre-exposure and exposure fecundity rate for both the negative and solvent control replicates, suggesting this difference may not be solvent related. Additionally, this was the only endpoint for which a notable difference between the negative and solvent control was observed.

Not all performance criteria for the OCSPP 890.1350 guideline were met for reported parameters. The EPA recommendation of at least 15 eggs/female/reproductive day/replicate or a spawning frequency of at least every four days in every replicate was not met (for one replicate in negative control and two replicates in solvent control); however, the overall mean fecundity in the controls was determined to be adequate for
interpretation of the results. While fertilization success was reported to be >95% in the control, these data were not provided in the study report. Additionally, there is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment groups within the blocks. In this study, there were 3 tanks in the exposure period in which the sex ratio was 3 males and 3 females instead of 2 males and 4 females as dictated by the guideline. Two of these tanks were in the solvent control and one was in the mid treatment group. All other performance and validity criteria were met.

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

Page 4 of 83

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

EPA MRID Number 48616106

Results Synopsis

Test Organism age at test initiation: ca . 6-7 months Mean body weight at test initiation (if measured): Males 2.14 g; Females 0.65 g. Mean length at test initiation (if measured): Males 4.8 cm; Females 3.5 cm. Test Type (Flow-through, Static, Static Renewal): Flow-through

Page 5 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Dichlobenil.Abbreviations:E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.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.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.021 No NA Yes No No No No No No No NA NA NA NA0.21 Yes NA Yes No No No No Yes No No NA NA NA NA2.1Yes NA Yes No No No No Yes No No NA NA NA NAPage6of83

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

EPA MRID Number 48616106

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. Prior to study initiation, the fecundity was 5.3-15.3 eggs/female/reproductive day/replicate, with only two replicates at 15 or greater. The guideline recommends at least two spawning events in the 7 days immediately prior to test initiation or 15 eggs/female/day/replicate. The study author reported that spawning occurred every fourth day; however, raw data to support this finding
were not reported. 2. The study author reported that fertilization success was “more than 99.9% in all test groups”, but these replicate data were not provided for confirmation. 3. The fecundity in the negative and solvent control groups were <15 eggs/female/day/replicate and spawning in every replicate did not occur at least every fourth day (one in the negative control and two in the solvent control).
4. The fish were between 6 and 7 months of age at the start of the study. The EPA guideline states that fish should be 4.5-6 months of age at the start of the study. 5. During the first 3 days of the pre-exposure period, light was continuous (no dark period). No adverse effects on fish or spawning were reported.
6. For one day during the pre-exposure period, the flow of dilution water was stopped to a group of 7 tanks (tanks 23-29). This caused a decrease in
temperature and dissolved oxygen during this period below the optimum levels. No adverse effects on fish or spawning were observed and eggs were produced on the effected day.

Page 7 of 83

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

EPA MRID Number 48616106

7. Food was not supplied in two equal portions on Days 4 and 5 of the exposure period. Amount supplied were sufficient to maintain health of the fish and no

adverse effects were observed. 8. In 3 replicates used in the exposure period, 3 females and 3 males were exposed instead of 4 females and 2 males. Two of these replicates were in the solvent control test group and one was in the 0.021 mg a.i./L test group. This deviation was noticed in the last week of the study. Fecundity calculations were adjusted based on the correct number of females. 9. There is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment groups within a block.

These deficiencies/deviations do not have an impact on the results or interpretation of this study.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims statements were provided. This study was conducted in accordance with GLP Standards as published by the OECD Principles of GLP [ENV/MC/CHEM(98)17].

A. TEST MATERIAL: Dichlobenil, CAS No. 1194-65-6

Description: White powder with lumps; solubility in water, 0.021 g/L.

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. : AM9K12A222 (Batch No.)

Purity: 99.3% (Expiration data October 12, 2012)

Impurities: None reported

Page 8 of 83

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

EPA MRID Number 48616106

Stability of Compound: Analytical verification at Days 0, 7, 14 and 21 of diluter stock fortified with 0.2, 2.0 and 20 mg a.i./L yielded recoveries ranging from 99 to 117% of nominal concentrations. Method validation recoveries fortified at 0.021 and 2.0 mg a.i./L yielded recoveries ranging from 81 to 111%. Analytical verification of the test solutions at Days -1, 0, 4, 7, 13/14 and 21 yielded
recoveries of 88 to 123% of nominal concentrations; there was one outlier that had been stored frozen with a recovery of 652%. There was no pattern of decline. The average %CV ranged from 8.9 to 11%. These data indicate the test material was generally stable under the test conditions.

Storage Conditions of

Test Chemicals: The test material was stored at room temperature in the dark and reported to be stable under these conditions. Storage stability was determined after 0, 3, 8, 15, and 21 days.

Page 9 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106B. 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):Teleostei, Cyprinidae Linnaeus, 1758Were fish obtained from a singlelaboratory stock?Yes EPA recommends that fish be from a single laboratory stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:At least 2 weeks EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Page10of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities during the 7 days prior to preexposure period was <5%.Fish did not receive any treatment fordisease during acclimation period.No behavioral abnormalities or clinical signswere noted. 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 reportedFrequency of feeding:2 times/dayAt least 3 hours apart, except on weekenddays.Details on feeding:NonePage11of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6-7 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):2.14±0.34 g Based on 10 males used to stock aquariafor 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):1.58-2.83 gMean weight of females at testinitiation (if determined):0.65±0.09 g Based on 10 females used to stock aquariafor pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):0.54-0.79 gMean length of males at testinitiation (if determined):4.8 ± 0.2 cm Based on 10 males used to stock aquariafor pre-exposure phase.Mean length of females at testinitiation (if determined):3.5 ± 0.1 cm Based on 10 females used to stock aquariafor pre-exposure phase.Duration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days.Page12of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes Exceptions:(1) During first 3 days of pre-exposure, testlight was continuous (no dark); dark-lightregime corrected for the following 11 daysand no adverse effect detected.(2) During a period of nearly one full day,the temperature was below the optimumrange in two tanks, oxygen was low andthere was no flow of dilution water; fish werenot affected and did produce eggs duringthis period. EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks: 30 tanks 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.Page13of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of males per tank: 2 males/tank3 tanks were found to have an incorrect sexratio (3 males and 3 females) during thepre-exposure phase and were not used forthe exposure phase of the study. Duringthe exposure phase, 3 additional tanks werefound to have the same incorrect sex ratio(3 males and 3 females).Number of females per tank: 4 females/tankPre-exposure fecundity:5.3-15.3 eggs/female/reproductive day/replicate in the 20 topperforming tanksThe EPA recommendation of at least 15eggs/female/reproductive day prior to testinitiation was not met. However, thefecundity was averaged over the entire 14day pre-exposure period and data were notbroken out for the 7 days prior to thedefinitive test. 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.Number of spawns during preexposure:Not reported No data provided, but spawning occurred atleast every fourth day according to the studyreport. EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:NonePage14of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilutionsystem:Proportional diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 21U.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).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Page15of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsFlow-through rate:3 L/hr (equivalent to 7volume exchanges per day) 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:Solution from mixing chamber split equallyamong replicate test vessels.Treatment recoveries prior to test initiation were86-101%.The flow splitting accuracy was 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? No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Page16of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Reverse-osmosis treatedtap water EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test

guideline.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?YesTest vessel type/materials: Glass aquaria with siliconesealant and covered withPerspex plateEPA 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 L (dimensions notreported) EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10 L EPA recommends 10 L solution per tank.Page17of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsSpawning substrate material: PVC pipe to form asemicircular arch 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: Not further described.Additional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.326.225Approximately every 4days; continuous incontrols EPA recommends temperature 25±1 oC; interreplicate and inter-treatment differentials should not exceed 1 o C.pH7.37.97.6Twice a week EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)5.88.77.2Twice a week EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mmol/L asCaCO3)0.8 1.0 0.9Weekly in control andhighest conc. only EPA recommends total alkalinity > 20 mg/L

as CaCO 3.Page18of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsHardness (mg/L as CaCO3) 161 196 181Weekly in control andhighest conc. onlyTotal organic carbon (mg/L)0.05 0.050.05 Once at start of test EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L) Not reported Not reported Not reported Not reported EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L) Not reported Not reported Not reported Not reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Conductivity (Pmhos/cm) Not reported Not reported Not reported Not reported 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.Page19of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?No EPA recommends conducting a range-finder if

96-hour LC 50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?NA EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Species:NALife stage:NA EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:NA EPA recommends a 96-hour exposure.Page20of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:NA EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21- day

test.Page21of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Not reported 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 test concentrations:Ten-fold EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:3 (3 treated, a negativecontrol and a solventcontrol) EPA recommends a minimum of 3 concentrations and a control, plus solvent control
if appropriate.Are nominal concentrationsadjusted for purity?YesPage22of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ): 0.001 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement: 0, 4, 7, 13, 14, and 21daysSamples taken on Day 4 were from the hightreatment group only as the stock solutionappeared not homogeneous; additionally, aDay 13 sample was taken from the lowtreatment as dilution water delivery wastemporarily disrupted. 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.Page23of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes While block design was used, it is uncertainof whether the treatments were randomizedwithin a block. EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to pre-exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):4 EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 During the last week of the definitive study,3 tanks were found to have an incorrect sexratio (3 males and 3 females). EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Page24of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?YesSolvent type (if applicable): AcetoneMaximum solvent concentration (ifapplicable):0.1 mL/L 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) (ifapplicable):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.Page25of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’s surface: 540 to 1080 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Details about test solution appearance werenot found in the study report.Page26of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Dichlobenil.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0.00 <LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control0.00<LOQNATreatment 10.0200.02111Mean measured and CVincludes Day 13 sampleTreatment 20.200.2111Treatment 32.02.18.9Mean measured and CVincludes Day 4 sampleAbbreviations:CVCoefficient of variation.LOQ=0.001 mg a.i./L.Page27of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106E. ObservationsBiological Endpoints:Survival, fecundity, and clinical signs were observed daily. At test termination (Day 21), secondary sex characterization (bodycolor, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology, and plasma vitellogeninwere evaluated.Were raw (individual) data provided?Yes, with the exception of fertility and spawning frequency data EPA recommends that observations of survival, fecundity, fertilization 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.Page28of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106II. RESULTS AND DISCUSSIONA. ResultsOne male died in the solvent control and two females died in the 2.1 mg a.i./L treatment group during the study. Overall survival values were 100,96, 100, 100 and 92% in the negative control, solvent control,and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively.Table 9: Adult Fish Survival in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesN1# Surviving% Survivaln# Surviving% SurvivalControl(<LOQ) 8 8 100 16 16 100Solvent control (<LOQ)1099014141000.021 9 9 10015 15 1000.21 8 8 1001616 1002.1 8 8 1001614 881Total number of fish at test initiation.LOQ=0.001 mg a.i./L.Page29of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Mean male body weight values were 2.07, 1.95, 2.01, 1.98 and 2.00 g and female body weight values were 0.79, 0.83, 0.83, 0.87 and 0.83 gin the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. Meanmale body lengthvalues were 47, 47, 47, 47 and 47 mm and female body length values were 35, 35, 35, 36 and 36 mm in the negative control, solvent control,and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively.Table 10: Size at Test Termination in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ) 4 2.07 0.32 4 0.79 0.02 4 47 1.5 4 35 0.6Solvent control(<LOQ)4 1.95 0.30 4 0.83 0.07 4 47 2.3 4 35 1.40.021 4 2.01 0.21 4 0.830.07 4 47 0.8 4 35 1.00.21 4 1.98 0.34 4 0.87 0.10 4 47 1.4 4 36 1.32.1 4 2.00 0.59 4 0.83 0.08 4 47 5.3 4 36 1.3LOQ=0.001 mg a.i./L.Page30of83

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

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Mean fecundity values were 12.1, 6.5, 7.5, 5.6 and 1.2 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. Fertilization success data were not reported, however the study author did report that fertility was >99.9% in all test groups.

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L) [mean-measured] Fecundity 1 Fertilization Success (%) 2

Mean ±SD

Control (<LOQ) 12.1 4.0 Not reported

Solvent control (<LOQ) 6.5 3.5 Not reported

0.021 7.5 1.2 Not reported

0.21 5.6 2.6 Not reported

2.1 1.2 1.1 Not reported

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.
LOQ=0.001 mg a.i./L.

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

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Median male tubercle scores were 26, 21, 22, 14 and 12 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. None of the surviving females were found to have tubercles, with the exception of one female fish in the 0.021 mg a.i./L test group, who had one tubercle on her snout that was not believed to be caused by exposure to dichlobenil.

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

.

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

n Median Tubercle

Score n Median Tubercle

Score

Control (<LOQ) 4 26 4 0

Solvent control (<LOQ) 4 18 4 0

0.021 4 21 4 0

0.21 4 14 4 0

2.1 4 12 4 0

LOQ=0.001 mg a.i./L.

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

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Average male GSI was 1.18, 1.27, 1.23, 1.26 and 1.53 and average female GSI was 12.31, 12.07, 10.51, 12.53 and 14.73 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively.

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( P imephales promelas)

.

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

n Mean GSI 1
(%) ±SD n Mean GSI 1
(%) ±SD

Control (<LOQ) 4 1.18 0.25 4 12.31 1.75

Solvent control (<LOQ) 2 3 1.27 0.21 4 12.07 1.24

0.021 4 1.23 0.46 4 10.51 1.20

0.21 4 1.26 0.25 4 12.53 1.95

2.1 4 1.53 0.07 4 14.73 2.01

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

2 The gonadal weights of two male fish in the solvent control replicate D were measured incorrectly and were not included in this analysis. LOQ=<0.001 mg a.i./L.

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

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Median male gonadal stage was 2, 2, 2, 2, and 2 and median female gonadal stage was 3, 3, 3, 4, and 3 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively.

Table 14: Gonadal Staging in Fathead Minnow ( P imephales promelas)

.

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. LOQ=0.001 mg a.i./L.

In males, there were minimal findings of increased granulomatous inflammation and no differences in stages of testes between the controls and treatment groups. In females, there was an increased incidence and/or severity (minimal to moderate) of oocyte atresia and a shift to more mature stages in the ovaries in the 0.21
and 2.1 mg a.i./L treatment groups. According to the study authors, both findings suggest a reduced spawning in females in these treatment groups. The study authors reported all other microscopic findings in both males and females to be within normal range of background pathology. Treatment (mg a.i./L) [mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Control (<LOQ) 4 2 4 3

Solvent control (<LOQ) 4 2 4 3

0.021 4 2 4 3

0.21 4 2 4 4

2.1 4 2 4 3

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

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Table 15: Gonadal Histopathology in Male Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased Proportion of Spermatogonia Presence of

Testis-Ova Increased

Testicular Degeneration Interstitial Cell Hyperplasia/ Hypertrophy Apoptotic Germ

Cell

n Incidence n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 NA NA NA NA NA NA NA NA 8 5

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 1

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

Solvent

control (<LOQ) 0 NA NA NA NA NA NA NA NA 9 6

1 NA NA NA NA NA NA NA NA 9 3

2 NA NA NA NA NA NA NA NA 9 0

3 NA NA NA NA NA NA NA NA 9 0

4 NA NA NA NA NA NA NA NA 9 0

0.021 0 NA NA NA NA NA NA NA NA 9 8

1 NA NA NA NA NA NA NA NA 9 1

2 NA NA NA NA NA NA NA NA 9 0

3 NA NA NA NA NA NA NA NA 9 0

4 NA NA NA NA NA NA NA NA 9 0

0.21 0 NA NA NA NA NA NA NA NA 8 6

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 0

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

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

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Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased Proportion of Spermatogonia Presence of

Testis-Ova Increased

Testicular Degeneration Interstitial Cell Hyperplasia/ Hypertrophy Apoptotic Germ

Cell

n Incidence n Incidence n Incidence n Incidence n Incidence

2.1 0 NA NA NA NA NA NA NA NA 8 5

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 1

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

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=0.001 mg a.i./L.

Page 36 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ)0 NA NA NANA NA NA NA NA 8 81 NA NA NA NA NA NA NA NA 8 02 NA NA NA NA NANA NA NA 8 03 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 0Solvent control (<LOQ) 0 NA NANA NA NA NA NA NA 9 81 NA NA NA NA NA NA NA NA 9 02 NA NA NA NA NA NA NA NA 9 13 NA NA NA NA NANA NA NA 9 04 NA NA NA NA NANA NA NA 9 0Page37of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.021 0NANANANANA NA NA NA 9 61 NA NA NA NA NA NA NA NA 9 02 NA NA NA NA NA NA NA NA 9 13 NA NA NA NA NA NA NA NA 9 14 NA NA NA NA NANA NA NA 9 05 NA NA NA NA NA NA NA NA 9 10.21 0NANANANANA NA NA NA 8 31 NA NA NA NA NA NA NA NA 8 22 NA NA NA NA NA NA NA NA 8 13 NA NA NA NA NA NA NA NA 8 14 NA NA NA NA NA NA NA NA 8 1Page38of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence2.10 NA NA NA NA NA NA NA NA 8 61 NA NA NA NA NA NA NA NA 8 12 NA NA NA NA NA NA NA NA 8 13 NA NA NA NA NANA NA NA 8 04 NA NA NA NA NANA NA NA 8 01Gonadal 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.LOQ=<0.001 mg a.i./L.Page39of83

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

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Table 17: Gonadal Histopathology in Female Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell Hyperplasia/ Hypertrophy Decreased Yolk

Formation Infection, Microsporidia

n Incidence n Incidence n Incidence n Incidence
Control (<LOQ) 0 16 12 NA NA NA NA NA NA

1 16 3 NA NA NA NA NA NA

2 16 1 NA NA NA NA NA NA

3 16 0 NA NA NA NA NA NA

4 16 0 NA NA NA NA NA NA

Solvent control (<LOQ) 0 14 10 NA NA NA NA NA NA

1 14 2 NA NA NA NA NA NA

2 14 1 NA NA NA NA NA NA

3 14 1 NA NA NA NA NA NA

4 14 0 NA NA NA NA NA NA

0.021 0 14 9 NA NA NA NA NA NA

1 14 4 NA NA NA NA NA NA

2 14 1 NA NA NA NA NA NA

3 14 0 NA NA NA NA NA NA

4 14 0 NA NA NA NA NA NA

0.21 0 16 6 NA NA NA NA NA NA

1 16 4 NA NA NA NA NA NA

2 16 2 NA NA NA NA NA NA

3 16 1 NA NA NA NA NA NA

4 16 0 NA NA NA NA NA NA

2.1 0 14 3 NA NA NA NA NA NA

1 14 6 NA NA NA NA NA NA

2 14 4 NA NA NA NA NA NA

3 14 1 NA NA NA NA NA NA

4 14 0 NA NA NA NA NA NA

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

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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=0.001 mg a.i./L

Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fluid Egg Debris in

Oviduct Granulomatous

Inflammation Macrophage Aggregate

n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 16 15 NA NA 16 15 16 9

1 16 1 NA NA 16 0 16 7

2 16 0 NA NA 16 1 16 0

3 16 0 NA NA 16 0 16 0

4 16 0 NA NA 16 0 16 0

Solvent control (<LOQ) 0 14 13 NA NA 14 0 14 13

1 14 0 NA NA 14 0 14 1

2 14 0 NA NA 14 0 14 0

3 14 1 NA NA 14 0 14 0

4 14 0 NA NA 14 0 14 0

0.021 0 14 11 NA NA 14 12 14 10

1 14 1 NA NA 14 1 14 3

2 14 1 NA NA 14 1 14 1

3 14 1 NA NA 14 0 14 0

4 14 0 NA NA 14 0 14 0

0.21 0 16 15 NA NA 16 15 16 13

1 16 1 NA NA 16 1 16 2

2 16 0 NA NA 16 0 16 1

3 16 0 NA NA 16 0 16 0

4 16 0 NA NA 16 0 16 0

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

EPA MRID Number 48616106

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fluid Egg Debris in

Oviduct Granulomatous

Inflammation Macrophage Aggregate

n Incidence n Incidence n Incidence n Incidence

2.1 0 14 12 NA NA 14 13 14 13

1 14 2 NA NA 14 0 14 0

2 14 0 NA NA 14 0 14 1

3 14 0 NA NA 14 1 14 0

4 14 0 NA NA 14 0 14 0

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=<0.001 mg a.i./L

Mean male VTG was 290, 875, 1078, 238 and 533 and female VTG was 1.27x10 6 , 1.42x10 6 , 1.30x10 6

, 1.05x10 6 and 8.80x10 5 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively.

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Table 19: Plasma Vitellogenin in Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Plasma Vitellogenin (VTG) 1

Males Females

n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD

Control (<LOQ) 4 290 299 4 1.27 x 10 6 4.27 x 10 5

Solvent control (<LOQ) 4 875 1551 4 1.42 x 10 6 5.96 x 10 5

0.021 4 1078 701 4 1.30 x 10 6 5.38 x 10 5

0.21 4 238 189 4 1.05 x 10 6 2.10 x 10 5

2.1 4 533 408 4 8.80 x 10 5 5.70 x 10 5

1 Average and standard deviations calculated by the reviewer using the individual data from the study appendices; these

values were not reported by the study author. LOQ=0.001 mg a.i./L.

Page 43 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Mean male and female plasma testosterone and plasma 17ȕ-estradiolwere not measured.Table 20: Plasma Sex Steroids in Fathead Minnow ( P imephales 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 NANA NA NA NA NA NA NA NASolvent control (<LOQ) NA NA NANA NA NA NA NA NA NA NA NA0.021 NA NA NA NA NA NANA NA NA NA NA NA0.21 NA NA NA NA NA NANA NA NA NA NA NA2.1 NA NA NA NA NA NANA NA NA NA NA NANANot applicable.LOQ=0.001 mg a.i./L.Page44of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106There were some minor effects at lower test levels that the study author did not consider treatment related (one female in solvent control with redovipositor on Days 4 and 5; one female with red ovipositor on Day 4 and one fish with bleeding then missing eye in 2.1 mg a.i./L; one fish in0.02 mg a.i./L with a stomage bedding on Day 19); however, the fish exposed to the 2.1 mg a.i./L treatment were observed to have a lighter,more yellowish color compared to the fish in the controls and other treatment levels (number of incidences not reported).Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceControl (<LOQ)None8 0None16 0Solvent control (<LOQ)None9 0None14 00.021 None 90 None 1500.21 None 80 None 1602.1 Yellowishcolor 8NotreportedYellowish color14NotreportedLOQ=0.001 mg a.i./L.Page45of83

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

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fecundity (#eggs/female/reproductive day), and VTG. Data for growth, tubercle score, GSI, and VTG were
analyzed separately by sex. For all parameters dichlobenil-treated conditions were compared to the

solvent control.

Some descriptive statistics (mean) were determined for some endpoints. Significant effects were detected for p<0.05 unless otherwise noted using TOXSTAT (release 3.5). The method used to
determine effects on survival were not reported. Effects on all other endpoints were determined by ANOVA and a one-tailed Dunnett’s test. The difference between negative and solvent control were determined by two-sided t-test. Prior to Dunnett’s, data were analyzed to test for normality using
Shapiro-Wilks test and homogeneity of variances using an F-test.

Adult survival (range 88-100%) was not significantly different compared to the controls (90-100%).
There was a statistically significant decrease in fecundity at the 2.1 mg a.i./L treatment level compared to the solvent control (p<0.05; Dunnett’s test). There was also an increased incidence
and/or severity of oocyte atresia and a shift towards more mature follicle stages at concentrations of 0.21 and 2.1 mg a.i./L, suggesting reduced spawning in females exposed at these levels.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via Toxstat 3.5. Female and male weight, female length, male mean vitellogenin (VTG), female GSI,
and female tubercle score were not consistent with a monotonic concentration-response. All data were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s
test. Data which met the assumptions of normality and homogeneity of variance were then analyzed using the parametric Dunnett’s test. Data that failed parametric assumptions were analyzed using the
non-parametric Mann-Whitney U test. Male length, female VTG, male GSI, male tubercle score and

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

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fecundity data exhibited monotonic trends and were analyzed using the non-parametric JonckheereTerpstra test, as recommended by the OCSPP 890.1350 guideline. The reviewer compared all dichlobenil-treated groups to the negative control group. The reviewer also compared the negative and solvent controls using two-sided t-tests. There were no significant differences between the negative and solvent control groups.

Median male tubercle score showed a treatment-related reduction from the negative control of 18%, 46% and 53% in the 0.021, 0.21 and 2.1 mg a.i./L treatment groups, respectively (p<0.05; Jonckheere-Terpstra). Fecundity exhibited a decreasing monotonic trend, with significant 53 and
90% reductions from the negative control at the 0.21 and 2.1 mg a.i./L treatment levels, compared to the negative control (p<0.05; Jonckheere-Terpstra). The reviewer did not provide fertility data,
but reported fertility was “more than 99.9% in all test groups” and that fertility of eggs from control
animals was at least 95%.

Fish exposed to the 2.1 mg a.i./L treatment were observed to have a lighter, more yellowish color compared to the fish in the controls and other treatment levels (number of incidence not reported). In females, there was an increased incidence and/or severity (minimal to moderate) of oocyte
atresia and a shift to more mature stages in the ovaries in the 0.21 and 2.1 mg a.i./L treatments.

Conclusions:

There was a significant decrease in fecundity (p<0.05) by 53% and 90% at the 0.21 and 2.1 mg
a.i./L treatment levels, respectively, accompanied by increased oocyte atresia and more mature ovarian stages. Male tubercle scores exhibited a monotonic increase (p<0.05) of 18-53% at 0.021,
0.21 and 2.1 mg a.i./L treatment levels, compared to the negative control. There were no significant differences in other endpoints.

There were no statistically significant differences detected by the study author (p>0.05) in mean
male or female body weights at any test level compared to the control. There were no significant

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

EPA MRID Number 48616106

differences (p>0.05) in male or female body length compared to the control. Fish at the 2.1 mg
a.i./L were reported to be more yellowish in color compared to other treatment groups and control.

While not statistically significant (p>0.05), the fecundity in the solvent control group was reduced by
46% compared to the negative control. Therefore, given this observed decrease there is concern that the use of a solvent may have influenced the interpretation of the study.

Page 48 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( P imephales promelas)in the FSTRA with Dichlobenil.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative Control (<LOQ) 26 NA 0 NA No 0 NA NA NA NA NASolvent control (<LOQ)318 0.236 7.63 0.646 No 203 0.485 NA NA NA NA0.021 210.0414.24 0.500 No 272 0.067 NA NA NA NA0.21 140.0086.99 0.304 No -18.1 0.996 NA NA NA NA2.1 120.00529.2 0.095 No 83.6 0.782 NA NA NA NAStatistical TestJonckheere-Terpstra Jonckheere-Terpstra NADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.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<0.05.3Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal varianceLOQ=0.001 mg a.i./L.Page49of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Table 23: Reproductive and HPG Endpoints1,2for Female Fathead minnow ( P imephales promelas)in the FSTRA with Dichlobenil.Treatment(mg a.i./L)[mean-measured]FecundityFert. Success4Tubercle Score3GSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p% Diff.pControl (<LOQ) 0 NA NA NA 0 NA0 NA No 0 NA NA NA NA NASolvent control(<LOQ)5-46.3 0.078 NA NA 0 NA -1.63 0.830No 11.52 0.703 NA NA NA NA0.021 -37.9 0.074 NA NA0 0.478 -14.6 0.371 No 1.93 0.614 NA NA NA NA0.21 -53.4 0.006 NA NA 0 >0.99 1.810.996 Yes -17.15 0.330 NA NA NA NA2.1 -90.1 <0.001 NA NA 0 >0.99 19.70.174 Yes -30.79 0.096 NA NA NA NAStatistical TestJonckheereNA Mann-Whitney Dunnett’s NA Jonckheere NANAAbbreviations: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<0.05.3One tubercle was noted for one female fish in the 0.021 mg a.i./L treatment level.4Replicate fertility data were not provided. Fertility was reported to be “more than 99.9% in all test groups” and “at least 95%” in the control animals.5Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal varianceLOQ=0.001 mg a.i./L.Page50of83

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

EPA MRID Number 48616106

Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproducti on Assay (FSTRA) with Dichlobenil.

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

Solvent control (<LOQ) 3 -5.80 0.601 5.06 0.270 0 0.868 0 0.791

0.021 -2.78 0.993 5.08 0.791 0.53 0.440 0.72 0.974

0.21 -4.59 0.972 9.84 0.360 0.53 0.470 5.07 0.089

2.1 -3.38 0.988 5.71 0.733 1.07 0.778 3.62 0.265

Statistical Test Dunnett’s Dunnett’s JonckheereTerpstra 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 Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal

variance LOQ=0.001 mg a.i./L.

E. Study Deficiencies

During the study the fecundity was 12.1 and 6.5 eggs/female/day in the negative control and solvent control, respectively. Additionally, during the 14 days prior to study initiation, the fecundity was 5.3-15.3 eggs/female/reproductive day/replicate, which the study author reported in general as spawning every fourth day. The EPA recommendation of at least 15 eggs/female/reproductive day or a spawning frequency of at least every four days in every replicate was not met in the controls (one in the negative and two in the solvent). Additionally, while it was reported that fertilization success exceeded 99% at

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

EPA MRID Number 48616106

every level, these data were not provided for confirmation. Temperature was below the optimum range 6 hours during the exposure period. No adverse effects were observed during this period. The remaining validity criteria for OCSPP 890.1350 were met.

The reviewer noted that performance criteria for the OCSPP 890.1350 guideline were met for reported
parameters, but the unionized ammonia and residual chlorine were not reported. EPA recommends that

unionized ammonia in the dilution water be <1 P g/L and residual chlorine in dilution water be <10

P g/L. There is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment

groups.

While not statistically significant (p=0.078), the fecundity in the solvent control group was reduced by
46% compared to the negative control. However, large variability was observed between the preexposure and exposure fecundity rate for both the negative and solvent control replicates, suggesting this difference may not be solvent related. Additionally, this was the only endpoint for which a notable difference between the negative and solvent control was observed.

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

F. Reviewer’s Comments

The reviewer’s and the study authors’ conclusions were generally in agreement; however, there weresome differences in the statistical results. The reviewer’s analysis using the nonparametric JonckheereTerpstra test detected significant reductions (53 and 90%) in fecundity at the 0.21 and 2.1 mg a.i./L treatment levels compared to the negative control, while the study author’s only detected a significant reduction at the 2.1 mg a.i./L treatment compared to the solvent control. The reviewer’s analysis detected significant reductions in median male tubercle scores at all test levels (18-53%) compared to the negative control, while the study author found no significant difference (p>0.05) compared to the

solvent control.

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

EPA MRID Number 48616106

There were three endpoints exhibiting a monotonic concentration response which also satisfied parametric assumptions (i.e., female VTG, male tubercle score, and fecundity). The OCSPP 890.1350
guidance recommends use of the non-parametric Jonckheere-Terpstra test to detect treatment-related effects for any monotonic response, while the historical EFED method suggests use of William’s test
when parametric assumptions are satisfied. Male tubercle score and fecundity produced different conclusions depending on the type of test. For male tubercle score, Jonckheere-Terpstra detected significant reductions (p<0.05) all test levels, while William’s test only detected significant reductions at
the 0.21 and 2.1 mg a.i./L levels. For fecundity, the pattern was reversed; Jonckheere-Terpstra detected significant reductions at the 0.21 and 2.1 mg a.i./L levels, while William’s test detected significant reductions (p<0.05) at all test levels.

There is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment groups. In the study report (Appendix I), the assignment of fish to the test groups is reported. The top five egg
producing tanks are used first in which the highest tank is assigned to the negative control replicate A, the second-highest to the solvent control replicate A, the third highest to the lowest treatment replicate A group and so on until the fifth highest group is assigned to the highest treatment level. Then the next five highest egg producers are used and the highest from this group is assigned to the highest treatment group replicate, B, the next highest to the middle treatment group, and so on until the lowest of these next five tanks is assigned to the negative control. This pattern was then repeated with the highest tank in the third group of five tanks assigned to the negative control. While such a pattern could occur randomly, it seems unlikely that this is the pattern that would emerge.

Based on the report, it appears that for VTG values that were non-detects, they were reported as 0.00 instead of the 1⁄2 LOQ as recommended in the 890.1350 guideline.

In this study, there were 3 tanks in the exposure period in which the sex ratio was 3 males and 3

females instead of 2 males and 4 females as dictated. Two of these tanks were in the solvent control and one was in the 0.02 mg a.i./L treatment group. Fecundity values were adjusted to account for the additional male. As it could not be determined from the study report which male was mistaken for a female, the analysis and results in the DER for male specific endpoints included all male data.

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

EPA MRID Number 48616106

While the solvent control fecundity was not significantly different from the negative control (p=0.078 using a two-sided student’s t-test), a 46% reduction compared to the negative was observed. The
reviewer compared the pre-exposure fecundity for both the negative and solvent control. The preexposure fecundity was very similar between the two groups (see table below); however it appears that
while the solvent control replicates either generally maintained or decreased fecundity rates during the exposure period relative to the pre-exposure period, the negative control replicates generally increased in fecundity compared to the pre-exposure period. While a potential solvent interference cannot be ruled out, this relative increase in fecundity rate in the negative control may have contributed to the observed difference between the negative and solvent control.

Negative

control Solvent

Control 0.021 0.21 2.1

Fecundity (eggs/female/reprod day/replicate)
Replicate PE E PE E PE E PE E PE E

A 15.3 6.6 15.0 4.5 12.1 6.8 11.8 8.7 10.3 1.2

B 8.8 11.8 9.8 11.6 9.9 6.4 10.1 5.5 10.1 2.7

C 8.6 15.8 8.1 4.3 8.1 7.8 7.9 6.1 7.6 0.9

D 5.3 14.3 6.1 5.6 7.0 9.1 7.1 2.3 7.1 0.0
PE = pre-exposure; E = exposure

III. REFERENCES

None reported.

Page 54 of 83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpoint Monotonic? Parametric? EDSP EFED CommentsCombined sexSurvivalNo NA Fisher’s Exacttest:n.s. p>0.05Fisher’s Exacttest:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale body weight NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body weight NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale body length No, increasing;median values,not meansYes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body length Yes, increasing NoJonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale VTG YesYesJonckheere:n.s. p>0.05William’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect forincreasing trendFemale GSI NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect forincreasing trendPage55of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Endpoint Monotonic? Parametric? EDSP EFED CommentsMale GSIYes, increasing NoJonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05EDSP and EFED same conclusions, no effect forincreasing trendFemale tuberclescoreNo No MannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05No score for control, 0.21, and 2.1 mg a.i./L groups;tubercles noted for one fish in the 0.021 mg a.i./L level.Male tubercle score Yes, medianvaluesYes Jonckheere:Dose 1 p=0.041Dose 2 p=0.008Dose 3 p=0.005William’s:Dose 2 p=0.021Dose 3 p=0.021EDSP and EFED conclusions differ. Median values showeda significant treatment-related reduction (18-53% lowerthan negative control) at all treatment levels, according toJonckheere-Terpstra. William’s test only detected significantreductions (p<0.05) at the 0.21 and 2.1 mg a.i./L levels.Fecundity Yes,decreasing Yes Jonckheere:Dose 1 p=0.074Dose 2 p=0.006Dose 3 p<0.001William’s:Dose 1 p=0.014Dose 2 p=0.002Dose 3 p<0.001EDSP and EFED conclusions differ. Mean values showed asignificant treatment-related reduction (38-90% lower thannegative control) at only two highest treatment levels,according to Jonckheere-Terpstra. William’s test detectedsignificant reductions (p<0.05) at all treated levels.FertilityNANANANAStudy author did not provide replicate data. Reported thatfertility was “at more than 99.9% in all test groups”.Ftestosterone NA NA NA NA NAMtestosterone NA NA NA NA NAFestradiol NA NA NA NA NAMestradiol NA NA NA NA NAPage56of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Control Comparisons% Mort.(all)% Mort.(male)% Mort.(female) Fecundity % Fert.Wt(male)Length(male)Wt.(female)Length(female)Neg Control000 6.6 NA 2.42 4.8 0.763.40 0 011.8NA2.274.80.793.50 0 015.8NA1.834.50.813.50 0 014.3NA1.764.60.793.4Average 0.0 0.0 0.000 12.1#DIV/0! 2.07 4.7 0.79 3.5SolventControl 0 0 0 4.5NA 2.36 5.0 0.73 3.30 0 011.6NA1.774.40.873.40 0 04.3NA1.984.70.863.550100 05.6NA 1.684.70.86 3.7Average 12.5 25.0 0.000 6.5 #DIV/0! 1.95 4.7 0.83 3.5t-test p value 0.356 0.356 #DIV/0! 0.078 #DIV/0! 0.601 0.868 0.270 0.791Page57of83Data Evaluation Record onthe Fish Short-Term Reproduction Assay with DichlobenilEPA MRID Number 48616106Control ComparisonsGSI(male) GSI (female)Tubercle(male)VTG(male) VTG (Female)Neg Control 1.17 10.2 28.5 0.00 1741.571.53 14.5 24.5 0.67 706.410.99 12.1 27.0 0.38 1287.481.03 12.5 24.5 0.11 1350.63Average 1.18 12.3 26.1 0.29 1271.52SolventControl 1.47 12.3 30.0 3.20 1839.461.28 12.9 12.7 0.03 1056.751.05 10.3 12.0 0.14 773.89. 12.9 23.0 0.14 2001.83Average 1.27 12.1 19.43 0.88 1417.98t-test p value 0.646 0.830 0.182 0.485 0.703Page58of83

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

EPA MRID Number 48616106

Dichlobenil Combined Sex Survival

Fisher's Exact Test

============================================================================ NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 24 0 24

Solvent Control 23 1 24

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

TOTAL 47 1 48

============================================================================
Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 23.

Since b is greater than 19.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 24 0 24

0.021 mg/L 24 0 24

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

TOTAL 48 0 48

============================================================================
Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24.

Since b is greater than 19.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 24 0 24

0.21 mg/L 24 0 24

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

EPA MRID Number 48616106

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

TOTAL 48 0 48

============================================================================
Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24.

Since b is greater than 19.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 24 0 24

2.1 mg/L 22 2 24

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

TOTAL 46 2 48

============================================================================
Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 22.

Since b is greater than 19.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 24 0 1 Solvent Control 24 1 2 0.021 mg/L 24 0 3 0.21 mg/L 24 0 4 2.1 mg/L 24 2

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

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

EPA MRID Number 48616106

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.983 0.984 1.490 0.267 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 0.79 0.02 0.01 2.62 0.75, 0.82 Dose1 4 0.83 0.07 0.03 8.45 0.72, 0.94 Dose2 4 0.87 0.10 0.05 11.54 0.71, 1.02 Dose3 4 0.83 0.08 0.04 10.02 0.70, 0.97

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

Ctrl 0.79 0.76 0.81 . . Dose1 0.83 0.74 0.91 105.08 -5.08 Dose2 0.83 0.79 1.01 109.84 -9.84 Dose3 0.87 0.71 0.89 105.71 -5.71

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.556

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 0.79 . 0.83 . . . . . . Dose1 0.83 0.791 0.83 0.849 . . . . . Dose2 0.87 0.360 0.83 0.876 0.891 . . . . Dose3 0.83 0.733 0.83 0.890 1.000 0.925 . . .

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

Degrees of Freedom TestStat P-value 3 2.81 0.422

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

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

EPA MRID Number 48616106

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.79 . . Dose1 0.83 0.343 0.877 Dose2 0.83 0.180 0.940 Dose3 0.87 0.343 0.933

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

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.556 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 -0.79 . -0.79 . . . . . . Dose1 -0.83 0.791 -0.83 0.276 . . . . . Dose2 -0.87 0.360 -0.85 0.170 0.891 . . . . Dose3 -0.83 0.733 -0.85 0.176 1.000 0.925 . . .

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

Degrees of Freedom TestStat P-value 3 2.81 0.422

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 -0.79 . . Dose1 -0.83 0.343 0.123 Dose2 -0.83 0.180 0.060 Dose3 -0.87 0.343 0.067

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

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

EPA MRID Number 48616106

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )** TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.950 0.494 1.113 0.382 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 2.07 0.32 0.16 15.68 1.55, 2.59 Dose1 4 2.01 0.21 0.10 10.26 1.68, 2.34 Dose2 4 1.98 0.34 0.17 17.16 1.44, 2.51 Dose3 4 2.00 0.59 0.29 29.29 1.07, 2.93

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

Ctrl 2.05 1.76 2.42 . . Dose1 1.95 1.84 2.31 97.22 2.78 Dose2 1.98 1.56 2.39 95.41 4.59 Dose3 1.84 1.50 2.83 96.62 3.38

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

Numerator df Denominator df F-stat P-value 3 12 0.04 0.988

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

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

Ctrl 2.07 . 2.07 . . . . . . Dose1 2.01 0.993 2.01 0.494 . . . . . Dose2 1.98 0.972 1.99 0.485 0.999 . . . . Dose3 2.00 0.988 1.99 0.502 1.000 1.000 . . .

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

Degrees of Freedom TestStat P-value 3 0.46 0.928

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

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

EPA MRID Number 48616106

Level Median MannWhit p-value Jonckheere p-value

Ctrl 2.05 . . Dose1 1.95 0.889 0.614 Dose2 1.98 0.889 0.529 Dose3 1.84 0.678 0.320

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

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

Numerator df Denominator df F-stat P-value 3 12 0.04 0.988

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

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

Ctrl -2.07 . -2.01 . . . . . . Dose1 -2.01 0.993 -2.01 0.666 . . . . . Dose2 -1.98 0.972 -2.01 0.701 0.999 . . . . Dose3 -2.00 0.988 -2.01 0.720 1.000 1.000 . . .

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

Degrees of Freedom TestStat P-value 3 0.46 0.928

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.05 . . Dose1 -1.95 0.889 0.386 Dose2 -1.98 0.889 0.471 Dose3 -1.84 0.678 0.680

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

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

EPA MRID Number 48616106

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.977 0.937 0.387 0.764 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 34.50 0.58 0.29 1.67 33.58, 35.42 Dose1 4 34.75 0.96 0.48 2.76 33.23, 36.27 Dose2 4 36.25 1.26 0.63 3.47 34.25, 38.25 Dose3 4 35.75 1.26 0.63 3.52 33.75, 37.75

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

Ctrl 34.50 34.00 35.00 . . Dose1 34.50 34.00 36.00 100.72 -0.72 Dose2 36.00 35.00 38.00 105.07 -5.07 Dose3 36.00 34.00 37.00 103.62 -3.62

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

Numerator df Denominator df F-stat P-value 3 12 2.47 0.112

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 34.50 . 35.31 . . . . . . Dose1 34.75 0.974 35.31 0.913 . . . . . Dose2 36.25 0.089 35.31 0.933 0.235 . . . . Dose3 35.75 0.265 35.31 0.942 0.554 0.905 . . .

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

Degrees of Freedom TestStat P-value 3 5.80 0.122

MannWhit - testing each trt median signif. different from control

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

EPA MRID Number 48616106

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 34.50 . . Dose1 34.50 0.879 0.624 Dose2 36.00 0.093 0.981 Dose3 36.00 0.220 0.982

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

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

Numerator df Denominator df F-stat P-value 3 12 2.47 0.112

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 -34.50 . -34.50 . . . . . . Dose1 -34.75 0.974 -34.75 0.440 . . . . . Dose2 -36.25 0.089 -36.00 0.041 0.235 . . . . Dose3 -35.75 0.265 -36.00 0.042 0.554 0.905 . . .

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

Degrees of Freedom TestStat P-value 3 5.80 0.122

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 -34.50 . . Dose1 -34.50 0.879 0.376 Dose2 -36.00 0.093 0.019 Dose3 -36.00 0.220 0.018

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose2 Jonckheere Dose2

Page 66 of 83

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

EPA MRID Number 48616106

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.845 0.012 3.891 0.037 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 46.75 1.50 0.75 3.21 44.36, 49.14 Dose1 4 47.00 0.82 0.41 1.74 45.70, 48.30 Dose2 4 47.00 1.41 0.71 3.01 44.75, 49.25 Dose3 4 47.25 5.32 2.66 11.25 38.79, 55.71

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

Ctrl 47.00 45.00 48.00 . . Dose1 47.00 46.00 48.00 100.53 -0.53 Dose2 46.50 46.00 49.00 100.53 -0.53 Dose3 45.50 43.00 55.00 101.07 -1.07

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

Numerator df Denominator df F-stat P-value 3 12 0.02 0.996

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 46.75 . 47.00 . . . . . . Dose1 47.00 0.999 47.00 0.634 . . . . . Dose2 47.00 0.999 47.00 0.669 1.000 . . . . Dose3 47.25 0.989 47.00 0.688 0.999 0.999 . . .

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

Degrees of Freedom TestStat P-value 3 1.23 0.747

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

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

EPA MRID Number 48616106

Level Median MannWhit p-value Jonckheere p-value

Ctrl 47.00 . . Dose1 47.00 1.000 0.560 Dose2 46.50 0.886 0.530 Dose3 45.50 0.673 0.222

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

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

Numerator df Denominator df F-stat P-value 3 12 0.02 0.996

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 -46.75 . -46.75 . . . . . . Dose1 -47.00 0.999 -47.00 0.531 . . . . . Dose2 -47.00 0.999 -47.00 0.564 1.000 . . . . Dose3 -47.25 0.989 -47.25 0.527 0.999 0.999 . . .

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

Degrees of Freedom TestStat P-value 3 1.23 0.747

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 -47.00 . . Dose1 -47.00 1.000 0.440 Dose2 -46.50 0.886 0.470 Dose3 -45.50 0.673 0.778

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

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

EPA MRID Number 48616106

Jonckheere >highest dose (no sign. differences)

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.961 0.672 0.891 0.474 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 1271523 426930.7 213465.3 33.58 592180.5, 1950864 Dose1 4 1296068 538855.4 269427.7 41.58 438628.3, 2153507 Dose2 4 1053518 210200.9 105100.4 19.95 719041.0, 1387994 Dose3 4 880077.5 569767.7 284883.8 64.74 -26550.0, 1786705

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

Ctrl 1319055 706410.0 1741570 . . Dose1 1189470 849070.0 1956260 101.93 -1.93 Dose2 1043280 869300.0 1258210 82.85 17.15 Dose3 793595.0 282160.0 1650960 69.21 30.79

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.551

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 1271523 . 1283795 . . . . . . Dose1 1296068 1.000 1283795 0.599 . . . . . Dose2 1053518 0.841 1053518 0.328 0.876 . . . . Dose3880077.5 0.506 880077.5 0.164 0.590 0.949 . . .

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

Degrees of Freedom TestStat P-value 3 2.01 0.571

Page 69 of 83

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

EPA MRID Number 48616106

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 1319055 . . Dose1 1189470 0.889 0.614 Dose2 1043280 0.346 0.330 Dose3 793595.0 0.494 0.096

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

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

Numerator df Denominator df F-stat P-value 3 12 0.73 0.551

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 -1271523 . -1125296 . . . . . . Dose1-1296068 1.000 -1125296 0.757 . . . . . Dose2-1053518 0.841 -1125296 0.790 0.876 . . . . Dose3 -880078 0.506 -1125296 0.807 0.590 0.949 . . .

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

Degrees of Freedom TestStat P-value 3 2.01 0.571

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 -1319055 . . Dose1 -1189470 0.889 0.386 Dose2 -1043280 0.346 0.670 Dose3 -793595 0.494 0.904

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL

Page 70 of 83

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

EPA MRID Number 48616106

Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.937 0.318 1.421 0.285 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 290.00 299.44 149.72 103.26 -186.48, 766.48 Dose1 4 1077.50 701.06 350.53 65.06 -38.05, 2193.05 Dose2 4 237.50 189.45 94.73 79.77 -63.96, 538.96 Dose3 4 532.50 408.03 204.02 76.63 -116.77, 1181.77

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

Ctrl 245.00 0.00 670.00 . . Dose1 895.00 460.00 2060.00 371.55 -271.55 Dose2 200.00 50.00 500.00 81.90 18.10 Dose3 450.00 130.00 1100.00 183.62 -83.62

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

Numerator df Denominator df F-stat P-value 3 12 3.01 0.072

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 290.00 . 683.75 . . . . . . Dose1 1077.50 0.067 683.75 0.936 . . . . . Dose2 237.50 0.996 385.00 0.739 0.081 . . . . Dose3 532.50 0.782 385.00 0.758 0.346 0.783 . . .

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

Page 71 of 83

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

EPA MRID Number 48616106

Degrees of Freedom TestStat P-value 3 6.11 0.106 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 245.00 . . Dose1 895.00 0.103 0.978 Dose2 200.00 1.000 0.500 Dose3 450.00 0.346 0.610

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

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

Numerator df Denominator df F-stat P-value 3 12 3.01 0.072

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 -290.00 . -290.00 . . . . . . Dose1-1077.50 0.067 -615.83 0.190 . . . . . Dose2 -237.50 0.996 -615.83 0.203 0.081 . . . . Dose3 -532.50 0.782 -615.83 0.210 0.346 0.783 . . .

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

Degrees of Freedom TestStat P-value 3 6.11 0.106

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 -245.00 . . Dose1 -895.00 0.103 0.022 Dose2 -200.00 1.000 0.500 Dose3 -450.00 0.346 0.390

Page 72 of 83

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

EPA MRID Number 48616106

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

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.934 0.279 0.423 0.740 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 12.31 1.75 0.88 14.22 9.52, 15.09 Dose1 4 10.51 1.20 0.60 11.38 8.61, 12.41 Dose2 4 12.53 1.95 0.97 15.53 9.43, 15.63 Dose3 4 14.73 2.01 1.01 13.67 11.53, 17.94

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

Ctrl 12.30 10.18 14.45 . . Dose1 10.68 8.90 11.79 85.40 14.60 Dose2 12.18 10.71 15.06 101.81 -1.81 Dose3 14.11 13.09 17.62 119.70 -19.70

******************************************************************************** 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 3 12 3.88 0.038

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 12.31 . 12.52 . . . . . . Dose1 10.51 0.371 12.52 0.654 . . . . . Dose2 12.53 0.996 12.52 0.689 0.401 . . . . Dose3 14.73 0.174 12.52 0.708 0.024 0.331 . . .

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

Page 73 of 83

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

EPA MRID Number 48616106

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

Degrees of Freedom TestStat P-value 3 7.61 0.055

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 12.30 . . Dose1 10.68 0.235 0.074 Dose2 12.18 0.889 0.558 Dose3 14.11 0.156 0.969

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

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

Numerator df Denominator df F-stat P-value 3 12 3.88 0.038

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 -12.31 . -11.41 . . . . . . Dose1 -10.51 0.371 -11.41 0.837 . . . . . Dose2 -12.53 0.996 -12.53 0.539 0.401 . . . . Dose3 -14.73 0.174 -14.73 0.047 0.024 0.331 . . .

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

Degrees of Freedom TestStat P-value 3 7.61 0.055

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 -12.30 . . Dose1 -10.68 0.235 0.926 Dose2 -12.18 0.889 0.442

Page 74 of 83

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

EPA MRID Number 48616106

Dose3 -14.11 0.156 0.031

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose3 Jonckheere Dose3

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.934 0.286 7.780 0.004 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 1.18 0.25 0.12 20.83 0.79, 1.57 Dose1 4 1.23 0.46 0.23 37.13 0.50, 1.96 Dose2 4 1.26 0.25 0.13 20.07 0.86, 1.67 Dose3 4 1.53 0.07 0.04 4.62 1.41, 1.64

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

Ctrl 1.10 0.99 1.53 . . Dose1 1.23 0.82 1.65 104.24 -4.24 Dose2 1.21 1.03 1.61 106.99 -6.99 Dose3 1.52 1.46 1.60 129.24 -29.24

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

Numerator df Denominator df F-stat P-value 3 12 1.12 0.378

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 1.18 . 1.30 . . . . . . Dose1 1.23 0.990 1.30 0.798 . . . . . Dose2 1.26 0.957 1.30 0.829 0.999 . . . . Dose3 1.53 0.267 1.30 0.845 0.503 0.594 . . .

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

Page 75 of 83

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

EPA MRID Number 48616106

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

Degrees of Freedom TestStat P-value 3 2.05 0.563

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 1.10 . . Dose1 1.23 1.000 0.500 Dose2 1.21 0.580 0.696 Dose3 1.52 0.156 0.905

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

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

Numerator df Denominator df F-stat P-value 3 12 1.12 0.378

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 -1.18 . -1.18 . . . . . . Dose1 -1.23 0.990 -1.23 0.480 . . . . . Dose2 -1.26 0.957 -1.26 0.441 0.999 . . . . Dose3 -1.53 0.267 -1.53 0.076 0.503 0.594 . . .

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

Degrees of Freedom TestStat P-value 3 2.05 0.563

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 -1.10 . . Dose1 -1.23 1.000 0.500 Dose2 -1.21 0.580 0.304

Page 76 of 83

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

EPA MRID Number 48616106

Dose3 -1.52 0.156 0.095

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

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.565 <.001 9.000 0.002 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.08 0.15 0.08 200.00 -0.16, 0.31 Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

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

Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.30 . . Dose2 0.00 0.00 0.00 . . Dose3 0.00 0.00 0.00 . .

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

Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

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 . 0.04 . . . . . . Dose1 0.08 0.389 0.04 0.833 . . . . . Dose2 0.00 1.000 0.00 0.618 0.515 . . . . Dose3 0.00 1.000 0.00 0.636 0.515 1.000 . . .

Page 77 of 83

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

EPA MRID Number 48616106

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

Degrees of Freedom TestStat P-value 3 3.00 0.392

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 0.478 0.841 Dose2 0.00 1.000 0.500 Dose3 0.00 1.000 0.327

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

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

Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

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 . 0.00 . . . . . . Dose1 -0.08 0.389 -0.03 0.384 . . . . . Dose2 0.00 1.000 -0.03 0.410 0.515 . . . . Dose3 0.00 1.000 -0.03 0.425 0.515 1.000 . . .

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

Degrees of Freedom TestStat P-value 3 3.00 0.392

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 . .

Page 78 of 83

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

EPA MRID Number 48616106

Dose1 0.00 0.478 0.159 Dose2 0.00 1.000 0.500 Dose3 0.00 1.000 0.673

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

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.931 0.257 1.601 0.241 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 26.13 1.97 0.99 7.56 22.98, 29.27 Dose1 4 17.93 10.33 5.17 57.65 1.48, 34.37 Dose2 4 14.38 6.39 3.20 44.49 4.20, 24.55 Dose3 4 14.75 5.84 2.92 39.58 5.46, 24.04

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

Ctrl 25.75 24.50 28.50 . . Dose1 21.10 3.00 26.50 68.61 31.39 Dose2 14.00 7.00 22.50 55.02 44.98 Dose3 12.00 11.50 23.50 56.46 43.54

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

Numerator df Denominator df F-stat P-value 3 12 2.57 0.103

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

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

Ctrl 26.13 . 26.13 . . . . . . Dose1 17.93 0.257 17.93 0.067 . . . . .

Page 79 of 83

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

EPA MRID Number 48616106

Dose2 14.38 0.077 14.56 0.021 0.880 . . . . Dose3 14.75 0.088 14.56 0.021 0.910 1.000 . . .

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

Degrees of Freedom TestStat P-value 3 7.73 0.052

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 25.75 . . Dose1 21.10 0.154 0.041 Dose2 14.00 0.066 0.008 Dose3 12.00 0.065 0.005

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 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 3 12 2.57 0.103

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

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

Ctrl -26.13 . -18.29 . . . . . . Dose1 -17.93 0.257 -18.29 0.969 . . . . . Dose2 -14.38 0.077 -18.29 0.978 0.880 . . . . Dose3 -14.75 0.088 -18.29 0.982 0.910 1.000 . . .

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

Degrees of Freedom TestStat P-value 3 7.73 0.052

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 -25.75 . .

Page 80 of 83

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

EPA MRID Number 48616106

Dose1 -21.10 0.154 0.959 Dose2 -14.00 0.066 0.992 Dose3 -12.00 0.065 0.995

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

027401 Dichlobenil 890.1350 48616106 **ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.945 0.409 1.847 0.192 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

Ctrl 4 12.13 4.04 2.02 33.29 5.70, 18.55 Dose1 4 7.53 1.20 0.60 16.00 5.61, 9.44 Dose2 4 5.65 2.63 1.31 46.55 1.47, 9.83 Dose3 4 1.20 1.12 0.56 93.54 -0.59, 2.99

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

Ctrl 13.05 6.60 15.80 . . Dose1 7.30 6.40 9.10 62.06 37.94 Dose2 5.80 2.30 8.70 46.60 53.40 Dose3 1.05 0.00 2.70 9.90 90.10

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

Numerator df Denominator df F-stat P-value 3 12 12.64 <.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 12.13 . 12.13 . . . . . . Dose1 7.53 0.063 7.53 0.014 . . . . .

Page 81 of 83

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

EPA MRID Number 48616106

Dose2 5.65 0.010 5.65 0.002 0.729 . . . . Dose3 1.20 <.001 1.20 <.001 0.019 0.116 . . .

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

Degrees of Freedom TestStat P-value 3 11.23 0.011

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 13.05 . . Dose1 7.30 0.235 0.074 Dose2 5.80 0.103 0.006 Dose3 1.05 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose2

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

Numerator df Denominator df F-stat P-value 3 12 12.64 <.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 -12.13 . -6.63 . . . . . . Dose1 -7.53 0.063 -6.63 0.999 . . . . . Dose2 -5.65 0.010 -6.63 0.999 0.729 . . . . Dose3 -1.20 <.001 -6.63 0.999 0.019 0.116 . . .

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

Degrees of Freedom TestStat P-value 3 11.23 0.011

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 -13.05 . .

Page 82 of 83

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

EPA MRID Number 48616106

Dose1 -7.30 0.235 0.926 Dose2 -5.80 0.103 0.994 Dose3 -1.05 0.067 1.000

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

Page 83 of 83

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616107)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer Signature: Rebecca L. Bryan, B.S. Date: 3/20/2012
Secondary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/30/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/05/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/05/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL/027401


### Primary Reviewer: Greg Akennan, Ph.D. 

### Health Effects Division Secondary Reviewer: John Liccione, Ph

. D. 

### Health Effects Division In vivo Hershberger Assay (2011)/ Page I of 16 OCSPP 890.1400/ OECD 441


### Signature:**-=--~...........-~--;---­** 

### Date:**~..._...,~~~rf15.-=** 

### S igna tu re: --+---1""-----+----r---r---- 

### D ate:**--~'----'-'"./-L-..L__--**


### DAT A EVALUATION RECORD

STUDY TYPE: Jn Vivo 

### Hershberger Assay (Rat) ; OCSPP 890.1400 ; OECD 441



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS#: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS: 2,6-dichlorobenzonitrile



### CITATION: Coder, P.S. (2011)

. The Hershberger Assay ofDichlobenil Administered Orally in Peripubertal Orchidoepidymectomized Rats. WIL Research Laboratories
, LLC, Ashland, OH. Laboratory Project Study ID: WIL-12434. December 7
, 2011. MRID 48616107. Unpublished.



### SPONSOR: Chemtura Corporation, 199 Benson Road
, Middlebury, CT



### TEST ORDER#: EDSP-027401-120



### EXECUTIVE SUMMARY: In a Hershberger assay (MRID 48616107) screening for androgenic activity
, dichlobenil (99.3% a.i
., Lot# AM9K12A222) in 1.0% gum tragacanth was administered daily via oral gavage to groups of 55-day old, castrated male Sprague Dawley rats (6 / group) at dose levels ofO (vehicle), 20
, 60
, or 180 mg / kg / day

. An androgenic positive control gro up consisting of six castrated rats exposed to 0.2 mg / kg / day testosterone propionate (TP) by

s ubcutaneous (s.c.) injection was also included as a positive control.

To screen for potential anti-androgenic activity
, dichlobenil in 1.0% gum tragacanth was also administered daily via oral gavage to 55-day old
, castrated male Sprague Dawley rats (6 / group)
at dose levels of 20, 60
, or 180 mg / kg / day in conjunction with a daily dose ofreference androgen T P at 0.2 mg / kg / da y b y s

. c. injection. The vehicle control groups consisted of six castrated male
rats dosed daily with TP (0.2 mg / kg / day) by s.c. injection in com oil, and the anti-androgenic positive control group con s isted of six castrated male rats dosed orally with flutamide (FT) in

com oil at 3 mg / kg / da y in conjugation with a daily s.c. dose of TP (0.2 mg / kg / day).

Fo r both components of the assay, the animals were dosed for 10 consecutive days and necropsied approximatel y 24 hours after the final dose administration to detennine weights of li ver, kidneys and the five androgen-dependent tissues

.

A ll animals survived until scheduled tennination

. There were no treatment-related clinical signs of toxicity

.In vivo Hershberger Assay (2011)/ Page 2 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441



### In the androgen agonist assa y, body weights and overall body we ight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, body weights were decreased (p<0.0 5) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body weight gains for 180 mg/kg/da y were decreased (p<0.05) by 44%. Liver weights were not affected in the low-dose a nd positive control groups, but were increased (p<0.05) in the 60 mg/kg/day ( ↑ 24%) and 180 mg/kg/day ( ↑ 32%) groups. Kidney weights were unaffected, except in the TP group, in which kidn ey weights increased (p<0.05) by 8%. There were no significant changes in weight of the accesso ry sex organs for dichlobenil-dosed animals. Animals in the TP group (positive control) ha d increases (p<0.05) in accessory sex organ weights as follows: 339% in seminal vesicles, 638% in ventral prosta te, 165% in levator anibulbocavernosus (LABC), 513% in Cowper’s gland, and 72% in glans penis. All %CV values were less than the maximum recommended guideline values.
In the anti-androgen assay, body weights and overall body weight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-11% on Days 1-10. The overall (Days 0-10) body weight gains for 180 mg /kg/day were decreased (p< 0.05) by 40%. For the positive control group (TP + FT), overall body weight gains were decreased (p<0.05) by 13%. No changes were noted in liver weight for any dos e group and kidneys were only affected in the positive control (TP + FT) group, in which kidney weights significantly decreased ( ↓ 10%).
Statistically significant decreases in 4 of the 5 target accessory sex organ weights were observed at the high dose (180 mg/kg/day). The followi ng decreases (p<0.05) in accessory sex organ weights were noted at 180 mg/kg/ day: 36% in seminal vesicles, 35% in ventral prostate, 27% in LABC, and 49% in Cowper’s gland. In addition, a decrease (21%; p<0.05) in seminal vesicle weight was observed at 60 mg/kg/day. Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as follows: 73% in seminal vesicles, 75% in ventral prostate, 48% in LABC, 71% in Cowper’s gland, and 20% in glans penis. The maximum guideline-specified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT (positive control) group. A ll other %CV values were less than the maximum recommended values.
Dichlobenil was negative for andr ogenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist as say. Dichlobenil was positive for anti-androgenicity in the Hershberger assay as statistically significant decreases in organ wei ghts were seen in four of the five androgen sensitive tissues in the andr ogen antagonist assay. However, bodyweights were decreased ≥ 10% at the dose that resulted in the ch anges in androgen-sensitive tissue weights indicating overt toxicity.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance, Data Confidentiality, Flagging, and Quality Assurance statements were provided.In vivo Hershberger Assay (2011)/ Page 3 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441



### I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: WIL Research Laboratories, LLC
Location: Ashland, OH Study Director: P.S. Coder Other Personnel: Les Freshwater, MS - Contributing Scientist, Statistical Analysis Katherine L. Gardner, PhD - Participating Scientist, Analyses of Dosing Formulations Gwendalyn M. Maginnis, DVM - Clinical Veterinarian

Study Period: May 23-December 7, 2011



### 2. Test Substance: Dichlobenil Description: White Powder Source: Notox B.V. Lot/Batch #: AM9K12A222, expires October 12, 2012 Purity: 99.3% a.i. Stability: Stable in 1.0% gum tragacanth for up to 3 days frozen and stable for 8 days refrigerated CAS #: 1194-65-6 Structure:

Cl Cl CN



### 3. Reference Androgen: Testosterone propionate (TP) Supplier: AK Scientific (Mountain View, CA) Lot/Batch #: 70321J, expires August 9, 2012 Purity: 98.3% CAS # : 57-85-2


### 4. Reference Anti-androgen: Flutamide (FT) Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ) Lot/Batch #: 2AC0144, expires March 1, 2013 Purity: 100.0% CAS # : 13311-84-7



### 5. Solvent/Vehicle Control: Tragacanth Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ) Lot/Batch #: 2AC3069, expires August 31, 2012 Rationale (if other than water) : Not soluble in water and 0.5% methylcellulose in deionized water created homogeneous suspension of test substance. Final concentration: Other solvents: 1.0% gum tragacanth in deionized water Corn Oil (used for positive control): expires July 1, 2012; Lot/Batch # ZT1301; Spectrum Ch emical Manufacturing Corp . (New Brunswick, NJ) Ethanol (used for positive control): 95% purity; expires August 2, 2013; Lot # ZT0426; Spectrum Ch emical Manufacturing Corp . (New Brunswick, NJ)In vivo Hershberger Assay (2011)/ Page 4 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441



### 6. Test Animals: Species: Rat (castrated males) Strain: Sprague Dawley (Crl:CD[SD]) Age/weight at dose initiation: Post-natal day (PND) 55; 214.6-258.5 g Source: Charles River Laboratories International, Inc., Raleigh, NC Housing: Rats were housed individually in cl ean, stainless steel, suspended, wiremesh cages with cage-board Diet: PMI Nutrition International LLC Certified Rodent LabDiet ®

, ad libitum Water: Reverse-osmosis purified tap water , ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 70.3-70.6 oF (21.3-21.4oC) 45.7-59.2% 10/hr 12 hrs light/12 hrs dark Acclimation period: 7 days



### B. STUDY DESIGN

1. In-life dates: Start: 6/7/11 End: 6/17/11
2. Study Design: This Hershberger Assay was conduc ted to screen for both potential androgenic and anti-androgenic ac tivity. In the test for andr ogenic activity, dichlobenil was administered daily via oral gavage to groups of castrated male rats. Positive androgenic activity is defined as a significant increase in tw o or more target organ weights compared to the vehicle control group. The positive cont rol group for androgenic activity consisted of castrated male rats dosed by s.c. injection with TP at 0.2 mg/kg/day. In the test for antiandrogenic activity, dichlobenil was administered daily via oral gavage to groups of castrated male rats in conjunction with TP at 0.2 mg/kg/day by s.c. injection. Anti-androgenic activity is indicated by a statis tically significant decrease in two or more target organ weights of the treated groups (t est substance + TP) comp ared to the TP-only control group. The positive control group for an ti-androgenic activity co nsisted of castrated male rats dosed by s.c. injection with TP ( 0.2 mg/kg/day) in conjunc tion with an oral dose of FT (3.0 mg/kg/day). For both assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after th e final dose administration for measurement of target organ weights.
3. Study Schedule: Following the initial 7-day acclima tion period, castrated male rats (surgical manipulation performed by Supplier at PND 42) were admi nistered the test substance from PND 55 to PND 64. Rats were euthanized on PND 65, approximately 24 hours after the last dose and necropsie d 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 means at study initiation.In vivo Hershberger Assay (2011)/ Page 5 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

Table 1. Study design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (corn oil) 0 6 Low (Dichlobenil) 20 6 Mid (Dichlobenil) 60 6 High (Dichlobenil) 180 6 Positive control (corn oil) +TP b 0.2 6 Anti-Androgen Assay Vehicle control (corn oil) +TP b 0 6 Low (Dichlobenil+TP) 20 6 Mid (Dichlobenil+TP) 60 6 High (Dichlobenil+TP) 180 6 Flutamide (oral gavage) + TP (s.c.), positive control 3 6 a Data were obtained from Table 2 on pages 18 of the study report. b Same animals dosed for both androgen agonist (serves as pos itive control) and anti-androgen (served as vehicle +TP control) assays. This did not affect th e outcome or conclusions of this study.



### 5. Dose Selection Rationale: It was stated that the doses we re selected based on the results of previous studies including a dose ra nge-finding study (MRID 48616113) which is summarized in an Appendix to this DER.
6. (a) Dose Preparation: Dose formulations were prepar ed by mixing the appropriate amounts of dichlobenil in 1.0% aqueous gum tragacanth. For test day one, the test suspensions were prepared on the day prior to treatment and stored refrigerated and protecte d from light. Starting on Day 2, the test suspensions were prepared daily and stored at room temperature prior to use

. The positive control was initially dissolved in 95% ethanol solution, and then added to corn oil.

Samples of dichlobenil from the first and last dose formulation preparations were analyzed for achieved concentration (reported for all fo rmulations from June 6 and 16). In a concurrent uterotrophic study (MRID 48616111) , dichlobenil samples at 0.02, 1, and 50 mg/mL were analyzed from samples collected on June 1 and June 9 for homogeneity (top, middle, bottom) and stability (both at 3 days frozen and at 8 days refrigerated).
( b) Dose Analysis

Results
Homogeneity (%RSD): 1.9-5.5% (with the exception of 26% RSD for the 0.02 mg/mL sample from June 1, 2011)
Stability (% of time 0): 94.1-98.6% following 3 days frozen storage; 89.9-97.9% following 8 days re frigerated storage

Concentration (% of nominal): 107-114%

These dose analyses were insufficient to allo w an unequivocal conclusion that the mixing procedure was adequate and the variation between no minal and actual dosage to the animalsIn vivo Hershberger Assay (2011)/ Page 6 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441


### was acceptable. Homogeneity was only evalua ted twice, and on one of these instances, homogeneity in the low dose was unaccepta ble. Stability was acceptable under the conditions tested (3 days frozen or 8 days refrigerated). Fr om Day 2, it was stated that the dose formulations were prepared daily and stored at room temp erature, protected from light, until use. It was not clear how long the samples were stored at room temperature; however, stability data should be submitted demonstratin g that dichlobenil was stable in the dose formulation over that storage in terval at room temperature.
7. Dosage administration: Test formulations of dichloben il or FT were administered to the animals daily via oral gavage (5 mL/kg bw ) for 10 consecutive days, beginning on Study Day 0. TP was given via s.c. injection at 0.5 mL/kg bw. The site of injection for TP was rotated between 4 distinct sites. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Statistical analyses were performed by BioSTAT Consultants, Inc. using SAS® version 9.2 or higher software (SAS Institute, Inc.). Data stat istically analyzed included the organ weights, daily body weights (incl uding necropsy body weight) and cumulative body weight gain. Testing for agonists was handled separately from antagonists. Each endpoint was tested for homogeneity of variance using Levene ’s test. If that te st was significant at p=0.01, then a log transform was applied and Levene’s test was conducted on the transformed data. If that test was still significant then the square root transformation was applied to the raw data and Levene’s test was conducted ag ain. If the test was still significant then a nonparametric test, as descri bed below, was used to analyze the data. Because negative values are possible, the l og and square root transformation were not evaluated for the cumulative body weight gain.

Organ Weights: Vehicle Control vs. TP
Organ Weights: TP vs. TP + FT If the variances were homogeneous then the one-sided t-test was performed on the organ weights, using p=0.05 to declare significance, looking for significant increases from the vehicle control. If the transformations were unsuccessful in making the variances homogeneous then the nonparametric Wilcoxon rank sum test was used to compare the two groups. The test was one-sided using p=0.05 to declare significance, looking for significant increases from the vehicle control.In vivo Hershberger Assay (2011)/ Page 7 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

Organ Weights: Vehicle Control vs. Test Substance Groups
Organ Weights: TP vs. Test Substance Groups + TP If the variances were homogeneous then an analysis of variance (ANOVA) was co nducted, on the raw or transformed organ weight data, as appropriate. A one-sided Dunnett’s test was conducted, regardless of the outcome of the ANOVA, looking for significant increases in the test subs tance groups when compared with the vehicle control. If the transformations were unsuccessful in making the variances homogeneous then the nonparametric Kruskal-Wallis test was used, followed by Dunn’s test, to compare each of the test substance groups with the vehicle control or test substance groups + TP with the TP group. Since these were pre-planned pairwise comparisons, Dunn’s test was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were one-sided, at the 0.05 significance level, looking for significant increases from the vehicle control. Daily Body Weight and Cumulative Body Weight Gain The statistical analysis wa s identical to the organ weights with the exception that the testing was 2- sided using p=0.05 to declare significance.


### The statistical analyses were considered appropriate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and moribundity were conducted twice daily. Clinical signs of t oxicity were recorded daily prior to treatment. Animals were also observed for signs of toxi city approximately 1-2 hours afte r dosing. The injection sites were examined daily from the initiation of dose administration for erythema, swelling, and other dermal findings. Erythema and swelling were evaluated in accordance with a 4-step grading system of very slight , slight, moderate, and severe.
2. Body Weight: Animals were weighed at randomiza tion, daily prior to dosing throughout the dosing period, and at termination.
3. Food Consumption (Optional): Food consumption was not determined.
4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted. Approximately 24 hours following each animal's last dose, blood samples were collected from the vena cava of each male following anesthesia by isoflurane inhalation. The blood samples were allowed to clot at room temperature and then placed on ice. Serum was separated in a centrifuge a nd stored frozen (approximately

− 70°C) for possible future hormone analysis.
5. Dissection and Measurement of Tissue and Organ Weights: On PND 65 (approximately 24 hours after the final administration of the test substance), all rats were weighed, euthanized by exsanguination, and subjected to a gross necropsy. The following tissues and organs were examined, collected, weighed, a nd placed in 10% neutral-buffered formalin: adrenal gland, seminal vesicles with coagula ting glands, Cowper's gland (bulbourethral), ventral prostate, glans penis, gross lesions, kidney, liver, a nd LABC muscle complex. With the exception of gross lesions, each of these organs was weighed fresh without blotting;In vivo Hershberger Assay (2011)/ Page 8 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441


### paired organs were weighed together. Care was taken to minimize fluid loss from the Cowper's gland, seminal vesicl es, and ventral prostate.

II. RESULTS

A. OBSERVATIONS
1. Mortality: All animals survived until scheduled termination.
2. Clinical signs of toxicity: No clinical signs of toxicity were observed in any of the dose groups at the daily examinations or approximately 1 to 2 hours following dose administration. No remarkable injection site findings were noted.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gains for the androgen agonist assay are presented in Table 2. Body weights and overall body weight gains for the 20 and 60 mg/kg/day treat ment groups were comparable to controls throughout the duration of the assay. At 180 mg/kg/day, body wei ghts were decreased (p<0.05) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 44%.In vivo Hershberger Assay (2011)/ Page 9 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)
Vehicle Control Positive Control Vehicle + TP (0.2) b Dichlobenil (20) Dichloben il (60) Dichlobenil (180)
N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 0 6 237.1 13.25 6 235.4 12.35 6 236.5 10.96 6 234.5 11.63 6 236.9 13.56 1 6 244.1 14.45 6 243.4 12.79 6 237.6 10.08 6 234.2 13.87 6 228.7 15.90 2 6 251.3 15.14 6 281.5 12.66 6 247. 5 11.08 6 239.7 15.93 6 226.7* ( ↓ 10) 14.38

3 6 257.3 16.05 6 260.1 14.47 6 257. 6 12.37 6 249.8 16.39 6 230.5* ( ↓ 10) 20.50

4 6 264.0 17.07 6 265.9 12.78 6 263. 4 13.31 6 256.9 17.75 6 239.5* ( ↓ 9) 15.02

5 6 271.6 17.88 6 275.0 13.95 6 271. 5 15.37 6 265.9 19.13 6 246.9* ( ↓ 9) 12.97

6 6 280.0 19.06 6 284.2 13.75 6 281. 1 14.45 6 275.0 19.45 6 253.3* ( ↓ 10) 12.93

7 6 286.0 21.33 6 293.7 14.64 6 288. 1 16.58 6 281.5 20.28 6 259.2* ( ↓ 9) 11.58

8 6 294.3 21.95 6 301.0 14.02 6 295.9 18.13 6 287.3 21.16 6 266.8 ( ↓ 9, NS) 13.72

9 6 300.3 22.48 6 309.5 13.51 6 302. 8 18.09 6 295.4 21.51 6 271.4* ( ↓ 10) 14.77

10 6 307.7 23.92 6 316.7 12.82 6 308.9 21.18 6 302.1 23.78 6 276.5 17.75

BWG (Days 0-10) 6 70.6 11.61 6 81.2 2.71 6 72. 4 10.65 6 67.7 12.55 6 39.6* ( ↓ 44) 11.09

a Data were obtained from Table 4 on page 25 and Tables S8 and S9 on pages 50-54 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The positive control group for the androgen agonist assay is same as the vehicle c ontrol group for the anti-androgen assay. This did not affect the outcome or conclusions of this study. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### Body weight and body weight gain data for the anti-androgen assa y are presented in Table 3. Body weights and overall body weight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the duration of the assay. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-11% on Days 1- 10. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 40% . For the positive control group (TP + FT), overall body weight gains were decreased (p<0.05) by 13%.In vivo Hershberger Assay (2011)/ Page 10 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean (±SD) Body Weights a nd Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control

+ TP (0.2) b Positive Control TP + FT (0.2 + 3.0) Dichlobenil+TP (20 + 0.2) Dichlobenil +TP (60 + 0.2) Dichlobenil +TP (180 + 0.2)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 0 6 235.4 12.35 6 235.1 11.15 6 235.0 12.85 6 237.3 15.45 6 235.1 12.42 1 6 243.4 12.79 6 242.3 10.04 6 239. 4 11.94 6 235.4 15.77 6 222.1* ( ↓ 9) 14.57

2 6 251.5 12.66 6 247.5 10.25 6 247. 2 11.61 6 244.1 16.99 6 225.8* ( ↓ 10) 14.88

3 6 260.1 14.47 6 254.7 12.87 6 254. 6 13.01 6 254.2 19.39 6 231.3* ( ↓ 11) 13.16

4 6 265.9 12.78 6 260.9 11.94 6 260. 2 13.16 6 260.6 19.37 6 238.1* ( ↓ 10) 8.89

5 6 275.0 13.95 6 269.8 12.35 6 268. 5 13.18 6 270.1 21.22 6 248.6* ( ↓ 10) 9.27

6 6 284.2 13.75 6 278.6 12.00 6 277. 4 12.82 6 279.8 21.87 6 255.3* ( ↓ 10) 10.44

7 6 293.7 14.64 6 286.4 11.31 6 285. 0 14.07 6 289.1 21.91 6 262.4* ( ↓ 11) 11.60

8 6 301.0 14.02 6 291.8 12.38 6 292. 5 13.59 6 296.9 24.17 6 270.6* ( ↓ 10) 13.46

9 6 309.5 13.51 6 299.9 12.67 6 299. 7 13.46 6 303.6 23.91 6 278.0* ( ↓ 10) 13.61

10 6 316.7 12.82 6 305.8 11.88 6 305. 0 14.08 6 311.2 26.17 6 284.2* ( ↓ 10) 13.79

BWG (days 0-10) 6 81.2 2.71 6 70.7* ( ↓ 13) 6.56 6 70.0 5.71 6 73.8 12.92 6 49.1* ( ↓ 40) 6.15

a Data were obtained from Table 5 on pages 26 and Table S11- S13 on pages 59-64 of the study report. Percent differences from vehicle controls (calculated by the reviewers) are included in parentheses. b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outcome or conclusions of this study. N Number of animals in the group SD Standard Deviation * Significantly different from ve hicle controls at p<0.05.



### C. FOOD CONSUMPTION (OPTIONAL): Food consumption was not determined.

D. SERUM HORMONE CONCE NTRATIONS (OPTIONAL): Serum hormone concentrations were not determined.

E. ORGAN WEIGHTS: Weights of liver, kidneys, and accessory sex organs for the androgen agonist assay are presented in Ta ble 4. There were no treatment-r elated effects in any of the accessory sex organs of animals dosed with di chlobenil. Liver weights were increased (p<0.05) in the 60 mg/kg/day ( ↑ 24%) and 180 mg/kg/day ( ↑ 32%) groups, and the kidney weights were increased (p <0.05) in the TP group ( ↑ 8%). Animals in the TP group (positive control) had increases (p<0.05) in accessory sex organ weights as follows: 339% in seminal vesicles; 638% in ventral prostate; 165% in LABC; 513% in Cowper’s gland; and 72% in glans penis. All %CV values were le ss than the maximum permissible values.In vivoHershberger Assay (2011)/ Page 11 of 16DICHLOBENIL/027401OCSPP 890.1400/ OECD 441Table 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague-DawleyRatsaOrganDose (mg/kg/day)Vehicle Control Dichlobenil (20)Dichlobenil (60)Dichlobenil (180)Positive ControlVehicle + TP (0.2)bN Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CVSeminal vesicles6 90.0 9.43 11 6 81.5 17.18 21 6 88.8 8.83 10 6 76.3 8.24 11 6 395.0*(↑339)19.14 5Ventral prostate6 18.4 2.05 11 6 15.1 5.45 36 6 18.9 2.85 15 6 19.4 4.93 25 6 135.7*(↑638)23.92 18LABC6 170.5 22.39 13 6 175.9 28.42 16 6 172.3 22.26 13 6 135.2 15.55 12 6 451.3*(↑165)37.83 8Cowper’s glands6 4.8 0.85 18 6 4.9 1.08 22 6 5.3 0.85 16 6 5.3 1.08 20 6 29.4*(↑513)4.99 17Glans penis6 87.0 8.36 10 6 84.9 9.02 11 6 85.2 9.71 11 6 85.3 8.22 10 6 149.9*(↑72)13.44 9Adrenal gland6 63.6 6.56 10 6 62.2 8.63 14 6 65.8 11.42 17 6 58.6 7.38 13 6 60.5 6.16 10Kidney6 2056.7 95.0 5 6 2197.9 96.10 4 6 2166.1241.44 11 6 2199.7 244.99 11 6 2224.1*(↑8)164.96 7Liver (g)6 12.7 1.11 9 6 14.8 2.01 14 6 15.7*(↑24)1.99 13 6 16.8*(↑32)1.66 10 6 13.2 0.86 7a Data were obtained from Table 6 on page 28 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.b The positive control group for the androgenagonist assay is same as the vehicle control group for the anti-androgen assay.This did not affectthe outcome or conclusions of this study.N Number of animals in the groupSD Standard DeviationCV Coefficient of Variation* Significantly different fromvehicle controls at p<0.05.In vivo Hershberger Assay (2011)/ Page 12 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441


### Weight of liver, kidneys and accessory sex orga ns for the anti-androgen assay are presented in Table 5. At 60 mg/kg/day, the weight of seminal vesicles was si gnificantly decreased ( ↓ 21%). At 180 mg/kg/day, accessory sex or gan weight decreases (p<0.05) were noted as follows: 36% in seminal vesicles, 35% in ventral prostate , 27% in LABC, and 49% in Cowper’s gland. Animals dosed with TP + FT (positive contro l) had decreased (p<0.05) accessory sex organ weights as follows: 73% in seminal vesicles; 75% in ventral prostate; 48% in LABC; 71% in Cowper’s gland; and 20% in glans penis. No changes were noted in liv er weights, but kidney weights for the TP +FT group were significantly decreased ( ↓ 10%). The maximum guidelinespecified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT (positive control) group. All other %CV values were less than the maximum recommended Guideline values.In vivoHershberger Assay (2011)/ Page 13 of 16DICHLOBENIL/027401OCSPP 890.1400/ OECD 441Table 5. Accessory Sex Organ Weights (mg) fromAnti-Androgen Agonist Assay in Sprague-DawleyRatsaOrganDose (mg/kg/day)Vehicle Control+TP (0.2)bDichlobenil (0.2 + 20) Dichlobenil(0.2 + 60) Dichlobenil (0.2 + 180)TP + FTPositive Control(0.2 + 3.0)N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CVSeminal vesicles6 395.0 19.14 5 6 394.6 54.55 14 6 312.2*(↓21)56.55 18 6 250.9*(↓36)39.61 16 6 106.8*(↓73)14.01 13Ventral prostate6 135.7 23.92 18 6 121.2 28.71 24 6 114.3 16.87 15 6 88.5*(↓35)16.29 18 6 34.2*(↓75)5.48 16LABC6 451.3 37.83 8 6 452.4 61.71 14 6 440.8 39.78 9 6 330.3*(↓27)18.49 6 6 233.8*(↓48)25.72 11Cowper’s glands6 29.4 4.99 17 6 27.9 4.98 18 6 28.1 5.96 21 6 15.1*(↓49)2.01 13 6 8.4*(↓71)1.70 20Glans penis6 149.9 13.44 9 6 146.2 12.30 8 6 152.9 19.46 13 6 142.0 15.70 11 6 120.5*(↓20)23.60 20cAdrenal gland6 60.5 6.16 10 6 57.2 5.34 9 6 55.8 4.17 7 6 54.7 11.21 21 6 61.8 6.82 11Kidney6 2224.1 164.96 7 6 2178.4 137.11 6 6 2287.9 195.39 9 6 2170.4 360.73 17 6 2002.4*(↓10)96.86 5Liver (g)6 13.2 0.86 7 6 13.3 1.65 12 6 16.8 2.78 17 6 18.5 1.08 6 6 12.8 0.60 5a Data were obtained from Tables 7 on page 28 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.b The vehicle control group for the anti-androgen assay is sameas the positive control group for the androgen agonist assay.This did not affect the outcomeor conclusions of this study.c The %CV exceeds the maximum Guideline recommended %CV.N Number of animals in the groupSD Standard DeviationCV Coefficient of Variation* Significantly different from vehicle controls at p<0.05.In vivo Hershberger Assay (2011)/ Page 14 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the results of this study, no evidence of androgen agonism was noted at any dose level in this study. Anti-andr ogenic activity of dichlobenil technical was demonstrated at 180 mg/kg/ day by lower mean bulbourethral gland, LABC muscle group, seminal vesicles/c oagulating gland, and ventral prostate weights compared to the TP positive control gr oup when administered orally by gavage to male Crl:CD(SD) rats. Androgen antagoni sm was not demonstrated at 20 and 60 mg/kg/day. Higher mean liver weights were noted in the 60 and 180 mg/kg/day dichlobenil technical-treated groups (with or without co-administration of TP). The anti-androgenic positive control substance, flutamide, generally elicited the expected responses (lower weights of androgen-dependent organs). The androgenic positive control substance, testosterone propionate, elic ited the expected responses (higher weights of androgendependent organs).

B. AGENCY COMMENTS: All animals survived until scheduled termination. There were no treatment-related clini cal signs of toxicity.
In the androgen agonist assa y, body weights and overall body we ight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the duration of the assay. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 44%. Liver weights were in creased (p<0.05) in the 60 mg/kg/day ( ↑ 24%) and 180 mg/kg/day ( ↑ 32%) groups, and the kidney weights we re increased (p<0.05) in the TP group ( ↑ 8%). There were no statistically significant increases in any of the accessory sex organ weights of dichlobenil-dosed animals. Animals in the TP group (positive control) had the expected increases (p<0.05) in all accessory sex organ weights. All %CV values were less than the maximum recommended values.
In the anti-androgen assay, body weights and overall body weight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, body weights were decreased (p< 0.05) by 9-11% on Days 1-10. The overall (Days 0-10) body weight gains for 180 mg/kg/ day were decreased (p<0.05) by 40%. For the positive control group (TP + FT), overall body weight gains were decreased (p<0.05) by 13%. There were no significant changes in liver weight, and kidne y weights were only affected in the TP +FT group ( ↓ 10%). Statistically significant d ecreases in 4 of the 5 target accessory sex organ weights were observed at the high dose (180 mg/kg/day). The following decreases (p<0.05) in accessory sex organ weights we re noted at 180 mg/kg/day: 36% in seminal vesicles, 35% in ventral pr ostate, 27% in LABC, and 49% in Cowper’s gland. In addition, a decreas e (21%; p<0.05) in seminal vesi cle weight was observed at 60 mg/kg/day. Animals dosed with TP + FT (posi tive control) had the expected decreases in all accessory sex organ weights. The ma ximum guideline-specified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT (positive control) group. All other %CV values were less than the maximum recommended values.
Dichlobenil was negative for andr ogenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Dichlobenil was positive for antiIn vivo Hershberger Assay (2011)/ Page 15 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441


### androgenicity in the Hershberger assay as stat istically significant decr eases in organ weights were seen in four of the five androgen sensitive tissues in the androgen antagonist assay. However, bodyweight was decreased ≥ 10% at the dose that resu lted in the changes in androgen-sensitive tissue weights indicating that the results are confounded by overt toxicity.

C. STUDY DEFICIENCIES: The following deficiency was noted that did not have an adverse effect on the results, interpreta tion or conclusions of this study:

 The maximum guideline-specified %CV was exceed ed for the ventral prostate (20% vs. 17%) in the TP +FT (positive control) group.In vivo Hershberger Assay (2011)/ Page 16 of 16 DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441



### APPENDIX
In a non-guideline range-fi nding study (MRID 48616113), 5 Sprague-Dawley (Crl:CD (SD)) rats/dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i., Lot #: AM9K12A222) in 1.0% gum trag acanth in deionized water at doses of 0, 20, 60, or 180 mg/kg/day for 3 (females) or 10 (males) consecu tive days. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded daily. A complete necropsy was c onducted on all rats at the scheduled euthanasia and selected organs were weighed. The purpose of this study was the selection of doses for the Hershberger and Uterotrophic assays. Results for th e females will not be discussed as part of the dose-selection rationale fo r the Hershberger assay.

No adverse treatment-related effect s were observed in mortality, cl inical signs of toxicity, or gross pathology.
An initial body weight loss was observed on the first day of treatment at 60 and 180 mg/kg/day, and body weight gain was decreased (p ≤ 0.01) by 50% on the second da y of treatment at 180 mg/kg/day. Body weights in the tr eated groups were similar to the controls throughout the study. No further decreases in body weight gains were observed after Day 2, and overall body weight gain (Days 0-10) in the treated groups were sim ilar to controls. The effect on body weight gain was considered transient and did not impact fi nal body weights. The decreases in body weight gain correlated with decreases (p ≤ 0.01) of 38-42% in food consumption (g/animal/day) at 60 and 180 mg/kg/day on the first day of treatment, and a decrease (p ≤ 0.01) of 18% at 180 mg/kg/day on the second day. There were decreases (p ≤ 0.05) in food consumption noted at 60 mg/kg/day on Days 2-3 and 8-9 ( ↓ 12-15%), but these differences were c onsidered transient and unrelated to dose. Overall food consumption (Days 0-10) was decreased at 60 ( ↓ 12%; p ≤ 0.05) and at 180 ( ↓ 9%; not statistically significant [NS]) mg/kg/day. As thes e changes in food consumption were minor and did not result in differences in the final body weight, the effe ct on food consumption was not considered adverse in males.
Absolute liver weight was increased (p ≤ 0.05, except NS at 60 mg/ kg/day) at all doses by 15-58%, and relative to body liver weights were increased (p ≤ 0.01) at all doses by 16-59%. No gross lesions were noted; however , this effect is considered equivocally adverse without microscopic pathology and c linical chemistry data.
Based on these results, the Sponsor considered it appropriate to use 20, 60, and 180 mg/kg/day doses in the Hershberger assay.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRIDs 48616108 and 48616112)

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: 03/27/2012
Secondary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/30/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/09/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/09/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL/ 027401


### Primary Reviewer: Kit Farwell D.V.M. 

### Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D. 

### Health Effects Division Female Pubertal Assay (2011) I Page I of 16 OCSPP 890.1450/ OECD None

# SignafuGV~~/"' Date:

_**~-** 

### _**Signature:~~-;.!Z!',f!~ra;!:£:fl.!~ 1**  

### Date:**'b/~k** Template J;.:;i60{)8/2011



### DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3%)



### SYNONYMS: 2,6-dichlorobenzonitrile



### CITATION: Sawhney Coder, P

. (2011). A Pubertal Development and Thyroid Function Assay of Dichlobenil Administered Orally in Intact Juvenile / Peripubertal Female Rats

. WIL Research Laboratories. LLC, Ashland, OH

. Laboratory Study No.: WIL12431
, December 7
, 2011. MRID 48616108. Unpublished.

Sawhney Code r , P

. (20 11). A 7-Day Dose Range-Finding Oral (Gavage) Toxicity Study of Dichlobenil Technical in Juvenile Rats for the Endocrine Disruption Screening Program. WIL Research La boratories

. LLC
, Ashland, OH. Laboratory Study No.: WIL-12437
, December 7
, 2011. MRID 48616112. Unpublished.



### SPONSOR: Chemtura Corporation, 199 Benson Road
, Middlebury
, CT



### TEST ORDER#: EDSP -027401-120



### EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48616108)
, 15 Sprague Dawley rats / dose group were treated daily via oral gavage (5 m L/ kg ) with dichlobenil (99.3% a.i., lot / batch # : AM9Kl2A222) in aqueous 1.0% gum tragacanth at doses ofO (vehicle), 20, or 60 mg / kg / day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22
, and age and weight at day of attainment was recorded. Blood

sa mples for chemical chemistry analyses were taken following sacrifice on PND 42. Total

seru m thyroxine (T 4) 

### and thyroid stimulating hormone (TSH) levels were determined using electroc hemiluminescent immunoassays and radioimmunoassays. The weights of adrenals, kidneys, liver
, pituitary, thyroid, and urogenital organs were recorded and microscopic examination of the ovaries, uterus , thyroid
, liver
, and kidneys were performed.

There were no dichlobenil-related effects observed on mortality
, clinical signs of toxicit y, body weights, body weight gains, age and weight at attainment of VO, mean age at first vaginal estrus,

mean cycle length
, or percent cycling and regularly cycling. Organ weights were similar between the control and dichlobenil treated groups with the exceptions of liver and uterus weights. Unadjusted, adjusted and related (to body) liver weight was increased (p < 0.05) by 

### 18

-Female Pubertal Assay (2011) / Page 2 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None


### 19% at 60 mg/kg/day, and relativ e liver weight was increased by 9% at 20 mg/kg/day. Blotted uterus weight was decreased by 12% (NS) and 21% (p<0.05) at 20 and 60 mg/kg/day, respectively, and wet uterus weight was decr eased by 26% (NS) at 60 mg/kg/day. However, no histological changes were noted in the liver, uter us or ovaries.

Serum T4 and TSH levels were altered in a dose dependent manner, but the changes did not attain significance. At 20 and 60 mg/kg/day, T 4 

### levels were decreased by 4% and 7%, respectively, and TSH levels were increased by 18 and 22%, respectively. The changes in thyroid hormone levels were not corroborated by changes in thyroid weight or histology.

The dose levels tested were determined to be adequate based on the dose dependent changes were seen in the uterus and the thyroid in th is assay, and the results of the dose range-finding study.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated GLP Compliance, Data Confidentiality, Flagging, and Quality Assurance statements were provided.Female Pubertal Assay (2011) / Page 3 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: WIL Research Laboratories. LLC
Location: Ashland, OH Study Director: P. Sawhney Coder Other Personnel: Midori Asakawa, BVSc, DACVP - Contributing Scientist, Pathology Les Freshwater, MS - Contributing Scientist, Statistical Analysis Katherine L. Gardner, PhD - Participating Sc ientist, Analyses of Dosing Formulations Gwendalyn M. Maginnis, DVM - Clinical Veterinarian Kathryn S. Marks, BS, HTL (ASCP), QIHC - Principal Investigator, Histology Study Period: June 22, 2011 through December 7, 2011



### 2. Test Substance: Dichlobenil Description: White powder Source: Notox B.V. (Hertogenbosch, Netherlands) Lot #: AM9K12A222 (expiration date: October 12, 2012) Purity: 99.3% a.i. Stability: Stable in the vehicle under refrigerated storage for up to 8 days. CAS #: 1194-65-6 Structure: Cl

Cl N



### 3. Vehicle: Aqueous 1% gum tragacanth

4. Test Animals: Species: Rat (females only) Strain: Sprague Dawley Age/Weight at Study Initiation: PND 22/ 44.6 g to 56.6 g Source: Charles River Laborat ories (Portage, MI) Housing: 2-3 per cage in plastic maternity cages with ne sting material (Aspen BedTM1, Cincinnati Lab Supply, Cincinnati, OH) Diet: Harlan Laboratories 2016CM Teklad Gl obal 16% Protein Rodent Diet; ad libitum ; with an average of 29.0 and 11.0 ppm total isoflavones (aglycone equivalent). Water: Reverse osmosis-purified water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 21.3-22.6 °C 34.5-60.5% 10/hr 14 hrs light/ 10 hrs dark



### B. STUDY DESIGN

1. In-Life Dates: Start: July 29, 2011 End: August 19, 2011
2. Mating: Pregnant dams were received from the supplier on gestation day (GD) 7. Dams were allowed to deliver naturally and rear their pups to PND 21. To reduce variability, litters were culled to 10 pups/litter (5/sex wh en possible) on PND 4 using a computerized randomization procedure. Litters with fewer th an 10 total pups and/or litters with fewer than 5 pups/sex were excluded from the study.Female Pubertal Assay (2011) / Page 4 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None



### 3. Animal Assignment: On PND 21, animals were randomly assigned (stratified by body weight) to the study groups noted in Ta ble 1 and no groups contained littermates.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females

Control 0 15 Low 20 15 High 60 15 a Data were obtained from page 19 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from a dose range-finding study (MRID 48616112, reviewed conc urrently) which is summarized in an Appendix to this DER.
5. Dose Preparation and Analysis: Dose formulations were pr epared approximately weekly by mixing appropriate amounts of test substa nce with 1% gum tragacanth in deionized water; formulations were st ored refrigerated and protec ted from light. Stability under refrigerated storage for up to 8 days was demonstrated for formulations between 0.02 mg/mL and 50 mg/mL (which bracketed those in the current study) in a separate study (MRID 48616111, reviewed concurrently). Ho mogeneity (top, middle, bottom) and achieved concentration were evaluated from each dosing formulation (4 and 12 mg/mL) prepared during the in-life phase.

Results of Dose Analysis
Stability (% of time zero): 89.9 to 97.9% after 8 days refrigerated storage

Concentration (% of nominal): 89.4 to 101%
Homogeneity (% RSD): 0.78 to 2.1%
The analytical data indicated that mixing procedure was adequate and the variation between nominal and actual dosage to the animals was acceptable.
6. Dosage Administration: All doses were administered once daily by gavage from PND 22 through PND 42 in a volume of 5 mL/kg body weight between 0700 and 0900 hours.

7. Statistics: The following statistical analyses we re conducted by the performing lab. Chisquare analysis was used to determine signi ficant difference between the cycling status (cycling vs. not cycling) and percent of animals cycling re gularly in the treated groups compared to the control group. Estrous cycl e length and the day of first estrus were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the treated groups to the contro l group. Pathology findi ngs presenting as a dichotomous response were analyzed with pair wise Fisher’s exact test to compare eachFemale Pubertal Assay (2011) / Page 5 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None


### treated group with the control. The tests were 1-sided at the 0.05 significance level testing for an increase.
The following parameters were statistically analyzed by BioSTAT Consultants, Inc. (Portage, MI) using SAS ® 

### version 9.2 or higher (SAS Institute, Inc., Cary, NC): absolute organ weights, relative (to body weight) organ weights (liver, kidneys, pituitary, and adrenal glands), daily body weights (including fina l body weight), cumulative body weight gain, clinical chemistry parameters , hormone levels, and VO data.
Each endpoint was tested for homogeneity of vari ance using Levene’s test . If that test was significant at p ≤ 0.01, then a log transform was applied and Levene’s test was conducted on the transformed data. If that test was still significant then the square root transformation was applied to the raw data and Levene’s test conducted again. If the test was still significant then a non-parametric test, as desc ribed below, was used to analyze the data.
If the variances were homogeneous then an analysis of variance (ANOVA) was conducted, on the raw or transformed data, as appropriate. The statistical model contained a factor for treatment group and a blocking factor based on the time of study necropsy data. A 2-sided Dunnett’s test was conducted, regardless of the outcome of the ANOVA, looking for significant differences in the test substance gr oups when compared with the vehicle control. If the transformations were unsuccessful in making the variances homogeneous then the non-parametric Kruskal-Wallis test was use d, ignoring the blocking factor, followed by Dunn’s test, to compare each of the test substanc e groups with the vehicle control. As these are pre-planned pairwise comparisons, Dunn’s te st was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, looking for significant differences from the vehicle control.
In addition, organ weights and VO data were subject to the fo llowing analyses if they met the homogeneity of variance criteria: 1) Anal ysis of covariance ( ANCOVA; covariate of PND 21 body weight) with Dunnett’s test. The model was as described above for ANOVA with the exception that PND 21 body weight was included as a covariate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model.
Histopathology findings presented as a graded response were analyzed with pairwise MannWhitney U tests to compare each treated group wi th the control. The tests were 1-sided at the 0.05 significance level testi ng for increased severity. Th e tests were two-sided for graded responses presented on a Grade 1-5 sc ale at the 0.05 significance level testing for increased or decreased severity. Exact p-va lues were calculated for the Mann-Whitney U test.
Significance was denoted at 5%, 1%, and 0.1% levels. The statistical analyses were considered appropriate.Female Pubertal Assay (2011) / Page 6 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None



### C. METHODS
1. Mortality and Clin ical Examinations: All animals were examined twice daily (morning and afternoon) for mortality and moribundity. Animals were also examined twice daily for clinical signs of toxicity (prior to dosing and 1- 2 hours post-dosing).
2. Body Weight: Animals were weighed at randomiza tion (PND 21), daily prior to dosing during PND 22 to 42, and at sacrifice. B ody weight gains were calculated for each corresponding interval and for the overall (PND 22-42) treatment period.
3. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of VO. Age and weight on the day of completion of VO were recorded.
4. Estrous Cyclicity: Beginning on the day of VO, up to and including the day of necropsy, daily vaginal smears were obtained for determinati on of the stage of estrus. The age at first vaginal estrus was recorded. The mean age at first vagina l estrus, mean cycle length, percent of each group cycling, and percent of each group cycling regularly were reported.
5. Sacrifice and Pathology: All females were sacrifi ced randomly across all groups by decapitation without anesthesia on PND 42 approximately 2 hours post-dosing

. It was stated that all animals were sacrificed by approximately 1300 hours due to normal diurnal fluctuation in thyroid hormone levels. Immedi ately following sacrifi ce, blood (minimum of 2 mL per animal) was collected for T 4 

### , TSH, and clinical chemistry analyses. Serum was separated, and the samples were analyzed fresh (T 4 

### ) or stored frozen ( ≤− 20°C) for subsequent analysis (TSH).
a. Hormone Analysis: An electrochemiluminescent im munoassay on the Cobas e411 (Roche Diagnostics, Indianapolis, IN) was used to analyze serum samples for the presence of T 4



### . To determine the concentration of TSH, the [ 125 

### I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary) was use d. This assay system relies on magnetic separation for quantitative measurement of TSH using a GAMMA-C12 gamma counter (Diagnostic Products Corporation, Los Angeles, CA). For both assays, multiple quality control samples were dispersed within the assay.Female Pubertal Assay (2011) / Page 7 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None


### b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol X Potassium X Globulin (calculated) X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Acetyl cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin/globulin ratio X Alanine aminotransferase (A LT/also SGPT) X Bile acids X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ XX Uterus*+ XX Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on guideline 890.1450


### All collected organs, except the thyroid, were weighed prior to fixa tion. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was separated from the vagina and weighed. The uter us was weighed again following removal of the fluid in the lumen (blotted weight). Following approxi mately 24 hours of fixation in 10% neutral buffered formalin, the ovaries, uterus, and kidneys were rinsed and retained in 70% ethanol. The thyroids, attached to the tr achea, were fixed for at least 24 hours in formalin, dissected from the trachea, weighed, and retained in 70% ethanol. The ti ssues were routinely processed in paraffin, sectione d at 4-6 microns, mounted, st ained with hematoxylin and eosin, and examined microscopically.
Thyroid sections were subjectively evaluated for follicular cell height and colloid area using a five point grading scale (1 = shortest/smallest amount; 5 = tallest/largest amount), and any abnormalities/lesions noted. At least two sect ions from each thyroid were examined.Female Pubertal Assay (2011) / Page 8 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None


### The uterus was evaluated for hypertrophy or hypotrophy by changes in uterine horn diameter as well as myometrial, stroma, or e ndometrial gland development. Estrus cycle stage at the time of necropsy was considered in the pathology assessment of the ovary and uterus.

II. RESULTS

A. Mortality: All animals survived until scheduled termination.

B. Clinical Signs of Toxicity: No treatment-related clinical si gns of toxicity were observed in any animals in any dose group.
C. General Growth and Vaginal Opening: Body weights, body weight gains, age of attainment of VO and weight at day of atta inment are presented in Table 2. In the 60 mg/kg/day group, body weight gain was transiently decreased following the first dose (PND 22-23), but did not attain stat istical significance. Additionally, mean body weight gains in this group were comparable to controls for the remainder of the dos ing period, and overall body weight gains were unaffected. Age and we ight at attainment of VO were similar across all groups. With th e exception of one 60 mg/kg/day animal (No. 17288-08), all animals achieved VO by PND 42.
The age at VO, body weight at VO, and final bo dy weight in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1450).

TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 15 51.5 2.82 5.48 15 50.9 3. 02 5.93 15 51.6 3.20 6.20 A NR NR NR NR NR NR NR NR NR NR NR NR Body weight at VO (g) U 15 122.1 11.05 9.05 15 124.2 11. 97 9.64 15 123.5 10.76 8.71 A 15 120.1 11.05 9.05 15 122.7 11. 97 9.64 15 121.7 10.76 8.71 Final body weight (g) U 15 156.0 10.46 6.70 15 152.6 10. 19 6.68 15 155.1 12.80 8.25 A NR NR NR NR NR NR NR NR NR NR NR NR Final body weight (% of control) U 15 NA NA NA 15 -2.18 NA NA 15 -0.58 NA NA A NR NR NR NR NR NR NR NR NR NR NR NR Body weight gain (final – initial; g) U 15 104.5 9.04 8.65 15 101.7 9. 43 9.28 15 103.5 11.97 11.56 A NR NR NR NR NR NR NR NR NR NR NR NR Age at VO (PND) U 15 35.4 1.68 4.75 15 36.5 1. 85 5.06 15 36.1 3.42 9.46 A 15 35.0 1.68 4.75 15 36.1 1. 85 5.06 15 35.7 3.42 9.46 Proportion unopened (#/N) 0/15 0/15 1/15 a Data were obtained from Table 4 on page 31 of the study report U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation NA Not applicable NR Not reported



### D. Organ Weights: Organ weights at necr opsy are presented in Ta ble 3. At 60 mg/kg/day,Female Pubertal Assay (2011) / Page 9 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None 

### mean absolute, adjusted, and relative (to body) liver weights were increased (p<0.001) compared to controls ( ↑ 18-19%). Additionally at this dose, mean absolute blotted and wet uterine weights were decreased by 21 (p<0.05) and 26% (not statistically significant, NS), respectively. The decreased abso lute blotted and wet uterine we ights were not considered to be not biologically adverse because adjusted uterine weights were comparable, there were no histopathological findings in the uterus, and there were no e ffects of treatment on the day of attainment of VO, estrous cyclicity, or the age at the first occurrence of estrus.
At 20 mg/kg/day, relative (t o body) liver weight was in creased (p<0.001) by 9% and absolute blotted uterine weight was decreased (NS) by 12%. In the absence of corroborating evidence of hepatotoxicity from gross and micr oscopic examination of the liver and clinical chemistry, the increased absolute and relative wei ghts were not considered to be adverse.
The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provid ed in the Guideline (890.1450), except for the adrenal gland weight (35.2 mg) which was be low the acceptable rang e (38.34 to 48.84 mg).Female Pubertal Assay (2011) / Page 10 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Adrenals (mg) U 15 35.2 4.32 12.27 15 33.9 5. 32 15.69 15 35.2 7.11 20.19 A 15 35.1 4.32 12.27 15 34.0 5. 32 15.69 15 35.2 7.11 20.19 R 15 22.6 2.91 12.86 15 22.2 3. 19 14.36 15 22.6 3.26 14.44 Kidneys (g) U 15 1.29 0.12 9.09 15 1.26 0. 12 9.17 15 1.32 0.16 12.26 A 15 1.30 0.12 9.09 15 1.29 0. 12 9.17 15 1.33 0.16 12.26 R 15 0.83 0.05 5.52 15 0.83 0. 05 5.57 15 0.85 0.06 7.46

Liver (g) U 15 7.00 0.82 11.78 15 7.45 0.69 9.20 15 8.29*** ( ↑ 18) 1.05 12.66

A 15 7.01 0.82 11.78 15 7.47 0.69 9.20 15 8.30*** ( ↑ 18) 1.05 12.66

R 15 4.48 0.32 7.15 15 4.88*** ( ↑ 9) 0.23 4.61 15 5.33*** ( ↑ 19) 0.35 6.55

Ovaries (mg) U 15 71.3 12.15 17.03 15 66.2 9. 48 14.31 15 66.4 18.49 27.84 A 15 74.0 12.15 17.03 15 69.3 9. 48 14.31 15 69.2 18.49 27.84 Pituitary (mg) U 15 9.8 1.18 12.09 15 8.9 1. 24 13.92 15 9.6 1.77 18.43 A 15 9.9 1.18 12.09 15 9.1 1. 24 13.92 15 9.8 1.77 18.43 R 15 6.3 0.71 11.36 15 5.8 0. 65 11.13 15 6.2 0.80 13.03 Thyroid, fixed (mg) U 15 9.14 1.27 13.90 15 10.20 1.69 16.52 15 9.71 1.57 16.18 A 15 9.22 1.27 13.90 15 10.30 1.69 16.52 15 9.79 1.57 16.18
Uterus, blotted (mg) U 15 260.6 71.89 27.59 15 228.8 ( ↓ 12) 71.86 31.41 15 205.4* ( ↓ 21) 68.90 33.55

A 15 263.0 71.89 27.59 15 234.1 71. 86 31.41 15 208.6 68.90 33.55
Uterus, wet (mg) U 15 299.3 105.18 35.14 15 285.2 149.27 52.35 15 222.0 ( ↓ 26) 81.80 36.85

A 15 296.4 105.18 35.14 15 287.6 149. 27 52.35 15 220.6 81.80 36.85 a Data were obtained from Tables 7 and S11 on pages 35 and 86- 91 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05. *** Significantly different fro m controls at p<0.001.



### E. Estrous Cyclicity: Estrous cycle data are provided in Table 4. There were no effects of treatment on any estrus cycle parameter. The mean age at first vaginal estrus, mean cycle length, percent cycling, and pe rcent regularly cycling were similar across all groups.Female Pubertal Assay (2011) / Page 11 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicity a

Dose Level (mg/kg/day) N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling Control (0) 15 36.8 4.9 100 88.9 10 1 4 0 Dichlobenil (20) 15 37.7 4.8 93.3 100 12 b 1 2 1 b

Dichlobenil (60) 14 37.1 4.7 100 87.5 11 0 3 0

a Data were obtained from Table 5 on page 32 of the study report. b One female in Diestrus was not cycling. N Number of animals



### F. Clinical Chemistry and Hormone Levels: Mean thyroid hormone levels and selected clinical chemistry parameters ar e presented in Table 5. Serum T 4 

### levels were slightly decreased and TSH levels were slightly incr eased at 20 and 60 mg/kg/day compared to controls. However, as these differences did no t attain statistical significance and there were no corresponding histopathological lesions in the thyroid or liver , they were not considered to be related to treatment.
At 60 mg/kg/day, increases in the following c linical chemistry parameters were noted: cholesterol ( ↑ 18%, p<0.05); albumin ( ↑ 5%, p<0.01); and total protein ( ↑ 4%, p<0.01). The changes in albumin and total pr otein were not considered to be adverse because they were small in magnitude. The small increase in cholesterol was not c onsidered biologically adverse. Levels for all other clinical chemis try parameters were similar across all groups. Historical control data were provided on pages 377-380 for clin ical chemistry parameters only.

The value for serum T 4 

### in the control group was within the acceptable range of the performance criteria provided in the Guideline (890.1450).Female Pubertal Assay (2011) / Page 12 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 , Total (μg/dL) 15 4.11 0.777 18.904 15 3.96 0. 697 17.583 15 3.81 0.633 16.625

Serum TSH (ng/mL) 15 5.15 1.399 27.191 15 6.07 1. 618 26.648 15 6.26 3.263 52.127

Clinical Chemistry A/G ratio 15 2.75 0.370 13.472 15 2. 79 0.280 10.050 15 2.86 0.336 11.730 Albumin (g/dL) 15 4.1 0.19 4. 74 15 4.2 0.23 5.45 15 4.3** ( ↑ 5) 0.22 5.15

ALP (U/L) 15 240 51.8 21.6 15 225 36.1 16.0 15 227 57.1 25.2 ALT (U/L) 15 69 13.8 20.1 15 67 19.4 28.9 15 71 11.2 15.8 AST (U/L) 15 188 46.3 24.6 15 181 50.4 27.8 15 197 70.7 35.9 Bile acids (umol/L) 15 45.6 34.16 74.85 15 18.6** 18. 83 101.1 15 27.4 24.84 90.58

Calcium (mg/dL) 15 12.4 0.41 3.32 15 12.7 0.41 3.22 15 12.5 0.39 3.14

Chloride (mEq/L) 15 102 1.7 1.7 15 102 1.4 1.4 15 102 1.2 1.2

Cholesterol (mg/dL) 15 85 10.8 12.7 15 91 11.1 12.2 15 100** ( ↑ 18) 14.2 14.2

Creatinine (mg/dL) 15 0.1 0.04 31.05 15 0.1 0.04 34.50 15 0.1 0.03 24.21

GGT (U/L) 15 0.3 0.25 97.3 15 0. 2 0.31 127.16 15 0.2 0.22 96.46 Globulin (g/dL) 15 1.5 0.16 10.61 15 1.5 0.13 8.38 15 1.5 0.20 12.98

Glucose (mg/dL) 15 147 6.0 4.1 15 147 8.2 5.6 15 148 13.2 9.0

Phosphorus (mg/dL) 15 9.9 0.89 9.02 15 9.6 0. 75 7.85 15 10 0.54 5.36

Potassium (mEq/L) 15 6.67 0.565 8.472 15 6.93 0. 405 5.853 15 6.79 0.540 7.955

Sodium (mEq/L) 15 143 1.8 1.3 15 142 1.8 1.2 15 143 1.7 1.2

Total Bilirubin (mg/dL) 15 0.06 0.016 26.767 15 0.05 0. 019 34.856 15 0.07 0.019 27.450

Total protein (g/dL) 15 5.6 0.15 2.71 15 5.7 0.28 4.96 15 5.8** ( ↑ 4) 0.3 5.16

Triglyceride (mg/dL) 15 56 13.7 24.7 15 65 19.8 30.7 15 64 19.2 29.9

Urea Nitrogen (mg/dL) 15 11.6 1.89 16.28 15 12.4 2.13 17.14 15 12.5 2.31 18.44

a Data were obtained from Table 6 on pages 33 and 34 of the st udy report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation ** Significantly different fro m controls at p<0.01.



### G. Histopathology: The incidences of histopathological findings of the thyroid gland and ovaries are presented in Tables 6 and 7, re spectively. No treatme nt-related microscopic findings were observed in the thyroid gland, ovaries, or in any other examined tissue at either dose.Female Pubertal Assay (2011) / Page 13 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

TABLE 6. Incidence of Histopathol ogical Lesions of the Thyroid Gland a

Dose (mg/kg/day) Parameter Evaluated Colloid Area Follicular Cell Height

Grade b Incidence Grade b Incidence O E O E Control (0) 1 0 15 1 15 15 2 0 15 2 0 15 3 0 15 3 0 15 4 15 15 4 0 15 5 0 15 5 0 15 Dichlobenil (20) 1 0 15 1 15 15 2 0 15 2 0 15 3 0 15 3 0 15 4 15 15 4 0 15 5 0 15 5 0 15 Dichlobenil (60) 1 0 15 1 15 15 2 0 15 2 0 15 3 0 15 3 0 15 4 15 15 4 0 15 5 0 15 5 0 15 a Data were obtained from Table 8 on page 37 of the study report. b The criteria for grades 1-5 we re listed on page 366 of the st udy report and are reported above. O Number observed E Number examinedFemale Pubertal Assay (2011) / Page 14 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopath ological Lesions in the Ovaries a

Tissue Grade b Dose (mg/kg/day) Control (0) Dichlobenil (20) Dichlobenil (60)
O E O E O E Corpora Lutea, Growth Phase 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 14 15 15 15 15 15 4 1 15 0 15 0 15 5 0 15 0 15 0 15 Corpora Lutea, Mature 1 0 15 0 15 0 15 2 2 15 1 15 1 15 3 12 15 14 15 14 15 4 1 15 0 15 0 15 5 0 15 0 15 0 15 Corpora Lutea, Regressive 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 13 15 15 15 15 15 4 2 15 0 15 0 15 5 0 15 0 15 0 15 Follicles, Atretic 1 0 15 0 15 0 15 2 0 15 1 15 1 15 3 15 15 14 15 14 15 4 0 15 0 15 0 15 5 0 15 0 15 0 15 Follicles, Primary 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 15 15 15 15 15 15 4 0 15 0 15 0 15 5 0 15 0 15 0 15 Follicles, Primordial 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 15 15 15 15 15 15 4 0 15 0 15 0 15 5 0 15 0 15 0 15 Follicles, Secondary 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 14 15 15 15 15 15 4 1 15 0 15 0 15 5 0 15 0 15 0 15 Follicles, Tertiary 1 0 15 0 15 0 15 2 0 15 0 15 0 15 3 14 15 15 15 14 15 4 1 15 0 15 1 15 5 0 15 0 15 0 15 a Data were obtained from Table 8 on page 37 of the study report. b The criteria for grades 1-5 we re listed on page 367 of the st udy report and are reported above. O Number observed E Number examinedFemale Pubertal Assay (2011) / Page 15 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The Investigators concl uded that there were no dichlobenil-related effects noted on the age a nd body weight at VO, the age at the first occurrence of estrus, and estr ous cyclicity. Alth ough lower mean wet and blotted uterus weights were noted at 20 and 60 mg/kg/day, there were no microscopic correlates. Also, there were no microscopic findings that co rresponded to higher mean TSH and lower T 4 

### levels observed in these groups. Therefore, no evidence of endocrine disruption was noted at 60 mg/kg/day, the highest level tested in this study.

B. AGENCY COMMENTS: There were no dichlobenil-related effects observed on mortality, clinical signs of toxicity, body weights, body weight gains, age and weight at attainment of VO, mean age at first vaginal estrus, mean cycl e length, and percent cycling or regularly cycling.
Organ weights were similar between the cont rol and dichlobenil treated groups with the exceptions of liver and uterus weights. Unadjusted, adjusted and related (to body) liver weight was increased (p<0.05) by 18–19% at 60 mg/kg/day, and relative liver weight was increased by 9% at 20 mg/kg/day. Blotted uter us weight was decrea sed by 12% (NS) and 21% (p<0.05) at 20 and 60 mg/kg/day, respectiv ely, and wet uterus weight was decreased by 26% (NS) at 60 mg/kg/day. However, no histologi cal changes were noted in the liver, uterus or ovaries.
Clinical chemistry parameters were generally unaffected by dichl obenil treatment, although bile acids were substantially reduced by 59% (p<0.05) and 40% (NS) at 20 and 60 mg/kg/day, respectively, and cholesterol wa s increased by 7% (NS) and 18% (p<0.05), respectively. Serum T4 and TSH levels were altered in a dose dependent manner, but the changes did not attain signifi cance. At 20 and 60 mg/kg/day, T4 levels were decreased by 4% and 7%, respectively, and TSH levels we re increased by 18 and 22%, respectively. The changes in thyroid hormone levels were not corroborated by changes in thyroid weight or histology.
Dose selection was based upon a range finding study, described in the Appendix, in which terminal body weights were decreased 8% a nd liver weights were increased 31-40% at 180 mg/kg/day, and liver wei ghts were increased 29-36% at 60 mg/kg/day.

C. STUDY DEFICIENCIES: The following minor deficien cy was noted that is not considered to have had an adverse impact on th e results, interpretation or conclusions of this study:
Control adrenal gland weight of 35.2 mg was outside the performance criteria acceptable range of 38.34 to 48.84 mg.Female Pubertal Assay (2011) / Page 16 of 16 DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None


## APPENDIX

In a dose range-finding study (MRID 48616112), juven ile male and female Crl:CD(SD) rats (5/sex/dose) were dosed daily vi a oral gavage (5 mL/kg) with dichlobenil (99.3% a.i.; Lot #: AM9K12A222) in aqueous 1% gum tragacanth at doses of 20, 60, or 180 mg/kg/day for 7 consecutive days.
All animals survived to scheduled necropsy. Th ere were no adverse treatment-related effects on clinical signs, food consumption, or macroscopic evaluations observe d at any dose in either sex.
At 180 mg/kg/day, body weight ga ins were decreased (p<0.05) by 30-46% in the males during Days 0-2. Mean body weight gain for the remai nder of the treatment pe riod was comparable to controls. Overall (Days 0-7) body weight gains in males of this group were decreased by 16% (not statistically significant, NS) compared to the controls, resulting in mean terminal body weights that were 8% lower than the controls. Additionally at this dose, mean liver weights (absolute and relative [to body weight]) were in creased (p<0.01) by 31-40 % in the males and by 45-47% in the females.
At 60 mg/kg/day, body weight gain s were decreased (p<0.05) by 34% in the males during Days 0-1. However, mean body weight gains were compar able to the controls for the remainder of the treatment period and for the overall treatment peri od. Mean body weights at the end of treatment were comparable to the controls. Additionally at this dose, mean relative (to body) liver weight was increased (p<0.05) by 10% in the males a nd absolute (NS) and relative (p<0.01) liver weights were increased by 29-36% in the females.

There were no adverse treatment-related effects in male rats at 20 mg/kg/day. However, absolute (NS) and relative (p<0.05) liver weights were increased by 16-18% in the females. There were no treatment-related effects on adrenal or ki dney weights at any dos e in either sex.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRIDs 48616109 and 48616112)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/24/2012
Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 3/28/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/09/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/09/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL I 027401 Male Pubertal Assay (2011) I Page I of 16 OCSPP 890.1500 / OECD None


### Primary Reviewer: Ayaad Assaad, D

. V

. M., Ph.D. 

### Health Effects Division Signature:

# fi;_/b:.<_~J_,.**Date ~,_----**  Secondary Reviewer: John Liccione, Ph.D. 

### Health Effects Division Signature.

### ~

# Date:**~.:: .:**  

# . .,

DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None.



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS:

CITATION: 2
, 6-dichlorobenzonitrile

Coder, P.S. (2011). A pubertal developmental and thyroid function assay of dichlobenil administered orally in intact juvenile / peripubertal male rats. WIL Research Laboratories, LLC
, Ashland, OH, Laboratory Study No

. : WIL-12432, December 7
, 2011. MRID 48616109

. Unpublished.

Coder, P.S

. (2011). A 7-day dose range-finding oral (gavage) toxicity study of dichlobenil technical in juvenile rats for the endocrine disruption screening program

. WIL Research Laboratories, LLC, Ashland, OH

. Laboratory Study No.: WIL-12437, December 7
, 2011. MRID 48616112

. Unpublished

.


### SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury
, CT



### TEST ORDER#: E DSP-027401-120



### EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48616109)
, 15 Sprague-Dawley (Cr l :CD(S D)) rats / dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i., Lot #: AM9K12A222) in aqueous 1.0% gum tragacanth at doses of 0, 20, or 60 mg / kg / day from post-natal day (PND) 23 to 53. The animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment was recorded

. Following sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using electrochemiluminescent immunoassays and radioimmunoassays

. The weights of adrenals, liver, pituitary
, thyroid, and urogenital organs
were recorded and micro scopic examinations of the testes , epididymides, thyroid
, liver
, and kidneys were performed.

No adverse treatment-related effects were observed in mortality, clinical signs of toxicity, bod y weight, body weight gain, age or weight at PPS
, clinical chemistry
, gross pathology
, or microscopic examination of the testes , epididymides, thyroid
, liver
, or kidney.Male Pubertal Assay (2011) / Page 2 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### The age and weight at attainment of PPS at all doses was within the acceptable range of the performance criteria. The unadjusted values for organ weights and hormone levels in the control group were within the acceptable range of the performance criter ia provided in the Guideline with the exception of a minor de parture in the thyroid weight of 13.44 mg compared to Guideline criterion of 14-26 mg. All thyroid glands examin ed in all dose groups we re considered within normal limits of control tissues.
Increased (p ≤ 0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-to-body weight ratio liver ( ↑ 35–36%) and kidney ( ↑ 9–10%) weights were observed at 60 mg/kg/day. Increased (p ≤ 0.05) organ-to-body weight ratio liver ( ↑ 16%) and kidney ( ↑ 7%) weights were observed at 20 mg/kg/ day. Increased (p ≤ 0.05) adjusted adrenal weight s were also noted at 60 mg/kg/day ( ↑ 13%). Except for the liver weights at 60 mg/kg/day, these differences were considered minor. Furthermore, in the absence of corroborating evidence of hepatotoxicity from gross and microscopic examination of the liver an d clinical chemistry, the increased weight was considered an adaptive response.
No biologically significant eff ect was noted on clinical chemistry parameters in the treated groups; however several clinical chemistry pa rameters in the 60 mg/kg/day group differed statistically (p ≤ 0.05) from controls.
Although no treatment-related effects were noted on pubertal development, the thyroid, or signs of systemic toxicity, the dosing levels utilized in the current study are considered appropriate based on the results of the dose range-finding study.
The assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated GLP, Data C onfidentiality, Flagging, and Quality Assurance statements were provided.Male Pubertal Assay (2011) / Page 3 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC
Location: Ashland, OH Study Directors: Coder, P.S. Other Personnel: Midori Asakawa, BVSc, DACVP - Con tributing Scientist, Pathology Les Freshwater, MS - Contributing Scientist, Statistical Analysis Katherine L. Gardner, PhD - Participating Scientist, Analyses of Dosing Formulations Gwendalyn M. Maginnis, DVM - Clinical Ve terinarian Kathryn S. Marks, BS, HTL (ASCP), QIHC - Principal Investigator, Histology Study Period: June 23, 2011 through December 7, 2011



### 2. Test Substance: Dichlobenil Description: White powder Source: Notox B.V. (Hertogenbosch, Netherlands) Lot #: AM9K12A222 (expires October 12, 2012) Purity: 99.3% a.i. Stability: Stabile in vehicle when stored for 8 days in the refrigerator CAS #: 1194-65-6 Structure:



### 3. Vehicle: 1.0% Gum tragacanth in deionized water (Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ, Lot # 2AC3069, expires August 31, 2012)Male Pubertal Assay (2011) / Page 4 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### 4. Test Animals: Species: Rat (males only) Strain: Sprague-Dawley (Crl:CD(SD)) Age/Mean Weight at Study Initiation: PND 23 / 49.2–62.4 g
Source: Charles River Laboratories, Portage, MI Housing: Parental dams were individually housed in plasti c maternity cages with aspen bedding; each dam was housed with her litter until weaning on PND 21. After weaning, up to 3 males were housed in plastic cages. Diet: 2016CM Teklad Global 16% Protein Rodent Diet (Harlan Teklad, Madison, WI), ad libitum . It was stated that an average of 29.0 and 11.0 ppm total isoflavones (genistein + daidzein + glycitein) was present. Water: Reverse osmosis-purified tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 21.3–22.6°C 34.5–60.5% 10/h 14 h dark / 10 h light



### B. STUDY DESIGN

1. In-Life Dates: Start: July 30, 2011 End: August 30, 2011
2. Mating: Time-mated pregnant dams (age not repo rted) were received from the supplier on gestation day (GD) 7 and were allowed to deliv er naturally and rear their pups to PND 21. Any litters with fewer than ten total pups and/ or less than five males or females (when possible) were excluded from the study. Litters were culled to 10 pups/litter on PND 4, with 5 pups/sex when possible.
3. Animal Assignment: Following weaning on PND 21, animals were assigned to study based on a computer-generated randomization sc heme based on body weight to test groups as noted in Table 1. Litter mate s were not placed in the same group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males
Control 0 15
Low 20 15 High 60 15 a Data were obtained from page 18 of the study report.



### 4. Dose Selection Rationale: In a dose range-finding study (MRID 48616112), juvenile male and female rats (5/sex/dose) were dosed daily via oral gavage (5 mL /kg) with dichlobenil (99.3% a.i.; Lot/batch#: AM9K12A222) in a queous 1% gum tragacanth at doses of 20, 60, or 180 mg/kg/day for 7 consecutive days. All animals survived to scheduled necropsy. There were no adverse treatment-related eff ects on clinical signs, food consumption, or macroscopic evaluations observed at any dose in either sex.
At 180 mg/kg/day, body weight gains were decreased (p<0.05) by 34-46% in the males during Days 0-2. Mean body weight gain for the remainder of the treatment period wasMale Pubertal Assay (2011) / Page 5 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### comparable to controls. Overall (Days 0-7) body weight gains in males of this group were decreased by 16% (not statistically significant, NS) compared to the controls, resulting in mean body weights that were up to 9% lower than the controls throughout the treatment period. Additionally at this dose, mean liv er weights (absolute and relative [to body weight]) were increased (p< 0.01) by 31-40% in the males a nd by 45-47% in the females.
At 60 mg/kg/day, body weight ga ins were decreased (p<0.05) by 34% in the males during Days 0-1. However, mean body weight gains were comparable to the controls for the remainder of the treatment period and for th e overall treatment period. Mean body weights at the end of treatment were co mparable to the controls. A dditionally at this dose, mean relative (to body) liver weight was increased (p<0.05) by 10% in the males and absolute (NS) and relative (p<0.01) liv er weights were increased by 29-36% in the females.

There were no adverse treatment-related effects in male rats at 20 mg/kg/day. However, absolute (NS) and relative (p<0.05) liver weight s were increased by 16-18% in the females. There were no treatment-related effects on adre nal or kidney weights at any dose in either

sex.
Based on these results, 60 mg/kg/day was select ed as the high-dose for the current study because it has the potential to s how signs of toxicity in both sexes. A dose of 20 mg/kg/day was maintained as the low-dose for the current study.
5. Dose Preparation and Analysis: Suspensions of test substa nce concentrations of 4 and 12 mg/mL in 1.0% gum tragacanth in deionized water were prepared approximately weekly with storage refrigerated and protected from light. Stability under re frigerated storage for up to 8 days was demonstrated for formula tions between 0.02 mg/mL and 50 mg/mL (which bracketed those in the current study) in a separate study (MRID 48616111, reviewed concurrently). Concentration and homogeneity (top, middle, and bottom) of the test mixture were evaluated from each dosing formulations prepared during the in-life phase.

Results
Stability (% of time zero): 89.9-97.9% after 8 days refrigerated storage

Concentration (% of nominal): 87.5–106%
Homogeneity (%RSD): 1.1–2.8
The analytical data indicate d that the mixing procedure wa s adequate and the variation between nominal and actual dosage to the animals was acceptable.
6. Dosage Administration: The dose formulations were ad ministered once daily from PND 23 through PND 53 by oral gavage at a dose volume of 5 mL /kg of body weight. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were genera lly dosed between 0700 and 0900 hours local time each day.Male Pubertal Assay (2011) / Page 6 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### 7. Statistics: All analyses were conduct ed using SAS® version 9.2 or higher software. Data that were statistically analy zed included the organ weight s, organ weight to final body weight ratio (liver, kidneys, pituitary, and adrenal glands ), daily body weights (including necropsy body weight), cumulative body weight gain, serum chemistries, and PPS data. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Each e ndpoint was tested for homogeneity of variance using Levene’s te st. If that test was significant at p ≤ 0.01, then a log transform was applied and Levene’s test con ducted on the transformed data. If that test was still significant, then the square root transformation was applie d to the raw data and Levene’s test conducted again. If the test was still significant , then a nonparametric test, as described below, was used to analyze the data.
If the variances were homogeneous then an analysis of variance (ANOVA) was conducted, on the raw or transformed data, as appropriate. The statistical model contained a factor for treatment group and a blocking factor based on the time of study start. A 2-sided Dunnett’s test was conducted, regardless of the out come of the ANOVA, looking for significant differences in the test substance groups when compared with the vehicle control. If the transformations were unsuccessful in ma king the variances homogeneous then the nonparametric Kruskal-Wallis test was use d, ignoring the blocking factor, followed by Dunn’s test, to compare each of the test substanc e groups with the vehicle control. As these were pre-planned pairwise comparisons, Dunn’s test was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, looking for significant differences from the vehicle control. In addition, the following analyses were conducted:

Classification Procedure Organ weights and PPS data If the data met the homogeneity of variance criteria: 1) Analysis of covariance (ANC OVA) with Dunnett’s test. The model was as describe d above for ANOVA with the exception that PND 21 body weight was included as a covariate; 2) lin ear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model. Histopathology findings presented as a graded response Da ta were analyzed with pairwise Mann-Whitney U tests to compare each treated group with the control. The tests were 1-sided at the 0.05 significance level testing for increased severity. The test s were two-sided for graded responses presented on a Gr ade 1-5 scale at the 0.05 significance level testing for increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U test. Histopathology presented as a dichotomous response Pairwise Fisher’s exac t tests was used to compare each treated group with the control. The tests were 1-sided at the 0.05 significance level testing for an increase.


### Significance was denoted at 5% or less. The statistical analyses were considered appropriate.Male Pubertal Assay (2011) / Page 7 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### C. METHODS
1. Mortality and Clin ical Examinations: Pups were sexed on PND 0, 4, and 21. Detailed physical examinations for each pup were recorded on PND 1, 4, 7, 14, and 21. All males were observed twice daily, once in the morni ng and once in the afternoon, for moribundity and mortality. Individual clinic al observations were recorded da ily (prior to test substance administration during the treatment period) th rough the day of euthanasia. Animals were also observed for signs of toxicity between1 and 2 hours following dose administration. The absence or presence of findings was recorded for individual animals. In addition, the presence of findings at the time of dose admi nistration was recorded for individual animals.
2. Body Weight: Pretest body weights for each pup were recorded on PND 1, 4, 7, 14, and 21. Individual body weights were recorded on the day of randomi zation, daily prior to test substance administration during the treatment period, and on the day of euthanasia. Mean body weight changes were calculated for each corresponding interval and also for PND 23–53.
3. Food Consumption: Food consumption was not measured.
4. Preputial Separation (PPS): Each male was observed fo r PPS beginning on PND 30. In addition, the appearance of a pa rtial and complete PPS or a pe rsistent thread of tissue between the glans and prepuce was recorded. The age and body weight on the day at which complete PPS was observed was recorded for each animal. However, if any animal within any group showed incomplete separation (persi stent threads) for greater than 3 days, a separate second analysis was conducted using the age at which incomplete separation was first observed. Examination of the males was continued daily until complete PPS was attained or until euthanasia. When an animal did not attain PPS prior to necropsy, PND 54 (last Study Day + 1) and body weight at necropsy was used as the age and body weight at PPS, respectively.
5. Sacrifice and Pathology: The dams were euthanized by carbon dioxide asphyxiation and discarded without examination. Excluded litters and culled pups were euthanized by carbon dioxide asphyxiation or intraperitoneal in jection of sodium pentobarbital without examination. All F1 males were euthanized randomly across all groups by decapitation without anesthesia on PND 53. Animals we re euthanized beginning at least 2 hours following dose administration that day and all animals were euthanized by approximately 1300 hours due to normal diurnal fluctuation in thyroid hormone leve ls. Immediately upon euthanasia by decapitation, trunk blood samples were collected for hormone analysis and serum chemistry. Serum was separated, and the samples were analyzed fresh or stored frozen ( ≤ -20°C) for subsequent analysis.
a. Hormone Analysis: An electrochemiluminescent im munoassay on the Cobas e411 (Roche Diagnostics, Indianapolis, IN) was used to analyze serum samples for the presence of testosterone and T 4 

### . To determine the concentration of TSH in serum samples, the [ 125 

### I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary) was used. This assay system relies on magnetic separation for quantitative measurement of TSHMale Pubertal Assay (2011) / Page 8 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### using a GAMMA-C12 gamma counter (Diagnos tic Products Corporation, Los Angeles, CA). For both assays, multiple quality control samples were dispersed within the assay.
b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol X Potassium X Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase X Albumin /globulin ratio (calculated) Lactic acid dehydrogenase (LDH) X Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in male rats based on guideline 890.1500.


### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed from all F1 main study an imals. The (XX) organs, in addition, were subjected to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)*+ XX Thyroid a * + XX Epididymides (left and right separately)*+ XX Liver* XX Seminal vesicle plus coagulating glands (with and without fluid)* XX Adrenals (paired)* XX Ventral prostate* XX Pituitary* XX Dorsolateral prostate* XX Levator ani/bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* +
X Gross lesions and masses b a Thyroid and parathyroids were collected and weighed together b Gross lesions and masses were collected but not weighed. * Weights required base d on guideline 890.1500. + Histopathological examination re quired based on guideline 890.1500.


### Necropsies were performed on all juvenile males at the scheduled necropsy on PND 53. Any tissues with gross lesions were saved in 10% neutral-buffered formalin for possible future histopathologic examina tion. The tissues listed above were excised, and the carcasses were then discarded. Tissues were weighed immediately to prevent drying out prior to weighing. All collected organs , except the thyroid/parathyroi ds, were weighed prior to fixation. Absolute organ weight and usually relative to body or gan weights were reported.Male Pubertal Assay (2011) / Page 9 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### In addition, organ weights were subject to ANCOVA (covariate of PND 21 body weight) when appropriate. Testes, epididymides, ventral prostate, and dorsola teral prostate were weighed separately. The seminal vesicle with and without coagulating glands fluid were weighed. The kidneys and adrenals were weighe d in pairs. The testes and epididymides were initially fixed in Bouin’ s fixative (for approximately 24 hours) and retained in 70% ethanol thereafter. The thyroid glands, attached to the trachea, and kidney were fixed for at least 24 hours in 10% neutral-bu ffered formalin, and then the liver and thyroid (dissected from the trachea) were each placed in 70% ethanol until histologica l processing. The liver was placed in 10% neutral-buffered forma lin for potential future histopathologic examination. After fixation, tissue samples of the thyroid, kidney, liver, testes, and epididymides were shipped to Biotechni cs, LLC. (a division of WIL Research), Hillsborough, NC, for routine processing into paraffin blocks, sect ioning, staining with hematoxylin and eosin generally, and examined microscopically. Hematoxylin and eosin were used for staining, except PAS and hematoxylin staining were used for the right testis and epididymis. The thyroid sections (minimum of 2 sections) were subjectively evaluated for follicular epithelial height and co lloid area and any abnormalities/lesion.
Testes and epididymides were evaluated as required by the EPA's Health Effects Test Guideline OCSPP 870.3800: Reproduction and Fertil ity Effects. Besides gross lesions such as atrophy or tumors, testicular histopathological examination was conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells into the lumen. The intact epididymis, including the caput, cor pus and cauda, was examined in longitudinal sections, in order to identify lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium.

II. RESULTS
A. Mortality: All animals survived until scheduled termination.
B. Clinical Signs of Toxicity: No treatment-related clinical si gns of toxicity were observed in animals for any dose group.
C. General Growth and Preputial Separation: Body weights, body weight gains, age of attainment of PPS, weight at PPS, and proportion/incidence of un-separated are presented in Table 2 below.
No treatment-related effects on general growth, or on age or weight at attainment of PPS were observed at any dose. Both the age and we ight at attainment of PPS at all doses was within the acceptable range of the pe rformance criteria for controls.
Differences from the control group values were s light and not statistica lly significant. When a second analysis was performed, adjusting the da y of attainment for those males with 3 or more consecutive days of incomplete separation (persistent threads), no statisticallyMale Pubertal Assay (2011) / Page 10 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### significant delays in the mean age of attain ment of PPS were noted for both the 20 and 60 mg/kg/day groups when compared to the control group.

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day

# of Males Mean SD CV (%) # of Males Mean SD CV (%) # of Male

s Mean SD CV (%) Age at PPS (PND) U 15 44.6 2.29 5.14 15 45.6 2. 53 5.55 15 45 1.46 3.25
A 15 44.6 2.29 5.14 15 45.6 2. 53 5.55 15 45 1.46 3.25 Body weight at PPS (g) U 15 211.8 19.97 9.43 15 213.4 21. 45 10.05 15 215.2 14.87 6.91

A 15 211.5 19.97 9.43 15 213.1 21. 45 10.05 15 214.9 14.87 6.91
Initial body weight (PND 23; g) U 15 57.1 3.1 5.43 15 56.6 3. 7 6.54 15 57.2 2.67 4.67

A 15 NR NR NR 15 NR NR NR 15 NR NR NR
Final body weight (g) U 15 280.7 24.67 8.79 15 267.2 17. 77 6.65 15 279.1 24.81 8.89
A 15 NR NR NR 15 NR NR NR 15 NR NR NR Final body weight (% of control) U 15 100 NR NR 15 95.2 b NR NR 15 99.4 b NR NR

A 15 NR NR NR 15 NR NR NR 15 NR NR NR
Body weight gain (final – initial; g) U 15 223.6 23.74 10.62 15 210.6 15. 92 7.56 15 221.9 24.04 10.83

A 15 NR NR NR 15 NR NR NR 15 NR NR NR
Proportion unseparated (#/N) 15 0/15 15 0/15 15 0/15

a Data were obtained from Tables 4 and 5 on pages 28 and 29 of the study report. b Calculated by reviewers U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 SD Standard Deviation CV Coefficient of Variation NA Not applicable NR Not reported



### D. Organ Weights: Organ weights at necr opsy are presented in Table 3 below. Increased (p ≤ 0.05) unadjusted, adjusted (covariate P ND 23 body weight), and organ-to-body weight ratio liver ( ↑ 35–36%) and kidney ( ↑ 9–10%) weights were observed at 60 mg/kg/day. Increased (p ≤ 0.05) organ-to-body weight ratio liver ( ↑ 16%) and kidney ( ↑ 7%) weights were observed at 20 mg/kg/ day. Increased (p ≤ 0.05) adjusted adrenal we ights were noted at 60 mg/kg/day ( ↑ 13%).
The mean values for the left and right epidi dymis weights for the control group were lower than the acceptable range in the performance criteria when the weights were combined; however, the CV values for the le ft and right epididymis were within the acceptable range in the performance criteria for the entire epididym is. In addition, the mean values of thyroidMale Pubertal Assay (2011) / Page 11 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### gland and kidney weights in the control gr oup were below the minimum values in the performance criteria; however, the CV values were within the acceptable range. There is no reported acceptable range for testis in the Spra gue Dawley rat in the performance criteria.

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day # of Males Mean SD CV # of Males Mean SD CV # of Males Mean SD CV
Liver (g) U 15 12.66 1.697 13.4 15 14.00 1.322 9.4 15 17.08* ( ↑ 35) 2.454 14.4

A 15 12.36 1.697 13.4 15 13.72 1.322 9.4 15 16.79* ( ↑ 36) 2.454 14.4

R 15 4.50 0.300 6.7 15 5.24* ( ↑ 16) 0.334 6.38 15 6.10* ( ↑ 36) 0.482 7.9

Kidneys (g) U 15 1.95 0.217 11.2 15 1.99 0.195 9.8 15 2.12* ( ↑ 9) 0.220 10.4

A 15 1.95 0.217 11.2 15 1.99 0.195 9.8 15 2.13* ( ↑ 9) 0.220 10.4

R 15 0.69 0.053 7.6 15 0.74* ( ↑ 7) 0.053 7.1 15 0.76* ( ↑ 10) 0.047 6.1

Pituitary (mg) U 15 11.6 1.61 13.9 15 10.7 2. 29 21.4 15 11.1 1.40 12.7
A 15 12.0 1.61 13.9 15 11 2. 29 21.4 15 11.4 1.40 12.7
R 15 4.1 0.45 10.9 15 4.0 0. 72 18.0 15 4.0 0.57 14.3
Adrenals (mg) U 15 44.3 5.81 13.1 15 45.0 8. 16 18.1 15 50.0 9.52 19.0

A 15 44.1 5.81 13.1 15 45.0 8.16 18.1 15 49.9* ( ↑ 13) 9.52 19.0

R 15 15.8 1.93 12.2 15 16.8 2. 84 16.9 15 17.9 2.61 14.6
Seminal vesicle + coagulating gland, with fluid (mg) U 15 614.3 110.72 18.0 15 576.9 124. 23 21.5 15 580.1 129.49 22.3

A 15 605.4 110.72 18.0 15 568.3 124. 23 21.5 15 571.2 129.49 22.3

Seminal vesicle + coagulating gland, without fluid (mg) U 15 396.1 63.33 16.0 15 366.5 58. 79 16.0 15 365.9 65.97 18.0

A 15 394.9 63.33 16.0 15 365.7 58. 79 16.0 15 364.7 65.97 18.0
Ventral prostate (mg) U 15 252.5 50.65 20.1 15 244.5 66. 65 27.3 15 223.5 26.85 12.0
A 15 NT NT NT 15 NT NT NT 15 NT NT NT
Dorsolateral prostate (mg) U 15 113.9 16.00 14.0 15 107.1 19. 29 18.0 15 105.1 17.88 17.0
A 15 112.4 16.00 14.0 15 105.7 19. 29 18.0 15 103.6 17.88 17.0
LABC (mg) U 15 523.3 88.41 16.9 15 501.6 85. 40 17.0 15 510.1 65.77 12.9
A 15 532.8 88.41 16.9 15 511.7 85. 40 17.0 15 519.6 65.77 12.9Male Pubertal Assay (2011) / Page 12 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day # of Males Mean SD CV # of Males Mean SD CV # of Males Mean SD CV Epididymis, left (mg) U 15 198.4 19.65 9.9 15 197.4 20. 21 10.2 15 194.5 20.69 10.6
A 15 200.1 19.65 9.9 15 199.8 20. 21 10.2 15 196.3 20.69 10.6 Epididymis, right (mg) U 15 207.5 20.33 9.8 15 207.1 22. 66 10.9 15 206.0 18.81 9.1
A 15 208.2 20.33 9.8 15 208.2 22. 66 10.9 15 206.6 18.81 9.1 Testis, left (mg) U 15 1432.2 116.92 8. 16 15 1390.8 321.17 23.1 15 1481.2 186.47 12.6
A 15 1458.8 116.92 8.16 15 1420.1 321.17 23.1 15 1508.0 186.47 12.6 Testis, right (mg) U 15 1449.3 123.78 8.54 15 1385.9 285.41 20.6 15 1468.1 115.43 7.9
A 15 1477.5 123.78 8.54 15 1415.6 285.41 20.6 15 1496.4 115.43 7.9 Thyroid, fixed (mg) U 15 13.44 2.012 15.0 15 13.31 1.812 13.6 15 13.89 2.508 18.0
A 15 13.25 2.012 15.0 15 13.13 1.812 13.6 15 13.70 2.508 18.0 a Data were obtained from Table 7 on pages 32–33 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 R Organ-to-body weight ratio (relative to body weight) SD Standard Deviation CV Coefficient of Variation NT Not Tested * Significantly different fro m controls at p<0.05.



### E. Clinical Chemistry and Hormone Levels: Clinical chemistry and hormone levels are presented in Table 4 below. Hormone levels in the treated animals were similar to controls. No biologically significant eff ect was noted on clinical chemistry parameters in the treated groups. The following clinical chemistry para meters in the 60 mg/kg/day group differed statistically (p ≤ 0.05) from controls: (i ) increased albumin ( ↑ 7%); (ii) decreased alkaline phosphatase ( ↓ 16%); (iii) increas ed cholesterol ( ↑ 18%); (iv) increased globulin ( ↑ 6%); and (v) increased total protein ( ↑ 7%). The hormone values for th e control group were within the acceptable range of the performance criteria provided in the Guideline (OCSPP 890.1500).

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day # of Males Mean SD CV # of Males Mean SD CV # of Males Mean SD CV
Hormones
Serum T 4 , Total (μg/dL) 15 5.92 0.764 12.9 15 5.99 0. 690 11.5 15 6.05 1.216 20.1

Serum TSH (ng/mL) 15 11.1 5.12 46.1 15 9.6 2. 92 30.3 15 10.2 3.87 37.8

Serum
testosterone (ng/mL) 15 2.2 1.46 67.3 15 2.4 1. 82 74.4 15 2.0 0.84 42.0Male Pubertal Assay (2011) / Page 13 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day # of Males Mean SD CV # of Males Mean SD CV # of Males Mean SD CV Clinical Chemistry A/G Ratio 15 2.22 0.152 6.9 15 2.25 0.185 8.2 15 2.22 0.199 8.98 Albumin (g/dL) 15 4.1 0.12 2.8 15 4.2 0.14 3.30 15 4.4* ( ↑ 7) 0.23 5.17
ALP (U/L) 15 290 55.5 19.1 15 261 57.0 21.8 15 245* ( ↓ 16) 32.6 13.3
ALT (U/L) 15 81 15.3 18.9 15 82 19.9 24.2 15 82 14.8 18.2 AST (U/L) 15 207 62.9 30.5 15 237 114.9 48.5 15 220 85.7 38.9 Bile Acids (umol/L) 15 33.6 15.82 47.1 15 33.4 20. 57 61.6 15 41.0 21.32 52.0

Calcium (mg/dL) 15 12.6 0.59 4.7 15 12.6 0.43 3.42 15 12.8 0.66 5.12

Chloride (mEq/L) 15 100 1.5 1.5 15 100 1.3 1.3 15 99 1.2 1.2

Cholesterol (mg/dL) 15 93 15.0 16.1 15 99 13.6 13.8 15 110* ( ↑ 18) 19.9 18.0

Creatinine (mg/dL) 15 0.1 0.05 36.6 15 0.2 0. 05 31.7 15 0.1 0.05 35.2
GGT (U/L) 15 0.1 0.00 0.0 15 0. 1 0.00 0.0 15 0.1 0.00 0.0 Globulin (g/dL) 15 1.9 0.11 6.1 15 1.9 0.15 8.2 15 2.0* ( ↑ 5) 0.19 9.7

Glucose (mg/dL) 15 153 10.1 6.6 15 151 7.2 4.8 15 149 8.7 5.8
Phosphorus (mg/dL) 15 10.1 0.53 5.3 15 10.1 0. 59 5.8 15 10.3 1.28 12.5

Potassium (mEq/L) 15 6.55 0.525 8.0 15 6.81 0. 705 10.3 15 6.52 0.439 6.7

Sodium (mEq/L) 15 142 1.0 0.7 15 143 1.6 1.1 15 143 1.6 1.1

Total Bili (mg/dL) 15 0.05 0.016 32.1 15 0.06 0. 024 43.6 15 0.06 0.015 24.4

Total Protein (g/dL) 15 5.9 0.18 2.99 15 6.1 0.23 3.8 15 6.3* ( ↑ 7) 0.33 5.3

Triglyceride (mg/dL) 15 121 38.5 31.7 15 117 44.3 37.8 15 149 60.1 40.4

Urea Nitrogen (mg/dL) 15 12.9 2.42 18.8 15 14.0 3. 14 22.4 15 12.1 2.38 19.6

a Data were obtained from Table 6 on pages 30–31 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. NR Not reportedMale Pubertal Assay (2011) / Page 14 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None


### F. Gross pathology: There were no effects of treatment observed at necropsy.
G. Histopathology: There were no treatmen t-related histopathological findings noted in the testes, epididymides, kidneys , liver or thyroid at any dose. The incidences of histopathological findings of the thyroid gland are presented below in Table 5. All thyroid glands examined in all dose groups were cons idered within normal lim its of control tissues.

TABLE 5. Incidence of Histopatholo gical Lesions of the Thyroid Gland a

Treatment

Groups Parameter Evaluated Colloid Quality Follicular Cell Height (Increase) Follicular Cell Height (Decrease) Follicular Cell Shape Severity b Incidence Severity Incidence Severity Incidence Severity Incidence O E O E O E O E

Vehicle Control 1 0 15 1 15 15 1 NR 15 1 NR 15 2 0 15 2 0 15 2 NR 15 2 NR 15 3 0 15 3 0 15 3 NR 15 3 NR 15 4 13 15 4 0 15 4 NR 15 4 NR 15 5 2 15 5 0 15 5 NR 15 5 NR 15

Dichlobenil (20 mg/kg/day) 1 0 15 1 13 15 1 NR 15 1 NR 15 2 0 15 2 2 15 2 NR 15 2 NR 15 3 1 15 3 0 15 3 NR 15 3 NR 15 4 14 15 4 0 15 4 NR 15 4 NR 15 5 0 15 5 0 15 5 NR 15 5 NR 15

Dichlobenil (60 mg/kg/day) 1 0 15 1 13 15 1 NR 15 1 NR 15 2 0 15 2 2 15 2 NR 15 2 NR 15 3 0 15 3 0 15 3 NR 15 3 NR 15 4 15 15 4 0 15 4 NR 15 4 NR 15 5 0 15 5 0 15 5 NR 15 5 NR 15 a Data were obtained from Table 9 on page 34 of the study report. b Thyroid histopathology is graded 1 – 5 based on severity; see Methods. O No. Observed E No. Examined NR Not Reported



### The incidence of histopathological findings of the testes, epid idymides, liver, thyroid, and kidneys are presented in Table 6.Male Pubertal Assay (2011) / Page 15 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None

TABLE 6. Incidence of Histopathological Lesions of the Testes, Epididymides , Liver, Thyroid, and Kidney a

Findings Dose Level (mg/kg bw/day) Vehicle Control Dichlobenil - 20 mg /kg/day Dichlobenil - 60 mg/kg/day
# Observed # Examined # Observed # Examined # Observed # Examined Testes, right Minimal degeneration of seminiferous tubules 2 15 1 15 0 15

Mild hypospermatogenesis 0 15 1 15 0 15 Epididymid es, right
Moderate hypospermia 0 15 1 15 0 15
Minimal mononuclear infiltrate 0 15 0 15 1 15

Liver
Minimal mononuclear infiltrate 9 15 4 15 11 15

Minimal acute inflammation 1 15 0 15 0 15

Mild coagulative necrosis 0 15 0 15 1 15 Thyroid Ultimobranchial cyst 5 15 2 15 2 15 Ectopic tissue 1 15 0 15 1 15 Kidneys Minimal basophilic tubules 7 15 3 15 1* 15 Cyst 1 15 1 15 3 15 Minimal mononuclear infiltrate 3 15 3 15 1 15

Minimal mineralization 1 15 0 15 0 15 Severe mineralization 1 15 0 15 0 15 Minimal chronic progressive nephropathy 5 15 5 15 4 15 Mild pyelonephritis 0 15 0 15 1 15
a Data were obtained from Table S 11 on pages 98–99 of the study report. * Significantly different from controls at p ≤ 0.05.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Test substance-related clinical chemistry alterations were limited to higher mean chol esterol, albumin, globu lin, and total protein levels in the 60 mg/kg/day group. However, no correlating histologic lesion was identified by histopathological examination in the liver or kidney. No test substance-related alterations in serum hormone parameters were noted at any dose leve l. Test substancerelated organ weight differences included highe r mean absolute and relative (to final body weight) adrenal gland, liver, and kidney we ights in the 20 and/or 60 mg/kg/day groups. However, no correlating histolog ic lesions were identified by histopathological examination in the liver or kidney; the adrenal gla nds were not examined microscopically.Male Pubertal Assay (2011) / Page 16 of 16 DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None



### B. AGENCY COMMENTS: No adverse treatment-related effects were observed in mortality, clinical signs of t oxicity, body weight, body weight ga in, age or weight at PPS, clinical chemistry, or gross a nd histopathology. The age and wei ght at attainment of PPS at all doses was within the acceptable range of the performance criteria. The unadjusted values for organ weights and hormone levels in the control group were within the acceptable range of the performance criteria .All thyroid glands examined in all dose groups were considered within normal limits of control tissues.
Increased (p ≤ 0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-tobody weight ratio liver ( ↑ 35–36%) and kidney ( ↑ 9–10%) weights were observed at 60 mg/kg/day. Increased (p ≤ 0.05) organ-to-body weight ratio liver ( ↑ 16%) and kidney ( ↑ 7%) weights were observed at 20 mg/kg/day. Increased (p ≤ 0.05) adjusted adrenal weights were noted at 60 mg/kg/day ( ↑ 13%). Except for the liver weights at 60 mg/kg/day, these differences were considered minor. Furthermor e, in the absence of corroborating evidence of hepatotoxicity from gross and microsc opic examination of the liver and clinical chemistry, the increased weight was considered an adaptive response.
No biologically significant eff ect was noted on clinical chemistry parameters in the treated groups. The following clinical chemistry para meters in the 60 mg/kg/day group differed statistically (p ≤ 0.05) from controls: (i ) increased albumin ( ↑ 7%); (ii) decreased alkaline phosphatase ( ↓ 16%); (iii) increas ed cholesterol ( ↑ 18%); (iv) increased globulin ( ↑ 6%); and (v) increased total protein ( ↑ 7%).
Although no treatment-related effects were noted on pubertal development, the thyroid, or signs of systemic toxicity, the dosing levels utilized in the current study are considered appropriate based on the results of the dose range-finding study.

C. STUDY DEFICIENCIES: The following measure was outside of the range specified in the performance criteria; however, this variat ion did not impact the interpretation of the assay:

 The thyroid weight of 13.44 mg compared to guidelin e criterion of 14-26 mg.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616110)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/21/2012
Secondary Reviewer Signature: Scott D. Studenberg, Ph. D., D.A.B.T. Date: 3/26/2012
Program Manager: Signature: Jack D. Early, M.S. Date: 4/05/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/05/2012

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

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).DICHLOBENIL I 027401 

### Primary Reviewer: Pati e nce Browne, Ph.D

. Health Effects Division Secondary Reviewer: 

### John Liccione Ph.D Health Effects Division Steroidogenesis Assay (2011) I Page I of 11 OCSPP 890.1550 / OECD None


### Signature:**-t:_.£------"~** 

### Date:

### -=----~CZ±.~;2-- 

### S ign 

### a tu re:**::::=:::;:;;~:Z:::;:==:::==::::;:::--** 

### Da te:**-=--L+~~'....,L..:.;L-_**



### DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890

. 1550



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS No.: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS: 2
, 6-dichlorobenzonitrile



### CITATION: Meerts, l.A.T.M

. (2011 ). Dichlobenil technical: steroidogenesis assay usin g the human cell lin e H295R

. NOTOX B.V
., The Netherlands, Laboratory Study No.: 495783
, December 9, 2011. MRID 48616110. Unpublished.



### SPONSOR: Chemtura Corporation, 199 Benson Road
, Middlebury, CT



### TEST ORDER#: EDSP-027401-120



### EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48616110)
, H295R cells cultured in vitro 

### in 24-well plates were incubated with dichlobenil (99.3% a.i., Batch # AM9K l 2A222) at serial log concentrations of 100 to 0.0001 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentratio n of 0.1 %

.

Testostero ne and estradiol levels were measured using ELISA. A Quality Control (QC) plate

was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properl y to positive control agents at two concentration levels. The positive
contro l s included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and
testosterone production.

Guide lin e acceptability recommendations and requirements were generally met , including adequate production of te stostero ne and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls

. Precipitation of the test compound

was not reported
, and cytotoxicity was not observed.

Treatment -rel ated effect from dichlobenil observed on testosterone or estradiol levels were not consistent across all runs. In Run 1
, there were significant reductions in testosterone levels at 0.0001
, 0

. 001
, 0.01
, 1and100 μM ofdichlobenil
, with the greatest reduction (0.71-fold) occurring at 0

. 01 μM. During Run 2, decreased (p :S 0.05) testosterone levels were also noted at 0.0001 and 0.01 μM. However
, no reductions in testosterone were noted in Run 3. No changesSteroidogenesis Assay (2011) / Page 2 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None


### in estradiol concentration we re observed during Runs 1 and 2, but estradiol was decreased (p ≤ 0.05) in Run 3 at all dich lobenil concentrations except 100 and 0.1 μM.
Due to the inconsistencies and variability in th e hormone alterations across the three runs, the results for dichlobenil in this assay were considered to be equivocal.
The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Steroidogenesis Assay (2011) / Page 3 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: NOTOX B.V. Location: The Netherlands Study Director: I.A.T.M. Meerts, Ph.D. Other Personnel: Dr. M.A. Thomson, study monitor M. Mulder, technical coordinator Study Period: July 27, 2011 through December 9, 2011



### 2. Test Substance: Dichlobenil Description: White powder Batch # (expiration date): AM9K12A222 (October 12, 2012) Purity: 99.3% Solubility: 50 mg/mL in DMSO 0.021 g/L in water Vapor pressure: 0.073 Pa at 20°C Stability: Not reported for assay conditions; stable as stored Storage conditions: Room temperature in the dark CAS #: 1194-65-6 Molecular weight: 172.0 Structure:



### 3. Positive Control: Forskolin Description (molecular weight): White powder (410.50) Source: Sigma-Aldrich (Steinheim, Germany) Batch # (expiration date): 109K5057V (December 13, 2014) Purity: 98% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature in the dark CAS #: 66575-29-9


### 4. Negative Control: Prochloraz Description (molecular weight): White powder (376.67) Source: Sigma-Aldrich (Steinheim, Germany) Batch # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature in the dark CAS #: 67747-09-5



### 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description (molecular weight): Clear liquid (78.13) Source: SeccoSolv, Merck (Darmstadt, Germany) (catalog # not reported) Lot # (expiration date): Not reported Purity: Not reported Storage conditions: Not reported CAS #: 67-68-5 Justification for choice of solvent: Not provided

Final concentration in assay: 0.1% v/vSteroidogenesis Assay (2011) / Page 4 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None



### 6. Stock Medium: Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham Source: Invitrogen Corporation (Breda, The Netherlands) Lot # (expiration date): Not reported
Sodium bicarbonate: Not reported Nu-Serum: BD Biosciences (Bedford, MA; catal og and lot nos. were not reported; background hormone concentrat ions were not reported) ITS+premix: Penicillin/streptomycin: BD Biosciences (Bedford, MA); catalog and lot nos. were not reported Invitrogen Corporation (catalog and lot nos. were not reported)



### 7. Test Cells: H295R human adrenocortical carcinom a cells (ATCC CLR-2128; Lot # not reported) at passage numbers 6.5, 7.5, and 8.5 were incubated in the stock medium at the time of plating, dosing, and harvest. Incubation conditions were at 5% CO 2 

### and 37 ± 1°C for approximately 24 hours prior to exposure.
The following performance criteria were met (indicated by an “x”):

x Cell passage identifier. Cell Passage #: 6.5, 7.5, and 8.5 x Cells frozen down at passage 5 x Frozen cells cultured for 5 additional passages x Total number of passages does not exceed 10



### B. METHODS

1. Pre-Test Information:
a. Hormone Assay Interference Test: Dichlobenil was tested for potential interference with the estradiol and testosterone hormone assa ys. Assays were conducted of the following samples of media containing: 0.1 mM dichlobenil; 0.1 mM dichlobenil with 2 × 10 -6 

### mg/mL testosterone and 2 × 10 -7 

### mg/mL estradiol; and 2 × 10 -6 

### mg/mL testosterone and 2 × 10 -7 

### mg/mL estradiol. The inte rference of dichlobenil with estradiol and testosterone hormone measurements was 16% and 0.38%, resp ectively. Since the interference observed for testosterone and estradiol hormone meas urement was very weak (below 20%), the Sponsor stated that results of hormone measur ements using the EIA kits were not corrected for this interference.Steroidogenesis Assay (2011) / Page 5 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None


### b. Hormone Extraction: Hormone extraction was not performe d, since it was determined in a validation study (NOTOX Project 493393) that the medium did not affect the hormone measurement system.
c. Laboratory Proficiency Test: No laboratory proficiency te st data were reported.

2. Test Solutions: Dichlobenil was dissolved in DMSO to make a 100 mM stock and then serially diluted in DMSO. Fo rskolin and prochloraz were diss olved in DMSO. Test agents were added to the cell culture plates, yielding final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μM for proc hloraz, and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM for dichlobenil. The final concentration of DM SO in the medium was held constant at 0.1% (v/v).
3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described above. Hormone concentrations in undiluted Nu-Serum were not reported. H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for at least five passages (number not reported). The cells were plated into wells of a 24-well cell culture plate at a density of approximately 200,000 cells/mL. The cells were then placed into a 5% CO 2 

### incubator at 37 ± 1°C for approximately 24 hours prior to chemical exposure. It was not reported that the cells were check ed microscopically for attachment and proper morphology prior to dosing,

4. Exposure: The medium was removed from the cells and replaced with medium containing concentrations of dichlobenil in triplicate, according to the schematic presented in Table 2.

TABLE 2. Dosing Schematic for the Exposure of H295 R Cells to Dichlobenil (Final Concentrations in μM). a 1 2 3 4 5 6 A DMSO DMSO DMSO 0.1 0.1 0.1 B 100 100 100 0.01 0.01 0.01 C 10 10 10 0.001 0.001 0.001 D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 20 of the study report.


### A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner with either forskolin or prochloraz, according to the schematic presented in Table 3.

TABLE 3. Dosing Schematic for the QC Plate for Po sitive Controls (Final Concentrations in μM). a 1 2 3 4 5 6 A Blank b Blank Blank Blank + MeOH c Blank + MeOH Blank + MeOH B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH C Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM D Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM a Data were obtained from page 19 of the study report. b Blank wells received medium only. c MeOH = 70% methanol was added to these wells for 30 mi nutes at room temperature following medium removal.Steroidogenesis Assay (2011) / Page 6 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None



### Following dosing, the plates were incubated for 48 hours under the conditions previously described. After the 48 hour exposure, each we ll was examined under the microscope for cell condition (attachment, mor phology, degree of confluence) and signs of cytotoxicity. In addition, digital photographs were taken from each well. The media was collected from all wells in two equal portions and stored at ≤− 75°C until shipment to the analytical laboratory for hormone measurement.
5. Cell Viability/Cytotoxicity Assay: After media removal, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide (MTT) assay. The medium from each well was replaced by 500 μ L fresh medium. Subsequently, the medium from wells designated for methanol treatment was removed and 70% methanol was added to these wells. The plates were incubated at room temperature for 30 minutes. After incubation, the methanol was removed, and these wells were rinsed carefully with medium, three times. After rinsing, medium was added to these wells. To all wells, MTT (5 mg/mL in PBS) was added. The plates were incubated at 37.0 ± 1.0°C and 5% CO 2 

### for 2-3 hours. After incubation, the medium in each well was replaced with DMSO. The plates were shaken on an orbital shaker at room temperat ure for 5 minutes. The formation of the blue formazan was measured at 560 nm against background absorption at 690 nm using a spectrophotometer. The change in cell viabil ity was determined by comparing treated wells to the solvent control wells. A greater than 20% reduction in cell vi ability was considered evidence of cytotoxicity.
6. Hormone Measurement System: The concentrations of test osterone and estradiol in the samples were determined using commercial EI A kits from Cayman Chemical Company. In addition, medium samples spiked with 500 a nd 2500 pg/mL testosterone or 50 and 250 pg/mL estradiol were prepared and analyzed in the EIA. Method detection limits were reported as 6.3–8.7 pg/mL for estradiol and 5.0–7.1 pg/ mL for testosterone in the QC plates.
The following performance criteria were met (indicated by an “x”):

x Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

NR Spiked sample recovery acceptable fo r two concentrations of testosterone and estradio l (mean measured amount from triplicate samples within 30% of nominal concentration) x Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) x Solvent control within 75% range be low maximum response on standard curve x Test compound tested for interfer ence with meas urement system



### C. DATA ANALYSIS: Results were normalized to the mean solvent control (SC) value for each assay, and results were expressed as change s relative to SC in each exposure plate. Prior to conducting statistical analysis, the assumptions of normality and variance homogeneity were evaluated. Normality was eval uated using a Shapiro-Wilk’s test. If data were not normally distributed, the data we re transformed to approximate a normal distribution. If the data were normally di stributed or approximated normal distribution, differences between chemical treatments and solvent controls were analyzed using the Dunnett’s Test. If the data were not norma lly distributed, the non- parametric Kruskal Wallis test was performed. Differences were considered significant at p ≤ 0.05. Statistical evaluation was done based on average values fo r each well that represents independent replicate data points. The SAS program (release 9.1 for Windows NT) was used forSteroidogenesis Assay (2011) / Page 7 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

### statistical evaluation. Also, Prism versi on 4.01 (Graphpad Software, San Diego, CA) was used for non-linear regression and statistical analysis.

II. RESULTS

A. TEST COMPOUND: Precipitation of dichlobenil was no t reported at any concentration, and no cytotoxicity was observed. Inter-plate variation (%CV) for the solvent controls in the test assays was 11% for testosterone and 34% for estradiol (criteria is ≤ 30%). Intra-plate variation (%CV) for the solvent controls was <11% for each run in both the testosterone and estradiol assays, and each were within the Guideline criteria.
In Run 1, there were statistically signifi cant reductions in test osterone at 0.0001, 0.001, 0.01, 1 and 100 μM, with the greatest reduction (0.71 -fold) occurring at 0.01 μM. During Run 2, decreased (p ≤ 0.05) testosterone levels were also noted at 0.0001 and 0.01 μM. However, no reductions in testosterone were noted in Run 3. No changes in estradiol concentration were observed during Runs 1 and 2, but estradiol was decreased (p ≤ 0.05) at all concentrations in Run 3, except at 100 and 0.1 μM.

TABLE 4. Mean (±SD) Hormone Concen trations Following Treatment with Dichlobenil for 48 Hours. a Nominal Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean b ± SD b Statistical Significance
Testosterone (pg/mL) Fold Difference DMSO 2006 2472 2187 — — — — — — 0.0001 1715 2094 2521 0.85 0.85 1.15 0.95 0.17 Runs 1 and 2 0.001 1725 2325 2407 0.86 0.94 1.10 0.97 0.12 Run 1 0.01 1430 1798 2135 0.71 0.73 0.98 0.81 0.15 Runs 1 and 2 0.1 1733 2477 2209 0.86 1.00 1.01 0.96 0.08 — 1 1643 2312 2416 0.82 0.94 1.10 0.95 0.14 Run 1 10 1920 2528 2693 0.96 1.02 1.23 1.07 0.14 — 100 1457 2200 2060 0.73 0.89 0.94 0.85 0.11 Run 1 Estradiol (pg/mL) Fold Difference DMSO 226 296 439 — — — — — — 0.0001 263 316 329 1.16 1.07 0.75 0.99 0.22 Run 3 0.001 247 304 338 1.09 1.03 0.77 0.96 0.17 Run 3 0.01 238 304 364 1.05 1.03 0.83 0.97 0.12 Run 3 0.1 231 285 377 1.02 0.96 0.86 0.95 0.08 — 1 247 323 331 1.09 1.09 0.75 0.98 0.20 Run 3 10 235 329 350 1.04 1.11 0.80 0.98 0.16 Run 3 100 247 343 429 1.10 1.16 0.98 1.08 0.09 — a Data were obtained from Tables 16 and 18 on pages 29–30 of the study report. Method detection limits were reported as 6.3–8.7 pg/mL for estradiol and 5.0–7.1 pg/mL for testosterone. b Calculated by the reviewers.Steroidogenesis Assay (2011) / Page 8 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None


### FIGURE 1. Change in Testosterone Production Relative to Dichlobenil Concentration.

FIGURE 2. Change in Estradiol Producti on Relative to Dichlobenil Concentration.

B. CYTOTOXICITY: No cytotoxicity was noted for the fo rskolin, prochloraz, blank, and SC wells; mean viability ranged from 93–129%, when solvent control was set at 100%. Methanol resulted in the expected response, approximately 0% viability. Data are summarized in Table 5.Steroidogenesis Assay (2011) / Page 9 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None
TABLE 5. Mean (±SD) MTT Cell Viability (% SC) Resu lts after Treatment with Forskolin, Prochloraz, or Dichlobenil for 48 Hours. a Compound Concen. (μM) Cell Viability – Run 1 Cell Viabilit y – Run 2 Cell Viability – Run 3 Mean SD Mean SD Mean SD Blank NA 107 10 104 9 96 7 SC NA 100 3 100 4 100 1 Forskolin 1 119 18 111 14 106 10 Forskolin 10 129 1 118 3 108 1 Blank + Methanol NA -1 4 -3 3 -4 5 SC + Methanol NA 0 7 0 7 0 7 Prochloraz 0.1 103 1 102 0 93 1 Prochloraz 1 109 2 111 9 103 6 Dichlobenil 0.0001 106 11 104 7 104 7 Dichlobenil 0.001 97 4 95 1 93 2 Dichlobenil 0.01 102 12 99 13 96 14 Dichlobenil 0.1 96 9 100 2 94 6 Dichlobenil 1 103 1 104 2 99 1 Dichlobenil 10 97 16 102 11 99 8 Dichlobenil 100 101 6 100 4 99 3 SC NA 100 10 100 11 100 1 a Data were obtained from Tables 20–25 on pages 36–37 of the study report. Viability of solven t control (SC) wells was set at 100%. NA Not applicable



### C. QC PLATE: The hormone concentrations after expos ure to the reference chemicals, SC, and blank samples, as well as the fold difference change relative to SC (individual and mean ± SD) for the three assay runs are presente d in Table 6. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells. OCSPP 890.1550 Guideline requirements for basal hormone production in SC wells, induction by 10 μM fo rskolin, and inhibiti on by 1 μM prochloraz were met for testosterone a nd estradiol, with the minor exception of induction of testosterone (1.9-fold in Run 2 instead of 2-fold). Thus, the Guideline requirements were generally met, indicating that the assay was sensitive to induction and inhibition of testosterone and estradiol.
The variability (%CV) between the runs (calculated by the re viewer) based on the absolute hormone concentrations in the SC were 17% fo r testosterone and 18% for estradiol, within the guideline criteria of ≤ 30% for the assay. The variability (CV) within each run for the SC in the testosterone and estradiol assays was 3.6-14.6%, which was within the guideline recommendation of <30%.Steroidogenesis Assay (2011) / Page 10 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None

TABLE 6. Mean (±SD) Hormone Concentrations Followi ng Treatment with Forskolin or Prochloraz for 48 Hours. a
Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean b ± SD b Testosterone (pg/mL) Fold Difference (Relative to DMSO) Blank 1848 2409 2266 --- --- --- --- --- DMSO 1671 2297 2311 --- --- --- --- --- 1 μM Forskolin 3295 3795 4060 2.0 1.7 1.8 1.8 0.15 10 μM Forskolin 3234 4395 4570 2.0 1.9 2.0 2.0 0.06 0.1 μM Prochloraz 602 832 832 0.4 0.4 0.4 0.4 0.00 1 μM Prochloraz 174 244 295 0.1 0.1 0.1 0.1 0.00 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Blank 250 279 361 --- --- --- --- --- DMSO 262 296 370 --- --- --- --- --- 1 μM Forskolin 3978 4021 2523 15.2 13.6 6.8 11.9 4.46 10 μM Forskolin 5434 6330 4414 20.8 21.4 11.9 18.0 5.32 0.1 μM Prochloraz 169 201 171 0.6 0.7 0.5 0.6 0.10 1 μM Prochloraz 74 68 36 0.3 0.2 0.1 0.2 0.10 a Data were obtained from Tables 39, 41, 43, 45, 47, and 49 on pages 62–67 of the study report. Method detecti on limits were reported as 6.3–8.7 pg/mL for estradio l and 5.0–7.1 pg/mL for testosterone. b Calculated by the reviewers.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Dichlobenil does not show a consistent pattern in estradiol release in H295R cells in one out of th ree experiments; but did have a statistically significant decrease in te stosterone release in H295R cells in two out of three experiments. This could indicate that dichl obenil might be able to inhibit one or more enzymes in the steroidogenesis pathway. However, since no cl ear patterns were observe d, the results were considered to be equivocal.

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements were generally met, except that induction of te stosterone was 1.9-fold in Run 2 instead of 2- fold. Precipitation of the test compound was not reporte d, and cytotoxicity was not observed.
In Run 1, there were significant reductions in testosterone levels at 0.0001, 0.001, 0.01, 1 and 100 μM of dichlobenil, w ith the greatest reduction (0.71 -fold) occurring at 0.01 μM. During Run 2, decreased (p ≤ 0.05) testosterone levels (0.73- and 0.85-fold) were also noted at 0.0001 and 0.01 μM. However, no reductions in testosterone were noted in Run 3. No changes in estradiol concentration were obs erved during Runs 1 and 2, but estradiol was decreased (p ≤ 0.05) in Run 3 at all dichlobenil concentrations except 100 and 0.1 μM. Due to the inconsistencies and variability in the hormone alterations across the three runs, the results for dichlobenil in this assa y were considered to be equivocal

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

 Stability of dichlobenil in the test system was not reported.

 Forskolin induction of testoste rone in Run 2 was below the Guideline limit (2-fold).

 The laboratory proficiency test data were not provided.Steroidogenesis Assay (2011) / Page 11 of 11 DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None



###  Mean ± SD for the fold-differences were not reported, but could be calculated from the data presented.

 Cell passage number was not reported.

 Recoveries of spiked estradiol samples we re outside the Guide line criteria of 30%

 Inter-plate variation was 34% for estrad iol exceeding the Guideline criteria of ≤ 30%.

 Validation study to determine the medium does not affect the hormone system (NOTOX Project 493393) was not submitted.

 Cross reactivity of antibodies used in EIA was not reported.

# DATA EVALUATION RECORD

DICHLOBENIL
Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-27-2012 (MRID 48616111)

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: Sandra Hastings Date: 3/26/2012

Secondary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 3/28/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/04/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/04/2012

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).DICHLOBENIL/ 027401 

### Primary Reviewer: Sheila Healy, Ph.D. 

### Health Effects Division Secondary Reviewer: --'J'-'e=s=-s -=-R=o

__ w'-'l=an=d=---

________ 

### Signa tu re:**--t:--~.:.....:::=-~='----** 

### Health Effects Division Date:**--- ......__'5j-=-l--'c._** **£_>** 

# __ Template version 09/2011 

### DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



### PC CODE: 027401 

### DP BARCODE: D397912



### TXR#: 0051691 

### CAS#: 1194-65-6



### TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.)



### SYNONYMS: 2,6-dichlorobenzonitrile



### CITATION: Coder, P.S. (2011). A Uterotrophic Assay ofDichlobenil Administered Orally in Ovariectomized Rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study ID.: WIL-12433, December 7, 2011. MRID 48616111. Unpublished.



### SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT



### TEST ORDER# : EDSP-027401-120



### EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48616111) conducted to screen for potential estrogenic activity, dichlobenil (99.3% a.i., Lot #AM9Kl2A222) in 1.0 % gum tragacanth in deionized water was administered daily via oral gavage to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 (vehicle), 20, 60 or 180 mg / kg / day on post-natal days (PND) 63-65

. A positive control group was treated with a daily dose of l 7aethynyl estradiol (EE) at 0.003 mg / kg / day by subcutaneous (s.c.) injection. Body weights were determined daily

. All animals were terminated and necropsied on PND 66 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termination and no clinical findings were observed in animals dosed with dichlobenil at any level. Body weights were decreased (p < 0.05) by 8% on Day 3 and 9% on Day 4 (termination) at 180 mg / kg / day compared to controls, resulting in a decrease (p < 0

. 001) of 129% in overall body weight gai n

. Body weights in the 20 and 60 mg / kg / day dose groups were comparable to controls throughout the duration of the assay. Uterine weights in the dichlobenil treated groups were comparable to the vehicle controls.

The body weights for the positive control (EE) group were decreased (p < 0.05) by 8% on Day 4 and overall body weight gain for the EE animals was decreased (p < 0.001) by 104% in comparedIn vivo Uterotrophic Assay (2011) / Page 2 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440


### with the controls. Absolute wet and blotted ut erus weights for the positive control group were increased (p<0.001) by 766% and 2 34%, respectively, as expected.
The highest dose tested (180 mg/kg/ day) was determined to be adequate based on the decrease in body weight gain (129%) at this dose.
No statistically significant changes were seen in uterine weight in this assay. Dichlobenil was negative in the uterotrophic assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Uterotrophic Assay (2011) / Page 3 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: WIL Research Laboratories, LLC Location: Ashland, OH Study Director: Prägati Sawhney Coder Other Personnel: L. Freshwater (Scientist); K. Gardner (Scientist); E. Bodle (Assistant Director); C. Kopp (Manager); G. Maginn is (Clinical Veterinarian ); J. Moore (Operations Manager);C. Wally (Group Supervisor) Study Period: May 23, 2011 to December 7, 2011



### 2. Test Substance: Dichlobenil Description: Technical; white powder Source: Notox B.V. (Chemtura Canada) Lot/Batch #: AM9K12A222 (expiration date: October 12, 2012) Purity: 99.3% a.i. Stability: Not reported CAS #: 1194-65-6 Structure:



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich (St. Louis, MO) Lot/Batch #: 028K1411 (expiration date: August 4, 2011) Purity: >98% CAS #: 57-63-6



### 4. Solvent/Vehicle Control: Gum Tragacanth in deionized water Supplier: Spectrum Chemical Manufacturing C orporation (New Brunswick, NJ) Lot/Batch #: 2AC3069 (expiration date: August 31, 2012) Rationale (if other than water): Test substance is not soluble in water at the concentrations tested and 1.0% gum tragacanth created a homogeneous suspension of the test substance. Final concentration: 1.0%



### 5. Test Animals: Species: Rat (ovariectomized) Strain: Sprague Dawley Age/weight at dose initiation: 63 days old/ 228.3 – 277.5 g females Source: Charles River Laboratories, Inc. (Raleigh, NC) Housing: Individually caged in stainless steel wire -mesh cages suspended above cage-board; the cage-board was changed at least 3 times/week. Diet: Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet , ad libitum Water: Reverse-osmosis purified water , ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 21.3-21.4oC 45.7-59.2% 10/hr 12 hrs light/ 12 hrs dark Acclimation period: 10 daysIn vivo Uterotrophic Assay (2011) / Page 4 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440


### B. STUDY DESIGN

1. In Life Dates: Start: June 10, 2011 End: June 13, 2011

2. Study Design: Sexually mature ovariectomized female rats were received from Charles River Laboratories; rats were ovariectomized at PND 49 by th e supplier. Animals were received 4 days following ovariectomy (PND 53) and acclimated for 10 days prior to initiation of dosing. Vaginal smears were take n daily during the last five days of the acclimation period to verify complete removal of the ovaries as indicated by a lack of estrous cycling. The dose administration pe riod was from PND 63 through 65. Rats were euthanized approximately 24 hours later on PND 66 and necropsied for uterine weight measurements.
3. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significant differences between group mean weight s at study initiation.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control 0 6 Low Dichlobenil 20 6 Mid Dichlobenil 60 6 High Dichlobenil 180 6 17 α -ethynyl estradiol (EE), Reference Estrogen 0.003 6 a Data were obtained from Table 1 on page 15 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from a rangefinding study (MRID 48616113) in which dichloben il in 1% gum tragac anth in deionized water was administered daily vi a oral gavage to 5 adult ( 61 days old) female Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 20, 60, or 180 mg/kg/day for 3 consecutive days. A summary of the study is included as an A ppendix to this DER. Based on the results, the dose levels selected fo r the main study were 0, 20, 60 and 180 mg/kg/day.

5. Dose Preparation and Analysis: Dose formulations of dichl obenil were prepared daily in 1% gum tragacanth and stored at room temper ature in the dark. A stock solution of the positive control (EE) was prepared in 95% ethanol, and diluted with corn oil daily for dosing. Homogeneity and stability of dichlobe nil suspensions in 1.0% gum tragacanth in deionized water at concentrations 0.02, 1, a nd 50 mg/mL were tested by the laboratory. Samples (duplicates from the top and bottom strata) were analyzed for stability following 3 days frozen storage; additionally stability was tested with QC samples in 1.0% gum tragacanth in deionized water following 3 days storage at room temperature.In vivo Uterotrophic Assay (2011) / Page 5 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440 

### Homogeneity of the test formulations pr epared (June 9, 2011) for Day 1 dosing was evaluated on the day of prep aration (duplicates from the t op, middle and bottom strata) and following 8 days refrigerated storage (resuspen sion samples). Additi onally, samples of dose formulations for Day 1 dosing were analyzed for achieved concentr ation on the day of preparation.

Results of Dose Analysis
Homogeneity (% RSD): 1.9-5.5% RSD on the day of preparation 0.33-2.9% RSD following 8 days refrigerated storage

Stability (% of initial): 90.7 - 106% following 3 days frozen storage 104-115% following 3 days at room temperature

Concentration (% of nominal): 98.0-114%
It is noted that samples of dichlobenil susp ensions prepared (June 1, 2011) 9 days prior to dosing initiation were also te sted for homogeneity (top, middl e, and bottom strata) and the % of nominal in samples from the low concentration (0.02 mg/mL) was low and outside the acceptable %RSD. A second preparation of th e samples achieved acceptable criteria for 0.02 mg/mL but not for 50 mg/mL. These formul ations were not used for dosing, and the reviewer has reported (above) homogeneity for the formulations actually used for dosing. The analytical data indicated that the mixing procedure was ad equate for the formulations used in dosing and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Animals were administered th e test formulations or vehicle control (1.0% gum tragacanth in deionized water) daily via oral gavage and EE via s.c. injection for three consecutive days in dose volumes of 5 mL/kg body weight. Dose volumes were adjusted daily based on the most recent body weight measurement.

7. Statistics: Mean values were reported with standard deviations a nd coefficient of variance. All analyses were conducted using Statistical Analysis System version 9.2 (SAS Institute Inc., 2002-2008). Each endpoint was tested fo r homogeneity of variance using Levene’s test. If the test was significant at p ≤ 0.01, then a log transformation was applied for reanalysis. If that test was stil l significant then the square ro ot transformation was applied to the raw data and Levene’s test conducted. If the test was st ill significant then a nonparametric test was conducte d to analyze the data.
Organ weights were compared to the vehicle control group. If variances were homogeneous then the analysis of covariance was perfor med using the body weight at necropsy as the covariate. Comparisons of each treatment gr oup with the vehicle control group were made with a 1-sided Dunnett’s test using p ≤ 0.05 to declare significance. If transformations were unsuccessful in making the variances homoge neous, then the nonparametric Kruskal-Wallis test was used followed by Dunn’s test to compare each treatment group with the vehicleIn vivo Uterotrophic Assay (2011) / Page 6 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440 

### control group. Body weights and body weight gains were analyzed similar to organ weights but there was no covariate in the statistical model (ANOVA) and the testing was 2-sided instead of 1-sided. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and morbidity were conducted twice daily and individual clinical signs of toxicity were conducted once daily. The injection site was examined for erythema, swelling and other dermal findings. Animals were also observed for signs of toxicity 1-2 hours after dose administration and at termination.

2. Body Weight: Animals were weighed at randomi zation, daily prior to dosing and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine Weight: On PND 66 (approximately 24 hours after final dose administration), all animals were euthanized by carbon dioxide inhalation and subjected to a gross necropsy. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just below the cervix in order to retain the luminal fluid in the uterus. The “w et” uterus (i.e., contai ning the luminal fluid) was weighed. Subsequently, the uterine horns we re cut longitudinally and blotted with filter paper to remove the luminal fluid while prev enting desiccation and th e blotted uterus was weighed. The uterus and vagina were pres erved in 10% neutral-buffered formalin for possible future histopathologic examination.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS
1. Mortality: All animals survived until scheduled termination.

2. Clinical Signs of Toxicity: No treatment-related clinical fi ndings were observed in animals dosed with dichlobenil. However, it wa s noted that red material around the nose, lacrimation of the eyes, and decreased defecatio n were observed in single animals in lowand high-dose group; these findings were not considered test s ubstance related as there was no dose-response. Clear material around th e nose was observed in two animals in the positive control group (EE) on Day 2 and 3 following dose administration. Clinical observations in the study ar e presented in Table 2.In vivo Uterotrophic Assay (2011) / Page 7 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440

TABLE 2. Incidence of Clinical Observ ations in the Estrogen Agonist Assay a

Observation Dose (mg/kg/day)

Vehicle Control Dichlobenil (20) Dichlobenil (60) Dichlobenil (180) Reference Estrogen EE (0.003) # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined Decreased defecation 0 6 0 6 0 6 1 6 0 6

Wet clear material around mouth 0 6 0 6 0 6 0 6 2 6 Lacrimation (eyes) 0 6 1 6 0 6 0 6 0 6 Dried red material around nose 0 6 0 6 0 6 1 6 0 6 a Data were obtained from Tables S1 and S2, on pages 30-31 of the study report.



### B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 3. Body weight s were decreased (p<0.05) by 8% on Day 3 and 9% on Day 4 (termination) in the high dose gro up compared to vehicle controls, resulting in a decrease (p<0.001) of 129% in overall body we ight gain. Body weights in the low- and mid-dose groups were comparable to contro ls throughout the durat ion of the assay.
The body weights for the positiv e control (EE) group were decreased (p<0.05) by 8% on Day 4 and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in comparison with the vehicle controls.

TABLE 3. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day Dose (mg/kg/day)

Vehicle Control Dichlobenil (20) Dichlobenil (60) Dichlobenil (180) Reference Estrogen EE (0.003) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 6 250.3 14.97 6 249.4 11.99 6 251.1 15.34 6 251.0 12.48 6 248.8 12.52

2 6 257.8 17.11 6 249.6 ( ↓ 3) 13.17 6 253.1 ( ↓ 2) 13.59 6 241.7 ( ↓ 6) 9.20 6 253.3 13.66

3 6 263.3 17.01 6 255.6 ( ↓ 3) 14.41 6 255.2 ( ↓ 3) 14.54 6 241.9* ( ↓ 8) 6.50 6 251.7 12.15

4 (termination) 6 270.0 16.61 6 263.0 ( ↓ 3) 13.09 6 262.3 ( ↓ 3) 17.60 6 245.3* ( ↓ 9) 8.50 6 248.0* ( ↓ 8) 11.51 Body Weight Gain (Days 1-4) 6 19.7 3.82 6 13.6 ( ↓ 31) 2.14 6 11.2 ( ↓ 43) 6.38 6 -5.7*** ( ↓ 129) 10.13 6 -0.7*** ( ↓ 104) 4.74 a Data were obtained from Table 2 on page 21 of the study report; note the study numbered days started with 0 (dose initiation), but the reviewer numbered the stud y days starting with 1 (dose initiation). Percent differences from controls calculated by the reviewers are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05 *** Significantly different from controls at p<0.001In vivo Uterotrophic Assay (2011) / Page 8 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440



### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY
1. Uterine Weights: Selected uterine weight data are presented in Tabl e 4. Uterine weights in the dichlobenil treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive cont rol (EE) group were increased (p<0.001) by 766% and 234%, respectively. The mean blotte d uterine weight for the vehicle control group was 0.03% of terminal body weight, which was deemed acceptable to yield sufficient assay sensitivity and was consistent with th e requirement (<0.09%) of the test guideline.

TABLE 4. Uterine Weights from Estr ogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day)

Vehicle Control Dichlobenil (20) Dichlobenil (60) Dichlobenil (180) Reference Estrogen EE (0.003) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 6 270 16.5 6 263 13.3 6 262 17.8 6 245 8.3 6 248 11.6 Wet, absolute (g) 6 0.1044 0.0131 6 0.1014 0.0092 6 0.1017 0.0106 6 0.1065 0.0094 6 0.9042*** ( ↑ 766) 0.1588 Wet, relative (%) 6 0.039 0.0048 6 0.039 0.0037 6 0.039 0.0030 6 0.043 0.0038 6 0.366 0.0737 Blotted, absolute (g) 6 0.0917 0.0132 6 0.0896 0.0064 6 0.0900 0.0113 6 0.0939 0.0089 6 0.3063*** ( ↑ 234) 0.0343 Blotted, relative (%) 6 0.034 0.0051 6 0.034 0.0026 6 0.034 0.0033 6 0.038 0.0038 6 0.124 0.0169 a Data were obtained from Table 3 on page 23, and Tables S11 and S14 on pages 42 and 45 of the study report; it is noted that relative weights in Table 3 were reported as mg/100 g, and relative weights as g/100 g were reported in Table S14 (and reported herein for % relative weights). Percent differences from controls calculated by the reviewer are included in parentheses. BW Body weight N Number of animals in the group SD Standard Deviation *** Significantly different from controls at p<0.001



### 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the lack of effects on mean uterine weights (wet and blotted), dichlobenil did not demonstrate or mimic biological activities consistent with agonism of natural estrogens when administered orally to ovariectomized female rats at dose levels of 20, 60, and 180 mg/kg/day. The positive control substance (17 α -ethynylestradiol) elicited the expected increases in uterin e (wet and blotted) weights.In vivo Uterotrophic Assay (2011) / Page 9 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440


### B. AGENCY COMMENTS: All animals survived until scheduled termination and no treatment-related clinical findings were obser ved in dichlobenil-dosed animals. Body weights were decreased (p<0.05) by 8 and 9% on Days 3 and 4 in the high dose group (180 mg/kg/day dichlobenil) compared to vehicle controls, resultin g in a decrease (p<0.001) of 129% in overall body weight gain. Body wei ghts in the low- and mid-dose groups were comparable to controls throughout the durati on of the assay. Ut erine weights in the dichlobenil treated groups were comp arable to the vehicle controls.
The body weights for the positiv e control (EE) group were decreased (p<0.05) by 8% on Day 4 and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in comparison with the vehicle controls. Absolu te wet and blotted ut erus weights for the positive control group were increased (p<0.001) by 766% and 234%, respectively, as expected. No statistically significant changes we re seen in uterine weights in this study. Dichlobenil was negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: NoneIn vivo Uterotrophic Assay (2011) / Page 10 of 10 DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440



### APPENDIX
Since this is a range-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in th e Uterotrophic Assay (MRID 48616113).
In a preliminary range-finding assay, dichlobe nil (99.3% a.i.; lot# AM9K12A222) in 1% gum tragacanth in deionized water was administered daily via oral gavage in a dose volume of 5 mL/kg body weight to 5 adult (61 days old) female Sprague Da wley [Crl:CD(SD)] rats/dose group at doses of 0, 20, 60, or 180 mg/kg/day. The animals were dosed for 3 consecutive days and necropsied approximately 24 hours af ter the final dose administration.

All animals survived to the scheduled terminatio n. No treatment-related clinical findings were noted at the daily examinations at any dose. Decreased defecation was noted for 2 animals each dosed at 60 and one animal dosed at 180 mg/kg/da y. Significant (p<0.01 or p<0.05) decreases in food consumption (g/animal/day) were observe d in animals dosed at 180 mg/kg on Day 1 ( ↓ 42%), 2 ( ↓ 32%), and Day 4 ( ↓ 39%) and in animals dosed at 60 mg/kg on Day 1 ( ↓ 35%) and 2 ( ↓ 24%). Decreased food consumption corresponded to the variability in the mean body weights and/or mean body weight gains for these dose groups. However, mean body weights and body weight gains were not significan tly different from c ontrols in dichlobenil dosed rats.
Mean absolute and relative (to final body weights) adrenal gland weights in all dichlobenil dose groups were comparable to the control group. Significant (p<0.01 or p< 0.05) increases in the mean absolute and relative weights of the kidneys ( ↑ 16% and ↑ 25%, respectively) and livers ( ↑ 27% and ↑ 36%, respectively) of animals dosed w ith dichlobenil at 180 mg/kg/day were observed. No significant differen ces in kidney and liver weights were observed in animals dosed at 20 or 60 mg/kg/day. Based on these data, dichlobenil dose le vels of 20, 60 and 180 mg/kg/day were selected for the uterotrophic assay.